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Bir ailenin primer enürezis noktürnalı bireylerindeki genetik değişimlerin araştırılması
Amaç: Enürezis noktürna (EN), idrar kontrolünün kazanılması gereken yaşta, bireyin uyku esnasında istemsiz olarak idrarını yapması anlamına gelmektedir. 5 yaş ve üzerinde, gece kuru kalma dönemi olmaksızın uykuda yatak ıslatma durumunun devam etmesi primer enürezis noktürna; 6 ayın üzerinde gece kuru kalma durumu sonrasında yatak ıslatmanın olması ise sekonder enürezis noktürna olarak adlandırılmaktadır. Dünya genelinde enürezis noktürna prevelansı %3-24.4 arasında olarak bildirilmekle birlikte, ülkemizde %10,5-17,5 arasında seyretmektedir. EN %2-3 oranında ise erişkin yaşta devam etmektedir. Anne ve babanın birinde enürezis varlığında çocukta hastalık oluşma riski %43 iken, ebeveynlerin her ikisinde de enürezis öyküsü varlığında risk %77'ye yükselmektedir. EN etiyopatogenezini aydınlatmak üzere idrar volümünde artış, mesane kapasitesinde azalma, uyku problemleri olmak üzere üç ana mekanizma üzerinde yoğunlaşılmış ve bunun yanında kan basıncı artışı, hiperkalsiüri, obezite, psikolojik ve çevresel etmenler üzerinde çalışmalar yapılmıştır. Genetik çalışmalar, kromozom 8q, 12q13-21, 13q13-q14.3, 22q11, 4p16.1 bölgeleri ve bu bölgeler ile ilişkili GNAZ, DRD5, D1B, DRD4, nNOS genleri üzerinde yoğunlaşmış fakat henüz net bir kanıya ulaşamamışlardır. Gereç ve Yöntem: Bu çalışmada bir ailenin primer enürezis noktürnalı bireylerinin ailevi segregasyonu gösterilmiş 6 (altı) bireyinden (Grup 1) tüm ekzom sekanslama yöntemi ile analiz yapılmıştır. Ekzom sekanslama ile elde edilen verilerden uygun görülen genetik değişimler, segregasyonu gösterilemeyen ailenin diğer 7 (yedi) bireyinde (Grup 2) araştırılmıştır. Bulgular: Aile bireylerinin ekzom sekanslamasından elde edilen ortak veriler analiz edilmiş ve daha önceki çalışmalarda enürezis noktürna ile ilişkilendirilmiş kromozom 8q, 12q13-21, 13q13-q14.3, 22q11, 4p16.1 bölgeleri ile GNAZ, DRD5, D1B, DRD4 genlerinde hastalık nedeni olabilecek 91 herhangi bir değişim taşımadıkları saptanmıştır. Enürezis noktürna etiyopatogenezinde suçlanan idrar miktarında artış, vazopressin eksikliği, azalmış mesane kapasitesi, uyku ve uykuda solunum bozuklukları, otonom sinir sistemi disfonksiyonu, obezite, hiperkalsiüri, dikkat eksikliği ve hiperaktivite bozukluğu nedenlerine yol açabilecek biyolojik yolaklarda yer alan tüm genlerdeki ortak değişimler incelenmiş, bu değişimlerin tamamının benign karakterde olduğu görülmüştür. Sonuç: Enürezis noktürnanın etiyolojisinin aydınlatılabilmesi için daha fazla vaka ile tüm genom çalışmaları yapılması; patofizyoloji ve epidemiyolojiye yönelik daha fazla çalışma yapılması gerekmektedir. Bu çalışma, enürezis noktürna genetiği ile ilgili daha önce yapılmış bağlantı çalışmalarından sonra DNA dizileme ile yapılan dünya literatüründeki ilk çalışma olma özelliği taşımaktadır.
Tinnitus ile beyin-kaynaklı nörotrofik faktör (BDNF) gen polimorfizmleri ve BDNF serum düzeyi arasındaki ilişkinin araştırılması
Patofizyolojisi hakkında halen çok az şey bilinen tinnitus işitme sisteminin yaygın bir semptomudur ve dışarıdan herhangi bir uyarı olmaksızın kulakta veya başının herhangi bir bölgesinde ses duyulması olarak tarif edilmektedir. Beyin kaynaklı nörotrofik faktör (BDNF), nöronların büyümesi, farklılaşması, yaşamlarını sürdürmesinde ve iç kulak duyu epitelyumu dahil gelişmekte olan işitsel yolda anahtar rol oynayan nörotrofik faktörlerden biridir. BDNF gen polimorfizmleri [rs6265 (Val66Met), rs2030324 ve rs1491850], BDNF salgılanmasının düzenlenmesindeki anormalliklerle ve işitsel uyarılmış beyin sapı potansiyel test (BAER) sonuçlarındaki değişmeler ile ilişkili olduğunu ve ayrıca BDNF düzeylerinin tinnitusta ve depresyon, stres ile ilişkili olabilecek distimik bozukluk, konversiyon bozukluğu ve yaşam krizlerine bağlı intihar girişimleri gibi psikiyatrik bozukluklar ve durumlarda azaldığını gösteren çalışmalar mevcuttur. Tinnitus sıklıkla depresyon gibi duygudurum bozuklukları ile yakından ilişkilidir. Tüm bu veriler, tinnitus ile BDNF gen polimorfizmleri ve BDNF serum düzeyleri arasında bir korelasyon olabileceğini akla getirmektedir. Bu çalışmada, BDNF polimorfizmleri ve BDNF serum düzeyi değişiklikleri ile tinnitus arasındaki ilişki incelenerek, BDNF değişikliklerinin tinnitus patofizyolojisinde herhangi bir rolü olup olmadığını araştırmak amaçlanmıştır. Bu çalışmada, en az 3 aydır devam eden, tinnitus tanısı almış kişilerde, tinnitusa yatkınlıkta BDNF varyantlarının olası etkileri incelendi. çalışmaya CBÜ Tıp Fakültesi Kulak BurunBoğaz (KBB) polikliniğine başvuran 18-55 yaş arası 52 hasta ve herhangi bir KBB ve sistemik hastalığı saptanmayan aynı yaş grubunda 42 kişi kontrol grubu olarak seçildi. Bu özelliklere uygun seçilen olgular değerlendirmeye alınarak, klinik muayeneleri ve psikiyatri polikliniğinde uygulanacak DSM-IV eksen I bozuklukları için Yapılandırılmış Klinik Görüşme ölçeği (SCID-I) kullanılarak psikiyatrik tanıların ayırıcı tanısı yapıldı. Venöz kanlar kullanılarak elde edilen DNA örneklerinden BDNF polimorfizmleri genetik incelemesi ve BDNF serum düzeyi ölçümleri yapıldı. Sonuç olarak Tinnituslu hastalarda kontrol grubuna göre serum BDNF protein düzeyi daha düşük olarak saptanmış olup BDNF gen polimorfizmleri ile hastalık arasında ise anlamlı bir ilişki olmadığı gözlenmiştir. Anahtar Kelimeler: Tinnitus, BDNF gene, polimorfizm, serum BDNF
Spondiloepi (meta) fizyel displazi ön tanılı hastalardaki genetik değişimin araştırılması
İskelet displazileri orantısız boy kısalığı ve çeşitli ortopedik komplikasyonlarla karakterize 436 farklı türden oluşan kompleks bir hastalık grubudur. Klinik, radyolojik ve moleküler farklılıklarına göre alt tiplere bölünmüştür. Bu alt tiplerden biri olan Spondiloepifizyel Displazi Tarda Sendromu ilerleyici spondiloepifizyel displaziye sıklıkla prematür osteoartrit bulgularının eşlik ettiği X'e bağlı resesif kalıtımlı bir iskelet displazisidir. Xp22.2 kromozomal bölgesinde lokalize, golgi aygıtından endoplazmik retikuluma vezikül transportunda rol oynayan sedlin proteininin sentezinde görevli SEDL (sedlin; OMIM*300202) genindeki mutasyonlar bu sendromdan sorumlu tutulmaktadır. İlerleyici Psödoromatoid Displazi (PPRD; Spondiloepifizyel Displazi Tarda ve ilerleyici artropati) ise elin küçük eklemlerinde genişleme ve artrit benzeri bulgularla karakterizedir. 6q21 kromozomal bölgesinde lokalize WISP3 (WNT1-İndüklenebilir Sinyal Yolağı Protein 3; OMIM*603400) genindeki mutasyonların neden olduğu otozomal resesif kalıtımlı bir iskelet displazisi alt grubudur. Tüm Ekzom Dizileme genotipi bilinmeyen hastalıkların genotipini belirleme, iskelet displazileri gibi nadir görülen alt tiplerden oluşan ve aynı fenotipe sebep olan farklı genotiplerin görüldüğü genetik heterojenite gösteren kompleks multifaktöriyel hastalıklara tanı olanağı sağlayan bir teknolojidir. Bu çalışmada ailede sadece erkek bireylerin etkilendiği, aile ağacı X' e bağlı kalıtımı ya da otozomal resesif kalıtımı düşündüren, akraba evliliği sonucu dünyaya gelmiş iskelet displazili 4 erkek kardeş tüm ekzom dizileme yöntemiyle taranmış ve WISP3 geninde NM_198239.1:c.210C>A; (p.Cys70Ter) nonsense değişimi ve NM_198239.1:c.302G>A (p.Gly101Glu) missense değişimi homozigot olarak saptanmıştır. Sonuç olarak X'e bağlı resesif kalıtım paternine benzer kalıtım sergileyen hastalara; otozomal resesif kalıtımlı İlerleyici Psödoromatoid Displazi tanısı konuldu ve genetik değişimi belirlendi. Saptanan p.Cys70Ter değişimi Türk hastalarda en sık saptanan patojenik varyant olup mevcut literatürle uyumlu olduğu görüldü. Aile öyküsü, klinik ve moleküler bulguların genetik heterojenite gösteren kompleks hastalık gruplarında yol gösterebileceği ancak kesin tanı ve ailelere preimplantasyon genetik tanı imkanı sunabilmek için moleküler çalışmalarla desteklenmesi gerektiği akılda tutulması gereken önemli bir noktadır.
Multipl skleroz (MS) patogenezinde YKL-40 ve CRTH2 gen polimorfizmlerinin rolü
Amaç: Multipl skleroz (MS), genç erişkinlerde engelliliğin önde gelen nedenlerinden biridir. MS hastalığı, kronik ve progresif seyir göstermesi nedeniyle hastalar ve sağlık hizmetleri üzerinde ciddi bir yük oluşturmaktadır. YKL-40 proteini proinflamatuar sitokinlere yanıt olarak salgılanır ve birçok inflamatuar hastalık ve kanserle ilişkilendirilmektedir. Ağırlıklı olarak inflamatuar süreçlerde rol oynayan CRTH2 reseptörünün ise periferik sinir sisteminde (PSS) miyelinin oluşumu ve korunmasında anahtar bir rol oynadığı bildirilmiştir. Bu bağlamda, çalışmamızda, MS hastalığının patogenezinde YKL-40 ve CRTH-2 gen polimorfizmlerinin rolünün araştırılması hedeflenmektedir. Gereç ve Yöntem: Çalışmaya 123 MS tanılı hasta ve 117 sağlıklı gönüllü birey dahil edilmiştir. YKL-40 genine ait rs10399805 ve rs871799 promotör bölge polimorfizmleri ile CRTH-2 genine ait rs533116 upstream (transkripsiyon başlangıç noktasından önceki dizi bölgesi) ve rs11571288 3'UTR (3'translasyon olmayan bölge) polimorfizmleri incelendi. Genotipleme analizleri qPCR ve PCR-RFLP yöntemleriyle gerçekleştirildi. Hasta ve kontrol grupları ile klinik alt gruplar arasında; genotip ve alel dağılımlar için otozomal dominant (OD) ve otozomal resesif (OR) kalıtım modellerine göre ilişkilendirme analizleri gerçekleştirildi. Bulgular: rs10399805 ve rs533116 polimorfizmleri ile MS hastalığı arasında anlamlı bir ilişki saptanmadı. Ancak rs871799 polimorfizmine ait genotip dağılımlarının, OD modele göre incelenmesinde MS grubunda, sağlıklı kontrollere göre CG+GG genotipinin anlamlı şekilde arttığı tespit edildi. MS klinik alt grupları ile sağlıklı kontroller arasındaki karşılaştırmada, rs871799 polimorfizmine ait GG genotipinin sekonder progresif MS (SPMS) grubunda, G alelinin ise SPMS ve primer progresif MS (PPMS) gruplarında sağlıklı kontrollere kıyasla istatistiksel olarak anlamlı düzeyde arttığı tespit edildi. OD modele göre yapılan karşılaştırmada ise rs871799 polimorfizmine ait CG+GG genotipinin SPMS ve PPMS gruplarında sağlıklı kontrollere göre istatiksel olarak anlamlı şekilde arttığı saptandı. OR kalıtım modeline göre yapılan karşılaştırmada, rs11571288 polimorfizmine ait GG genotipinin atak ve remisyonlar ile karakterize (RRMS) hastalarında, kontrol grubuna kıyasla istatiksel olarak anlamlı şekilde arttığı tespit edildi. MS klinik alt gruplarının karşılaştırılmasında, rs871799 varyantına ait G alelinin PPMS grubunda anlamlı şekilde arttığı saptandı. Ek olarak rs871799 polimorfizmine ait GG genotipinin ve G alelinin, OD modeline göre CG+GG genotipinin ve OR modeline göre GG genotipinin, EDSS skoru ≥4 olan hastalarda anlamlı bir artış gösterdiği saptandı. Tartışma ve Sonuç: YKL-40 geni rs871799 polimorfizminin, YKL-40 ekspresyonuna etki ederek, MS hastalığının gelişimine ve ilerlemesine, nörolojik defektlerin daha şiddetli bir şekilde ortaya çıkmasına neden olabileceği sonucuna varıldı. Ek olarak, CRTH2 geni rs11571288 polimorfizminin inflamatuar hücresel yanıtların artışına yol açarak hastalığın ilerleyişine etki edebileceği düşünüldü. Bulgularımız, rs871799 ve rs11571288 varyantlarının, hastalık riski, klinik seyri izleme ve nörolojik hasarın şiddetini belirlemede potansiyel rolleri olduğunu düşündürmektedir. Bu bağlamda sonuçlarımızın potansiyel rolünü ortaya koyacak daha kapsamlı araştırmalar ile doğrulanmasına ihtiyaç duyulmaktadır.
