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Türkiye sularına ait siyanobakteri türlerinden izole edilen ekzopolisakkaritlerin (EPS) karakterizasyonu ve kozmetik sektöründe kullanılma potansiyellerinin araştırılması
Bu yüksek lisans tez çalışmasının amacı, Türkiye'nin farklı bölgelerinden izole edilen siyanobakteri türlerinin EPS'lerini araştırmak, karakterize etmek ve kozmetik endüstrisinde kullanılabilme potansiyelini araştırmaktır. Siyanobakteri türleri (55 ve 72) tarafından üretilen EPS'lerin monosakkarit kompozisyonları, fonksiyonel grupları ve kristalografik özellikleri sırasıyla HPLC, FT-IR ve XRD ile belirlenmiştir. Ayrıca, antioksidan aktiviteleri, nem emme ve nem tutma kabiliyetleri ve tirozinaz inhibisyon aktiviteleri değerlendirilmiştir. 16S rRNA dizileme sonucu suşlar >%98,9 benzerlik oranıyla Chlorogloeopsis fritschii (55) ve Pseudanabaenaceae cyanobacterium (72) olarak belirlenmiştir. 55 ve 72 kodlu suşların EPS üretim miktarları sırasıyla 888,0 mg/L ve 776,8 mg/L olarak bulunmuştur. İlk defa monosakkarit kompozisyonları belirlenen C. fritschii (55 kodlu suş) ve P. cyanobacterium (72 kodlu suş) suşlarından elde edilen EPS'lerde ramnoz, glukoz, arabinoz, mannoz, ksiloz ve galaktoz türlere göre farklı oranlarda bulunmuştur. En baskın monosakkarit ramnoz olarak tespit edilmiştir. EPS'lerin amorf yapıda olduğu ve farklı fonksiyonel gruplar içerdiği tespit edilmiştir. Her iki EPS'nin de %80 DPPH süpürme potansiyeline ve %8-11 arasında değişen toplam antioksidan kapasitesine sahip olduğu bulunmuştur. Ayrıca, her iki EPS'de kozmetik sektöründe yaygın olan üreye ve kitosana benzer nem tutuma kapasitesi gösterirken, onlardan daha iyi nem emilim kapasitesine sahip olduğu tespit edilmiştir. Ek olarak, 55 kodlu EPS'nin literatüre kıyasla düşük bir konsantrasyonda (2 mg/mL) %10'luk bir tirozinaz inhibisyon aktivitesine sahip olduğu bulunmuştur. Bu sonuçlar, siyanobakterilerin yüksek miktarda EPS ürettiğini ve siyanobakteriyel EPS'lerin antioksidan ve anti-tirozinaz aktivitelerinin yanı sıra nem emme ve nem tutma kapasiteleri ile kozmetikte potansiyel bir doğal bileşen olarak kullanım için umut verici bir aday olduğunu göstermektedir.
Kuraklık stresine maruz bırakılan biberde (Capsicum annuum L.) eksojen strigolakton uygulamasının fizyolojik ve biyokimyasal etkileri
Bu tez çalışmasında, kuraklık stresi ve stressiz koşullar altında yetiştirilen iki biber çeşidinde (C. annuum L. cv. Oğuz ve C. annuum cv. Çözüm), 10 µM eksojen strigolakton uygulamasının fizyolojik [çimlenme yüzdesi (%), kök uzunluğu (cm), gövde uzunluğu (cm), gövde çapı (mm), dallanma sayısı, yaprak genişliği (mm), bitki boyu (cm) ve taze ağırlık (g)] ve biyokimyasal [total protein miktarı, prolin, lipid peroksidaz (MDA), superoksit dismutaz (SOD), katalaz (CAT), askorbat peroksidaz (APX)] etkileri karşılaştırmalı olarak değerlendirilmiştir. Yapılan çalışmada, %10 PEG-6000 kuraklık stresinin etkisini hafifletmek için, sentetik strigolakton analoğu olan GR24'ün 10 µM eksojen uygulamasının her iki biber çeşidinde de çimlenmeyi teşvik ettiği, kök uzunluğu, gövde uzunluğu, bitki boyu, yaprak genişliği ve gövde çapı parametreleri üzerine olumlu etki gösterdiği ve büyüme/gelişmeyi desteklediği belirlenmiştir. Bununla birlikte, biyokimyasal ve aktioksidan aktivite üzerine çalışılan parametrelerde total protein miktarı, prolin, MDA, SOD, APX aktivelerinin kuraklık stresi koşullarında arttığı, kuraklığın olumsuz etkisini hafifletmede 10 µM eksojen strigolakton uygulamasının iyileştirici etki gösterdiği ortaya konulmuştur. Buna karşın, kuraklık stresi koşullarında CAT aktivitesi her iki çeşit içinde, tüm parametrelere oranla stressiz koşullarda azalma gösterirken Çözüm çeşidinde kuraklık stresi koşullarında 10 µM eksojen strigolakton uygulamasının stressiz koşullar altındaki strigolakton uygulamasına göre, 2 katından fazla oranda azalma gösterdiği belirlenmiştir. Tüm parametreler bir arada değerlendirildiğinde, Çözüm çeşidinin Oğuz çeşidine oranlar kuraklık stresine karşı daha dirençli olduğu, eksojen stigolakton uygulamasının uygun konsantrasyonlarda kuraklık stresinin bitki büyüme/gelişmesi üzerindeki olumsuz etkilerini hafifletmede yaprağa püskürtme yöntemiyle uygulanmasının daha başarılı sonuçlar sunduğu ortaya konulmuştur.
Yeni paladyum komplekslerinin kolon kanseri hücrelerindeki anti-kanser aktivitesinin araştırılması
Kolon kanseri, dünyada en sık rastlanan 3. kanser türüdür. Dünya genelinde her yıl bir milyon yeni kolon kanseri vakası tespit edilmektedir ve bunların yarısından fazlası hayatını kaybetmektedir. Kolon kanseri yavaş ilerler ve kolon kanserine yakalanan hastaların sadece %40'ı erken teşhis edilebilmektedir. Kolon kanseri tedavisinde çok sayıda ilaç kullanılmaktadır ancak erken teşhis yapılamayan hastalarda bu ilaçlar çok etkili olamamaktadır. Bu nedenle kolon kanseri tedavisi için yeni anti-kanser ajanların geliştirilmesine ihtiyaç duyulmaktadır. Kanser hücreleri, birden fazla mekanizma ile hayatta kalmayı ve uygulanan tedavilere karşı direnç geliştirmeyi başarmaktadır. Kanser hücrelerinin bu özelliği dikkate alındığında etkili bir tedavi yöntemi geliştirmek zordur ve yeni yaklaşımlara ihtiyaç duyulmaktadır. Kolon kanseri tedavisinde birçok farklı yolakta görev alan enzim ya da proteinler hedef alınmaktadır. Bu yolaklar hedef alınırken birçok farklı bileşik türü kullanılmaktadır. Metal iyonu içeren bileşikler kanser tedavisinde yaygın olarak kullanılmaktadır. Bu çalışma kapsamında Fen-Edebiyat Fakültesi Kimya Bölümü öğretim üyesi Prof. Dr. Fatih Mehmet Emen ve ekibi tarafından ilk defa sentezlenmiş 4 farklı paladyum kompleksinin, SW620 kolon kanseri hücre hattı ve CCD-18Co normal kolon hücresi üzerindeki anti-kanser etkileri araştırılmıştır. Öncelikle her iki hücre hattı için bileşikler 1-200 µM aralığında hücrelere uygulanmış ve hücre canlılık testi yapılarak IC50 değerleri hesaplanmıştır. Devamında P4 olarak isimlendirilen bileşiğinin normal hücre hattı ve kolon kanseri hücre hattında IC50 değerinde ve daha düşük dozlarda uygulamasının apoptozis, otofaji ve hücre döngüsü üzerine etkileri araştırılmıştır. Apoptozis, otofaji ve hücre döngüsü belirteç genlerinin ifade değişimleri western blot yöntemi ile analiz edilmiştir. Ayrıca akış sitometrisi yöntemi ile hem ölü hücre dağılımı hem de hücre döngüsü aşamaları analiz edilmiştir. Sonuç olarak P4 bileşiğinin düşük dozlarda kolon kanseri hücrelerinde apoptozisi uyardığı ve hücre döngüsünü durdurduğu gözlenmiştir.
Fisetin'in Drosophila melanogaster'de yaşlanmayla ilişkili bazı genlerin ifadeleri üzerine etkisi
Yaşlanma canlılar için doğal ve fizyolojik bir olaydır. Hücresel yaşlanma olan senesens metabolik olarak hücre aktivitesinin devam ettiği ancak hücrelerin bölünmediği bir süreci oluşturmaktadır. Metabolik aktiflikleri devam eden senesent hücreler senesensle ilişkili salgı fenotipleri (SASP) salgılar. Bu salgı bileşenleri organizmaların kronik hastalıklar başta olmak üzere çeşitli hastalıklara yakalanma riskinin artmasına neden olur. Senolitikler senesense uğramış olan hücreleri çeşitli özelliklerine göre hedef alarak onları spesifik bir şekilde ortadan kaldıran bileşiklerdir. Bu tez çalışması kapsamında Bcl-2 ailesi hedefli bir senolitik madde olan fisetin ile çalışılmıştır. Fisetin, moleküler biyolojide model organizmalardan biri olan Drosophila melanogater olarak bilinen meyve sineğine uygulanmıştır. Uygulama 2 farklı dozda (0,002 M ve 0,005M) ve 4 farklı gün grubunda (7, 14,21 ve 28 gün) yapılmıştır. Farklı sürelerde ve fisetin dozlarında yaşlanmayla ilişkilendirilen memeli sirtüin 1 homoloğu dsir2, dFOXO ve dRPD3 genlerinin ifadeleri RT-qPCR yöntemiyle analiz edilmiştir. Elde edilen tüm verilerin istatistiksel analizi SPSS yazılımı 20.0 (IBM, Armonk, NY, ABD) kullanılarak %95 güven aralığında Duncan testi (p < 0,05) ile analiz edilmiştir. Gen ifadeleri incelenen genlerin ayrıca Cytoscape yazılımı kullanılarak protein protein etkileşimleri ve TBtools programı ile farklı dokularda dijital ifade profilleri çıkarılmıştır. Yapılan ifade analizleri sonucunda dsir2 geni ifadesi en yüksek 7. Günde 0,005M fisetin uygulaması yapılan grupta, 14. günde 0,002 M fisetin uygulaması yapılan grupta, 21. günde 0,005M fisetin uygulaması yapılan grupta ve 28. günde fisetin uygulanmamış kontrol grubunda olduğu görülmüştür. dFOXO geni için en yüksek gen ifadesinin 7. günde 0,002 M fisetin uygulaması yapılan grupta, 14. günde fisetin uygulanmamış kontrol grubunda, 21. günde 0,005M fisetin uygulaması yapılan grupta ve 28. günde fisetin uygulanmamış kontrol grubunda olduğu ve dRPD3 geni için en yüksek gen ifadesinin 7. Günde 0,005M fisetin uygulaması yapılan grupta, 14. günde 0,002 M fisetin uygulaması yapılan grupta, 21. günde 0,005M fisetin uygulaması yapılan grupta ve 28. günde fisetin uygulanmamış kontrol grubunda olduğu görülmüştür.
