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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.
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.
Dissection of in vitro behavior and microtubule association of centriolar satellites
Centriolar satellites are membrane-less granules that localize around the main microtubule organizing center of animal cells, the centrosome. Centriolar satellites interact with microtubules and molecular motors and move around centrosomes in a microtubule-dependent way. They are scaffolded by pericentriolar material 1 (PCM1) protein, which interacts over 200 proteins including the ones mutated in ciliopathies, primary microcephaly, and schizophrenia. In agreement with their disease links, they have been reported to regulate a diverse array of cellular processes such as primary cilium assembly, Hedgehog signaling, ciliary motility, autophagy and proteostasis. They mediate their functions by acting as cellular machines that store, traffic, modify and assemble their residents. Although centriolar satellite functions and molecular mechanism of action are evident, little is known about how they are assembled and maintained. To address this question, I dissected the biophysical the biophysical properties of PCM1 because it is the only essential protein for centriolar satellite assembly and maintenance. Phase separation has emerged as a mechanism by which a variety of membrane-less organelles assemble. Therefore, my central hypothesis was that PCM1 phase separates to form centriolar satellites. Supporting my hypothesis, centriolar satellites are spherical in shape in cells, dissolve during mitosis and exhibit fusion and fission events. To test my hypothesis, I used condensation assays, removal of solubility tag and treatments of manipulating the external conditions of the solvent. Results from these experiments showed that PCM1 assembles solid spherical condensates and scaffold-like mesh networks in vitro. To gain further insight into the PCM1 domains that contribute to its assembly; localization and microtubule association experiments identified a 923-1200 aa. region in central PCM1 that binds and bundles microtubules in vitro and in vivo. The mutant PCM1 lacking the microtubule binding domain was compromised for microtubule association. The microtubule binding fragment of PCM1 formed condensates upon disruption of microtubule network in cells. In in vitro assays, this fragment formed liquid-like condensates in the absence of microtubules. Taken together, these results identify phase separation as a mechanism that contributes to the assembly of the centriolar satellite scaffold and provide insight into how centriolar satellites interact with microtubules.
Structural studies of wild-type and Val120Thr mutant candida boidinii formate dehydrogenase
Candida boidinii NAD+-dependent formate dehydrogenase (CbFDH) has attracted considerable interest owing to its potential in generating biofuels and various industrial chemicals from carbon dioxide (CO2). In an endeavor to enhance its utility, this study introduces essential insights regarding the molecular structures of CbFDH variants. These insights provide details that will help our understanding of the enzyme's functional mechanisms and its inherent potential for protein engineer- ing. Employing advanced methodologies at the Turkish Light Source "Turkish DeLight", the atomic X-ray crystal structures of both the wild-type CbFDH and the Val120Thr mutant were determined. These structures were obtained at different temperatures, cryogenic and ambient, thereby offering a holistic comprehension of the enzyme's dynamics under diverse environmental settings. A key aspect of importance centers around the newly discovered hydrogen bonds within the active site of the Val120Thr mutant. The formation of these bonds between Thr120 and water molecules implies a potential enhancement of the active site's stability. Additionally, it suggests the likelihood of an improved electron transfer during the enzymatic reaction. These structural differences provide a foundation for understanding the mutant's increased effectiveness in catalyzing formate conversion — a characteristic with significant promise for industrial applications. It is important to note that while these structural observations offer exciting prospects, further experimental data are required to validate the hypothesized mechanisms. Co-crystallization with the coenzyme and substrate has the potential to offer further insights, allowing a comprehensive understanding of the role of the identified hydrogen bonds in enhancing CbFDH's capabilities. In conclusion, the knowledge gained from the X-ray crystal structures of CbFDH in its wild-type and Val120Thr mutant forms, provides a foundation for future advancements in the field of protein engineering. As researchers delve further into the functional implications of the observed structural changes, the potential for harnessing CbFDH's catalytic abilities in biotechnological applications becomes increasingly feasible. This study highlights the dynamic relationship between structural insights and practical applications, underscoring the exciting possibilities for optimizing enzymatic processes in service of a more sustainable future.