Multiple skleroz ile PARP-1 geni arasındaki ilişkinin araştırılması
Multiple skleroz (MS), genç yaşlardaki engelliliğin önemli bir nedenidir. Vakaların büyük kısmı relaps ve remisyonlarla (RRMS) seyreden formda başlamakla birlikte ilerleyen dönemde sekonder progresif (SPMS) forma dönüşebilmektedir. Ataklar için çeşitli farmakolojik tedavi seçenekleri mevcuttur; fakat progresyon sürecinde bu tedavilere yanıt oldukça kısıtlıdır. Progresyona geçişte santral sinir sistemi (SSS) inflamatuvar karakterinin de değiştiği gösterilmiş ve progresyon patogenizinin ataklardan farklı olduğu fikri öne sürülmüştür. Poli (ADP-riboz) polimeraz-1 (PARP-1); gen ekspresyonu, hücre ölümü ve DNA onarımı gibi hücresel süreçte görevli bir enzimdir ve otoimmün-otoinflamatuvar yolaklardaki etkinliği de birçok çalışma ile bildirilmiştir. Çalışmamızda inflamatuvar bir hastalık olan MS ile PARP-1 geni ilişkisinin araştırılması hedeflenmiştir. Çalışma grubu 123 MS hastası ve 168 sağlıklı kontrolden oluşturuldu. PARP-1 genine ait, protein düzeyi ve fonksiyonu üzerine muhtemel etkisi olabilecek rs1136410 missense polimorfizmi, rs7527192 promotör bölge polimorfizmi ve rs8679 3'UTR polimorfizmi PCR-RFLP yöntemi ile çalışıldı. Hasta ve kontrol grupları ve klinik alt gruplar arasında; genotip ve allel frekansı dağılımları, haplotip dağılımları, otozomal dominant (OD) ve otozomal resesif (OR) kalıtım modeline göre ilişkilendirme analizleri gerçekleştirildi. rs1136410 ve rs8679 ile MS arasında anlamlı bir ilişki bulunamadı. rs7527192 CC genotipinin SPMS hastalarında kontrollere kıyasla istatiksel olarak anlamlı şekilde arttığı saptandı. OD kalıtım modeline göre SPMS hastalarında CC genotipinin sağlıklı kontrollere göre istatiksel olarak anlamlı şekilde arttığı saptandı. EDSS skoru >3-6 olanlarda CC genotipi ve C allel frekansının sağlıklı kontrollere göre istatiksel olarak anlamlı şekilde arttığı saptandı. EDSS skoru >3-6 arasında olan grubun ≤3 ve ≥6 olanlara kıyasında C allel frekansının istatiksel olarak anlamlı şekilde arttığı saptandı. PARP-1 geni promotor bölge varyantı rs7527192, PARP-1 ekspresyonunu etkileyerek SPMS'de progresyon mekanizması üzerinde etkili olabilir. PARP-1'in inflmazom yolağı aktivitesini arttırarak, süreklilik kazanan bir doğal immün yanıta neden olabileceği düşünülmüştür. Bu noktada PARP-1 inhibitörlerinin oldukça kısıtlı tedavi seçeneği bulunan SPMS'de umut vadedici bir tedavici seçeneği olabileceği göz önünde bulundurularak yapılacak ileri çalışmalara ihtiyaç vardır.
Metamfetamin bağımlılığı patogenezinde HCRTR ve NPSR genlerinin rolü
Bağımlılık, bireyler üzerindeki tüm ciddi fiziksel ve psikolojik olumsuz etkilerine rağmen, bazı madde ve kimyasalları kompulsif arama ve alma davranışı ile karakterize nörogelişimsel, genetik ve sosyokültürel etmenlerin rol oynadığı kronik bir hastalıktır. En sık suistimal edilen yasadışı psikostimulanlardan biri olan metamfetamin, merkezi sinir sistemini etkileyen güçlü ve bağımlılık yapma riski yüksek bir uyarıcıdır. Metamfetamin, merkezi ve periferik sinir sistemindeki sinir terminallerinden dopamin, serotonin, noradrenalin ve adrenalin salınımını tetiklemektedir. Ayrıca oksidatif stres, nöroinflamasyon ve nörotoksisite olmak üzere en az üç farklı moleküler mekanizmayı modüle ettiği anlaşılmıştır. Beyindeki birçok nöronal devre bağımlılık gelişimi ile ilişkilendirilmiş olup, yapılan son çalışmalar hipotalamustaki oreksinerjik nöronlar ve bu nöronlarla yakın ilişkide olan nöropeptid S sisteminin bağımlılık gelişiminde rol oynayabileceğini düşündürmüştür. Çalışmamızda metamfetamin kullanım bozukluğu (MKB) olan hastalarda ve sağlıklı gönüllülerde HCRTR1 rs2271933 ile rs41263963, HCRTR2 rs2653349, NPSR rs324981 gen polimorfizmlerinin frekansları ve Oreksin A serum düzeyleri belirlenmiştir. Bu çalışmada söz konusu parametrelerin MKB patogenezindeki rollerinin araştırılması amaçlanmıştır. MKB tanısı konan 173 hasta ile 102 sağlıklı gönüllü çalışmaya dahil edilmiştir. Gruplar arasında serum Oreksin A düzeylerinin MKB grubunda, kontrol grubuna göre anlamlı düzeyde düşük olduğu belirlenmiştir. Ayrıca MKB hastalarının detoksifikasyon sonrası aşerme dönemlerinde, detoksifikasyon öncesi dönemlerine göre daha yüksek oreksin A düzeylerine sahip olduğu saptanmıştır. HCRTR1 rs2271933 ile rs41263963, HCRTR2 rs2653349, NPSR rs324981 gen polimorfizmlerinin genotip ve allel frekansları dağılımı açısından hasta ve kontrol grubu arasında istatistiksel olarak anlamlı farklılık bulunamamıştır. Sonuç olarak; Oreksin A düzeyinin MKB ve dönemlerinin patogenezinde etkili olduğunu düşündürmekte olup, gelecekte daha geniş popülasyonlar üzerinde yapılacak olan çalışmalar ile hipokretin ve nöropeptid S reseptör polimorfizmlerinin ve oreksin A düzeyinin MKB patogenezindeki rolünün daha net olarak anlaşılması beklenmektedir.
Mitokondriyal DNA Mutasyonları
Mitokondriyal hastalıklar, genellikle çocukluk çağında başlayan ve değişken klinik özelliklere sahip bir hastalık grubudur. Mitokondri, kendi genetik materyaline sahip, hücre içinde oksidatif fosforilasyon ve hücresel enerji üretimden primer olarak sorumlu bir organeldir. Mitokondriyal fonksiyonlardan sorumlu alt ünitelerin bir kısmı mitokondriyal DNA'da, kalanı ise nükleer DNA'da kodlanmaktadır.Mitokondriyal DNA'daki nokta mutasyonlar, mitokondriyal hastalıkların büyük bir kısmından sorumludur. Mutasyonların tespitinde sekans analizi, PZR/RFLP, DHPLC gibi yöntemlerin yanı sıra son yıllarda mikroarray tabanlı yeniden sekanslama teknikleri kullanılmaktadır. Mikroarray tabanlı olarak geliştirilen Mitochip V2.0 yeniden sekanslama sistemi, kısa sürede sonuç verme, tüm mitokondriyal genomun tek analizde taranabilmesi, uygun maliyetli olması ve güvenilirlik oranının yüksekliği nedeniyle mitokondriyal hastalıkların moleküler tanısında oldukça etkin ve tercih edilebilir bir yöntemdir.Bu çalışmada, klinik bulgularıyla mitokondriyal hastalık tanısı almış 6 olgu ve annelerinin mitokondriyal genomu, mikroarray tabanlı yeniden sekanslama yöntemiyle analiz edilmiş, elde edilen sonuçlarla literatürdeki veriler karşılaştırılarak genotip fenotip ilişkisi kurulmaya çalışılmıştır.
Kronik myeloid lösemi ile IL-3, IL-6 ve IL-11 gen ekspresyonları arasındaki ilişki
Giriş ve Amaç: KML hematopoetik kök hücrelerin transformasyonundan köken alan hematopoetik bir malignensidir. KML hastalarının çoğunda 9. ve 22. kromozomlar arasında bir translokasyon sonucu oluşan BCR-ABL geni saptanır. IL-3, IL-6 ve IL-11 myeloid seri hücrelerinin gelişimini etkileyen sitokinlerdir. Çalışmamızın amacı KML gelişimi ile IL-3, IL-6 ve IL-11 arasında bir ilişki olup olmadığının değerlendirilmesidir. Materyal ve Yöntem: Bu çalışmada Karadeniz Teknik Üniversitesi Tıp Fakültesi İç Hastalıkları Anabilim Dalı'na bağlı Hematoloji Polikliniği'nde takip edilen 26 ile 82 yaşlaı arasında 33 KML hastasından ve hastanemiz poliklinik bünyesinde yapılan muayene ve tetkikler sonucu sağlıklı olduğu tespit edilen, yaşları 29 ile 81 arasında değişen 34 bireyden kan alınmıştır. Bu kanlardan RNA izoleedilerek cDNA'lar sentezlendi. Bu cDNA'lar kullanılarak IL-3, IL-6 ve IL-11 gen ekspresyon analizleri yapıldı. Bulgular: İlk tanı anında gelen ve BCR-ABL ekspresyonu yüksek olan KML hastalarındaki IL-3, IL-6 ve IL-11 gen ekspresyonları, sağlıklı bireylere oranla önemli derecede azalmıştı. BCR-ABL ekspresyonu olmayan, majör moleküler yanıt tespit edilen KML hastalarında ise ilk tanı anına göre IL-3, IL-6 ve IL-11 gen ekspresyonlarında artış söz konusuydu. Tartışma: Elde ettiğimiz sonuçlara göre KML'deki BCR-ABL ekspresyonunun artışı normal hematopoezde etkili olan IL-3, IL-6 ve IL-11 gen ekspresyonuna ihtiyaç duymadan, neoplastik hücrelerin çoğalmasını sağlayabilmektedir. KML hastalarımızda BCR-ABL ekspresyonu ile IL-3, IL-6 ve IL-11 ekspresyonu arasında ters orantı mevcuttur. IL-3, IL-6 ve IL-11 gen ekspresyonlarının; BCR-ABL ekspresyon seviyesi ile ilişkisini belirlemek ve KML hastalarının takibinde kullanılmak üzere daha ileri çalışmalara ihtiyaç vardır.
Mikrorna'ların hashimoto tiroiditi'ndeki rolü
MikroRNA (miRNA) 'lar son yıllarda ortaya çıkan, 18-24 nükleotid uzunluğunda küçük kodlama yapmayan (noncoding) ve büyük bir çoğunluğu hücre içinde bulunmakla birlikte ekstrasellüler alanda, vücut sıvılarında ve kanda bulunabilen RNA molekülleridir. miRNA'lar hücre proliferasyonu, farklılaşması, metabolik süreçler, apopitoz, inflamasyon ve immünolojik süreçlerin düzenlenmesi gibi yolaklarda düzenleyicidirler. Çeşitli çalışmalarda yapılan gözlemler, insan plazmasında bulunan miRNA'ların yüksek bir stabiliteye sahip olduğunu ve bu örneklerde yapılan miRNA ekspresyon kalıbı analizinin çeşitli hastalıkların durumları hakkında yararlı bilgiler sunabileceğini ortaya koymuştur. miRNA'ların solid dokulardan elde edilen örneklere ek olarak hasta serum/plazmasında da belirlenebilmesi, tanısında mutlaka girişimsel tekniklerin kullanılmasını gerektiren hastalıklarda oldukça kolay bir örnek eldesini sağlar. Potansiyonel olarak gen veya protein sentez bozukluğu aracılı her hastalık, hücre tipi veya doku tipi miRNA tabanlı hedefleri oluşturabilirler. miRNA'lar gelişimsel açıdan mesajcı RNA'ların protein üretmesini düzenleyici rol üstlendiğinden, terapötik açıdan önemli olmalarını sağlamaktadır. Hashimoto Tiroiditi (HT), T ve B lenfositlerin tiroid dokusunu istila ederek parçalaması sonucu Anti-TPO ve Anti-TG antikorlarının üretimi ile karakterize bir hastalıktır. En yaygın görülen otoimmun ve endokrin hastalıklardan birisidir. miRNA'ların immün sistemi kontrol etmede önemli bir yeri olduğunu son yıllarda yapılan bilimsel çalışmalar açıkça göstermektedir. HT'de bunu tiroid dokusuna infiltre olan lenfositleri aktive ederek yaptıkları düşünülmektedir. Bu sebeple sirkülatuar miRNA'nın tanımlanması hastalığın kliniği ve patogenezinde önemli ve yeni bilgiler sağlayabilir. Hashimoto Tiroiditi'nde yeni tanı anında ve aynı hastaların ötroid dönemdeki miRNA profili bu güne kadar henüz çalışılmamış bir konudur. Biz de bunu göz önünde bulundurarak TSH'ın yüksek olduğu tanı anında ve aynı hastaların ötroid oldukları kontrol dönemindeki miRNA ekspresyon düzeylerini belirleyip sağlıklı kontrol grubu ile aralarında ekspresyon düzeyi açısından bir fark olup olmadığını tespit etmeyi amaçladık. Çalışmamız ile HT'nin şimdiye dek tam olarak aydınlığa kavuşamamış olan patogenezine yeni bir bakış açısı sunarak hem literatüre katkı yapmayı planlıyoruz; hem de bu yeni bakış açısının HT güncel tedavisine yeni ufuklar açmasını bekliyoruz. Çalışmamızda Karadeniz Teknik Üniversitesi Tıp Fakültesi İç Hastalıkları Anabilim Dalı'na bağlı Endokrinoloji Polikliniği'ne gelen ve yeni tanı Hashimoto Tiroiditi olup TSH değerleri yüksek olan 34 hastadan tanı anında ve 3 ay sonra ötroid oldukları dönemdeki halleri ile endokrinoloji polikliniği'nde yapılan muayene ve tetkikler sonucu herhangi bir sağlık problemi tespit edilmeyen, hasta grubu cinsiyet ve yaşı ile uyumlu 33 gönüllü birey çalışmaya dahil edilmiştir. Hasta ve sağlıklı bireylerden EDTA'lı kan örnekleri alınmıştır. Plazmada eksprese olduğu bilinen ve otoimmunite ile ilişkisi daha önceki çalışmalar ile ortaya konmuş 5 farklı miRNA'nın (mir-22, miR-141, miR-155, miR-375, miR-451) ekspresyon düzeyleri hasta ve kontrol grubunda çalışılmıştır. Hasta ve kontrol grupları arasındaki miRNA ekspresyon düzeylerinin kantitatif olarak belirlenmesi amacıyla RT-PCR verileri Qiagene Gene Globe Veri Analizi Merkezi portalı ΔΔCt modülü kullanılarak analiz edilmiştir. (http://www.qiagen.com/us/shop/genes-and-pathways/data-analysis-center-overview-page/). Veri normalleştirmek için yine aynı analiz merkezinde bulunan "Global CT Mean Of expressed miRNAs" kullanıldı. Gruplar arasındaki miRNA ekspresyon düzeyleri arasındaki ilişkisi Student T testi ile analiz edildi (p<0.05 anlamlı olarak kabul edildi). Hasta grubun ilk tanı anı ve üç ay sonraki kontrol dönemi arasındaki TSH ve miRNA ekspresyon düzeyi arasındaki ilişki Spearman korelasyon analizi ile hesaplanmıştır. Veriler IBM SPSS Statistics for Windows 23.0 istatistik paket programında analiz edilmiştir. Yapmış olduğumuz bu çalışma plazmadaki sirkülatuvar miRNA'ların otoimmun bir hastalık olan HT'de hastalık aktivitesi ile ilişkili olabileceğini ortaya koymaktadır. Özellikle miR-451'in gen ekspresyonunda yeni tanı anındaki TSH'ı normal sınırın üzerinde olan hasta grubunda, sağlıklı kontrol grubuna göre 1,495 kat artış (p=0,434) saptanması; bununla birlikte hastaların tedavi sonrası TSH değerlerinin normale döndüğü kontrol döneminde sağlıklı kontrollere göre aynı genlerin ekspresyonunda 0,545 kat azalma (p=0,042) gözlenmesi ve hasta grubunun kendi arasındaki karşılaştırmasında da TSH'ın yüksek olduğu dönemde normal olduğu döneme göre miR-451 gen ekspresyonları arasında 2,74 katlık artış (p= 0,00262) tespit edilmesi miR-451'in hastalık aktivitesi ile yakından ilişkili olduğunu düşündürmektedir. Otoimmun bir hastalık olan HT, patogenezi hala aydınlatılamamış bir hastalıktır. miRNA'ların son yıllardaki hastalık patogenezlerindeki önemli etkilerinin ortaya konması ile birçok hastalık patogenezi aydınlatılmış, tedavi alternatifleri sunulmuştur. Bizim çalışmamız ile ortaya konulan sonuçlar ışığında HT patogenezini aydınlatmada ve yeni tedavi yaklaşımlarının geliştirilmesinde öncülük edecektir.