Vitamin D3'ün multiple myeloma hücrelerinde uzun kodlayıcı olmayan RNA (lncRNA) ifadesine etkisinin incelenmesi
Multiple myeloma (MM), anormal plazma hücrelerinin kemik iliğinde aşırı çoğalmasıyla gelişen hematolojik bir malignitedir. Hematolojik kanserlerin %10'unu oluşturan MM, genellikle 69 yaş ve üzeri bireylerde görülmektedir. MM hastalarına kombine ilaç terapisi ve uygun bireylerde kök hücre transplantasyonu uygulansa da, zamanla gelişen ilaç direnci sonucu hastalık nüksetmekte ve hastalar kaybedilmektedir. Bu nedenle hastalığın erken tanısı, önleyici tıp yaklaşımlarının geliştirilebilmesi ve karşılaşılan ilaç direncinin aşılması için hastalığın gelişiminde rolü olan moleküler mekanizmaların aydınlatılması son derece önemlidir. MM, hem genetik hem epigenetik düzensizliklerin sonucu ortaya çıkmaktadır. Epigenetik mekanizmanın bir parçası olan uzun kodlayıcı olmayan RNA (lncRNA)'ların anormal ifadesi, birçok patolojide ve MM'de sık rastlanan bir durumdur. Tümör baskılayıcı veya onkogenik karaktere sahip olabilen lncRNA'ların anormal ifadeleri, MM'de tümör gelişimini ve ilaç direncini teşvik etmektedir. lncRNA ifadesini düzenleyici rolü olduğu bilinen vitamin D, bir kemik iliği mikroçevresi elemanı olup, MM hastalarının %44'ünde yetersiz seviyededir. Promotör bölgesinde vitamin D yanıt dizileri (VDRE) bulunan lncRNA'ların transkripsiyonunu yönlendirebilen vitamin D'nin ortamda yeterli miktarda bulunmaması, lncRNA ifade düzensizliklerine yol açabilir. Bu bağlamda, bu tez çalışmasında insan MM hücre hattı olan NCI-H929 hücrelerine 24 ve 48 saat sürelerle tek başına veya proteazom inhibitörü carfilzomib ile birlikte uygulanan vitamin D'nin, MM'de farklı fonksiyonları olan hedef lncRNA (H19, NEAT1, MEG3, TUG1, UCA1) düzeylerine etkisi incelenmiştir. Bu çalışmanın bulgularına göre, vitamin D, lncRNA UCA1'in ifadesini artırırken, diğer lncRNA seviyelerinde önemli bir değişime yol açmamıştır. Vitamin D-carfilzomib kombine uygulaması ile yine UCA1 ifadesinde önemli bir artış tespit edilmiştir. Sonuç olarak, vitamin D, NCI-H929 hücrelerinde onkogenik lncRNA UCA1'in transkript seviyesini artırmaktadır. Bu etkinin mekanizması ve MM hücre işlevleri üzerindeki etkilerini inceleyen ek çalışmalar, vitamin D'nin MM hastalarında güvenle kullanılabilmesi için kritik öneme sahiptir.
Centriolar satellites are required for efficient ciliogenesis and ciliary content regulation.
Centrosome is the main microtubule organizing center of the cell. It has the role in cell division, cell shape and migration. Other than these roles, centrosome has an important function which is forming primary cilium. Primary cilium is a microtubule-based organelle serving as signaling hub for the cell. Structural or functional defects associated with centrosome/cilium complex lead to genetic diseases called ciliopathies. In order to understand molecular mechanism under ciliopathies, it is important to understand how centrosome/cilium complex is regulated in time and space. Centriolar satellites are the third component of the centrosome/cilium complex In the second chapter of my thesis, I characterized the cells without centriolar satellites in terms of ciliogenesis and ciliary function. I established the satellite-less cells by knocking-out PCM1 gene in inner medullary collecting duct (IMCD3) cells. PCM1 is the scaffolding protein of the centriolar satellites. Satellite-less IMCD3 cells do not ciliate efficiently as much as control cells. Content and function of cilia formed by satellite-less cells are also different. To understand how cellular process are affected in PCM1 knock-out cells, I applied tandem mass tag (TMT) labeling‐based quantitative analysis to compare the global proteome control and satellite-less cells. This analysis revealed that many processes such as actin cytoskeleton, cell migration and adhesion, endocytosis, neuronal processes are affected in the absence of PCM1 protein. With these observations, I showed that centriolar satellites are important to regulate ciliogenesis, ciliary content and function. However, the mechanism behind this regulation is poorly understood. In the third chapter of my thesis, I applied the miniTurbo labeling method to compare interaction partners of PCM1 in asynchronous and ciliated cells. The aim of this chapter to find interaction partners of PCM1 specific to ciliated cells to explain mechanism of regulation by centriolar satellites during ciliogenesis. After miniTurbo experiments and mass spectrometry analysis, we determined a set of protein which interact with PCM1 in ciliated cells. Among these proteins, we checked the localization and function of the TBC1D31 protein by loss of function experiments. In this chapter, we determined the set of protein might have a role in regulation of ciliogenesis with centriolar satellites. The interactions between these proteins with PCM1 and their relationship in the regulation of ciliogenesis are going to be explored in our future studies.
Chemically induced assay for centriolar satellite mispositioning reveals their functions at the primary cilium
Centriolar satellites are membrane-less, electron dense granular structures that localize and move around centrosomes and cilia. The satellite proteome is composed of over 200 protein components, which were implicated in a wide range of functions such as centriole duplication, cell division, cellular signaling, primary cilium biogenesis and microtubule dynamics. Importantly, various proteins mutated in ciliopathies and primary microcephaly were also identified as part of the satellite proteome, suggesting an intimate link between centriolar satellite function and development. Although centriolar satellites have remained as understudied structures since their discovery more than 60 years ago, recent work showed that satellites store, modify and traffic centrosome/cilium proteins and play important roles in the regulation of centrosome/cilium biogenesis and function. To mediate their trafficking function, satellite cluster and move around centrosomes in most cell types. However, satellite distribution varies in response to different stimuli such as cell cycle cues and across different cell types, suggesting context-dependent functions for satellites. Dissecting these spatial and temporal functions have been challenging using traditional approaches such as loss-of-function studies, in particular, in ciliated cells. To overcome these challenges, I developed a chemical based inducible trafficking assay that allows efficient redistribution of centriolar satellites to cell periphery or cell center. Using this assay, I showed that satellite mispositioning disrupts centrosomal targeting of key regulators of cilium biogenesis, identifying a direct role for satellites in centrosomal protein targeting and sequestration. To identify the functional consequences of satellite mispositioning, I used functional assays to probe cilium biogenesis and microtubule dynamics in cells where satellites were mispositioned at the membrane. The results of these assays showed that satellites regulate primary cilium assembly, maintenance and disassembly. Taken together, our results showed a direct link between satellite functions and their pericentrosomal clustering in ciliated cells and also provided a new tool for studying acute functions of satellites in a context-dependent way. Finally, given the crucial roles of the primary cilium as the signaling center for developmentally important signaling pathways such as Hedgehog signaling, our results sheds light into why satellite proteins are mutated in developmental disorders.
A novel control mechanism of mitotic exit in Saccharomyces cerevisiae
Mitotic exit is the cell cycle stage in which the cell transits from M phase to a new G1 phase. While mitosis is under control of cyclin-dependent kinases (CDK), mitotic exit depends on inactivation of CDKs. Timely coordination of mitotic CDK inactivation with respect to chromosome segregation is important to avoid aneuploidy and maintain ploidy. A comprehensive understanding of regulation of mitotic exit is missing. In budding yeast, mitotic exit is achieved by the use of a special network, called Mitotic Exit Network. An inhibitor to Mitotic Exit Network, Kin4 kinase, leads to lethality for the cells when is expressed in high doses. Our goal is to find out novel mechanisms of mitotic exit control. In this thesis, we identified a temperature sensitive mutant that rescues the lethality of Kin4 overexpression. Through a "dosage suppressive genetic screening", we found two genes, SAN1 and PHO2, that retarded growth this temperature sensitive mutant when Kin4 was overexpressed. We further characterized the effect of these genes on mitotic exit, with a focus on SAN1. Our results indicate a novel regulatory mechanism for mitotic exit. Data presented in this thesis will pave the way to illuminate a new section in the process of exiting from mitosis. Owing to the fact that the basic cellular tasks are preserved from yeast to human, we envisage that characterization of this newly emerged role in the mitotic exit of S. cerevisiae would contribute to the understanding of analogous control mechanisms in more complex organisms.
Hücre yüzeyi proteinlerinin sitokinezdeki rollerinin belirlenmesi
Cell division requires many sequential and coordinated actions of chromosomes, cell cycle machinery, cytoskeleton, and cellular membranes. These events have been extensively studied in many cell biology laboratories. Among those, how cell surface components and intracellular cell cycle machinery communicate, especially during cytokinesis, is much understudied. In this project, we aim to determine the roles of cell surface proteins during cytokinesis. Previous studies in the Eggert lab have shown that Glypicans 3 and 5 might have a role in cytokinesis since silencing their expression causes multinuclear cells. Glypicans are members of heparan sulphate proteoglycans that are highly conserved among eukaryotes. They are covalently attached to the plasma membrane via GPI anchors where they entirely face the extracellular matrix. In this thesis, I determined cellular localization of Glypican 3 and 5 during cell division and characterized their cell division phenotype. We performed a mass spectrometry-based proximity interactome analyses of Glypicans 3 and 5 at interphase and dividing cells. Our study revealed that Glypican 5 might have important roles in cortical actin stability and midbody abscission. In addition, we identified several proteins as Glypican 3 and 5 interactors that might give insights about the molecular mechanisms underlying their role in cytokinesis.
Structural analysis of phosphorylation sites at a proteome level and their functional relevance
Phosphorylation is an essential post-translational modification for the regulation of almost all cellular processes. Several global phosphoproteomics analyses revealed proteome-wide phosphorylation events and changes in phosphorylation profiles under different conditions. Beyond phospho-site sequence positions identified by proteomic approaches, protein structures add another layer of information to assess the biological relevance of phosphorylation events. In this study, we systematically characterize phosphorylation sites based on their 3D locations in the protein and establish a location map for phospho-sites. More than 250,000 phospho-sites have been analyzed of which 8,686 sites match at least one structure and are stratified based on their respective 3D positions. Core phosphorylation sites possess two distinct groups based on their dynamicity. Dynamic core phosphorylations are significantly more functional compared to static ones. Dynamic core and the interface phospho-sites are the most functional among all 3D phosphorylation groups. Our analysis provides global characterization and stratification of phosphorylation sites from a structural perspective that can be utilized for predicting the functional relevance and filtering out false positives in phosphoproteomic studies.
Structural dynamics of wild type and mutant forms of CLIC4 and ezrin
Cell division is an extremely vital process in life that occurs continuously within all living beings to maintain genomic integrity, cell growth, reproduction and regeneration. It enables the cells to survive by replacing old, damaged, or dead ones with newly formed healthy daughter cells. Therefore, various types of cellular proteins have a role in this process to ensure to complete the division successfully. It has been demonstrated that during cytokinesis, the interaction of ezrin with Chloride Intracellular Channel 4 (CLIC4) protein at the plasma membrane is required for the completion of cytoplasmic division. Their co-localization anchors the plasma membrane and actin cytoskeleton at the cleavage furrow and the midbody providing cortical stability. The successful translocation of CLIC4 to the cleavage furrow and the midbody requires its conserved residues Cys35 and Phe37. When these two residues are mutated to C35A and F37D, respectively, the localization of CLIC4 to the mitotic cell surface and cleavage furrow is abolished. Moreover, ezrin needs to be activated to interact with CLIC4. First, it should bind to phosphatidylinositol-(4,5)-biphosphate (PIP2) and then be phosphorylated from its conserved residue threonine (Thr567) at the carboxyl terminus. However, the mutual interaction between ezrin and CLIC4 is still not well understood. Also, it is not clear how a mutation at the conserved residue Cys35 of CLIC4 or at the phosphorylation site of ezrin changes their structures. Since this interaction might have an important role in cancer therapy, this study aimed at unveiling the mystery behind structural information of these two proteins. Although the wild type structures of CLIC4 and ezrin proteins were previously determined, the structures of their mutant forms, CLIC4 C35A and ezrin T567D, are not available. Thus, this study has been motivated to analyze the structural dynamics of wild type and mutant forms of CLIC4 and ezrin with their mutual interaction.
Identification of oncostatin M target genes by RNA-seq in mouse primary myotube cells
Cancer cachexia is a debilitating wasting syndrome marked by inflammation and loss of adipose and skeletal muscle tissues. While about 20% of cancer deaths are caused by cachexia, there are no effective treatments available, and molecular mechanisms underlying cancer cachexia remain elusive. Oncostatin M, belonging to the gp130/IL6 cytokine family, is mainly released by macrophages, monocytes, and T-cells and exert pleiotropic activities in cell differentiation, proliferation, and inflammatory network. In various cell and cancer types, OSM was paradoxically reported to both promote and inhibit inflammation. Even though cytokine IL-6 is known to be associated with muscle atrophy, the functional significance of cytokine OSM in skeletal muscle cells is poorly understood. The advent of high-throughput sequencing technologies presented an unprecedented approach to study RNA. Therefore, it has become the prime choice to characterize and quantify cell transcripts. Here, we aim to investigate tumor signaling to muscle tissue by identifying cytokine Oncostatin M target genes in skeletal muscle cells using high- throughput RNA sequencing (RNA-seq) technology. Using "Tailored Pipeline," we have analyzed IL6 family cytokines, specifically OSM, IL6 and, LIF. We treated mouse primary myotube cells with these cytokines and deciphered the differentially expressed genes that were significantly up-and down-regulated and show association with muscle atrophy. RNA sequencing showed widespread alterations in cytokine treated samples compared to control samples, with the largest differences in OSM treated samples compared to other conditions. OSM significantly induced muscle atrophy-related genes such as Ampd3, Sln, Murf-1, Atrogin-1, and Serpina3n. Consistent with this, these signature genes were validated using qRT-PCR. GSEA analysis revealed that top commonly enriched pathways include hypoxia, inflammatory response pathway, glycolysis, and IL6-JAK-STAT3 pathways. These results provide a global view of OSM, IL6, and LIF expression responses in mouse primary myotube cells with cytokine-specific response patterns. Our results suggest these cytokines may use similar signaling mechanisms in mouse primary myotube cells. Compared to IL6 and LIF, OSM potently promotes cellular atrophy in cultured myotubes. Elucidating cytokine-induced mechanisms may establish potential therapeutic targets for the treatment of the cachectic syndrome. Our findings support an important role for OSM in cellular muscle atrophy and suggest that OSM could be targeted as a novel factor in fighting cachexia associated with cancer or other chronic diseases.
Quantitative proteomics analysis of clear cell renal cell carcinoma for the identification of diagnostic and prognostic biomarker panels
Clear cell Renal Cell Carcinoma (ccRCC) is the third most common and most malignant urological cancer, with a 5-year survival rate of 10% for patients with advanced tumors. Despite an increasing rate of early detections, one third of the patients already show metastasis at diagnosis. Inherent and acquired resistance to chemo- and radiotherapies complicate the treatment of the disease, leaving the current treatment option primarily to surgical resection of the tumor. Biomarkers can help to monitor and to target tumor progression and growth, and to make a better prognosis on patient outcome. However, no universal biomarkers are in clinical use for ccRCC. Here, a rigorous quantitative dimethylation-based proteomics approach is described to identify biomarker panels for the diagnosis (part I) and for the stratification (part II) of ccRCC tumors, and to illuminate the driving phosphosignaling events in renal cancers (part III). The comprehensive characterization of the ccRCC global proteome and phosphoproteome revealed that the candidate marker proteins PLOD2, FERMT3, SPARC and SIPRα are overexpressed, and that diverse kinases of the groups CDK, PAK and MAPK are highly activated in the tumor tissues compared to normal adjacent tissues. The associated phosphosignaling cascades are linked to tumor growth and metastasis. Furthermore, our analysis suggested that due to interpatient heterogeneity, ccRCC tumors distinguish into two groups with distinct overall survival of patients and different enriched malignant pathways. Overall, the suggested biomarkers can serve as targets for future treatment strategies of ccRCC tumors in combination with approved therapeutics.
Characterization of the single nucleotide polymorphisms that effect repression activity and stability of cryptochrome2
The daily rhythm of sleep/wake cycles, body temperature, metabolism, hormone secretion and many other physiological activities are in control of the circadian mechanism which is conserved from simple cyanobacteria to complex humans. In mammals, the circadian clock is endogenous and highly regulated with complex transcriptional and translational feedback loops (TTFL). Entrainment of the rhythm through the whole body is maintained by suprachiasmatic nuclei (SCN) located in the anterior hypothalamus with the cues from the environment. Many physiological processes are linked to the circadian clock, and it is not surprising that disruption of the clock can lead to certain disorders as demonstrated in various studies. At the molecular circadian mechanism of mammals, Circadian Locomotor Output Cycles Kaput (CLOCK) and Brain and Muscle ARNT-Like-1 (BMAL1) heterodimerizes and transactivates the expression of clock controlled genes including Period (Per) and Cryptochrome (Cry) by binding to E-box(CACGTG). PER and CRY accumulate in the cytoplasm and translocate into the nucleus, where they repress BMAL1/CLOCK-driven transactivation. The inhibition of BMAL1/CLOCK is relieved by the breakdown of CRY and PER proteins, resetting the cycle. In this study, the effect of the rare CRY2 variants in the central clock mechanism is aimed to be investigated. To this end, the fifteen rare Cry2 variants are identified from the database of 1000 Genomes Project and Ensembl. The initial assessment of these fifteen variants is predicated to be pathogenic. Further experimental and structural studies lead us to study p.Pro123Leu CRY2, p.Ser210Ile CRY2, and p.Asp406His CRY2 variants, which are located at a functionally important domain of the CRY2 called the secondary pocket, plays a role in CLOCK binding. CRY2 variants were unable to repress BMAL1/CLOCK driven transcription. Further biochemical studies indicated these variants were less stable than the wild type CRY2. Additionally, compared to wild type CRY2 the p.Pro123Leu CRY2, the p.Asp406His CRY2 and p.Ser410Ile had reduced affinity to CLOCK assessed by co-immunoprecipitation. Finally, it has been observed that p.Ser210Ile CRY2 and p.Asp406His CRY2 are not capable of rescuing the circadian rhythm in Cry1-/-Cry2-/- double knockout mouse embryonic fibroblast cell line by complementation test. Collectively our data suggest that the secondary pocket of CRY2 plays a significant role not only for the CLOCK binding but also for the stability and the proper circadian rhythm at the cellular level.
Complex genetics and disease mechanisms in a Turkish ataxia cohort
Ataxias are a clinically, genetically, and mechanistically heterogeneous group of disorders, characterized by degeneration of the cerebellum. There are significant subtypes of ataxias which also overlap with other neurological disorders, making the precise diagnosis challenging. The developments in next generation sequencing in recent years contributed to the molecular diagnosis of ataxias. It became possible to sequence the coding regions of the genome in a time-saving and cost-effective manner by whole exome sequencing (WES). WES evolved to a powerful tool to identify genetic causes of complex ataxias. In the framework of this thesis, 83 index patients with complex ataxia phenotypes were investigated by WES. Mutations in 18 different genes were identified as the genetic cause in 26 families, which corresponds to a diagnostic yield of 31%. Identification of mutations in hereditary spastic paraplegias and other neurodegenerative disease genes showed that there is a significant overlap among different neurodegenerative disorders. The adaptation of rapidly improving sequencing approaches and detailed clinical information of the remaining patients will help to identify the disease genes in unsolved families. The results presented here complement the picture of the molecular basis of ataxias in Turkey. This will hopefully pave the ways for more precise diagnosis in future and will also contribute to the development of therapeutic approaches in ataxias.
Characterization of the SPOC regulatory function of BUD14
Saccharomyces cerevisiae, also known as the budding yeast, is a commonly used model organism to study the orientation of mitotic spindle and its effects on asymmetric cell division. The mitotic spindle of budding yeast needs to be oriented in the mother-to-daughter polarity axis in order to segregate one copy of its genomic DNA to the daughter cell during the cell cycle. Spindle Position Checkpoint (SPOC) is a surveillance mechanism that inhibits the progression of cell cycle upon spindle mispositioning. Cells with deficient SPOC lose their genomic integrity, eventually resulting in multiploidy and aneuploidy. Therefore, SPOC is necessary for the maintenance of ploidy in budding yeast. However, so far it has not been completely enlightened how the SPOC mechanism functions. In this thesis, we showed that Bud14 is a novel component of SPOC. We showed that deletion of BUD14 promotes the growth of cells with impaired mitotic exit, indicating Bud14 is an inhibitor of mitotic exit. We demonstrated that bud14Δ cells are SPOC deficient, accumulating multinucleated cell populations in the presence of spindle misalignment. Our data showed that SPOC regulatory function of Bud14 is not dependent on its role in formin-dependent actin polymerization regulation. We identified that Bud14-Glc7 interaction is required for the mitotic exit inhibitory function of Bud14 because a temperature sensitive mutant version of Glc7 (glc7-12) that cannot bind to Bud14 resulted in SPOC deficiency in the presence of BUD14 upon spindle misalignment. Our data indicate that Bud14-Glc7 limits the SPB-bound levels of Bfa1-Bub2 complex in metaphase and anaphase. We identified that Bud14 falls off from the bud cortex, spreading through the cell during late anaphase. We showed that phosphatase Bud14-Glc7 and the SPOC kinase Kin4 have different and additive molecular roles in SPOC. We proposed a model in which Bud14-Glc7 complex acts on Bfa1 with a parallel pathway to Kin4 in order to block the cell cycle progression in response to spindle misorientation.
PAS components are required for timely mitotic exit
Phosphoinositides are evolutionary conserved phospholipid derivatives found in cellular membranes. Each phosphoinositide interacts with specific proteins to perform cellular functions such as membrane trafficking, signal transduction and cytoskeletal organization. Phosphotidylinositol (3,5) Biphosphate (PtdIns(3,5)P2) is the least abundant phosphoinositide derivative in the cell. PtdIns(3,5)P2 deficiency is associated with neurological diseases such as ALS and Charcot-Marie-Tooth Disease. Although PtdIns(3,5)P2 plays roles in membrane trafficking, stress response, vacuole/endolysosome structure/function and transcriptional regulation, no mitotic function has been attributed to PtdIns(3,5)P2. In this thesis we asked whether PtdIns(3,5)P2 has a function in exit from mitosis. We first applied genetic approaches to analyze contribution of PtdIns(3,5)P2 to mitotic exit. We further analysed anaphase duration and actomyosin ring contraction in PtdIns(3,5)P2 deficient cells. We also investigated the localization of Mitotic Exit Network (MEN) components and its regulators in cells lacking PtdIns(3,5)P2. Our genetic analysis demonstrated that PtdIns(3,5)P2 synthesis becomes essential in cells with impaired MEN activity. Consistent with this result, anaphase and actomyosin ring contraction lasted longer in PtdIns(3,5)P2 deficient cells than in wild type cells. Release of the conserved phosphatase Cdc14 (the most downstream component of the MEN) was also delayed in the absence of PtdIns(3,5)P2. Taken together, our results suggest that PtdIns(3,5)P2 synthesis contributes to mitotic exit in budding yeast through yet an unknown mechanism.
Dissecting the function and regulation of the dual specificity kinase DYRK at the centriolar satellites and primary cilium
The centrosome/cilium complex plays critical roles during important cellular and organismal processes. In mammalian cells, it is composed of the centrosome, the primary cilium and the centriolar satellites. At the core of the centrosome are two centrioles, which are essential for the assembly of cilia including the primary cilium, which functions as a hub for developmentally important signaling pathways. Centriolar satellites are granules that localize and move around centrosomes. These vertebrate-specific structures have emerged as important regulators of primary cilium assembly and function. Importantly, structural and functional defects of centrosomes, cilia and centriolar satellites cause various human diseases including ciliopathies, primary microcephaly and cancer. In order to uncover the pathogenesis of these diseases, a complete understanding of the mechanisms by which they are assembled, maintained and remodelled are required. To this end, I investigated the function and regulation of Dual specificity tyrosine-phosphorylation-regulated kinase 3 (DYRK3) during primary cilium assembly based on its identified in proteomic datasets of the motile cilia and regulatory functions during centriolar satellite biogenesis. First, I used proximity-based labelling approach, BioID, and generated the first in vivo proximity interaction map for wild type DYRK3 and its kinase-dead mutant. This map revealed proximity interactions between DYRK3 and regulators of primary cilium assembly and function. To test this, I inhibited DYRK3 activity in mammalian cell lines and multiciliated epithelial cultures, and showed that DYRK3 is required for primary and motile cilia assembly, respectively. These results identify DYRK3 as a new player of cilia biogenesis and advances our understanding of kinase-dependent regulation of this process.