Türk toplumunda çocukluk çağı obezitesinin MC4R geni ve bu gene komşu bölgelerde meydana gelen mutasyon ve polimorfizmlerle olan ilişkisinin araştırılması
Çocukluk çağı obezitesi, özellikle gelişmiş ülkelerde olmakla beraber bütün dünyada artan bir prevalansa sahiptir. Halk sağlığına olan önemli etkisi açısından, çocukluk çağı obezitesi yakından takip edilmelidir. Birçok araştırıcı MC4R polimorfizmlerinin ve güncel olarak da MC4R yakın bölge değişikliklerinin erişkin ve çocuklarda ve monogenik obezitenin nedeni olabileceğini gösteren çalışmalar yapmıştır.Bu tez çalışmasında Türk toplumunda çocukluk çağı obezitesinin MC4R geni ile bu gene komşu bölgede bulunan Ser58Cys, Val50Met, Ile102Ser, Val103Ile, Asn274Ser, rs17782313 ve rs17700633 polimorfizmleri arasındaki ilişki araştırılmıştır.Val50Met polimorfizmi, kontrol grubunda daha yüksek bulunmuş ve obeziteyle arasında negatif ilişki kurulmuştur. MC4R yakın bölge polimorfizmlerinden rs17700633 çocuk obez hastalarda insülin direnciyle ilişkili olarak bulunmuş ve yine araştırılan MC4R yakın bölge polimorfizmlerinden rs17782313 polimorfizminde ise polimorfik alleli taşıma sıklığı çocuk obez hasta grubunda daha yüksek bulunmuştur. Son olarak, rs17782313 polimorfizmini taşıyanların %40.4'ünün aynı anda rs17700633 polimorfizmini de taşıdığı saptanmıştır. Bu konuda yaptığımız çalışma ve gelecekte daha çok hasta üzerinde yapılacak yeni çalışmalar obezitenin genetik nedenlerini aydınlatılabilecek ve saptanan genetik bozukluklar riskli guruplarda bir test paneli şeklinde taranabilecektir.
Gelişimsel kalça displazisi ile ilişkili gen/ genlerin araştırılması
Gelişimsel Kalça Displazisi (GKD), femur başının asetabuluma yerleşmesi ile ilgili sık rastlanılan bir anomalidir. Kapsüler laksisite ile femoral başın hafif insitabilitesinden, femoral başın asetabulumdan tam çıkığına kadar değişen şiddette görülebilmektedir. Genelde bilateral olup, kadınlarda, erkeklere oranla daha sık (5:1) görülmektedir. GKD daha çok izole olarak karşımıza çıkmakla birlikte, farklı sendromların bulgusu ya da kromozomal anomalilerle birlikte de görülebilmektedir. Görülme sıklığını etkileyen önemli bir diğer faktör de, coğrafik ve ırksal dağılımlardır.Literatürde, bu hastalığının kalıtım modeli olarak sıklıkla otozomal dominant ve/veya multifaktöriyel kalıtım ifade edilmekle beraber, ortak olarak tek bir gen veya birkaç gen tanımlanmamaktadır. Bu tez çalışması ile bağlantı analizi tekniği kullanılarak GKD hastalığının etiyolojisinde sorumlu olabilecek aday gen ve/veya genlerin belirlenmesi hedeflenmiştir. Çalışma kapsamına, penetrans eksikliği ile birlikte giden otozomal dominant kalıtım modeline sahip, 16 hasta ve 15 sağlıklı bireyden oluşan iki geniş aile alınmıştır. Hastalık ile ilişkili olabileceği düşünülen 3 aday bölgenin (4q32, 6q16 ve 13q23) çalışılması yapılmış ve yapılan haplotip ve iki noktalı LOD skor analizleri sonucunda, GKD ile bu bölgelerin hiçbirine bağlantı gösterilememiştir.Sonuç olarak, bu tez ile iki büyük ailede bağlantı analiz çalışması yapılarak, günümüze kadar GKD ile birlikteliği sunulan 3 aday bölgenin ilişkili olmadığı gösterilmiştir. Bu da genetik heterojen olan bu hastalıkta, başka aday genlerin sorumlu olabileceği düşüncesini doğrulamaktadır. Bu nedenle hastalıkla ilişkili olabilecek genleri belirlemek için, bağlantı analiz çalışmalarının devam edilmesinin veya tüm genomun rastgele taranması prensibine dayalı genom-boyu tarama çalışmalarının yapılmasının anlamlı olabileceği düşünülmektedir.
Identification of diagnostic and prognostic indicators of hypercoagulability in patients with pseudoexfoliation syndrome
Patients diagnosed with Pseudoexfoliation (PEX) syndrome are highly detected in the group of people over 50 years old. The risk of thrombotic incidents increases in the PEX patients due to the impact of abnormal intraocular vascular deposition. This study aims to clarify the fundamental mechanism of possible hypercoagulability state in the PEX patients leading to venous thrombosis and secondary disease in this study group. Eighteen ocular PEX syndrome cases (mean age 63.6 ± 6.5 years) attended as a study group and nineteen cases without PEX syndrome (mean age 69.7 ± 4.4 years) as a control group participated in this study. Blood samples were analyzed by ROTEM®, Complete Blood Count test and Flow cytometry. A one-tailed Mann-Whitney was performed for statistical assessment of the data presented in this study. The mean values of the ROTEM analysis were significantly decreased in the FIBTEM clotting time (CT) (60.3 vs 56.9; P <0.05) and increased FIBTEM alpha-angle (70.0 vs 73.3; P <0.05) when comparing the control group with PEX patients. In flow cytometric measurement, data showed a considerable difference in the expression level of CD62P (P < 0.0001) and PAC-1 (P <0.05) between the study groups. Antigen-binding capacity (ABC) analysis revealed Furthermore, a significant difference in the CD62p marker (P <0.05). Complete Blood Count parameters of the PEX group did not show any significant differences compared to the controls. The result from the study suggests the higher contribution of functional fibrinogen in clot formation and a high level of platelet activation markers that expressed in the PEX patients. These findings support the idea that PEX syndrome increase the chance of hypercoagulability in PEX patients. Further investigations are recommended to broaden our knowledge of the exact role of platelets and functional fibrinogen in the initiation or development of PEX syndrome. Keywords: Hypercoagulability, ROTEM, Pseudoexfoliation syndrome, Coagulation
Assessing the epigenetic modifiers of drug resistance in human astrocyte and glioblastoma co-cultures
Glioblastoma (GBM) is the most common and malignant of all primary CNS tumours. Unfortunately, GBM has very low survival. The standard treatment regimen consists of surgery, ionizing radiation, and chemotherapy. The most common chemotherapeutic used for the treatment of GBM is temozolomide, followed by carmustine, which could not change the overall survival rates in the last 25 years. Unfortunately, most drug trials fail at the initial phases. For this failure in progress, tumour-microenvironment interactions play an important role, beside others causes. Therefore, understanding the nature of therapy resistance in relation to tumour-microenvironment interactions is essential. In this thesis, a new co-culture model was established to examine temozolomide response of tumour cells in the context of astrocyte microenvironment. Contact-dependent and contact-independent co-culture models were examined with cell viability and imaging methods. Contact-dependent astrocyte-U87MG co-cultures presented temozolomide resistance. Transcriptomic differences between U87MG cells alone and in co-cultures with astrocytes were examined by cell sorting, followed by RNA sequencing. As a testament to increased cell-to-cell interaction, co-culture models were found to express high levels of several cell-extracellular matrix interaction pathway components. Specifically, increased levels of collagen (COL), matrix-metalloproteins (MMP), and tubulin (TUB) families were observed. These adaptive changes were most likely vital elements of drug resistance in co-cultures. As contact-dependent temozolomide resistance may involve genetic and epigenetic changes, epigenetic vulnerabilities of tumour cells in co-cultures were examined. Cancers show altered epigenetic regulation; global changes in DNA and histone methylation. Yet, the epigenetic regulation of temozolomide response in co-cultures is not established. We performed a small molecule epigenetic inhibitor probe library screen to examine and identify epigenetic regulators of temozolomide response in co-cultures. We identified several histone demethylase inhibitors, in combination with temozolomide, as potent agents to target U87MG cells cultured with astrocytes. We then focused on GSK-J4, a histone demethylase inhibitor, as a potent agent that could affect GBM cells that are grown in co-cultures. GSK-J4 is a known agent that inhibits KDM6A and KDM6B, whose roles have not been studied in the context of temozolomide response in GBM. To understand the mechanisms of GSK-J4 in co-cultures, cell viability assays with chemical inhibition and CRISPR/Cas9-targeted gene silencing of KDM6A or KDM6B were applied. Further CITE-single cell sequencing was performed in U87MG-astrocyte co-cultures in the presence and absence of GSK-J4. We showed that both cell compartments were differentially represented at their transcriptome levels. Hypoxia and glycolysis-related genes MT-CYB, MT-CO2, MT-ND2, and MTRNR2L10 were upregulated and apoptosis-related genes were down-regulated in both tumour cells and astrocytes in response to GSK-J4. In conclusion, by generating and characterising co-culture models of GBM, we were able to find epigenetic regulators of temozolomide response and present transcriptomic differences that may serve as potential therapeutic intervention points for GBM in the future.
Development of brain-mimetic hydrogels for modelling neuronal differentiation
Biomechanically and biochemically tunable brain tissue models are notably essential for tissue engineering applications and neuroscience studies. Derivation of hydrogels through decellularization of native tissues is a promising strategy to reconstitute the native brain extracellular matrix for use in in vitro human models. Due to distinct features of the brain tissue and its implications on cellular behavior, it is particularly important to characterize and modulate the biochemical and biomechanical properties of constructed hydrogels from decellularized tissues. In the present study, we investigated the use of bovine brain tissue as a biomaterial carrier for neuroscience studies, assessed whether it could be an advantageous replacement for the human brain with easy accessibility, reproducibility and microenvironmental resemblance. We established and examined different methods for decellularization of bovine brain tissue and fabrication of reconstituted hydrogels. The decellularized tissues were evaluated with histological assessments and biochemical assays to both confirm elimination of cellular material and conservation of extracellular matrix components. Afterwards, decellularized tissues were solubilized with enzymatic digestion and reconstituted under physiological conditions in order to form hydrogels with thermal crosslinking capability. Mechanical characterization of hydrogels was performed to assess their stiffness and viscoelastic properties. Hydrogels were then tested for their three-dimensional cell encapsulation efficiency and their cytocompatibility with neuroblastoma cell line (SH-SY5Y) in culture. Collectively, it was shown that each decellularization technique resulted in different biochemical and biomechanical properties and these factors affected cell growth and behavior such as the degree of neurite formation. Given that mechanical microenvironment acts as an important parameter in cancer and neurodegenerative diseases, the results of this study provide significant insights. In the second part of the study, neuronal differentiation of neuroblastoma cells was investigated under 2D and 3D cell culture conditions to assess the effect of culture dimensionality and the presence of native brain matrix ligands on cellular fate. For this purpose, neuroblastoma cells were either grown on cell culture plate or encapsulated within decellularized brain-derived hydrogels. Following a neuronal differentiation regime, cells were evaluated morphologically through brightfield microscopy to determine neurite formation. Then, the expression of neuronal markers was assessed on both protein level by immunostainings and gene level by qRT-PCR. In conclusion, it was shown that synaptogenesis was improved by differentiated cells with elongated neurite formation in both 2D and 3D cultures. The proliferation rate was reduced and the gene expression levels of neuronal markers, including TUBB3 and CHAT were increased. Besides the common trends, significant differences were also observed between 2D and 3D cultured differentiated cells, whereas in 3D culture an increase in GFAP, glial cell marker, was detected.
Discovery and analysis of novel microRNAs playing a role in cancer dormancy
Recurrence after cancer treatment accounts for most cancer-related deaths. Disseminated tumor cells can settle in tissues and organs in a quiescence-like state for years or even decades. These quiescent cells are called dormant cells. Dormant cells, when reactivated, form more aggressive and treatment-resistant tumors. Therefore, study of cancer dormancy is very important from a basic science and a clinical perspective. MicroRNAs (miRNAs) are non-coding RNAs playing crucial roles in gene expression regulation. miRNAs generally interact with the 3' untranslated region (UTR) of the gene their target mRNAs, and lead to their degradation or block their translation (3,4). The role of microRNAs in dormant cancer cell behavior is not clear. In our lab, a Matrigel 3D cell culture system was optimized as an in vitro model of cancer dormancy. To discover new players in cancer dormancy using this system, miRNA Sequencing (miR-Seq), RNA Sequencing (RNA-Seq) and proteomics analyses were performed. Comparison of miRSeq results of actively dividing cancer cells with their dormant counterparts revealed a list of dormancy-associated novel miRNAs. As a result of the genetic manipulation of two novel dormancy microRNAs in cancer cell lines, we demonstrated the importance of the miRNAs for dormant cell behavior. Direct gene targets of the miRNAs playing a role in the dormant phenotype were discovered. Study of dormancy-associated miRNAs will allow a better understanding of molecular pathways of cancer dormancy and contribute to the development of new diagnosis, follow-up, and treatment strategies.
Ailesel olan ve ailesel olmayan şizofreni hastalarında kromozomal yeni düzenlenimlerin konvansiyonel sitogenetik ve moleküler sitogenetik yöntemlerle incelenmesi
Aile, ikiz ve evlatlık çalışmaları, şizofreni gelişimi için en önemli risk faktörünün bireyin yakınlarında şizofeni hastalığının bulunması olduğunu göstermiştir. Bu tip çalışmalar genetik faktörlerin şizofreni etiyolojisinde ne kadar etkin olduğu göstermektedir.Özellikle idiyopatik zeka geriliği olan hastaların önemli bir kısmına (%5) tanı koydurması sebebiyle yaygınlaşan subtelomerik FISH uygulamasının başka hastalıklar açısından da önemli olabileceğini düşünmek anlamlıdır. Çünkü subtelomerik bölgeler, gen içeriği bakımından çok zengin olup santral sinir sisteminin gelişimiyle ilişkili genleri içeriyor olabilirler. Yakın zamanda yapılan, şizofreni tanısına eşlik eden subtelomerik delesyonu olan olgu bildirimleri, bu hasta grubunda subtelomerik yeniden düzenlenimleri araştırmamızı sağlamıştır. Yine FISH çalışması ile tanısı konulabilen, şizofreni ile ilişkili 22q11.2 delesyon sendromunu teşhis edebilmek için, bu sendroma özgü prob da çalışmamızda kullanılmıştır.Ailesel şizofreni olgularının katıldığı çalışmamızda, hastaların ve 1. derece akrabalarının dismorfik muayeneleri ve HRB analizleri yapılmış, ayrıca hastalara subtelomerik ve 22q11.2'ye özgü problarla FISH uygulanmıştır. FISH çalışmasına konu olan ilgili bölgelerde yeniden düzenlenimle karşılaşılmamıştır. Hastaların klinik değerlendirmesinde literatürle uyumlu olarak obezite, kulak, damak anomalilerinin nispeten sık olması dikkat çekmiştir. Bu bulgulara ek olarak hastaların bir bölümünde daha önceden bildirilmemiş olan açık/soluk ten renginin görülmesi dopamin yolakları ve pigmentasyon ilişkisi düşünüldüğünde ilgi çekicidir. Hastalarımızdan şizofreni tanısı olan bir anne ve kızında mozaik tipte Turner Sendromu ve trizomi X tespit edilmesi, ve literatürde bildirilen şizofreninin eşlik ettiği Turner Sendromu olgularının neredeyse tamamının mozaik karyotipe sahip olması dikkate değerdir.Çalışmamız şizofreni hastalarında temel genetik yaklaşımın gerekliliğini göstermiştir. Subtelomerik yeniden düzenlenim ve şizofreninin ilişkili olduğunu veya olmadığını gösterebilmek için daha geniş çaplı çalışmalara gerek vardır.
Türk populasyonunda erkek faktörlü infertilitede kromozomal anomali ve y kromozom mikrodelesyonları insidansının belirlenmesi
?nfertilite, çiftlerin en az bir yıl süreyle, hiçbir kontrasepsiyon yöntemi kullanmaksızın, düzenli cinsel iliskide bulunmalarına rağmen, çocuk sahibi olamama durumudur ve üretken çağdaki çiftlerin %10-15'inde görülen major bir sağlık problemidir. Erkek faktörü, infertil çiftlerin yaklasık olarak %50'sinden sorumludur. ?nsan Y kromozomu, spermatogenez için gerekli olan ve gonadal farklılasmanın testis yönünde gelismesi için gerekli olan genleri içermektedir. Bu çalısmanın temel amacı, ciddi erkek faktör infertilitesi olan hastalarda ve fertil kontrol grubunda hem kromozomal anomali hem de Y kromozom mikrodelesyonlarının frekansını ve tipini belirlemektir. Çalısmada 90 infertil hasta, 75 fertil erkek kontrol grubu olarak incelendi. 90 infertil hastadan 30'u nonobstruktif azospermik, 30'u oligospermik, 30'u ise normospermik infertil hasta idi. Bunlardan 5 azospermik hastada (%16.7), 4 ciddi oligospermik hastada (%13.3), 2 de normospermik infertil hastada (%6.7) Y kromozom mikrodelesyonu saptandı. AZFc lokusu en fazla delesyona uğrayan bölge (%63.6) idi. 10 azospermik, 4 oligospermik, 3 de normospermik infertil hastada kromozomal anomali tespit edildi. 75 fertil erkek ise genetik olarak normal bulundu. Sonuç olarak çesitli kromozomal anomaliler ve Y kromozom mikrodelesyonları infertiliteye neden olabilir, bu yüzden infertil hastalara genetik inceleme mutlaka önerilmelidir. Anahtar Kelimeler: Erkek infertilitesi, Y kromozom mikrodelesyonu, kromozomal anomali.
Optimization of epigenome-wide CRISPR-CAS9 knockout screen analysis to prioritize cancer therapeutics
Cancer target identification has been expanded by genome-wide, high-throughput CRISPR knockout screens. Using CRISPR-Cas9 knockdown screening, cancer cell survival genes are identified, and new targeted treatments are developed. Genome-scale knockout screens help with the discovery of essential genes that are needed for cancer cell growth. The examination of the screening data produced by this newly developed technology offers several difficulties. A variety of epigenetic modifiers are the focus of the epigenetic knockout library EPIKOL. Five EPIKOL screenings in two separate cell lines were employed in this study (Prostate and triple-negative breast cancer). Variations in sample size, sgRNA knockout efficiency, and the distribution of read counts make it difficult to interpret findings from CRISPR-Cas9 knockout screen data. Since off-target effects can cause drug development to progress in the wrong direction, it is particularly crucial to comprehend what the results of a genetic screen indicate. Therefore, a promising computational algorithm capable of handling various screening library types and read counts is required. There are multiple methods for genome-wide CRISPR screen analysis; however, not all of them are suitable for small-scale screening libraries. False positives appear to be widespread in small-scale library screen analysis. Our objective is to create the most efficient method for screening small-scale CRISPR libraries. To achieve this objective using EPIKOL data, we compared the outcomes of three distinct methods. The implemented algorithms are MAGeCK, CRISPRcleanR, and BAGEL2. This study illustrates that altering the method of normalization or differential expression analysis can enhance the number of hit genes in small-scale libraries. According to the results of the analysis, BAGEL2 discovers more hit genes than other approaches.
The role of deubiquitinating enzyme USP22 in human somatic cell reprogramming
Human somatic cells can be reprogrammed to induced pluripotent stem cells (iPSC) by overexpressing OCT4, SOX2, KLF4 and MYC (OSKM). There are cell intrinsic barriers to reprogramming. We conducted a CRISPR-Cas9-mediated knockout screen during reprogramming to reveal chromatin pathways acting as barriers to reprogramming. Other than DNMT3A and EP300 which were already known to be barriers to reprogramming, this screen revealed a barrier role for USP22 during reprogramming. In this thesis, I validated the barrier role of USP22 in reprogramming by loss-of-function assay. In addition to this, overexpression of various USP22 mutants revealed that USP22 deubiquitinase activity or its integration into the SAGA complex does not affect reprogramming. Interestingly, CRISPR-Cas9-mediated knockout of SAGA deubiquitinase members, ATXN7L3 and ENY2 had no positive impact on reprogramming efficiency. To investigate the effect of USP22 on human pluripotency, I obtained single USP22 knockout clones. These clones expressed pluripotency markers, contributed to tissues from three germ layers when subjected to teratoma formation assay and showed normal karyotyping as in control pluripotent stem cells. To understand the defects in specific lineage specifications, these clones were subjected to in vitro embryoid body formation assay. There seem problems in pluripotency exit as revealed by unsuccessful downregulation of pluripotency markers such as OCT4 and SOX2. Furthermore, mesoderm and endoderm differentiation defects were observed in one of USP22 knockout clones compared to wild-type clones as judged by the lower expression levels of marker genes. To gain more mechanistic insight on the USP22 loss-mediated enhanced reprogramming, we performed an RNA-Seq experiment. USP22 knockout and wild-type fibroblasts were reprogrammed by OSKM expression and on day 6 of reprogramming RNA-Seq was performed. Expectedly, we observed that development-related genesets were negatively enriched whereas pluripotency-related genesets were positively enriched by USP22 loss. SOX2 target geneset was among positively enriched genesets and we hypothesized that USP22 loss activates endogenous pluripotency network earlier during reprogramming to increase its efficiency. To test this hypothesis, we collected RNA at different days of reprogramming and revealed that endogenous SOX2 levels increased up to 3-fold upon USP22 loss during reprogramming. These results show that USP22 acts as a barrier to reprogramming by suppressing endogenous pluripotency network independent from its catalytic activity and SAGA incorporation.
3B kültür modelinden içgörüler: Doku sertliği ve matris bileşiminin tümör ilerlemesi üzerindeki sinerjistik etkisini keşfetmek
Extracellular Matrix (ECM) is a highly versatile network that regulates essential cellular processes. The matrix contains a wide array of proteins and carbohydrates, the composition and organization of which determines the mechanical and chemical signature of the ECM. The architecture of the ECM is tightly modulated in physiological events, whereas the chemical and mechanical properties are altered aberrantly during malignancy. The elevated stiffening of lung tissues due to excessive production of the matrix components, increased crosslinking, as well as the deregulation of enzymes involved in matrix remodeling and degradation accompany tumor progression. The aberrant increase in stiffness of tumor tissues is received through mechano sensitive receptors whose activation is required for the transmission of mechanical inputs into biochemical signals. As tumor tissues stiffen, elevated activation of surface receptors leads to epithelial to mesenchymal transition of cancer cells as well as promotes their stem cell like characteristics. Recapitulating the biochemical and biomechanical properties of the tumor microenvironment is of utmost importance in modelling cancer progression and enhancing the efficacy of therapeutic regimes. Hence, generating an in vitro model which enables alteration of tissue stiffening independently from ligand composition and concentration is of great value for examining the sole impact of stiffening on tumor progression. In addition, mimicking in vivo characteristics of tumor cells with respect to organ-specific cues in a 3D model where tissue stiffness is decoupled from other parameters enables comprehensive disease modeling. The impact of elevating tissue stiffness on modulating the malignancy of cancer cells in different ECM compositions has not been studied in an in vitro 3D culture model. In this study, a fully defined double network of alginate and two different matrices, decellularized healthy bovine lung ECM and tumorigenic basement membrane, is generated. Utilizing this model, it has been revealed that EMT activation and stemness abilities of cancer cells are promoted through synergic interactions between the stiffness of the microenvironment and tumorigenic composition of the matrix. In addition, the absence of PI3K gene, a well-known mediator of mechanotransduction process, is compensated through elevated stiffness of the tumor microenvironment.
Achieving synergism in combination chemotherapy for gastric cancer treatment
Gastric cancer is the fifth most prevalent malignancy and the fourth-leading cause of cancer-associated deaths worldwide. Combination chemotherapy in adjunct to surgery is the mainstay of treatment in gastric cancer. However, survival rates are still very low, despite the administration of potent chemotherapeutics with different mechanisms of action in combination regimens, due to dose-limiting toxicities and chemoresistance. Endeavors in the molecular characterization of cancer enabled the incorporation of molecular-targeted agents into combination regimens and improved treatment outcomes in several cancers. However, the number of approved targeted therapies and the benefit they provide is still limited in gastric cancer. In this thesis study, we aimed to identify the molecular-targeted agent and conventional chemotherapeutic combinations with synergistic action in gastric cancer cell models and dissect the mechanisms of synergism employing powerful functional genomic approaches. Screening the dual combinations of small molecule inhibitors that target EGFR, mTOR, and cMET with five conventional chemotherapeutics with diverse mechanisms of action revealed erlotinib (EGFR inhibitor) and SN38 (topoisomerase I poison) as the most synergistic combination in all four cell models we tested. The synergism was much more robust in the gastric cancer cell model resistant to chemotherapy and stronger than that for combination regimens used in the clinic. Assessment of growth rate and cell death kinetics with FLICK assay validated that the synergism was due to increased cell death and a significant decrease in population size. With a genome-wide perturbation screen and an RNAi-based signature assay, we revealed that the synergism was mainly due to the inhibition of the ABCG2 efflux pump by erlotinib, which enhanced the action of SN38. Hence this study marks the first discovery that functional genomics methodologies identify ABCG2 as an off-target of erlotinib and a potential mechanism of drug resistance against topoisomerase I poisons in gastric cancer. Based on the insights provided by this thesis study, we propose that ABCG2 inhibition by erlotinib in the presence of topoisomerase I poisons represents a promising strategy for the treatment of both naïve and chemoresistant gastric tumors.
Identification of the transcriptional regulators of ATB7B gene by genomic locus proteomics and their effect on cisplatin resistance
ATP7B is a copper pump that plays a vital part in cellular homeostasis. It removes the excess metal from the cells to prevent the toxic results of copper accumulation. Insufficient ATP7B activity results in copper gathering in the body, culminating in Wilson's disease. Upregulation of ATP7B conversely causes the removal of platinum-based drugs along with copper, leading to drug resistance. Understanding the transcriptional regulation of ATP7B may shed light on the mechanisms of disease progression. Metal Regulatory Transcription Factor 1 (MTF1) is a well-studied regulator of ATP7B; however, its expression does not always correlate with ATP7B expression in cancers. The expression of a transcription factor does not always have to be correlated with the expression of the target gene. But the probability that ATP7B is regulated by a single transcription factor is also very low, thus indicating the necessity to identify novel regulators of ATP7B. An in-silico analysis using TRANSFAC/PROMO software was performed to find these additional factors. It was found that several transcription factors may bind to the ATP7B promoter, which was focused around -3000 to +1, indicating that the regulatory sequences are most likely located in this region. A novel proximity labeling methodology called "Genomic Locus Proteomics" was used in this thesis to identify the transcription factors that regulate ATP7B expression. In this technique, the deadCas9 (dCas9) protein is fused to the APEX2 enzyme, and the protein is guided to the ATP7B promoter via specific gRNAs. The efficient gRNAs spanning the -3000 to +1 region were determined by T7E assay and ChIP-qPCR experiments. The proteins near the dCas9-targeted regions of the ATP7B promoter were subsequently biotinylated with the APEX2 enzyme and pulled down with streptavidin-magnetic beads. The marked proteins were then identified with mass spectrometry, and several proteins were selected based on their enrichment scores and their associations with cancer progression and drug resistance in cancers. The target gene PINX1 (Pin2/TRF1-Interacting Protein) was analyzed to illuminate its relationship with ATP7B activity, and it was shown that overexpression of this gene leads to an increase in ATP7B expression. Furthermore, PINX1 overexpression in the HEK293T and Huh7 cells increased the cell viability against cisplatin treatment. This resistance to cisplatin is attributed to the heightened ATP7B activity in the cells. Our findings indicate a transcriptional connection between PINX1 and ATP7B. This relationship may play a role in the progression of the disease and could potentially be utilized to develop more effective therapies.