Investigating the roles of CREBBP/EP300 and BRD9 in somatic cell reprogramming to pluripotency
Somatic cells can be reprogrammed to induced pluripotent stem cells (iPSC) by overexpressing four transcription factors, OCT4, SOX2, KLF4 and MYC (OSKM). Epigenetic pathways that safeguard somatic cell identity are barriers for somatic cell reprogramming. Chemical or genetic inhibition of such pathways increases the efficiency of reprogramming. In this thesis, I investigated the effects of three chromatin factors CREB (cyclic-AMP response element binding protein) binding protein (CREBBP), E1A binding protein of 300 kDa (EP300) and bromodomain containing 9 (BRD9) during reprogramming using a combination of chemical probes and genetic loss of function experiments. In the first chapter, I investigated barrier function of bromodomain-mediated interaction of CREBBP/EP300 in reprogramming with specific bromodomain inhibitors. RNA-Sequencing showed that CREBBP/EP300 bromodomain inhibition in fibroblasts decreases the expression of genes that are highly expressed in the cells. However, inhibition of the acetyltransferase activity of CREBBP/EP300 by another small molecule, A485, decreased the reprogramming efficiency by downregulating pluripotency-associated genes. Assay for transposase-accessible chromatin (ATAC-Sequencing) and H3K4me1 and H3K27ac chromatin immune-precipitation (ChIP-Sequencing) results indicated that CREBBP/EP300 bromodomain inhibition decreases H3K27ac at accessible chromatin regions and at putative enhancers. Additionally, I functionally showed that continuous expression of PRRX1, a downregulated transcription factor upon CREBBP/EP300 bromodomain inhibition, impairs iPSC generation by suppressing pluripotency-related gene expression. These results uncovered a role for the interaction of CREBBP/EP300 with acetylated lysines in safeguarding somatic cell identity. In the second chapter of my thesis, I performed a SWI/SNF-focused CRISPR-Cas9-mediated knockout screen to identify regulatory subunits of reprogramming. This screen identified non-canonical BRG1- or BRM-associated factors (ncBAF) specific subunits, BRD9 and BRD4 Interacting Chromatin Remodeling Complex Associated Protein (BICRA, GLTSCR1) as barriers to reprogramming. Additionally, I showed that three structurally distinct BRD9 bromodomain inhibitors and a PROteolysis Targeting Chimera (PROTAC) degrader of BRD9 increase reprogramming efficiency and generate iPSCs with only OCT4 and SOX2 expression. BRD9 knockout iPSCs expressed pluripotency markers and short term BRD9 inhibition did not decrease cell proliferation and OCT4-positive cells indicating that BRD9 is dispensable for pluripotency induction and maintenance. However, BRD9 depletion and inhibition impaired mesoderm differentiation of iPSCs. RNA-Seq was performed on fibroblasts and results showed that BRD9 inhibition downregulates highly expressed genes in fibroblasts. ATAC-Seq results showed that chromatin accessibility around putative active enhancers rather than promoters was decreased upon BRD9 inhibition. Additionally, overexpression of MN1 and ZBTB38, which were downregulated upon BRD9 perturbations, decreased reprogramming efficiency. These results indicate a role for BRD9 and ncBAF in maintaining somatic cell identity through maintenance of active enhancers. The findings presented in this thesis show that chromatin factors safeguarding somatic cell identity are barriers to cellular reprogramming and inhibition of such factors can be utilized to increase the low efficiency of human iPSC generation.
Stabilization of the CLOCK:BMAL1 heterodimer decreases circadian rhythm amplitude
Rhythmic changes in behaviour and/or physiology are governed by the daily light-dark cycle in most organisms to increase their fitness to the environment. These rhythmic changes are generated from the biological clock and last about 24 hours. In mammals, a hierarchically ordered circadian clock that is highly regulated by complex transcriptional-translational feedback loops (TTFLs) is observed. At the molecular level, CLOCK and BMAL1 form a heterodimer to bind E-box sequences within the promoter region of the clock-controlled genes (including Cryptochrome (Cry) and Period (Per)) to initiate the transcription. Then CRYs and PERs which are accumulated within the cytosol, translocate into the nucleus along with Casein Kinase Iε to represses BMAL1:CLOCK-driven transcription. Small molecules regulating the activities of these core-clock proteins could offer temporal control over the circadian clock. Considering the pathologies- such as cancer, metabolic diseases, and accelerated aging- that can arise from the lack of a robust circadian clock, identification of such molecules carries great importance. Therefore, I characterized the CLK95, which is previously identified by our group, as a novel CLOCK and BMAL1 binding small molecule. I showed that CLK95 stabilizes the interaction between CLOCK and BMAL1 which, in turn, enhances their nuclear localization both in vitro and in vivo. Further experiments revealed that the CLK95 dampens the amplitude and increases the period of the circadian rhythm by stabilizing the positive loop of the primary TTFL. Considering elevated CLOCK and BMAL1 levels inhibit cell growth, CLK95 may show therapeutic effects against cancer cells due to its enhancing effect on CLOCK and BMAL1.
ENKD1 is a centrosomal and ciliary microtubule-associated protein important for primary cilium content regulation
Centrioles and cilia are conserved, microtubule-based structures critical for cell function and development. Their dysfunction cause cancer and developmental disorders. How microtubules are organized into ordered structures by microtubule-associated proteins (MAPs) and tubulin modifications is best understood during mitosis but is largely unexplored for the centrioles and the ciliary axoneme, which are composed of stable microtubules that maintain their length at steady state. In particular, we know little about the identity of the centriolar and ciliary MAPs and how they work together during the assembly and maintenance of the cilium and centriole. Here, we identified Enkurin domain containing 1 (ENKD1) as a component of the centriole wall and the axoneme in mammalian cells and showed that it has extensive proximity interactions with these compartments and MAPs. Using in vitro and cellular assays, we found that ENKD1 is a new MAP that promotes microtubule polymerization and regulates microtubule organization and stability. Consistently, ENKD1 overexpression increased tubulin polymerization and acetylation and disrupted microtubule organization. Cells depleted for ENKD1 were defective in ciliary length and content regulation and failed to respond to Hedgehog pathway activation. Together, our results advance our understanding of the functional and regulatory relationship between MAPs and the primary cilium.
CCDC66 controls mitotic progression and cytokinesis by promoting centrosome maturation and microtubule bundling
Precise spatiotemporal control of microtubule nucleation and organization during mitosis and cytokinesis is critical for faithful segregation of cytoplasmic and genetic material. Microtubule-associated proteins (MAPs) govern the assembly, maintenance, and remodelling of microtubule arrays of cell division. Their deregulation causes cancer and developmental disorders. Despite having a nearly complete list of mitotic and cytokinetic MAPs, key questions remain about their functions and mechanisms, their regulation in different contexts and the nature of their crosstalk with different cellular structures. In this study, we identified the centrosomal and ciliary MAP, CCDC66, as a component of the bipolar spindle, central spindle and midbody. CCDC66 regulates mitotic microtubule nucleation via centrosomal recruitment of CDK5RAP2, Pericentrin and gamma-tubulin. Accordingly, CCDC66-depleted cells have defective spindle assembly, organization, and positioning, unstable K-fibers and chromosome alignment defects. Phenotypic rescue experiments revealed that its microtubule and centrosome pools mediate its mitotic functions. Furthermore, CCDC66 bundles microtubules in vitro and governs cytokinesis by regulating organization of the central spindle and midbody microtubule arrays. Our findings identify CCDC66 as a multifaceted regulator of cell cycle progression and advances our understanding of how nucleation and organization of distinct microtubule arrays are regulated.
Structural studies of N. maritimus acetyl-CoA/Propionyl-CoA carboxylase with X-Ray crystallography approach
The Archaea domain is among one of the most broadly distributed prokaryotic life forms on our planet. Most of the members of this domain are known to live in extreme environments and are involved in the fixation of inorganic compounds into organic compounds. Many ammonia-oxidizing archaea belonging to the phyla Crenarchaeota and Thaumarchaeota were found to assimilate inorganic carbon into organic carbon through a carbon fixation pathway called 3-hydroxypropionate/4-hydroxybutyrate (HP/HB) cycle. Interestingly, the marine species of the phylum Thaumarcheota were found to use a modified version of the HP/HB cycle. Among these organisms, the species N. maritimus was discovered to use the most energy-efficient carbon fixation mechanism under aerobic conditions. This feature provides N. maritimus several advantages to thrive in extreme oligotrophic environments. In the HP/HB cycle, the carboxylation of acetyl-CoA and propionyl-CoA is of great importance as its products are essential precursors for the maintenance of this cycle. Importantly, in N. maritimus, acetyl-CoA and propionyl-CoA are carboxylated by a biotin-dependent single bifunctional enzyme called Acetyl- CoA/Propionyl-CoA carboxylase. This enzyme is composed of three subunits: a carboxyltransferase Nmar_0272, carbamoyl phosphate synthase Nmar_0273, and a biotin-lipoyl attachment domain-containing protein Nmar_0274. Together, these subunits form a protein complex to become functional. In this study, we have purified and crystallized Nmar_0272 and Nmar_0274 subunits and determined the first ever structure of carboxyltransferase subunit Nmar_0272. The structure of Nmar_0272 exhibited an apo form dimer structure at 2.26 Å resolution and in P63 space group symmetry. Additionally, certain undetermined regions were reconstructed using homology and AI-based tools. Overall, our study presents a novel protein structure and serves as a starting point for elucidating the carbon fixation mechanisms in marine Thaumarchaeota.
Understanding how loss of histone H3 lysine 36 trimethylation enhances somatic cell reprogramming
Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) using a set of transcription factors (OCT4, SOX2, KLF4 and c-MYC). However, reprogramming of somatic cells to iPSCs suffers from a very low efficiency indicating the presence of barriers against reprogramming. Epigenetic factors regulating post-translational modifications of DNA and histones are among such barriers of reprogramming. In a preliminary study, SETD2 which is the only enzyme catalyzing histone H3 lysine 36 tri-methylation (H3K36me3) in mammals was shown to be a barrier of reprogramming. H3K36me3 is a histone mark deposited throughout gene bodies of actively transcribed genes. In this thesis, I investigated the molecular mechanisms of how H3K36me3 loss enhances iPSC generation. I proposed several hypotheses to understand how H3K36me3 loss increases reprogramming efficiency. First, by analyzing previously generated RNA-sequencing data, I considered four transcription factors (CITED2, EBF3, SMAD3 and FOSL1) downregulated by SETD2 knockdown as candidate SETD2-downstream genes. I tested whether overexpression of these transcription factors could reverse the increased reprogramming phenotype of SETD2 knockdown. I showed that none of these transcription factors could alone decrease the increased reprogramming efficiency by SETD2 knockdown. In a second line of experiments, I carried out chromatin immunoprecipitation (ChIP) for active H3K36me3 and repressive H3K27me3 marks, which are deposited in a mutually exclusively manner on the tails of histone H3. I analyzed the distribution of H3K27me3 on the genes downregulated upon SETD2 knockdown to investigate whether H3K36me3 loss could lead to H3K27me3 deposition. However, ChIP-qPCR results did not indicate a reciprocal relationship between these two marks on the selected genes such as SMAD3, LPAR1, EBF3 and SFRP1. Lastly, I performed a CRISPR/Cas9-based knockout screen targeting all known H3K36me3 readers in mammals to define the roles of these readers in somatic cell reprogramming. H3K36me3 readers are involved in diverse cellular processes such as alternative splicing, DNA repair, transcription elongation and DNA and histone methylation. Therefore, I aimed to find which H3K36me3 reader could phenocopy SETD2 depletion in reprogramming. CRISPR-based screen demonstrated that PSIP1, MRG15, MSH6, MSL3, NSD2 and NSD3 are barriers against reprogramming as their knockout led to increased reprogramming efficiency. These H3K36me3 readers were validated as barriers to reprogramming and MRG15, the H3K36me3 reader associated with regulation of alternative splicing was chosen for function-related assays. Therefore, I analyzed the effect of SETD2 knockdown on alternative splicing of key factors known to be significant for reprogramming with the goal of understanding whether SETD2 inhibition promotes reprogramming by regulating alternative splicing. Through RT-qPCR analysis, I observed that both SETD2 knockdown and MRG15 knockout individually led to a significant increase in the pluripotent cell-specific variant of MBD2 expression. Taken together, these findings identified the factors serving as barrier against somatic cell reprogramming through reading trimethylation of H3K36 residue and alternative splicing of MBD2 gene as an underlying mechanism for enhanced reprogramming by H3K36me3 loss.