Identification of chromatin modifiers regulating vincristine resistance in medulloblastoma
Medulloblastoma (MB) is the most prevalent brain cancer in children, typically occurring between the ages of six and eight. While primary MB treatments have a success rate of over 50%, relapse affects more than 30% of the patients, resulting in a poor prognosis with survival rates dropping below 25%. Furthermore, acquired-drug resistance is widely observed in patients with relapse, posing a significant challenge for the treatment of MB. Recent cohorts revealed mutations and differential regulation of chromatin modifiers in distinct subgroups of MB, emphasizing the importance of epigenetic regulations in MB tumorigenesis. In this study, we aimed to identify the epigenetic vulnerabilities of MB by employing loss-of-function screening approaches. Using paired parental and vincristine-resistant MB cells previously generated in our lab, we first conducted a chemical screen with an epigenetic probe library. Second, we performed genetic perturbation screens using our CRISPR/Cas9-based epigenome-wide knockout library (EPIKOL). The ultimate goal was to discover new epigenetic mechanisms of drug response in MB. The established vincristine-resistant MB cells showed up to 100-fold resistance to vincristine, displaying Multi-Drug Resistance (MDR) characteristics primarily attributed to a significant upregulation of ABCB1. With chemical screens, we demonstrated that inhibition of the bromodomain of CBP/p300 histone acetyltransferases leads to sensitization of vincristine-resistant MB by regulating the expression of ABC and SLC transporters. With genetic screens using EPIKOL, we further validated the sensitizing effect of CBP inhibition on drug resistance. Additionally, we identified KEAP1, the regulator of NRF2, as a novel target. Accordingly, KEAP1 was identified from 3 different screens on vincristine-resistant MB, where the negative selection was applied with increasing doses of vincristine, but not on parental MB cells. KEAP1 loss by CRISPR/Cas9 using multiple sgRNAs also confirmed the sensitizing effect through viability assays. Moreover, upon exposure to vincristine, a significant increase in apoptosis was also observed in KEAP1-KO vincristine-resistant cells. Additionally, the transcriptomic analysis revealed the downregulation of ABCB1, the main efflux pump of vincristine, on KEAP1-KO vincristine-resistant cells, further supporting the sensitizing effect of KEAP1 loss. This thesis identified two major regulators of acquired vincristine resistance in MB, which have the potential to serve as therapeutic intervention points in the future.
Functional genetic screens to uncover the roles of chromatin modifiers in IDH-mutant glioma
Gliomas, a malignant group of central nervous system tumors, pose a significant health threat, particularly grade IV glioblastoma, which is the most aggressive type. Among gliomas, IDH-mutant gliomas exhibit a more favorable response to therapies, making them an intriguing target for precision treatments. This thesis delves into the investigation of essential epigenetic modifiers that may underlie vulnerabilities in IDH-mutant gliomas using a custom designed CRISPR-Cas9 epigenetic library called EPIKOL. In the first part of the thesis, IDH-wildtype and IDH-mutant cells were generated from the A172 glioma cell line. Subsequently, CRISPR-Cas9 mediated screening with the EPIKOL library was performed on these cell lines to identify epigenetic modifiers that are essential for IDH-wildtype and/or IDH-mutant glioma cells. The candidates were then validated through functional in vitro assays, focusing on cell viability and colony formation ability. Among the validated genes, RBBP7, which plays a role in complexes involved in histone modification and gene expression regulation, emerged as a potential essential regulator of IDH-mutant glioma cells. Overall, the findings from this study lay the foundation for identifying novel therapeutic targets for IDH-mutant glioma through further experiments and analyses. Future work will dissect out the gene regulatory function of RBBP7 in IDH-mutant cells using RNA sequencing. The investigation of essential epigenetic modifiers offers promising avenues for precision treatments in combating this challenging form of brain tumor.
Evaluation of a cell migration- and autophagy-related kinase as a novel drug target in pediatric glioma
Pediatric gliomas are aggressive brain tumors that pose serious difficulties in terms of prognosis and treatment options. The purpose of this thesis is to assess a novel therapeutic target for pediatric glioma, a cell migration- and autophagy-related kinase. This kinase was discovered as a prospective therapeutic target by a thorough study of proteomic datasets due to its altered expression and interaction with important pathways involved in cell migration and autophagy in glioblastoma. This thesis examines the functional importance and expression of the discovered kinase in pediatric glioma cell lines and patient tumour samples. We observed that migration and drug resistance of pediatric glioma cells are more sensitive to changes in autophagic flux. Also, its expression and localization in patient samples is evaluated using quantitative PCR, Western blotting, and immunohistochemistry. To determine the influence of targeting this kinase on glioma cell migration and autophagy, functional tests such as cell migration assays, autophagy flux analysis, and cell viability assays were carried out. The findings from this study could aid in facilitating the improvement of pediatric glioma-targeted treatments. Understanding the functions of this kinase protein will help researchers develop novel pharmaceutical candidates that selectively limit the growth of gliomas while minimizing side effects. The interaction between the interested kinase protein and one of the main autophagy proteins, ATG5, was displayed. The changes in the autophagic degradation with wild-type, kinase overexpressing and CRISPR-Cas9 mediated knockout cell lines were revealed. Moreover, cellular migration was significantly influenced upon depletion of the protein. Besides, drug resistance against various commonly used chemotherapeutic agents was not changed with gene deletion and kinase inhibition. To summarize, this research on the interested kinase protein as a druggable target may pave the door for individualized treatment plans that develop the prognosis and quality of life for children with gliomas.
Reversion of taxane resistance through PRMT5 inhibition in castration-resistant prostate cancer
The most common cancer in men is prostate cancer (PCa), and the first-line treatment for PCa is the surgical intervention of the tumor and/or chemical castration and radiotherapy. Despite the initial efficacy of this treatment, a stage known as castration-resistant prostate cancer (CRPCa) is eventually reached by a majority of patients within 2 to 3 years. At this stage, chemotherapeutic drugs like taxanes (such as Docetaxel and Cabazitaxel) are considered as treatment options. However, a significant clinical challenge emerges withathe development of drug resistance over time. Epigenetic regulation in cancer has a significant impact on tumor development and progression, as well as chemotherapy resistance. Therefore, targeting epigenetic mechanisms emerges as a promising strategy to prevent drug resistance. Previously, CRPCa cell lines resistant to Dtx and Cbz were generated to identify potential epigenetic targets that could overcome resistance. The Epigenetic Drug Library screen was conducted to uncover the reversing effects of epigenetic modifiers on resistance mechanism. In this thesis, the screen revealed Protein Arginine Methyltransferase 5 (PRMT5) as a re-sensitizer to taxane treatment. RNA sequencing analyses revealed that PRMT5 silencing led to the negative enrichment of four signaling pathways, including MYC, E2F, and G2/M. Indeed, MYC inhibition led to a stronger suppression of growth in resistant cells contrast to parental cells. Furthermore, the treatment of resistant cell lines with PRMT5 inhibitors induced cell cycle arrest in the G2/M phase, suggesting the restoration of taxane susceptibility. This effect was accompanied by an increase in apoptotic markers (Caspase 3/7 activation and Annexin-V staining). Pharmacological inhibition of PRMT5 reduced ABCB1 activity, as determined by the Calcein efflux assay, indicating that PRMT5 may be involved in an efflux-related mechanism. These results offered valuable insights into potential avenues for addressing taxane resistance in CRPCa treatment, paving the way for the development of more effective therapeutic strategies.
Turboid reveals NEK2a's semi-dynamic cell cycle interactions and Nusap1 as a novel partner
Nek2A is a cell cycle regulated kinase, which is involved in several cellular processes and overexpressed in numerous cancer types. It has been associated with chromosome instability, increased cell proliferation and drug resistance in cancers. Our study primarily aims to identify the specific partners that interact with Nek2A during the cell cycle, deepening our understanding of Nek2's role in cancer. To meet this objective, we employed the TurboID proximity labeling technique on synchronized cell groups, exploring the dynamic interactions of Nek2A as the cell cycle advances. We synchronized the cells using a double thymidine block and harvested them at designated release time points, focusing on G1/S, late S, and G2/M cell cycle phases. Through mass spectrometry, we identified biotinylated proteins, which we further analyzed using MaxQuant, Cassiopeia, and Amica tools. Our experiments reliably identified previously recognized Nek2A partners like the Anaphase Promoting Complex (APC) and Kif24. We confirmed proteins closely interacting with Nek2A during the G1/S, late S, and G2/M stages using western blotting. Co-immunoprecipitation unveiled direct interactions of Nek2A with proteins such as Nusap1, Kif2c, Mapre3, and Mpg. We also confirmed the colocalization of these proteins with Nek2A using fluorescence microscopy. We took a special interest in Nusap1 due to its unique role, distinct from other Nek2A partners. Nusap1 acts as a stabilizer for DNA damage proteins, and its depletion can lead to DNA strand breaks, negatively impacting cell survival. Our analysis highlighted a prevalent occurrence of ubiquitin-driven proteolysis during the cell cycle's progression. We initially studied Nusap1's expression in cells with either diminished or amplified Nek2A activity. Remarkably, there was an increase in Nusap1 levels post-siRNA treatment and in two distinct Nek2 null (KO) cell variants. Conversely, overexpression of Nek2A led to a short-lived reduction in Nusap1 amounts, lasting just 6 hours, before other cell mechanisms presumably balanced it. Using the well- known proteasome inhibitor, MG132, we were able to counteract this reduction. Moreover, we observed a notable reduction in Nusap1 ubiquitination in Nek2A KO cells, pointing to the possible role of proteasomal breakdown processes. A broad protein analysis in Nek2A deficient cells confirmed many proteins, including Nusap1, showed decreased expression. In essence, our findings indicate Nusap1 as a new Nek2A partner, with Nek2A possibly affecting Nusap1's degradation through ubiquitination. The exact nature of this interaction and the clinical significance require further exploration.
Sentrozom kümesi üzerine odakli Nek2a kinaz hedeflerinin incelenmesi
Cancer cells, unlike normal cells, usually have extra centrosomes, which form multipolar spindles (MPS) and cause cell death. Nonetheless, they divide successfully and avoid the lethal implications of uneven genetic material segregation by clustering their extra centrosomes into two poles. Nek2A is a mitotic kinase that regulates a variety of mitotic events. In this study, we demonstrate that while reduction of Nek2A activity via knock-out, silencing or using specific inhibitors favours centrosome clustering, its overexpression unclusters extra centrosomes making cancer cells vulnerable to cell death. Interestingly, none of the centrosomal targets of Nek2 (C-Nap1, Rootletin or Gas2L1) or its targets that can induce genomic instability (TRF1 or HEC1) appeared to responsible for its action on clustering, suggesting that other targets may be involved in this process. To investigate whether the effect of Nek2A on MPS is a novel pathway or acted in concert with other known unclustering factors, we tested HSET and NuMA. Our findings revealed that HSET's unclustering activity was complementary to NEK2A, implying an independent process, whereas the suppression of NuMA could reverse this effect. We also performed TurboID proximity labelling analysis, uncovering several potential Nek2A targets that were either situated at the centrosome or along microtubules, including NuMA. Although NuMA was in proximity, it did not co-IP with Nek2A. Intriguingly, we identified KIF2C as a new interaction partner of Nek2A and our subsequent analysis indicated that silencing it attenuated Nek2A activity on centrosome clustering. In conclusion, Nek2A's role in centrosome clustering and the identification of new interaction partners like KIF2C provides a deeper comprehension of cancer cell biology and may offer innovative avenues for targeted cancer therapy.
Examining chromatin modifiers essential for glioma growth and drug response
Glioblastoma (GBM) is a highly aggressive primary brain tumor associated with low survival rates. Standard-of-care involves surgery, irradiation, and chemotherapy utilizing Temozolomide (TMZ), a DNA alkylating agent. Despite its high effectiveness, the efficacy of TMZ can be compromised by various epigenetic mechanisms, including the transcriptional regulation of 0-6-methylguanine methyltransferase (MGMT) enzyme expression. The promoter methylation status of MGMT is a crucial prognostic factor, as its epigenetic suppression enhances the response to TMZ. Novel epigenetic factors regulating the survival and resistance to therapy in GBM remain undiscovered. To explore resistance mechanisms in GBM, we first generated TMZ-resistant cell lines starting from naïve cells and escalating TMZ doses over a long period. We demonstrated that TMZ-resistance phenotype was sustainable both in vitro and in vivo. Transcriptome analysis revealed MGMT as an upregulated gene in TMZ-resistant models along with many differentially expressed genes. Considering that resistance may be associated with adaptive epigenetic changes, we investigated the functional roles of chromatin regulators in TMZ-resistant cells. To this end, we employed a targeted CRISPR/Cas9-based screen with our Epigenetic Knock-Out sgRNA Library (EPIKOL), focusing on various chromatin modifiers and epigenetic enzymes. Applying EPIKOL screens in multiple naïve and TMZ-resistant cell lines, we first identified key epigenetic factors regulating GBM cell viability. We then explored selective vulnerability of TMZ-resistant cell lines and identified Retinoblastoma Binding Protein 4 (RBBP4) as a regulator of acquired TMZ resistance. Knock-out of RBBP4 in resistant models resulted in increased apoptosis and G2/M arrest. RNA sequencing of control and RPPB4 knock-out cells revealed G2/M checkpoint and E2F targets as downregulated pathways. We demonstrated that the expression of MGMT was not significantly altered by RBBP4 in TMZ-resistant models, providing strong evidence for RBBP4-mediated regulation of cell proliferation and TMZ response in an MGMT-independent manner. Overall, our findings hold promise for the development of innovative epigenetic-based therapeutic strategies targeting GBM in the future.