The molecular architecture of ataxias in Turkey
Ataxias are a clinically, genetically, and mechanistically heterogeneous group of neurological disorders characterized by motor incoordination, resulting from dysfunction of the cerebellum and its connections. Ataxias with different phenotypes overlap and make a precise clinical diagnosis challenging. In recent years, advances in next generation sequencing (NGS) have drastically contributed to the molecular diagnosis of ataxias. With whole exome sequencing (WES), one of the NGS technologies, it has become possible to analyse the coding regions of the genome in a time-saving and cost effective manner. WES, which is a state-of-the-art method, has evolved into an efficient tool for identifying genetic causes of complex ataxias. In the framework of this thesis, 95 index patients with complex ataxia phenotypes were investigated by WES. Causative mutations in 28 different genes were identified in 40 families, this corresponds to a diagnostic yield of 42%. A significant overlap among different neurodegenerative disorders has been demonstrated by identifying mutations in hereditary spastic paraplegias and other neurodegenerative disease genes. Rapidly improving sequencing technologies, collaborative projects, and detailed clinical information will help to identify disease genes in remaining unsolved families. The results presented in this thesis will hopefully contribute to pave the ways for more definitive diagnosis of ataxias in the future and to developing molecular therapies.
Identification of critical epigenetic modifiers in prostate cancer
Prostate cancer (PCa) is the second leading cause of cancer-related death in men. The introduction of potent second-generation antiandrogens such as enzalutamide (ENZA) has improved overall survival for patients with recurrent or metastatic PCa. Yet, while patients initially respond to treatment, the vast majority eventually develop resistance. ENZA-resistant PCa is almost invariably lethal, and few treatment options are available. Clinical and basic studies have demonstrated that epigenetic events play a critical role in the development of ENZA resistance by changing the genome accessibility through DNA methylation, histone modifications, and chromatin remodeling. Considering their critical function and druggability, epigenetic modifiers offer a promising pharmacological target to treat ENZA-resistant PCa. This study aims to identify critical epigenetic modifiers in late-stage ENZA-resistant PCa. Due to the availability of lysine demethylase (KDM) inhibitors, we tested the essentiality of all KDM enzymes to provide preclinical evidence that would support further drug development. We performed an arrayed screen using a KDM-focused shRNA library in multiple PCa cell lines. Despite previous literature, we observed that the knockdown of KDM enzymes largely had little effect on PCa proliferation, potentially due to genetic redundancies. To expand the focus of the project and screen all epigenetic modifiers, we, therefore, generated an epigenetic-focused CRISPR library and performed unbiased CRISPR-Cas9 dropout screens in multiple PCa and ENZA-resistant cell lines. Although shRNA screen results were inconclusive, our epigenome-wide CRISPR-Cas9 screens (EPIKOL) identified both known and novel epigenetic targets in ENZA-resistant PCa. From this, we focused on SMARCC2, a core subunit of the SWI/SNF complex as a potential candidate for ENZA-resistant prostate cancer. We confirmed SMARCC2-dependency on multiple ENZA-resistant models with orthogonal proliferation assays. We found that the SMARCC2 cistrome was significantly expanded in ENZA-resistant cells with marked differences in transcription factor occupancy. These gained binding sites correlated with increased chromatin structures in PDX and clinical CRPC samples. Collectively, these findings suggest that the SMARCC2-dependent SWI/SNF complex gains an essential role in ENZA resistance by expanding its chromatin regulation. This dependency could be exploited for the treatment of ENZA-resistant CRPC by SWI/SNF inhibitors.
The regulation of keratin 8 by aurora B during the cell cycle
Cell division is required for living organisms to carry out their most basic functions, such as transmitting genetic information to the future generation. The morphology and biochemistry of a cell changes dramatically during cell division. Dynamic changes occur in multiple components of the cell including chromosomes, membranous organelles and cytoskeletal elements during mitosis and cytokinesis. Various cell cycle-dependent kinases strictly regulate these intricate activities. The molecular details of regulatory mechanisms during cell division and master regulators and their dynamic targets are active research questions in cell division. Microtubule and actin filaments are two main elements of the cytoskeleton that play critical roles in mitosis and cytokinesis and have relatively well-understood in terms of their functions and regulatory mechanisms. In contrast, intermediate filaments, a type of cytoskeletal element, are a less well-studied component of cell division. Keratins are the most diverse family of intermediate filaments. They are expressed in tissue-specific manner. Additionally, they are used as a biomarker in cancer diagnosis because of their differential expression in malignant tissues. The primary function of keratins is to provide mechanical strength to the cell. However, how the Keratins are re-organized during cell division and their regulatory mechanisms are not well studied. I aimed to investigate how the master mitotic kinases regulate keratins during mitosis and cytokinesis by using both proteomic and cell biology-based approaches. Our mass spectrometry analysis revealed six Aurora B kinase-dependent phosphorylation sites of Keratin 8. By creating phospho-mutant versions, I analyzed and characterized the roles of the phosphorylation site of Keratin 8 during mitosis and cytokinesis. Our study provides insight into the function and regulation of Keratin 8 during cell division. We showed that cytokinesis-specific phosphorylation of Keratin 8 is required for successful cytokinesis by reorganizing Keratin 8 fibers by Aurora B kinase.
Novel mechanisms in control of mitotic exit
Mitotic exit network is a G-protein signaling process that regulates the exit from mitosis. This process must be tightly regulated for proper cell division. The aim of this thesis is to characterize novel mechanisms controlling mitotic exit. For this purpose, two independent but related mechanisms are studied in Saccharomyces cerevisiae, budding yeast. The first mechanism focuses on an unanticipated effect of the daughter cortex localized bud site selection protein Bud14 on Cdc14 which is the main protein phosphatase that triggers the mitotic exit in budding yeast. Our data shows that in the absence of BUD14 or if Bud14 cannot bind to the protein phosphatase 1 (bud14-F379A), Cdc14 stays longer at the spindle pole body (SPB). SPB localization of Cdc14 was dependent on the Cdc14 release mechanisms FEAR and MEN as well as the mitotic exit inhibitor protein Bfa1. Thus, Bud14-Glc7 is part of the mechanism that controls Cdc14 localization to SPBs. The second part of the thesis investigates the mechanisms by which Cdc36 influences mitotic exit. Cdc36 is a component involved in CCR4-NOT complex which have roles in a variety of mechanisms in mRNA regulation and translation. Our data show, cdc36-16 mutant rescues a mitotic exit defective yeast strain. We performed RNAseq analysis of WT and cdc36-16 cells during their synchronous progression in the cell cycle from anaphase onset until mitotic exit. Many cell cycle related genes were found to be differentially regulated in cdc36-16. Our data thus suggest Cdc36 dependent expression is important for a timely mitotic exit. Taken together, in this thesis, we discovered novel conserved proteins (Bud14-Glc7 and Cdc36) that take part in mitotic exit control. We anticipate that these conserved proteins may have similar functions in higher eukaryotes.
Structural comparison study of (6-4) photolyase from Vibrio cholerae
Upon Ultraviolet (UV) irradiation to the DNA, pyrimidine dimers are formed. These mutagenic lesions are either in form of cyclobutene pyrimidine dimers (CPD) or (6-4) photoproducts ((6-4) PPs). Photolyases (PLs) are DNA repair enzymes that use blue light energy to catalyze pyrimidine dimers back to their original state. PLs are named after their specific substrates and their repair functions. (6-4) PLs repair (6-4) PPs with the help of catalytic cofactor flavin adenine dinucleotide (FAD) and an additional antenna chromophore. Bacterial (6-4) PLs contain another cofactor called [4Fe-4S] cluster with an unknown function. In this study, the crystal structure of (6-4) photolyase from Vibrio cholerae (O1 bivar Tor str. N16961) (Vc) has been identified at 2.5 Å resolution. The presence and location of FAD, 6,7-dimethyl-8-ribityllumazine (DMRL), and [4Fe-4S] cofactors were shown by the electron clouds. Their interactions with the Vc(6-4) PL were investigated with the comparison of other structurally available bacterial (6-4) PLs and Drosophila melanogaster (Dm) (6-4) PL. The comparison revealed a conserved water molecule that might be stabilizing the DMRL in bacterial PLs, which makes tighter interaction in Vc(6- 4) PL. In a conserved motif located at the catalytic domain, a different conformation was observed between Vc(6-4) PL and Dm(6-4) PL residue. A disordered region in the Vc(6- 4) PL was observed and suggested as a DNA binding site. A structural comparison with Dm(6-4) PL revealed that Vc(6-4) PL has two additional helixes in C-terminal. Further computational studies suggest either DNA binds to Vc(6-4) PL in a different manner than Dm(6-4) PL or Vc(6-4) PL might be subjected to a conformational change upon DNA binding.
Memeli sirkadyen saatinde kriptokrom 1'i stabilize eden bir küçük molekülün karakterizasyonu
In most organisms, several physiological and behavioral mechanisms are regulated in a circadian rhythm-dependent manner. The mammalian circadian clock is generated by negative and positive transcriptional-translational feedback loops (TTFL), which consists of BMAL1, CLOCK, Periods (PER) and Cryptochromes (CRY) at the molecular level. At the positive arm of the TTFL loops, BMAL1 and CLOCK form a dimer and bind to E-box elements to initiate the transcription of the clock-controlled genes (CCGs) including the Per and Cry genes. Within the time Cry1/2 and Per1/2/3 accumulate in the cytosol and then translocate into the nucleus along with Casein kinase I epsilon/delta. They interact with BMAL1:CLOCK heterodimer and repress their activity and, in turn, repress the transcription of the CCGs. In mammals, 43% of the protein-coding genes are clock-controlled and transcribed rhythmically in at least one tissue. Therefore, disruption of the circadian clock as a result of circadian misalignment or genetic factors will be expected to greatly affect most of the physiological systems. In fact, several different diseases are shown to be linked to circadian disruption such as sleep disorders, metabolic and neural diseases, and cancer progression. That's why several groups have initiated drug discovery studies to find molecules that regulate the circadian clock for the treatment of these diseases. In this thesis, I characterized a novel small molecule (M54) specific for the core clock protein CRY1 in vitro level. M54 is discovered by an in silico approach called structure-based drug designing (SBDD) and predicted to bind to the primary pocket of CRY1. The primary pocket is important for the regulation of CRY stability due to interactions with the E3 ubiquitin ligases (FBXL3 and 21). I showed that M54 significantly increases the circadian period in a dose-dependent manner at the cellular level. Further biochemical studies demonstrated that M54 increases the stability of the CRY1. This study offers a new avenue as a therapeutic approach for patients who suffer from circadian clock-related disorders associated with dampened CRY1 levels.
IFITM1 modulates circadian rhythm by regulating CRY1 dtability
Circadian rhythm, generated by the central and peripheral clocks, regulates nearly all physiological and behavioral processes by synchronizing internal time to the earth's rotation within a period of 24 hours. Hence, disruption of circadian rhythm causes different types of diseases such as cancer, cognitive impairments, metabolic diseases, psychiatric illnesses, and immune system-related disorders. At the molecular level, the circadian clock is controlled by the interlocking of positive and negative transcriptional- translational feedback loops (TTFL). In the positive loop, BMAL1 and CLOCK form a heterodimer and initiate the transcription of clock-controlled genes (CCG), including Per and Cry, by binding E-BOX elements. In the negative loop of TTFL, Later, a heterodimer of CRY and PER interact with casein kinase Ie (CKIe) and translocates to the nucleus to repress the transactivation of BMAL1:CLOCK, thus transcription of CCG. In this thesis, I demonstrated that IFITM1, a transmembrane protein taking a role in the immune defense under viral attack and identified by machine learning, functionally interacts with CRY1 through the C-terminal domain of CRY1 in the nucleus. Knockout of IFITM1 by CRISPR/Cas9 in U2-OS Bmal1-dluc cells leads to period lengthening by 1.28 h and dampening of the amplitude. Further, biochemical studies showed that IFITM1 increases the half-life of CRY1 significantly and represses the BMAL1:CLOCK-driven transcription, hence modulating circadian rhythmicity. Moreover, treatment of U2OS cells with poly (I: C) result in an increased level of IFITM1 and a change in circadian period length. This suggests that IFITM1-CRY1 interaction may be important for regulating cellular circadian physiology and circadian clock-driven immune system regulation.
Conformational changes of wild-type SOD1 affect the dimerization at ambient temperature
Amyotrophic lateral sclerosis is a neurodegenerative disease caused by the degeneration of lower and upper motor neurons in the brain and spinal cord. Although ALS is rare, it is the third most common neurodegenerative disease comes after Alzheimer's and Parkinson's. Since ALS has a complex molecular background, there is no effective treatment for this disease. 90% of ALS patients are sporadic, and 10% have familial disease. Mutations in more than 25 genes account for 70% of familial ALS and also explain <10% of sporadic ALS. SOD1, the second most common ALS-causative gene, encodes the dimeric antioxidant superoxide dismutase 1 protein. More than 180 mutations in the SOD1 gene are associated today with ALS. However, their mechanisms leading to ALS are not fully known. Although most of the mutations appear to have similar pathological effects on protein structure and motor neurons, they lead to different consequences in terms of disease progression. Mutations in SOD1 cause misfolding of immature intermediate form of SOD1 and disrupt the stable dimer structure of the protein; oligomerization and accumulation of the protein occurs in the cytosolic region and at the outer mitochondrial membrane of motor neurons. It has been suggested that misfolded SOD1 may interact with a voltage-dependent anion channel (VDAC1) located at the outer membrane of the mitochondria and prevents entrance of NADH, anions and ATP required for oxidative phosphorylation. On the other hand, macrophage migration inhibitory factor (MIF) is suggested to act as a chaperone for the SOD1 protein, helping misfolded SOD1 fold correctly. This thesis includes i) overexpression and purification of wild-type SOD1, SOD1G93A, MIF and VDAC1, ii) crystallization of both wild-type and mutant forms of SOD1 and iii) diffraction data collection from wild-type SOD1. In this study, we attempted to determine the first ambient temperature X-ray structure of SOD1 and identified a wild-type apo SOD1 protein structure at 4.00 Å resolution and P 21 21 21 space group.
PI(3,5)P2'nun mitoz boyunca analizi
Phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) is a low abundant phosphatidylinositol derivative involved in several signaling and regulatory pathways such as membrane fusion/fission, Ca+2 signaling, vacuolar acidification, selective and bulk autophagy, stress response, Multi vesicular body (MVB) pathway and RNA granule transportation . Previous genetic screens in budding yeast identified upstream regulators of PI(3,5)P2 which are VAC7, VAC14 and FAB1 among genes necessary for growth of mitotic exit defective cells, which suggest a novel role for PI(3,5)P2 in mitosis, specifically during mitotic exit. In this thesis we aimed investigating the interplay between mitosis and PI(3,5)P2 .We first asked whether PI(3,5)P2 synthesis is regulated by the cell cycle. To this end, we analyzed localization and levels of Atg18, a known PI(3,5)P2 effector, throughout the cell cycle by using fluorescence live cell microscopy. Atg18-GFP localized to the vacuole membrane dependent on PI(3,5)P2 and Vac7. We showed that Atg18-GFP predominantly localized at the periphery of the daughter vacuole rather than mother vacuole during mitosis. We next asked which proteins PI(3,5)P2 interacts with during mitosis. We employed a pull-down approach using mitotic cell extracts and PI(3,5)P2 coated beads. Net1, a nucleolar protein that binds the mitotic exit triggering phosphatase Cdc14, was pulled down with PI(3,5)P2. In addition, several ribosomal and rRNA related proteins such as; RPS11B, RPL24A, RPL5, NSR1, RVB2 came out as hits of our assay. Taken together, daughter specific localization of Atg18 and thus PI(3,5)P2 may indicate a daughter-specific role in the asymmetric cell division of budding yeast. In addition, potential PI(3,5)P2 interactors may be the link to the functions of PI(3,5)P2 in mitotic exit, rRNA granule transportation and translation. Further studies such as using Western Blotting approaches, lipid strips, colocalization assays and growth assays will reveal the functional significance of the asymmetric PI(3,5)P2 synthesis as well as the specificity and significance of identified novel PI(3,5)P2 interactors.
The role of ANKIB1 in regulating cell death signaling versus geneactivation following TLR3 activation
Biochemical reactions in cells are necessary to fulfill underlying cellular processes and signal transduction from both, the extracellular and intracellular environment. Their dysregulation can lead to an increase of morbidity and mortality in organisms. Because of that, they are strictly regulated to hamper adverse outcomes. One of the most common regulatory mechanisms are post-translational modifications (PTMs) of proteins, including by ubiquitination. Different pairs of E2 ubiquitin conjugating enzymes and E3 ubiquitin ligases can generate different types of ubiquitination that lead to diverse outcomes for the substrate protein. Nonetheless, their implication in the control of the different immune and inflammatory signaling pathways needs to be further studied. In the current thesis, I dissected the role of a new E3 ubiquitin ligase, Ankyrin repeat and IBR domain- containing protein 1 (ANKIB1), in the Toll-like Receptor 3 (TLR3) signaling pathway and in the antiviral response. The Walczak group previously showed that ANKIB1 has an active catalytic domain and interacts with the TLR3 signaling complex (TLR3-SC) upon stimulation. I have demonstrated that ANKIB1, especially its catalytic activity, is required for the activation of TANK-Binding Kinase 1 (TBK1) and, consequently, type I interferon (IFN) production following TLR3 activation. Oppositely, the deletion of ANKIB1 does not have any consequences on Tumor Necrosis Factor (TNF) signaling. I have shown that, upon TLR3 stimulation, ANKIB1 does not only interact with activated TBK1 but also with optineurin (OPTN) and the NF-kappa-B Essential Modulator (NEMO), the primordial protein for TBK1 activation. To further elucidate the regulation of this process, I have performed an unbiased proteomic analysis of the ubiquitinated proteins upon TLR3 stimulation and found that TBK1, Interferon regulatory factor 3 (IRF3), and TNF Induced Protein 3 (A20) contain ubiquitinated sites that are dependent on the presence of ANKIB1. Interestingly, I have also seen the same consequence on Gasdermin E suggesting a potential role of this new E3 ligase in the regulation of cell death. Therefore, I dissected the potential role of ANKIB1 on TLR3-induced cell death in vitro using WT and ANKIB1 KO cells. This analysis revealed that ANKIB1 deficiency in HT29 cells results in higher susceptibility to TLR3-induced cell death. Overall, this study highlights the requirement of ANKIB1, a previously uncharacterized E3 ligase, for TBK1 activation and, consequently, type I IFN production in TLR3 signaling. Several components of this signaling complex are impacted by the absence of ANKIB1 in terms of ubiquitination, implying an important contribution of ANKIB1 to TLR3-induced gene activation and regulation of cell death, primordial to the induction of an efficient immune response against pathogens.
Structural dynamics studies of wild-type and mutant candida boidinii nad+-dependent formate dehydrogenase
Candida boidinii NAD+-dependent formate dehydrogenase (cbFDH) is a biotechnologically invaluable enzyme that recently gained more attention on biosynthetic applications for biofuel production. Structure-based mutants of this enzyme performed highly promising results of enhanced catalytic activity and thermal stability. In this study, we aimed to employ a holistic approach and combine X-ray crystallographic analysis of NAD+-dependent cboFDH with Gaussian Network Model analysis to determine the underpinnings of the cboFDH catalytic mechanism and allostery. Additionally, Alphafold2 predictions of a mutant form of cbFDH enzyme were discussed for mutation-based structure prediction efficiency. The 1.4 A resolution X-ray crystallography data of NAD+-dependent cbFDH revealed the structural dynamics of the enzyme with the help of the computational approach. This understanding of enzymatic dynamics and the effect of mutations on structure and function may provide new insight for future biotechnology studies.
Elucidation of the mechanisms that underlie centriolar satellite biogenesis and dynamics
Centriolar satellites are the third novel component of the mammalian centrosome/cilium complex. They are membrane-less electron-dense granules clustered around the main microtubule organizing center. These granules are heterogeneous in terms of composition, size, and shape and have a variety of roles in different cellular processes such as cilium assembly/disassembly, centriole duplication, stress response, and autophagy. Therefore understanding the centriolar satellite composition, dynamics and behavior are essential to elucidate their implications for health and diseases such as cancer and ciliopathies. The main scaffolding protein of the centriolar satellites is Pericentriolar Material 1 (PCM1), which maintains the satellite integrity since depletion of PCM1 leads to loss of centriolar satellites. Therefore understanding the biochemical and biophysical properties of the PCM1 is an essential first step to uncover the biochemistry of centriolar satellites. I found that PCM1 is a highly disordered protein, suggesting that it might undergo liquid-liquid phase separation (LLPS) to form centriolar satellite granules. Even though they are spherical and dynamic, which aligns with the membrane-less granule properties that go under the LLPS, mature satellite granules are in a gel-like state or become gelified over time. The global RNA depletion leads to the dispersal of the PCM1 granules showing that they have a stable RNA core that helps with the granule integrity. Structure-function analysis of PCM1 showed that three different fragments within the PCM1 N-terminus form granules, whereas the fragments from the C-terminal act cytoplasmic. This result shows that N-terminal has a role in the granule formation, whereas the C-terminal is more likely to have a regulatory role. The PCM1 fragments that form granules exhibit dynamic events including fusion, fission, kiss, and split events. Lastly, the de novo PCM1 disassembly experiment shows that with decreasing protein concentration, the granules are broken into smaller granules until no visible granule is seen. Our findings show that PCM1 goes under the LLPS and shows liquid-like behavior however there is an equilibrium between both the liquid and gel-like state.
Determining demographic and genetic structure of sedentary and migratory bears (Ursus arctos) within Eastern Anatolia using genome-wide genetic markers
Large carnivores are known for altering their life-history strategies in response to environmental change. One such shift was recently discovered in brown bears (Ursus arctos) within Eastern Anatolia, where the availability of city dumps as a food source has led to the evolution of two distinct life strategies: sedentary bears that use city dumps as a primary food source and migratory bears that avoid the dump and migrate in search of food. Understanding the demographic and genetic processes that have led to the establishment of these life-history strategies is vital for predicting which life history strategy will dominate in the future and the overall impact of anthropogenic pressures on wild carnivores forced to live in human-dominated landscapes. Adaptive and genomic processes responsible for the two life-history strategies were determined using genotype data from 57 blood samples collected from captured bears, 31 of which were fitted with satellite collars and tracked almost for one year, providing information on bears' movement ecology and migratory behavior. We found that the Eastern Anatolian brown bear population is genetically highly differentiated and isolated from other world populations but contains high genetic diversity. Historical demography showed a recent dramatic increase in the effective population size of the bear population in Eastern Anatolia while relatedness analysis showed higher coefficient of kinship and sib-ship in sedentary bears utilizing the garbage dump. We also determined several genomic regions and distinct genotypes associated with sedentary and migratory behavior and strong signatures of positive selection at these loci. Outlier loci were associated with a number of transcript modifier genes, including the first exon of CCRL2, a gene that regulates immune response. Moreover, polygenic discrimination studies revealed 464 SNPs distributed throughout the genome actively contributing to difference in movement behaviorand could reliablt predict movement behavior in un-classified individuals. Gene ontology for these discriminatory SNPs revealed that the detection of sensory and chemical stimuli was the most important function that differentiated sedentary and migratory bears. Collectively our results indicate that adaptation to human-oriented landscapes in the East Anatolian brown bear population may have a strong genetic basis and emphasizes the importance of evolutionary genomics for understanding how species survive and adapt to human-mediated global change.
Memeli hücrelerinde Glypican 5'in hücre bölünmesi rolü
Glypicans (GPCs) are members of the Heparan Sulphate Proteoglycans (HSPGs) family of glycosaminoglycans. They are extracellular proteins anchored to the plasma membrane through their GPI anchor. Glypicans play role in developmental processes, cell differentiation, cancer progression, and stem cell function by regulating the activity of morphogens like Hedgehog and Wnt. An early RNAi screen has shown that the knockdown of Glypican 5 (GPC5) led to an increase in multinucleation suggesting a function in cell division. However, their role in cell division is not investigated. Our previous proximity dependent proteomic analysis in cytokinesis revealed that GPC5 interacts with proteins that are involved in cell adhesion, migration, actin reorganization, and RhoA activation. Even though cell division was extensively studied in terms of chromosome segregation and cytoskeleton dynamics, the function of cell surface proteins in cytokinesis remains elusive. In this thesis, I investigate the role of Glypican 5 in cell division by performing live cell imaging in GPC5 knock-out cells. In parallel, I investigated the functionality of previously identified interaction partners of GPC5. My analysis revealed that GPC5 have a role in the late cytokinesis by regulating adhesion properties of dividing cells specifically during abscission. This study illuminates the mechanism of how the outside and the inner part of the cell communicates during division.