The role of molecular markers and clinicopathological features in predicting central lymph node metastasis of papillary thyroid microcarcinoma
Arka Plan: Papiller tiroid mikrokarsinomunun (PTMK) olumlu prognozuna rağmen, santral lenf nodu metastazının (SLNM) sık görülmesi (%60'a kadar), nüks ve sağkalımı etkileyen önemli bir sorun teşkil etmektedir. Preoperatif boyun ultrasonografisinin SLNM'yi tespit etmedeki sınırlı kullanımı, SLNM'nin etkili bir şekilde öngörülmesi için radyolojik, histopatolojik ve klinik faktörlerin araştırılmasını gerektirir. Bu çalışma, PTMK hastalarında moleküler belirteçlerin ve klinikopatolojik özelliklerin SLNM'yi öngörmedeki rolünü değerlendirmeyi amaçlamaktadır. Yöntemler: Şubat 2019 ile Aralık 2023 tarihleri arasında Koç Üniversitesi Hastanesi veya Amerikan Hastanesi'nde tiroidektomi operasyonu geçiren hastalar arasından tümör boyutu ≤10 mm (PTMK) olan 145 hasta çalışmaya dahil edildi. Profilaktik veya terapötik santral boyun diseksiyonu olmayanlar ve eksize edilen lenf nodu sayısı 3'ün altında olanlar çalışma dışı bırakılmıştır. Hastalar SLNM'ye göre iki çalışma grubuna ayrılmıştır. Hastaların demografik özellikleri ve klinikopatolojik özellikleri, BRAFV600E mutasyonu, dezmoplazi ve Masson trikrom boyaması değerlendirilmiştir. Hem tek değişkenli hem de çok değişkenli lojistik regresyon analizleri yapılmıştır. Bulgular: 65 katılımcının 35'inde (%53,8) SLNM görülmüştür. Tek değişkenli analizlerde tümör boyutlarının büyük olmasının, lenfovasküler invazyonun, agresif alt tip varlığının, BRAFV600E mutasyon pozitifliğinin, desmoplazi ve trikrom yayılım ve yoğunluk skorlarının yüksek olmasının lenf nodu metastazı varlığını öngörmede anlamlı değişkenler olduğu gösterilmiştir (p<0,001). SLNM varlığını tahmin etmek için çok değişkenli lojistik analizler yapılmıştır. Diffüz desmoplazi ve trikrom yayılımının lenf nodu metastazı olasılığını sırasıyla 13,6 ve 9,8 kat arttırdığı belirlenmiştir. Sonuç: Bu çalışmanın bulguları, artan desmoplazi ve trikrom boyama skorları ile lenf nodu metastazı olasılığı arasında anlamlı ilişkiler olduğunu ortaya koymuştur. Ancak bu ön bulguları doğrulamak için daha büyük gruplarla ve kapsamlı moleküler analizlerle daha ileri prospektif çalışmalar yapılması gerekmektedir. Anahtar Kelimeler: Desmoplazi; Lenf nodu metastazı; Papiller tiroit mikrokarsinomu
Targeting poor prognostic CAF markers in gastric cancer
Gastric cancer is the 5th most common cancer worldwide and the 4th leading cause of cancer deaths. Although highly cytotoxic chemotherapies are used in the clinic, curative strategies for advanced gastric cancer remain limited. Cancer-associated fibroblasts (CAFs) are the key elements of the tumor microenvironment that secrete various extracellular matrix proteins to increase tumor aggressiveness. Patients with CAF phenotype in their tumor microenvironment exhibit poor survival, prognosis, and chemoresistance. Recently through a bioinformatic study we identified poor prognostic markers associated with CAFs in gastric cancer, that decrease the patient survival significantly. Unraveling tumor-driving phenotypes associated with these markers of CAFs can show great promise in the treatment of gastric cancer. Therefore, in this study, we investigate the poor prognostic effects of CAFs using co-culture models of fibroblasts or patient-derived CAFs with gastric cancer cells.
Oligodendrojenez modellemesi için 3 Boyutlu (3B) çoklu ve mikropaternli GelMA Platformu
The occurrence of disturbances in myelination can lead to the development of a range of disorders. A reliable and biologically mimicking in vitro model of oligodendrogenesis is of necessity to aid in elucidating the pathogenesis of demyelinating diseases. Oligodendrogenesis and myelination are highly dependent on the function of extracellular matrix (ECM) components. The recent advancements in biomaterials illustrated that hydrogels have tremendous potential to mimic the ECM of central nervous system (CNS). It remains elusive whether the characteristics of hydrogels can manage to regulate the differentiation and maturation of oligodendrocyte (OL) lineage cells and provide a suitable model without the need for primary cells or in vivo models. Here, this study examines the impact of a multiplexed and micropatterned 3 Dimensional (3D) Gelatin Methacrylate (GelMA) platform on the viability, and spheroid formation of Human Oligodendroglioma (HOG) cells under different UV exposure conditions, while also exploring the maturation status. Both proliferation and spheroid formation were enhanced with prolonged UV exposure durations, illustrating the importance of ECM dynamics on cellular behaviour. The cell viability on day 5 was the greatest at 50 seconds of exposure condition. The formation of spheroids was discernibly observed on day 5 at exposure conditions of 40 and 50 seconds, with the latter showing a notable effect. Maintenance in GelMA induced the mRNA expression of transcription factors OLIG2, SOX2 and SOX10; OPC markers PDGFRα and NG2; and OL markers CNPase, MBP, and MOG. The expression of GFAP was not detected in the case of 3D whereas it was found to be traceably expressed in 2D cultured cells, suggesting a possible rescue from astrocyte lineage in 3D. Immunofluorescence microscopy confirmed the expression of MBP and MOG at the protein level. These results imply the regulatory effect of the GelMA environment on the expression profile of HOG cells, altering their fate towards OL lineage. To ascertain whether alterations in the expression of OL genes are exclusive to 3D maintenance, a separate treatment of UV and GelMA was applied to HOG cells cultured in a 2D environment. Neither UV treatment nor GelMA addition made significant changes on the expression profile of HOG cells. In conclusion, this platform appears to be a noteworthy culture model for oligodendrogenesis, warranting further investigation. Coculture of SH-SY5Y cells with HOG cells was conducted to see if any interaction would occur. Although SH-SY5Y cells were not differentiated into mature neurons, their interaction with HOG cells was evident, leading to accumulation near HOG spheroids with the latter crosslinking exhibiting a notable effect. Further investigation with the usage of mature neurons and oligodendrocyte lineage cells within the hydrogel may aid in enlightening whether the platform also serve as a model for myelination. Key words: Oligodendrocytes, oligodendrogenesis, 3D-hydrogel Model, GelMA, myelination
Exploration of biological effects and AUTAC drug approach potential of antibiotics
Lung cancer, a pervasive and lethal disease, requires more effective treatments due to the limitations of existing options. Innovative approaches, such as AUTACs (Autophagy Targeting Chimeras) targeting autophagy modulation, hold promise in disrupting cancer cell survival mechanisms. AUTAC, inspired by selective autophagy, allows selective destruction of disease-related targets. In this system, a chimeric molecule is formed by combining a drug that specifically binds to the target and another that binds to autophagosomes. This chimeric molecule facilitates the accumulation of targeted structures in autophagosomes, leading to their destruction in autolysosomes. Consequently, target molecules are separated from the cell and degraded, presenting a novel approach for targeted therapeutic interventions. The main subject of this thesis is the development of chimeric chemical drugs that will provide targeted destruction of disease-related structures and organelles by the autophagy-lysosome system. Initially, cellular targets and two antibiotics binding to these targets were identified. Subsequently, the impact of these antibiotics was investigated in various cell lines. Non-small cell lung cancer (NSCLC) cell lines and the BEAS-2B bronchial epithelial cell line were employed to assess and compare the effects of these drugs on cancer and normal cells. The comparative analysis included an examination of the drugs' binding capabilities to targets, their influence on autophagy, and their impact on other cellular process in both cancer and normal cells. Furthermore, the anti-cancer properties of these drugs were explored, particularly in combination with chemotherapy drug. This investigation yielded insights into the targeting capabilities and applicability of the two drugs, particularly within the AUTAC system. The combination of these drugs with a linker has the potential to expand therapeutic applications. This innovative approach holds promise for its utilization in the treatment of various diseases.
Investigation of anti-cancer effects of a palladium compound (Pd(bpma)(barb).Cl • H2O) in colorectal cancer cell lines
Background and Objective: Colon cancer ranks as the third most prevalent form of cancer globally. An eminent issue that arises during and after the treatment procedure is the development of drug resistance in cancer cells, rendering them unresponsive to therapy. Therefore, it is crucial to develop novel therapeutic targets for colon cancer therapy by thoroughly investigating all molecular pathways associated with the disease. Recent studies have shown that some metal compounds have intriguing therapeutic benefits, making them potential candidates for medication development. Studies on colon cancer indicate that apoptosis often functions as a pathway for survival. Methods: This thesis research aimed to explore the anti-cancer and cytotoxic effects of the Palladium (II) compound [[Pd(bpma)(barb)]Cl.H2O] on human colon cancer cell lines (HCT-15, HCT-116, and HT 29). The impact of the Pd (II) compound on cell viability was assessed using the MTT viability assay. To ascertain the cellular process by which the substance induces cell death, we investigated the impact of oxidative stress on DNA, autophagy, and the annexin V pathways using the flow cytometry technique. The process of programmed cell death, known as apoptosis, was seen in cells using the Hoechst 33342/Propidium Iodide staining technique, with the use of a fluorescence microscope. Ultimately, the RT-qPCR technique was used to analyze the gene expression levels of a total of 82 genes that are linked to these pathways. Results and Conclusion: The study revealed that the Pd (II) compound led to enhanced anti apoptotic activity and apoptosis in the HCT-15 cell line. Given the prediction that the Pd (II) molecule has potential as a candidate for an anti-cancer therapy, it is believed that doing more in vivo trials will provide valuable guidance for both clinical applications and future cancer research investigations.
Detection of the red blood cell adhesion to the vascular endothelium using microfluidic chip in beta thalassemia patients
Beta thalassemia is a hereditary genetic disorder affecting the hematopoietic system, primarily due to mutations in the beta globin gene. The disease is characterized by ineffective erythropoiesis resulting from reduced or absent beta globin production, leading to low or no production of hemoglobin A. This deficiency causes an accumulation of free alpha globin chains in the cytoplasm of erythroid cells, which disrupts their physiology. The free α-globin chains aggregate and precipitate, causing cytotoxic effects, oxidative stress, and decreased survival of erythroid cells, leading to high levels of hemolysis. The hemolysis of red blood cells (RBCs) releases hemoglobin and heme into plasma. Extracellular hemes are highly proinflammatory, activating immune and endothelial cells (ECs), contributing to the disease's pathogenesis. EC activation leads to the overexpression of adhesion molecules and increased RBC adhesion to ECs. Beta thalassemia patients exhibit altered RBC flow characteristics, causing microcirculatory issues. However, the mechanisms and molecules involved in RBC adhesion in beta thalassemia remain not fully understood. This study investigated RBC adhesion in beta thalassemia major (BTM) patients under flow conditions using a microfluidic chip with microchannels functionalized with 40 μM heme-activated HUVECs, fibronectin, and laminin. Increased RBC adhesion in BTM patients in microchannels functionalized with 40 μM heme-activated HUVECs and fibronectin was demonstrated compared to laminin. There were significant positive correlations between hemoglobin, WBC, platelet values, and adherent RBC number in microchannels functionalized with fibronectin. Application of Forskolin (activator of adenylyl cyclase) and SQ22536 (inhibitor of adenylyl cyclase) indicated that RBC adhesion could be altered by targeting the AC-cAMP-PKA signaling pathway. RBC deformability was also assessed, showing a slight decrease after shear stress at high shear stress levels for untreated patient blood, with Forskolin and SQ22536 impairing deformability compared to the vehicle after shear stress. Lastly, plasma hemoglobin concentration was higher in beta thalassemia major patients' plasma samples than healthy controls. These data highlight the importance of VCAM-1 and fibronectin for RBC adhesion in BTM patients and the effects of targeting the AC-cAMP-PKA signaling pathway on RBC deformability and adhesion. This study has contributed to understanding the pathophysiology of beta thalassemia major patients and importance of targeting AC-cAMP-PKA signaling pathway. This underscores the need for these studies to be conducted with a larger patient cohort to gain more thorough insights.
Regulation of acquired drug resistance by mirnas in high grade serous ovarian cancer
Epithelial ovarian cancer is the seventh most common malignancy diagnosed in women worldwide and the fifth most common cause of cancer-related deaths. Limited improvements in outcomes have been made over the past decades, and the relative five-year survival rate is less than 50%. High-grade serous ovarian cancer (HGSOC) is the predominant histological subtype. PARP inhibitors such as Olaparib (Lynparza™), and niraparib (Zejula™) have been approved for the treatment of platinum sensitive, recurrent HGSOC. However, like many other targeted agents, the efficacy of PARP inhibitors is limited by the development of drug resistance. miRNAs are small (21-23 nucleotide long) non-coding RNA molecules that play a role in post-translational regulation of gene expression. Several cellular pathways are affected by miRNAs, such as cell differentiation, proliferation, and apoptosis. The aim of this project is to explore the role of miRNAs in the context of treatment response and resistance in high grade serous ovarian cancer. To this end, drug resistant HGSOC cells established with ex vivo cultures of primary cells obtained from resistant (patients with poor prognosis after maximal cytoreductive surgery) and sensitive (patients with good prognosis after maximal cytoreductive surgery) patients diagnosed with HGSOC to investigate the underlying mechanisms of drug resistance towards clinically used drugs. 188-5p gene expression level was found significantly downregulated in Olaparib resistant OVCAR-3 cells and resistant FFPE patient samples with poor prognosis after cytoreductive surgery. Combinational treatment studies showed that synergistic effect of Olaparib and 188-5p mimic decreased the survival rate of the resistant cells. Downstream effects of synergistic treatment of the 188-5p mimic and Olaparib exhibited an increase in caspase-8 mediated apoptosis levels in resistant cells. Moreover, in cell cycle experiments, subG1 arrest was observed upon 188-5p transfection. Results revealed that upregulating mir188-5p level in resistant cells significantly suppressed the epithelial-to-mesenchymal transition of resistant cells by decreasing N-cadherin and SNAIL and increasing E-cadherin protein levels.
Investigation of n-terminus mll complexes on the reversion of taxane resistance in castration-resistant prostate cancer
Prostate cancer (PC) typically relies on androgen for abnormal growth, and androgen deprivation therapy (ADT) is preferred as the primary treatment. However, patients frequently progress to a castration-resistant (CR) stage, where tumor growth becomes unresponsive to ADT. Despite the common use of conventional chemotherapeutics like Taxanes (Docetaxel-Dtx, Cabazitaxel-Cbz), either alone or in conjunction with hormone therapies, certain tumors may experience recurrence. This work focuses on the epigenetic regulations conferring Dtx resistance in CRPC. Firstly, the generation of Dtx-resistant cells was conducted using the dose increment method, and subsequently, our model was confirmed via in vitro and in vivo models. Epigenetic drug screening identified MLL-Menin and MLL-WDR5 inhibitors as hit molecules that effectively reverse drug resistance through G2/M arrest and apoptosis induction. N- and C-terminal binding partners of MLL were individually knocked out via CRISPR-cas9. Our analysis led to the discovery of sensitivity on DtxR cells upon Menin depletion, while no effect was observed on parental counterparts. On the other hand, parental cells lacking Menin expression showed reduced capacity to develop drug resistance, suggesting an indispensable role of Menin for the drug refractory phenotype. Restoring several Menin mutants led to the identification of another effective factor, LEDGF, which specifically diminishes colony growth on the DtxR model. RNA-seq analysis was conducted on parental and DtxR cells, upon Menin and LEDGF ablation. GSEA analysis revealed positively enriched mTOR signaling, E2F targets, and G2M checkpoints gene sets in DtxR cells. Interestingly, Menin and LEDGF depletion significantly reversed the enrichment profile of the interested gene sets. Initially, we revealed the essentiality of the mTOR pathway in DtxR maintenance and cell growth. Furthermore, mTOR expression was significantly reduced in Menin knockout cells. On the other hand, Menin depletion triggered a significant synergy with Torin (mTOR inhibitor) and Dtx in our resistant model. Recovering Menin also induced mTOR expression in our DtxR model and abolished the observed synergy. Menin and mTOR correlation was also increased in metastatic CRPC in patient-derived clinical data. Furthermore, our ChIP-qPCR experiments demonstrated Menin enrichment on mTOR promoter region in DtxR CRPC cells. Competition experiments exhibited significant domination by control cells; indicating Menin depletion results in a slower rate of cell division. A higher proportion of Menin knockout cells accumulated in the G1 cell cycle state. Furthermore, two important factors promoting G1-S transition, Cyclin D1 and CDK20 were significantly lower in Menin-depleted cells. Restoring Menin also rescued expression patterns of these targets; furthermore, Menin occupied the promoter region of Cyclin D1 and CDK20. This slow growth rate observed in Menin ablated cells also provided a slight resistance against CDK4/6 inhibitors. Overall, Menin appears as a key regulator that confers drug resistance through mTOR upregulation and controls G1-S progression via Cyclin D1 and CDK20 in our DtxR model. Menin and LEDGF both contribute to essentiality in DtxR cells, while Menin shows enrichment on the promoter regions of specific targets. Our study provides a detailed analysis of Dtx resistance in CRPC, through epigenetic regulation.
Functional roles of two chromatin factors (USP22 ve MENIN) in reprogramming and pluripotency
Overexpression of OCT4, SOX2, KLF4 and MYC (OSKM) factors can reprogram somatic cells to induced pluripotent cells (iPSC). Process of somatic cell reprogramming is inherently inefficient, pointing to the cell's intrinsic barriers that safeguards somatic cell identity. Previously conducted CRISPR-Cas9-based knockout screens during reprogramming revealed USP22 and MLL1 as barriers to reprogramming. In the first part of this thesis, overexpression of wild-type and catalytic mutant USP22 in USP22 KO cell lines were performed which showed that increased reprogramming efficiency is indeed related to USP22 loss, and this effect is not related to its deubiquitination activity. Reprogramming of USP22 knock-out primary fibroblasts under different primed pluripotency culture conditions proved that increased reprogramming efficiency is independent of cell line or culture condition of choice. Transcriptome analysis at specific time-points during reprogramming revealed that loss of USP22 represses fibroblast-specific genes such as COL1A2, POSTN, FOXC2 that occurs throughout reprogramming. In addition, USP22 loss results in upregulation of general pluripotency markers such as SOX2, LIN28A and naïve pluripotency markers such as DNMT3L, GDF3, ALPPL2, ARGFX as early as 3 days after OSKM expression. To investigate a potential role of USP22 in attaining naïve pluripotency, I reprogrammed somatic cells under naïve culture conditions which resulted in an increased number of naïve colonies as quantified by TRA 1-60/KLF17 double staining. In addition, USP22 loss enhanced conversion from primed to naive pluripotency indicated by higher expression levels of naive pluripotency markers DNMT3L and ALPPL2. In the second part of the thesis, I showed that MEN1 loss enhances reprogramming efficiency in primary fibroblasts. Rescue experiments utilizing overexpression of H433A mutant MENIN which cannot recognize H3K79me2, indicated that MENIN acts as a barrier partially through its ability to read this chromatin mark. To test whether knocking-out MEN1 affects pluripotency, single clone iPSCs were generated from MEN1 KO fibroblasts. Characterization assays showed that these clones express endogenous OCT4, SOX2 and KLF4 while silencing C-MYC and other exogenous transgenes. Also, MEN1 iPSCs stably express pluripotency related proteins and contribute to all three germ layers when subjected to teratoma formation assay. Finally, MENIN-MLL1 Complex inhibition via VTP50469 enhanced reprogramming efficiency in primary fibroblast lines. In addition, VTP50469 supplementation increased reprogramming efficiency of 3-factor (OSK) reprogramming, and enabled iPSC generation with only 2 factos (OS). Taken together, this work demonstrates important roles for two distinct chromatin factors in reprogramming and pluripotency.
In Vitro primary neuron and Ex Vivo retina stimulation with optoelectronic biointerfaces
Progressive vision impairment generally arise due to irreversible photoreceptor damage in retinal degenerative diseases. Although there are applications aimed at improving patients' life quality, the exact treatment of these diseases has not been found. Optoelectronic approaches aim to perceive the incoming light and convert it into electrical signals by performing an artificially similar function of photoreceptors, and to activate retinal neurons to send signals for perceiving light in brain. Development of optoelectronic biointerfaces provides new strategies for therapeutic application in vision-related diseases. This thesis aims to investigate the design of photovoltaic devices, biocompatibility and functional applications. Fabricated novel biointerfaces were tested in in vitro culture conditions and ex vivo retinal stimulation. The final part of the thesis includes the optimization of an in vivo optic nerve injury model for further prosthesis testing in the future. The first part of the results consists of in vitro tests including cell viability, intracellular stress level, mitochondrial health, calcium influx of primary embryonic hippocampal neurons cultured on different photovoltaic device designs based on P3HT:ITIC and AgBiS2 quantum dots. More than 80% cell viability and minimal stress under light illumination made the biointerfaces suitable candidates for further studies. The next part includes the final design of AgBiS2-based devices with return layer of RuO2 and interlayer of ZnO nanowires to improve photocurrent and photovoltage values. In addition to the biocompatibility and functionality tests on primary hippocampal neurons, the stimulation capacity of AgBiS2-based biointerface was validated by ex vivo retinal recordings from rats with retinal degeneration under repetitive near-infrared light illumination. The last part includes the optimization of animal studies for optic nerve crush model. Histological and functional analysis showed that injury-related conditions such as retinal layer deformation, gliosis, fibrosis and decrease in retinal ganglion cell activity progressed with increasing injury duration. The in vivo model provided a new platform to study the neuroprotective and neuroregeneration capacity of photovoltaic devices in the future.
The diagnostic role of next-generation sequencing on facioscapulohumeral muscular dystrophy(FSHD) and related phenotypes
Facioscapulohumeral muscular dystrophy (FSHD) is the third most common genetic neuromuscular disorder exhibited by progressive muscle weakness, primarily affecting the face, scapular, and especially upper arm muscles. In approximately 95% of cases, classified as type 1 (FSHD1), the disease results from contractions of the D4Z4 macrosatellite repeats on chromosome 4q35. The remaining cases, classified as type 2 (FSHD2), are primarily caused by pathogenic variants in SMCHD1, with a smaller subset involving DNMT3B and LRIF1 genes. These variants not only lead to D4Z4 hypomethylation and aberrant DUX4 activation but may also influence the disease severity in FSHD1 patients with borderline D4Z4 repeat contractions. The diagnostic algorithm of FSHD involves the examination of D4Z4 Repeat Units (RU) and the analysis of the FSHD-associated genes to confirm or exclude diagnosis. The Molecular Combing (MC) technique has been a crucial tool for the precise and effective analysis of the D4Z4 region, while NGS provides a broader genetic landscape, specific to FSHD2 cases. This approach enables the identification of variants in SMCHD1, LRIF1, and DNMT3B, as well as the detection of variants in other neuromuscular disease-related genes. It enhances differential diagnosis, prevents misclassification, and improves the interpretation of genotype-phenotype correlations. A total of 96 unrelated patients' D4Z4 RUs were determined using MC. In this group, 82 patients had contracted D4Z4 RUs (67/82 with pathogenic 1–7 RUs and 15/82 with borderline 8–10 RUs). Of the remaining 14 patients, 12 presented with ≥11 RUs while in the remaining 2 cases, only the 4qB allele was detected. Twelve patients from this uncontracted group were selected for Whole Exome Sequencing (WES) analysis based on their disease severity and complex MC results. One unaffected and two affected relatives of one patient were included in this study group for segregation and confirmation purposes. Pathogenic variants in SMCHD1 were detected in three patients confirming the clinical diagnosis of FSHD. Pathogenic and/or likely pathogenic CAPN3 variants were identified in two patients, leading to a revised diagnosis of Limb-Girdle Muscular Dystrophy 2A (LGMD2A). In one patient, variant of unknown significance in FHL1 gene were identified. The results of this study largely align with the literature regarding the prevalence of contracted D4Z4 alleles in patients with a clinical diagnosis of FSHD and underscore the significance of NGS for ascertaining a definitive diagnosis in the small subset without contracted D4Z4 alleles. It is crucial to explore the impact of NGS to reveal overlapping genetic factors that may contribute to the clinical heterogeneity of FSHD and its differential diagnoses.
Fasiyoskapulohumeral musküler distrofi (FSHD)'de bisülfit dizilemesi ile DNA metilasyon profillemesi
Facioscapulohumeral Muscular Dystrophy (FSHD) is a progressive neuromuscular disorder caused by the aberrant expression of the DUX4 gene, which is normally epigenetically silenced in somatic cells. This aberrant expression is directly linked to hypomethylation of the D4Z4 macrosatellite repeat region at chromosome 4q35, a key epigenetic alteration that plays a critical role in disease pathogenesis. FSHD is classified into two subtypes: FSHD1, which results from contraction of the D4Z4 array from 11-150 repeat units down to 1-10 repeat units, and FSHD2, which is associated with pathogenic variants in epigenetic regulators such as SMCHD1, DNMT3B, and LRIF1. Despite their distinct genetic origins, both forms exhibit a significant loss of DNA methylation at the D4Z4 locus, leading to DUX4 activation and muscle degeneration. This study aimed to implement and optimize PCR-based bisulfite sequencing, a high-resolution, single-base method for assessing cytosine methylation patterns, in order to characterize D4Z4 methylation profiles in FSHD patients, enhance diagnostic accuracy, and evaluate CpG profiling as a biomarker for disease severity. A subset of 33 patients from a larger cohort of 112 individuals was selected for detailed methylation analysis, stratified by D4Z4 repeat size and clinical subtype. Six healthy individuals were included as controls. Methylation status was evaluated in the DR1 and DR2 regions, with special focus on CpG density and correlation to clinical and genetic backgrounds. Our findings showed that 17 FSHD1 and 6 FSHD2 patients exhibited significantly reduced methylation compared to controls, while 10 patients had borderline or normal methylation levels. These results support the utility of methylation profiling in distinguishing between FSHD subtypes and clarifying cases with ambiguous genetic findings. Additionally, integration of next-generation sequencing (NGS) data from a selected group of patients enabled further investigation into potential genetic modifiers. Methylation analysis in FSHD is crucial, as it reveals the epigenetic alterations that drive aberrant DUX4 expression, enabling precise differentiation between FSHD subtypes and enhancing diagnostic accuracy. Our study highlights the value of bisulfite sequencing as a complementary clinical and research tool that not only improves diagnostic precision but also contributes to a deeper understanding of the epigenetic landscape in FSHD, thereby informing targeted patient management and guiding potential therapeutic interventions.
Enhancing radiotherapy efficacy in glioblastoma by inhibiting BRD9
Glioblastoma (GBM) remains one of the most aggressive and treatment-resistant forms of brain cancer, marked by high heterogeneity, invasive growth, and limited therapeutic options. Despite current standard therapies such as surgical resection, radiotherapy, and temozolomide chemotherapy (Stupp protocol), recurrence and resistance are still frequently seen. In this study, we aimed to enhance the radiotherapeutic response in GBM by targeting the epigenetic regulator BRD9, a bromodomain-containing protein implicated in chromatin remodeling and transcriptional control. Using U373 and U87MG cells, we combined a selective inhibitor (I‑BRD9) and CRISPR‑mediated BRD9 knockout with ionizing radiation (IR). BRD9 loss impaired viability and further reduced clonogenic survival in combination with IR. To capture early chromatin effects, we performed a histone Western‑blot panel that included BRD9‑linked acetylation marks, revealing a global reduction in active‑chromatin signatures upon BRD9 inhibition/knockout. These data indicated that BRD9 remodeling the epigenetic landscape may underlie the observed radiosensitization. To uncover the pathways altered by BRD9 loss, we next carried out transcriptomic profiling in two glioblastoma models (U373 and U87MG) exposed either to BRD9 inhibition or genetic knockout. Due to the downregulatory effect of BRD9, our studies focused more on genes with decreased expression that are intersecting in four different RNA sequencings. 31 downregulated genes were common in different conditions, which were linked to MYC signaling and translation machinery as a result of pathway analysis. BRD9 perturbation consistently rewired translation, MYC signaling, and ribosome biogenesis programs, with 31 genes reproducibly downregulated; many encode components of the translational machinery and tRNA aminoacylation. We focused on a MYC‑associated axis involving aminoacyl‑tRNA synthetases (AARS1, CARS1, GARS1, WARS1, YARS1) and EIF2S2. qPCR validated the coordinated suppression of these targets. Mechanistically, our data support a functional BRD9–MYC axis that coordinates aaRS/EIF2S2 expression and ribosome biogenesis. MYC overexpression restored translation‑related transcripts and ribosome‑biogenesis markers, reversing the effects of BRD9 inhibition and reinforcing this dependency. In parallel, transcripts for rRNA species essential to ribosome production (47S, 28S, 18S, 5.8S, 5S) decreased upon BRD9 inhibition/knockout, aligning with the epigenetic shift and supporting a model in which BRD9 sustains translational capacity. Together, these findings nominate BRD9 as an epigenetic regulator that regulates chromatin accessibility for protein synthesis in GBM and suggest that targeting BRD9 can disrupt the translational machinery and enhance radiotherapeutic response. Further studies will elucidate the direct genomic occupancy for BRD9 and MYC while also revealing the interacting partners of BRD9. Moreover, test in vivo whether disrupting the BRD9–MYC–aaRS/EIF2S2 circuit durably sensitizes GBM to radiation.
Partial chemical reprogramming for induced cellular rejuvenation
Aging is caused in part by epigenetic dysregulation, making cellular reprogramming a promising strategy to restore youthful function. Full induction of pluripotency resets cellular age but erases somatic identity, limiting therapeutic use. Partial reprogramming offers a compromise by transiently engaging reprogramming pathways to reverse hallmarks of aging while maintaining cellular identity. Chemical reprogramming has emerged as an alternative approach, using small molecules to remodel signaling and chromatin states in a more controllable and homogeneous manner than transcription factor induction. Here we compare genetic and chemical reprogramming modalities for cellular rejuvenation and show that chemical cocktails can reproduce and, in some respects, surpass genetic partial reprogramming, reducing senescence and improving mitochondrial performance while avoiding induction of pluripotency. Chemical reprogramming led to a homogeneous early change in identity and pluripotency markers, while factor induction produced mixed subpopulations and prolonged treatment increased stress. Screening by systematic removal of cocktail components identified MLL-menin inhibition and chromatin regulators such as p300/CBP as necessary for senescence suppression. By contrast, RAR signaling was indispensable. Its removal not only elevated senescence but also diminished identity markers. These results establish chemical partial reprogramming as a promising alternative strategy for epigenetic rejuvenation. They highlight the balance between senescence reduction and lineage stability and set the stage for protocols that may translate into safer, tunable therapeutic approaches.