Transcription factor-mediated gene regulation of chromosome 1q in breast cancer
Breast cancer is the most commonly diagnosed cancer among women and the fifth cause of cancer deaths in the world. Approximately 80% of breast cancer patients are estrogen receptor alpha (ERα) positive, which is a sequence-specific transcription factor that interacts with DNA either directly by estrogen response elements (EREs) or recruited by other transcription factors (TFs). Forkhead Box A1 (FOXA1) and GATA Binding Protein 3 (GATA3) are two of the major factors that regulate ERα binding and their abnormal expression is associated specifically with luminal A subtype breast cancer. Copy Number Variation (CNV) is a structural variant in the human genome, and chromosome 1 is shown to be one of the most altered regions of breast cancer cells. Consequently, more than 70% of breast tumors display copy number gain in the 1q arm. In this study, the aim is to investigate the association of ERα-GATA3-FOXA1 cistrome with copy number amplifications of chromosome 1q and, finally, gene regulation. For this purpose, chromatin immunoprecipitation followed by sequencing (ChIP-seq) focused analysis of ERα, FOXA1, and GATA3 binding sites, RNA-seq data for the gene expression in MCF7, T47D, and ZR75-1 cell lines, and copy number data collected from Cancer Cell Line Encyclopedia are used. After obtaining the direct and indirect binding sites of targeted TFs, along with the overlapping binding sites among the cell lines, chromosome 1q cytobands with distinguished binding patterns are determined. Custom-made CRISPR/Cas9 libraries are generated in order to target those genomic regions in distinct ways. ChIP-seq and RNA-seq data association focused on specific cytobands revealed upregulated genes that might be promising candidates for further studies.
Interrogation of the functionality of ERα binding sites with STARR-seq
Breast cancer (BCa) and Endometrial cancer (EnCa) are the most common cancer types worldwide in the female population. The common point of these cancer types is Estrogen Receptor α (ERα) activity which is a transcriptional factor controlling a variety of genes important for drug response, cell proliferation and survival. Active ERα translocates from cytoplasm to the nucleus where it can bind thousands of regions, ERα binding sites (ERBSs). These regions are known as regulatory elements controlling the genes related to the initiation and progression of BCa and EnCa. Considering the critical role of ERα in both cancer types, a better understanding of how ERα-bound enhancers drive the transcription of target genes is promising to clarify unique pathways and to develop effective treatment strategies. To characterize the ERBSs in BCa and EnCa, we aimed to assess the enhancer activity of clinical ERBSs in a quantitative manner with a novel massively parallel reporter assay, STARR-seq. This will allow us to generate the first functional "map" of transcriptional activity of ERα in two systems, from these maps ERα regulated enhancers can be classified as inducible ones, constitutively active ones, and inactive ones. As a first step of this collaborative project, clinically relevant ERBSs were identified by ERα ChIP-seq from ERα positive BCa and EnCa patients to create a custom STARR-Seq library. As a model, MCF7 cells (breast cancer) and Ishikawa cells (endometrial cancer) were used to assess the transcriptional activity of candidate ERBSs to assess pathologically important enhancer regions collected from patients. Within the scope of this thesis, we optimized the experimental conditions to deliver a custom STARR-Seq library to cell lines models, MCF7 and Ishikawa, and after optimization; we prepared samples required for next-generation sequencing and bioinformatic analysis. The bioinformatics analysis will be performed as the final step. Overall, the final goal is to identify and categorize the activity of clinically detected ERBSs and generate a functional mapping of ERα enhancer activity.
Towards In Vitro synthesis of oxygen-tolerant CpI hydrogenase variants for photosynthetic hydrogen production
Hydrogen is an attractive alternative to non-renewable energy sources like fossil fuels due to its carbon-free nature and high energy density. However, most of hydrogen production uses feedstock chemicals derived from fossil fuels, which leads to carbon dioxide emissions and thus contributes to the climate crisis. Photosynthetic hydrogen technology can provide an alternative, sustainable and entirely carbon-free energy cycle. However, oxygen sensitivity of the hydrogenase enzymes that catalyze hydrogen production is one of the most significant barriers against photosynthetic hydrogen production, as oxygen is a by-product of photosynthesis. Therefore, the hydrogenase enzyme to be used in the production of photosynthetic hydrogen must tolerate gaseous oxygen. In this thesis, four locations known to be important for oxygen tolerance in the Clostridium pasteurianum (CpI) hydrogenase were replaced with the amber (TAG) stop codon for non-canonical amino acid (ncAA) incorporation. CpI was chosen as it is one of the most efficient and fast [Fe-Fe] hydrogenases characterized in terms of hydrogen production. Significant progress was made toward ncAA insertion using cell free protein synthesis (CFPS) to the selected spots on the enzyme. The genes of six M. jannaschii-derived aminoacyl-tRNA synthetases (aaRSs) required for cysteine and tyrosine analog incorporation were first cloned into the high- copy pY71 vector. These aaRSs were then produced in vivo and purified with IMAC as they have genetically fused N-terminal 6xHis-tags. The orthogonal tRNACUA template necessary for ncAA insertion was also synthesized with PCR and purified. In the next part of the thesis, experiments were performed to synthesize M. barkeri- derived aminoacyl tRNA synthetase, HRS, necessary for histidine analog incorporation. Different conditions were tested in vivo and in vitro (CFPS) synthesis of the enzyme. However, HRS formed insoluble aggregates and could not be purified. Lastly, the M. barkeri-derived orthogonal tRNA template was produced by PCR assembly and purified. In the last part, wild-type CpI was anaerobically produced and matured in vivo, followed by purification using StrepTactin affinity chromatography. The components required for the two biochemical assays to measure the hydrogen production activity of CpI, namely NADPH- and DTH-driven assays, were synthesized. With the activity assays, the anaerobically produced and purified CpI enzyme was shown to be in the active state. Attempts were made to produce CpI anaerobically in CFPS as well, however this was unsuccessful. But everything necessary for CFPS production of both wild-type and mutant CpI was prepared.
Identification of epigenetic factors that will overcome therapy resistance in triple-negative breast cancer
Triple Negative Breast Cancer (TNBC) is the most aggressive and recurrent type of breast cancer with a poor prognosis. Due to the lack of expression of hormone receptors (HR) and Her2, TNBC cannot be treated with targeted therapies, leaving chemotherapy as the mainstay treatment. However, acquired resistance to chemotherapy is a major challenge that, in part, causes relapse, which is thought to be driven by coordinated actions of genetic and epigenetic events. In this study, we aimed to elucidate the roles of the full spectrum of epigenetic modifiers in both naïve and chemotherapy resistant TNBC cell lines. For this, we generated an epigenome-wide CRISPR knockout library (EPIKOL) targeting all writers, readers and erasers, as well as the chromatin remodelers and structural subunits of epigenetic complexes. First, we discovered novel epigenetic modifiers that regulate TNBC cell fitness and confirmed the effects of NSL complex members (KANSL2, KANSL3, and KAT8) and SS18L2 on cell growth. Notably, in vivo screen with EPIKOL on MDA-MB-231 cell line also revealed SS18L2 as a fitness gene in two different time points of tumor growth. To generate in vitro models of chemoresistant TNBC, we exposed three different TNBC cell lines to escalating doses of an anthracycline (doxorubicin) or a taxane (paclitaxel, taxol). SUM159PT taxol resistant cells had elevated levels of ABCB1 and showed the characteristics of multidrug resistance. EPIKOL screen and a complementary epigenetic probe library screen in one of the taxol resistant SUM159PT cells in the presence of taxol identified BRPF1, a bromodomain-containing reader, as a taxol sensitizer. Upon BRPF1 inhibition or loss, transcriptome analysis revealed a significant downregulation of ribosome biogenesis pathways and a decrease in ABCB1 expression. Additionally, we demonstrated the binding of BRPF1 to the ABCB1 promoter, possibly regulating its expression in drug resistant state. Collectively, these findings provide a basis for developing combination therapies targeting specific chromatin-based epigenetic factors to counteract TNBC cell viability and chemoresistant phenotype.
Plasmodium vivax'da laktat dehidrogenaz enziminin aktif bölge halkası amino asitlerinin yönlendirilmiş mutagenez çalışmalarıyla analizi
ÖZETYÜKSEK LİSANS TEZİPlasmodium vivax'da LAKTAT DEHİDROGENAZ ENZİMİNİN AKTİFBÖLGE HALKASI AMİNO ASİTLERİNİN YÖNLENDİRİLMİŞMUTAGENEZ ÇALIŞMALARI İLE ANALİZİDilek SADAKFırat ÜniversitesiFen Bilimleri EnstitüsüBiyoloji Anabilim Dalı2006, Sayfa: 48Mevcut antimalariallara karşı direncin ortaya çıkması yeni ilaçların geliştirilmesinigerekli kılmıştır. Bu tezde; Plasmodium vivax'ın glikolitik enzimi olan laktat dehidrogenazhedef olarak seçilmiştir. Enzimin aktif bölge halkasında ilave 5 amino asit uzantısı vardır. Builave uzantının insan laktat dehidrogenaz enzimlerinde kopyası mevcut değildir. Kinetikçalışmaları ile beraber bu ilave, yapıya dayandırılmış ilaç tasarım çalışmaları için bölgeyi idealbir hale getirmektedir. Laktat dehidrogenaz enzimi bu tezde yönlendirilmiş mutagenezçalışmalarıyla Plasmodium vivax'da çalışılmıştır. Aktif bölge halkasından ilk iki amino asitçıkarıldığında enzim aktivitesi devam etmiştir ama 5 amino asit çıkarıldığında enzim aktivitesidurmuştur. Bu sonuçlar yeni antimalarialların tasarımında bu bölgenin ideal bir hedef olmasıfikrini desteklemiştir.Anahtar Kelimeler: Plasmodium vivax, laktat dehidrogenaz, aktif bölge halkası,yönlendirilmiş mutagenez, antimalarial.
Functional characterization of p.Ser420Phe CRY2 variant
The circadian clock is an innate mechanism that enables organisms to adapt to the rhythmic changes of the environment. In mammalian cells, this mechanism is achieved by the interaction of four core clock proteins (CRYs, PERs, CLOCK, and BMAL1). Human clock-gene variations are associated with numerous behavioral and physiological diseases, such as sleep, metabolism, addiction, neurological disorders, and endocrine and metabolic. However, little is known about the possible effects of variations at the molecular level. Currently, the functional consequence of these variations and the strength of their association with the disease remains unclear. The functional analyses of variants help us to understand the role of amino acid residues in the structure and function of proteins. In this study, rare CRY2 variants were selected from the 1000 Genomes Project, and the Ensembl database. SNPs were filtered based on the prediction of pathogenicity using in silico tools. Using the 3D structure of the CRY2 protein, the variant (p.Ser420Phe CRY2) located in the vicinity of the coiled-coil-like helix (CC helix) was functionally characterized. We have found that the p.Ser420Phe CRY2 had reduced repression activity on CLOCK/BMAL1-driven transcription and showed rescued circadian rhythm with a shorter period than wild type in Cry1-/-Cry2-/- double knockout mouse embryonic fibroblast cell line. Moreover, the variant had reduced affinity to other core clock proteins CLOCK and PER2 and could not properly localize in the nucleus using biochemical methods. Further stability results suggested that the p.Ser420Phe CRY2 variant was degraded by a noncanonical mechanism. Collectively, our results show that the CC helix region plays an important role in the degradation of CRY2, interaction with other core clock proteins, also nuclear shuttling.
Identification of ABE2A as a circadian amplitude enhancer with drug-like potency
Many species have an adaptation system to the period of the solar day. This endogenous mechanism is called the circadian clock, which can be defined as an autonomous 24h cycle controlling daily rhythms in physiology and behavior. Thus, a robust circadian clock is required for many processes in our body to work smoothly. Our body's control center of circadian clock operation is SCN (suprachiasmatic nucleus), which uses the phoetic input sensed by intrinsically photosensitive retinal ganglion cells (ipRGCs). SCN regulates cellular clocks and peripheral tissues and coordinates them with the help of neural or hormonal signals. This oscillatory system depends upon transcriptional translational regulatory feedback loops (TTFLs). The main loop consists of CLOCK, BMAL1 (positive arm), and CRYs, PERs (negative arm). Mechanistically, CLOCK and BMAL1 interact with each other in the cytosol. Then this heterodimer translocates into the nucleus and binds to E-Box elements within the promoter of CRYPTOCHROME (CRY) and PERIOD (PER). Cryptochrome (CRY), PER, and the epsilon isoform of casein kinase I (CK1ε) form a complex in the cytosol that enters the nucleus and inhibits their own transcription regulated by CLOCK and BMAL1 heterodimer. Any disruption in this mechanism may result in diseases or disorders. Small molecule modifiers are efficient tools to repair this perturbed mechanism and have many advantages over other therapeutic approaches. In my study, I characterized a novel small molecule called ABE2A, 100 times more effective derivative of the CLK8 molecule, which increases the amplitude of circadian rhythm by specifically interacting with CLOCK and decreasing the interaction between CLOCK and BMAL1. Further experiments revealed that ABE2A decreases the nuclear abundance of CLOCK, BMAL1 and increases the cytosolic abundance of BMAL1. Additionally, it reduces the total cell protein level of CLOCK and increases PER2. ABE2A also affects the transcriptional levels of E-box and D-box-regulated genes. For instance, it drops the expression of core clock genes such as DBP, BMAL1, and PER2. Overall, similar to CLK8, ABE2A reduces the positive arm components in the nucleus, limiting the transcription of the negative arm. Thus, the repression activity in the TTFL is stabilized, and the amplitude of the circadian rhythm enhances. For these reasons, and also by being effective at nanomolar doses, ABE2A has a drug-like potency against various health problems related to decreased amplitude, such as some metabolic diseases, mood disorders, and accelerated aging.