Approaching mosaicism for the genomic odyssey of rare and undiagnosed diseases
Mosaicism is a fundamental biological phenomenon with broad implications for human health and disease. This thesis firmly establishes that postzygotic genetic variation arising at distinct developmental stages can lead to a diverse array of phenotypes, ranging from subtle pigmentary changes to severe multisystem disorders. The difficulties and limitations in detecting somatic mosaicism hinder definitive diagnosis of the affected individuals, yet the knowledge and data regarding its genetic basis remain limited. With a particular focus on selected syndromes/cases that exhibit segmental and/or cutaneous findings, we aimed to address these challenges by confirming clinical diagnoses of individuals in this patient group at the genomic level using advanced molecular and cytogenetic diagnostic techniques. The study is structured around two key components: a cohort presumptive for PIK3CA-related overgrowth spectrum syndromes and a series of single-case studies involving diverse mosaic phenotypes. Patients with clinical findings suggestive of mosaicism were evaluated using a multimodal diagnostic approach. By integrating thorough clinical phenotyping with genomic analyses, we identified, characterized, and redefined the molecular basis of various rare mosaic disorders. These findings encompass variants in crucial biological pathways, including the PI3K-AKT-mTOR and RAS-RAF-MAPK pathways, along with the components of cell-cycle regulation, mitochondrial dynamics, and chromosomal architecture. This study highlights the significant impact of timing and distribution of postzygotic variants on phenotypic expression and challenges conventional genotype-phenotype paradigms. The findings support the implementation of comprehensive clinical-genomic workflows and suggest that mosaicism can be viewed as a unifying framework for interpreting rare syndromes and complex phenotypes. Ultimately, this work reinforces our understanding of mosaicism as a fundamental, yet poorly understood and poorly explored concept in medical genetics, demonstrating the importance of personalized diagnosis for improved genetic counseling and care for patients and their families. The unresolved cases provide valuable resources for future research and fuel our continued desire to explore the unknown in cell and human biology.
Development of systems to produce antibodies to target tumor-stroma crosstalk
Cancer cells can program their tumor microenvironment (TME) in a manner that facilitates their adaptation, thereby influencing cancer survival, progression, and metastasis. In this context, it is crucial to elucidate the stroma components and their roles in various cancer types, as well as to investigate their potential applications in diagnosis, treatment, and follow-up. Previously, we demonstrated that cancer-derived secreted factors, cytokines, alter tumor fate through regulating stromal autophagy, cancer-stroma signaling. This thesis aims to develop systems for producing antibody-based drugs that target cytokines in the tumor microenvironment. To achieve this, we developed systems to produce recombinant cytokines and established a mouse single-chain variable fragment (scFv) library. To obtain these, we first cloned cDNA of cytokines into different IPTG-inducible, lac-promoter-containing vectors, including pGEX4T2 and pGEX4T1-3xFLAG. We validated our positive clones and further optimized the protocols to produce recombinant GST- and GST-FLAG-tagged cytokines using BL21 E. coli cells. In the second part, we focus on optimizing protocols and establishing recombinant scFv antibodies through phage display technology. Hence, we first determined the sequences of the antibody variable light (VL) and variable heavy (VH) chains. To establish a diverse scFv antibody library, we designed at least 36 oligos to produce VL and VH fragments. Fragments were further combined by SOE-PCR (splicing by overlap extension) and cloned into the pCANTAB-5E phage display plasmid for further phage display studies against recombinant cytokines. Overall, we established systems for producing recombinant cytokines and recombinant scFv antibodies. This study could lead to the discovery of new, specific, and drug-potential antibodies that target specific cytokines in the tumor stroma context, potentially serving as a new cancer therapeutic.
Epigenome-targeted CRISPR screen reveals stage-specific dependencies in human erythropoiesis
Erythropoiesis is a tightly regulated differentiation process that proceeds through discrete cellular states and culminates in terminal maturation and enucleation. While lineage-defining transcription factors are essential to erythroid gene regulation, including the master erythroid transcription factor GATA1, the mechanism of how epigenetic regulators shape cell state–specific transcriptional programs during erythroid maturation remains poorly understood. In this thesis, we systematically interrogated the epigenetic regulatory landscape of human erythropoiesis through CRISPR/Cas9-based functional screens. This work revealed two principal chromatin regulatory pathways that play central roles in erythroid differentiation: the histone acetyltransferase EP300 and the SMARCB1-containing cBAF (SWI/SNF) chromatin remodeling complex. Using synchronized human erythroid differentiation models, including BEL-A cells and primary CD34⁺ progenitors, we demonstrate that EP300 and SMARCB1 regulate erythropoiesis through distinct, cell state–dependent mechanisms. We show that EP300 is required at early stages of erythroid differentiation. Loss of EP300 disrupts early erythroid cell state transitions, leading to a broad failure in erythroid gene induction and impaired commitment to the erythroid lineage. In contrast, SMARCB1 is essential during terminal maturation. In primary CD34⁺ hematopoietic stem/progenitor cells, EP300 loss blocked progression at the basophilic–polychromatic transition, while SMARCB1 deficiency significantly reduced erythrocyte output and enucleation efficiency. Furthermore, using single-cell and bulk RNA sequencing, we further validated that EP300 governs differentiation-associated transcriptional programs. To understand the role of SMARCB1 in terminal erythropoiesis, we assessed chromatin accessibility across the genome and found that SMARCB1 loss does not alter global chromatin accessibility. We also found that GATA1 protein expression does not change with SMARCB1 perturbation. These findings indicated that SMARCB1 does not regulate erythroid maturation through changes in chromatin accessibility or transcription factor expression. To investigate the protein–DNA interactions underlying SMARCB1-dependent erythroid transcriptional programs, we performed Docking & Deamination-Seq (D&D-Seq), a deaminase-based footprinting approach that enables high-resolution mapping of transcription factor occupancy on chromatin. The results indicated that SMARCB1 knockout alters GATA1 occupancy at a subset of erythroid regulatory loci, including reduced binding at the SNCA locus and increased binding at TFR2. As SNCA is robustly induced during normal late-stage erythroid differentiation, these results demonstrate that SMARCB1 is required for proper GATA1-mediated activation of terminal maturation gene programs. Together, these findings establish EP300 as an early differentiation dependency and SMARCB1/cBAF as a late enucleation dependency, uncovering distinct chromatin-based mechanisms that coordinate sequential stages of human erythropoiesis.
Kronik lenfositer lösemi ile IL-10, IL-17A, IL-17F ve TGFß1 gen polimorfizmleri arasındaki ilişki
Amaç: Bu çalışmada, IL-10 geni -592A/C, IL-17A geni -197A/G , IL-17F geni 7383A/G ve TGFß1 geni +915G/C polimorfizmlerine ait genotip dağılımlarının ve allel sıklıklarının saptanması ve bu polimorfizmler ile KLL arasındaki ilişkinin tespit edilmesi amaçlanmaktadır.Materyal ve Metot: 2011 Nisan ile 2012 Nisan dönemleri arasında Karadeniz Teknik Üniversitesi Tıp Fakültesi İç Hastalıkları Anabilim Dalı Hematoloji Bilim Dalı'nda tanısı konulan ve klinik takibi yapılan yaşları 32 ile 84 arasında değişen 35'i erkek, 15'i kadın 50 KLL hastasından ve yapılan muayene ve tetkikler sonucu sağlıklı olduğu tespit edilen yaşları 45 ile 75 arasında değişen 35'i erkek, 15'i kadın 50 bireyden alınan periferik venöz kandan DNA izole edilmiş ve PCR-RFLP yöntemi ile ilgili genotipleri belirlenmiştir. Rastgele örneklerden yapılan DNA dizi analizi ile PCR-RFLP yöntemi konfirme edilmiştir.Bulgular: IL-10 geni -592A/C, IL-17A geni -197A/G , IL-17F geni 7383A/G ve TGFß1 geni +915G/C polimorfizmleri için saptanan genotip dağılımları ve allel sıklıkları açısından hastalar ile kontrol grubu arasında bir fark saptanmamıştır. Bu polimorfizmlerin hasta tanı yaşı, tanı anında lenfosit sayısı, IgG, IgA ve IgM seviyeleri, FISH bulguları ve hastaların klinik evresi gibi klinik ve laboratuvar özellikleri üzerindeki etkisi incelendiğinde, TGFß1 geni +915GC genotipi ile trizomi 12 arasında istatistiksel olarak anlamlı ilişki saptandı. Ayrıca kadın hastalarda IL-17A -197AG genotipi sıklığı erkek hastalara göre istatistiksel olarak anlamlı şekilde daha fazla saptandı.Sonuç: IL-10 geni -592A/C, IL-17A geni -197A/G , IL-17F geni 7383A/G ve TGFß1 geni +915G/C polimorfizmleri ile KLL riski arasında istatistiksel olarak anlamlı bir ilişki bulunmamakla birlikte; TGFß1 geni +915GC genotipinin, normal karyotipe göre daha uzun sağ kalımın görüldüğü trizomi 12 ile ilişkili olabileceği, ayrıca, IL-17A -197AG genotipine sahip kadınların aynı genotipe sahip erkeklere göre daha fazla KLL riskine sahip olabileceği düşünüldü.
Ankilozan spondilit ile IL-17 gen polimorfizmleri arasındaki ilişki
Amaç: Ankilozan Spondilit (AS), patogenezi tam olarak anlaşılamayan inflamatuar bir hastalıktır. İnflamatuar sitokin interlökin-17 (IL-17), hücresel yanıtın oluşturulmasından sorumludur. IL-17'nin altı aile üyesi vardır (A, B, C, D, E ve F). Bu çalışmada Ankilozan spondilit ile IL-17A ve IL-17F gen polimorfizmi arasında bir ilişki olabileceğini amaçladık. Ayrıca BASDAI skorları, aile öyküleri ve HLA-B27 değerleri allel frekansı ile hesaplandı. Gereç ve Yöntem: 89 Ankilozan Spondilit hastasını ve 89 sağlıklı kontrolü inceledik. RFLP yöntemi kullanarak IL17A (-197A / G) ve IL 17F (7383A / G) polimorfizmlerini inceledik. Bulgular: IL17A (-197A / G) polimorfizmi ile Ankilozan Spondilit arasında anlamlı bir ilişki bulduk. IL-17A geni -197AG polimorfizmlerinin genotip dağılımları ve BASDAI skorları ile aile öyküsü arasında istatistiksel olarak anlamlı bir ilişki yoktu. Bununla birlikte, hastaların genotip dağılımlarında HLA-B27 sonuçlarıyla istatistiksel bir ilişki vardı. IL-17F (7383A / G) ile AS, BASDAI puanları, aile geçmişi ve HLA-B27 sonuçları arasında bir ilişki bulamadık. Sonuç: Sonuç olarak, AS ile genetik ve sitokin etkisi yıllara göre incelenmiştir. Çalışmamızda AS ile IL-17A gen polimorfizmi arasında önemli bir bağlantı vardır. Bu, IL-17A gen polimorfizmi ile AS arasında ilişki bulan ilk çalışmadır. Bu ilişkiden emin olmak için daha ileri çalışmalar yapılmalıdır.
Mide kanserli hastalarda Ku70 gen polimorfizmi araştırılması
Genomda; DNA çift zincir kırıklarının tamirinde homolog olmayan uç birleştirme onarım sisteminin önemli bir üyesi olan DNA tamir geni Ku70?in önemli bir rol oynadığı düşünülmektedir. Bu çift zincir kırık onarımı kapasite bozukluklarının geri dönüşümsüz olarak genomik kararsızlığa yol açtığı bilinmektedir. Ancak, Ku70 genine ait polimorfik varyantlar ve mide kanseri duyarlılığı arasındaki bağlantı Dünya?da ikinci, ülkemizde ise ilk kez tarafımızca çalışılmıştır. Hastanemiz Genel Cerrahi Bölümü?nde değerlendirilen mide kanserli 92 hasta ve kontrol grubu olarak ailesinde ve kendisinde kanser öyküsü olmayan 194 gönüllü çalışmaya dahil edilmiştir. Bu kişilerden DNA tamirinde görevli Ku70 proteini ile ilgili iki polimorfizm araştırılmıştır. Bu polimorfizmler intron 3 (rs132774) ve G-57C (rs2267437) polimorfizmleridir. Çalışma yöntemi olarak gerçek zamanlı polimeraz zincir işlemi kullanılmıştır. Sonuçlar Ki-Kare testi ile değerlendirilmiştir. Değerlendirilen polimorfizmlerde kanserli hasta örnekleri ve sağlıklı kontrol grubuna ait örnekler arasında anlamlı bir fark saptanmamıştır. Özet olarak, Ku70 promotör G-57C (rs2267437) ve intron 3 (rs132774) polimorfizmleri ve mide kanseri gelişiminin birbiriyle ilişkili olmadığı görülmüştür.
İdiyopatik adölesan skolyozunda vitamin D reseptör geni Bsm1 polimorfizmi
Dokuz Eylül Üniversitesi Tıp Fakültesi, Tıbbi Genetik Anabilim Dalı, İzmirOmurgada koronal planda yana doğru oluşan eğrilik nedeniyle direkt grafide ölçülen Cobb açısının on derecenin üzerinde olmasıyla tanımlanan Skolyoz; toplumda %3 gibi yüksek sıklıkta görülen, olguların %85 kadarında altta yatan neden bulunamadığı için idiyopatik olarak değerlendirilen, en sık adölesanlığa karşılık gelen 10 ? 20 yaş aralığında ortaya çıkan, her iki cinsi eşit oranda tutmasına rağmen kızlarda on kat daha fazla ilerleyici bulunan, ikiz kardeşlerdeki konkordans ve olguların aile ağaçlarının incelendiği epidemiyolojik çalışmalar sonucunda birden fazla genin etkileşmesi sonucu poligenik kalıtımla oluştuğu düşünülen, etyolojisinde genetiğin büyük rol aldığı bir hastalıktır. Vitamin D reseptör geninin kemik dokusu üzerinde düzenleyici etkileri vardır ve BsmI polimorfizmi osteopeni ile ilişkilendirilmiştir. Osteopeni skolyozun klasik bir komponentidir. Çalışmamızın amacı İdiyopatik Adölesan Skolyozu ile Vitamin D reseptör geni BsmI polimorfizmi arasında etyolojik olarak ilişki olup olmadığını araştırmaktır.Adölesan İdiyopatik Skolyozu tanısı koyulmuş, birbirleriyle akraba olmayan yedisi erkek 45'i kız toplam 52 hasta ve 20 ? 35 yaş arasındaki, birbirleriyle akraba olmayan, yedisi erkek toplam 53 gönüllü kontrol grubu olarak çalışmaya dahil edilmiştir ve olguların periferik kan örneklerinden DNA izolasyonu yapılarak VDR BsmI genotipi strip test kullanılarak belirlenmiştir.Hasta grubunda genotip dağılımı bb %25, Bb %59,6, BB%15,4 iken kontrol grubundaki dağılımın bb %28,3, Bb %37,3, BB %34 olduğu görülmüş ve iki grubun genotip frekanslarının farkının anlamlılığını gösteren Ki-kare değeri: 6,35 ve p değeri: 0,0417 olarak bulunmuştur.Sonuçlar Vitamin D reseptör geni BsmI polimorfizminde Bb genotipi ile İdiyopatik Adölesan Skolyozu arasında ilişki olabileceğini düşündürmüştür.