The molecular mechanism of BRPF proteins for the reversion of taxane resistance in prostate cancer
Prostate cancer (PCa) is a major cause of cancer-related death in men. Localized PCa can be treated with androgen suppression and surgery, nonetheless, many patients progress to the castration-resistant PCa (CRPCa), which is more aggressive and/or metastatic. The standard of care for CRPCa patients are taxanes (namely Docetaxel and Cabazitaxel), however resistance against these drugs develops over time. Taxane resistance makes clinical application of taxanes inefficient. Epigenetic regulation of cancer not only affects tumor development and progression but also resistance for chemotherapy. Hence, targeting these mechanisms appears as an alternative strategy to overcome drug resistance. Previous studies using an epigenetic drug library and epi-targeted CRISPR screens using taxane resistant CRPCa cells (Du145 and 22Rv1) have shown that inhibition of BRPF proteins (epigenetic reader proteins containing bromodomain group) efficiently reverses taxane resistance. To this end, we aimed to elucidate the molecular pathways involved in the reversion of taxane resistance through BRPFs in CRPCa cells. In this thesis, several approaches were combined including RNA sequencing, chromatin immunoprecipitation, bioinformatics and genome editing methods in order to characterize the biological significance of the BRPF proteins in taxane-resistant CRPCa. BRPF inhibition with small molecules resensitized taxane-resistant CRPCa cells to both taxanes under study. Efficient silencing of both BRPF1 and -2 were able to revert taxane resistance, albeit only mildly. On the other hand, knockout of BRPF2 effectively reversed taxane-resistance and induced cell death. BRPF1 was essential for the viability of resistant cells, hence BRPF1 knockout in resistant cells could not be generated despite several attempts. Furthermore, silencing or knocking out of BRPFs led to downregulation of ABCB1, which encodes a permeability glycoprotein (Pgp), and suppressed its function, potentially explaining how cells may be resensitized to taxane treatment. This supression seems to involve direct binding of BRPF1 to the ABCB1 promoter, as determined by ChIP-qPCR analysis. Furthermore, we observed that the promoter region of ABCB1 in taxane-resistant CRPCa cells exhibited occupancy by H3K27ac. Taken together, our results uncovered molecular players involved in BRPF mediated drug resistance. BRPF inhibition appears as a promising anticancer strategy in taxane resistant CRPCa and the mechanism seems to involve inhibition of drug efflux. BRPF inhibition can be utilized in the development of effective therapies against taxane resistant CRPCa. Keywords: BRPF, castration-resistant prostate cancer, drug resistance, epigenetics, taxane resistance.
Discovery of small molecule: Regulates the cryptochrome stability and controls blood glucose levels in diabetic mice
The circadian rhythm controls the behavioral, biochemical, and physiological variables in most living organisms, from bacteria to mammals. The circadian rhythm is an endogenous, the most well-known biological rhythm, and refers to a 24-hour cycle that regulates various physiological processes like hormone secretion, food intake, sleep-wake cycles, body temperature regulation, and metabolism. The mammalian circadian clock mechanism is endogenous, although it is synchronized with the environmental cues through the suprachiasmatic nuclei (SCN) located in the anterior part of the hypothalamus. Circadian clock disruption influences metabolic health and is closely associated with many diseases, including sleep disorders, neurological conditions, immune dysregulation, obesity, diabetes, and cancer. Cryptochromes (CRYs), one of the core clock proteins, are transcriptional repressors of the circadian clock in mammals. The stability of CRYs is important because of the ability to alter the period and amplitude of the circadian rhythm. The gluconeogenesis pathway is regulated by a variety of factors, including hormones such as glucagon and insulin and the circadian clock mechanisms. On the molecular level, CRY interacts directly with the glucagon-mediated G-protein coupled receptors, in turn, inhibits the gluconeogenesis pathway that controls glucose metabolism. Therefore, we aimed to discover small organic molecules which increase the stability of CRYs and inhibit gluconeogenesis. Such molecules could be used as anti-diabetic drugs, which control blood glucose levels. To this end, we used a structure-based drug design approach against the primary pocket of CRY, which is responsible for their degradation. Around 2 million small molecules with non-characterized functions were screened through molecular docking in silico. The candidate small molecules were tested for cellular toxicity and their effects on CRYs stability, circadian clock, and gluconeogenesis. We identified a novel molecule, TW68, which increases the stability of both CRY1 and CRY2, lengthens the period of circadian rhythm, and represses gluconeogenic genes in further characterization of pre-clinical studies. In transgenic and fat-induced diabetic animal models, TW68 regulated the fasting blood glucose levels in biochemical and physiological studies. These results signify the therapeutic potential of TW68 on the circadian clock – gluconeogenesis-related metabolic diseases, including type 2 diabetes mellitus.
The role of MLL1-MENIN complex in somatic cell reprogramming
Expression of OCT4, SOX2, KLF4 and MYC (OSKM) can reprogram somatic cells to pluripotency. This process involves a complete reset of the somatic cell identity. However, chromatin-based mechanisms that safeguard cellular identities act as barriers to reprogramming, resulting in low efficiency of cell fate conversions. To identify such barriers, a loss of function genetic screen has previously been performed and MLL1 (KMT2A) was identified as a significant roadblock for reprogramming. In the scope of this work, we employed chemical and genetic approaches to characterize the effects of MLL1 in complex with MENIN on reprogramming of human somatic cells. Firstly, we validated the barrier function of this complex via utilizing an inhibitor, VTP50469, that selectively targets MLL1-MENIN complex, which led to increased reprogramming efficiency. Next, we generated fibroblast cell lines that express Cas9 and gRNAs targeting MLL1 and MEN1 genes. Reprogramming experiments with these cell lines showed that deletion of both MEN1 and MLL1 increase the efficiency of reprogramming significantly. To further understand the barrier function of this complex, we tested the rescue capabilities different MEN1 point mutants by overexpressing their cDNAs in MEN1 KO cells. These experiments showed that while MLL1 interaction is crucial for MENIN barrier function, MENIN might be interacting with additional proteins such as JUND to prevent reprogramming. Furthermore, we observed that DOT1L inhibition does not further enhance the reprogramming efficiency when combined with VTP50469 treatment or MEN1 depletion. This suggests that DOT1L-related mechanisms might be involved in the prevention of somatic cell reprogramming. Moreover, we performed RNA-sequencing to identify the transcriptional changes that place in MENIN inhibition and depletion. RNA-sequencing results showed that, compared to the controls, several of pluripotency genes which include genes that are usually expressed in late reprogramming are upregulated both in the context of MENIN inhibition and depletion. In line with that, we observed that a large set of fibroblast-specific genes are downregulated in the absence of MENIN activity. Additionally, we also showed that this complex imposes a barrier function during the resetting of primed pluripotent stem cells to naive pluripotency. The supplementation of the naive culture conditions with VTP50469 enhanced the induction naive pluripotent stem cells. This suggests that MLL1-MENIN inhibition is a feasible target for modulating human naive pluripotency. Taken together, the results represented herein shows that MLL1-MENIN complex imposes barriers in the acquisition of cell identities of higher developmental potential.
Phylogenetic analysis of brown bear (Ursus arctos) populations across Türkiye
Brown bears (Ursus arctos) are omnivores distributed across Eurasia and North America. Previous studies based on mitochondrial DNA (mtDNA) revealed significant differences in genetic variation between European and Middle Eastern brown bears, with each region characterized by distinct haplogroups. Türkiye, as a crossroads between three continents, is an important region for brown bear diversity, as it harbors haplotypes from both regions. However, previous information on brown bear genetic diversity in Türkiye was based on only mtDNA sequences and limited sample sizes. Therefore, the role Türkiye has played in the evolutionary history of brown bears is still largely unknown. Here, using genome-wide sequencing data together with the mtDNA D-loop region sequences, we aim to fill this gap by investigating the patterns of brown bear genetic diversity and differentiation across Türkiye and nearby regions. Our results demonstrate the geographical distribution of haplogroups across Türkiye and show that Turkish populations have three distinct mtDNA haplogroups clustered into western and eastern geographic groups. Genome-wide SNP analysis supported this conclusion, with high patterns of genetic diversity and distinct geographic structuring. Collectively, our results indicate that the evolutionary dynamics influencing brown bear populations across Türkiye and the Middle East are distinct and Türkiye's brown bears have higher genetic diversity than other world populations.
Role of extracellular microenvironment on lung cancer cell metabolism
Lung cancer is the second most prevalent type of cancer with the highest mortality rate among all cancer types. Despite significant progress in cancer drug development and therapeutic strategies targeting cancer metabolism, a lack of understanding about the intricate complexities of the tumor microenvironment continues to hinder effective treatment approaches. This study aims to tackle these challenges by using advanced three dimensional (3D) in vitro cancer models to explore the role of interactions between cancer cells and their microenvironment in regulation of cancer metabolism. Due to limitations in conventional two-dimensional (2D) cultures, this work focused on creating two distinct hydrogel models representing healthy lung tissue and tumor conditions. These models were developed by combining decellularized native lung extracellular matrix with inert or tumor-mimetic biomaterials, aligning with the research objectives. In the first phase of the study, an in-depth analysis of gene expression profiles in lung tumor cells was conducted, comparing metabolic and tumorigenic regulators between 2D culture and the novel 3D healthy lung-mimetic model. The results showed that the 3D healthy-mimetic environment influenced the expression of epithelial-mesenchymal transition (EMT) markers in tumor cells in response to changing glucose levels. The microenvironment also played a significant role in regulating the stemness of tumor cells and affected the expression of metabolic markers, providing valuable insights into cellular behavior and metabolism. The second phase of the research explored the impact of elevated sulfation, mimicking the aberrant glycosaminoglycan increase within the tumor microenvironment, under varying glucose levels on tumor cell behavior. The investigation provided deeper insights into the complex interplay between sulfation, glucose availability, and cellular responses. Increased sulfation in the tumor-mimicking environment significantly affected cell proliferation and metabolic activity, while the effect of glucose levels varied depending on the microenvironmental conditions. The interplay between glucose levels and the expression of EMT markers was also observed, with high glucose in the tumor mimetic environment leading to significant upregulation of mesenchymal markers in tumor cells. The study highlighted the complexity of metabolic regulation in the tumor microenvironment. The last phase of the study focused on investigating the role of the PIK3CA gene as a key cellular signaling regulator in the observed phenomena. Comprehensive analysis of PIK3CA's effect provided valuable insights into the processes influencing cellular responses in the novel hydrogel models under different microenvironmental conditions. The study revealed a significant role of PIK3CA in regulating cell proliferation in tumor mimetic environments, with glucose levels modulating this effect, validated with PIK3CA knock-down cells. High glucose exposure partially compensated for the effects of PIK3CA knockdown, influencing EMT marker expression, stemness markers, and various metabolic pathways in tumor cells. The complex interplay between glucose metabolism, extracellular microenvironment and PIK3CA signaling was evident, offering potential therapeutic targets for PIK3CA-associated cancers. In conclusion, this comprehensive research underscores the importance of sophisticated 3D in vitro cancer models to accurately simulate the tumor microenvironment and investigate cellular behavior. The findings provide valuable insights into cancer cell metabolism, the impact of microenvironmental factors, and potential therapeutic targets, advancing lung cancer treatment strategies.