Investigating genetic and epigenetic regulators of human naive pluripotent stem cells
Pluripotent stem cells can exist in two primary cellular states referred to as naive or primed. Naive pluripotent stem cells possess enhanced developmental potency compared to conventional primed states. However, generating and maintaining human naive pluripotent stem cells remains challenging. Developing tools to isolate naive cells and elucidate associated genetic and epigenetic regulators is therefore essential. We aimed to create a reporter for isolating naive cells from heterogeneous cultures, optimize naive induction protocols, and uncover regulators of naive status acquisition. Based on expression data, we selected KLF17 as a gene specifically expressed in naive pluripotent stem cells. Therefore, we engineered a KLF17-T2A-EGFP fluorescent reporter in human induced pluripotent stem cells (iPSCs) using CRISPR/Cas9-based genome editing. We tested this GFP-based reporter system during naive conversion steps. Quantitative PCR analysis of EGFP expression showed naive-specific activation, indicating successful construction of the reporter. Optimizing the conversion of naive stem cells is an ongoing challenge. We tested the effects of overexpressing KLF17 and NANOG/LIN28A on naive induction. qPCR results demonstrated these transcription factors increased activation of naive markers compared to controls, suggesting they may accelerate resetting. We also aimed to assess the effects of KLF17 and NANOG overexpression on naive and primed reprogramming efficiency from somatic cells. The results showed that overexpression of KLF17 did not affect reprogramming efficiency. However, NANOG overexpression increased the efficiency of both primed and naive reprogramming. In addition, we investigated the impacts of knocking out transcription factors KLF5, KLF17, and LIN28A on naive and primed reprogramming. KLF5 knockout did not affect either reprogramming system. But KLF17 knockout specifically decreased naive reprogramming efficiency while not affecting primed reprogramming. In contrast, LIN28A knockout reduced the efficiency of both primed and naive reprogramming. Lastly, we tested small molecule inhibitors of epigenetic regulators on primed and naive reprogramming. The inhibitors iCBP112, iBRD9, RN1, A83-01, and EPZ5676 all significantly increased reprogramming efficiency under both primed and naive conditions. Taken together, these results demonstrated that both transcription factors and epigenetic regulators affect the acquisition of primed vs. naive identity from somatic cells.
Quantification of Atg18 localization and asymmetric localization of Atg18 on the daughter vacuole of budding yeast
Phospholipids play a key role in intracellular signaling. Membrane-bound phosphatidylinositols (PIs) are examples of those signaling lipids. They are phosphorylated from three different places, and these phosphorylations attract different proteins. In this thesis, phosphatidylinositol 3, 5 biphosphate PI(3,5)P2 was investigated. The autophagy protein Atg18 localize on vacuole membranes in a way that correlates with PI(3,5)P2 levels on the vacuoles. Therefore, if Atg18 can be utilized as a PI(3,5)P2 marker is inquired. To quantify Atg18 localization on the vacuoles, the EzColocalization plugin was used in ImageJ-FIJI. This plugin provided us with a Pearson Correlation Coefficient, which indicates the amplitude of Atg18 localization on the vacuole. The deletion of PI(3,5)P2 regulatory complex proteins, VAC7, VAC14, and FAB1, was investigated along with Atg18 mutants that either can only bind PI(3,5)P2, or cannot bind any PI at all. The quantified results showed that Atg18 localization on vacuoles, indeed, increases with elevated PI(3,5)P2 levels. Moreover, it was observed that Atg18 localizes only to the daughter vacuole after the vacuole segregates to the daughter cell. Using the EzColocalization plugin, I was able to show this phenomenon in numbers. Through several analyses, I found that daughter-specific Atg18 localization to the daughter vacuole mostly starts right after vacuole segregation ends, within 5 minutes around metaphase-anaphase transition, and ends 5 minutes after the spindle breakdown, persisting on daughter vacuole for 30 minutes on average. 49% of the mid-log phase population showed sole Atg18 daughter-specific localization from the end of vacuole segregation until cytokinesis, while more than 95% of the population showed daughter-specific Atg18 localization at some point. Whether other PI(3,5)P2 regulators show asymmetry was also investigated. Timelapse of Vac14 showed no asymmetry. Population analysis of cells by measuring fluorescence intensity of still images also showed that Vac7, Vac14, and Fab1 do not localize asymmetrically. On the other hand, two more proteins, Sch9 and Ivy1, were also analyzed since they are relevant to PI(3,5)P2 signaling. Sch9 was slightly enriched on the mother vacuole, whereas Ivy1 was strongly enriched on the daughter vacuole. Atg18-Sloop mutant that only binds PI(3,5)P2, was enriched on the daughter vacuole as well. Altogether, the findings of this thesis suggest that Atg18 vacuole localization correlates with the PI(3,5)P2 production and it is likely that it may reflect PI(3,5)P2 levels. This thesis also further presents evidence that the daughter and the mother vacuole exhibits compartment specific behavior in terms of some PI(3,5)P effectors (Atg18, Sch9) and regulators (Atg18 and Ivy1).
Characterization of Bud14 function in yeast centrosome size maintenance
The cell cycle is an extensively conserved process that governs DNA duplication and chromosome segregation. Eukaryotic cells distribute their chromosomes into daughter cells by the mitotic spindle, formed between spindle poles named centrosome. The Spindle Pole Body (SPB) is a functional equivalent of mammalian centrosome in S. cerevisiae. SPB consists of distinct structural layers and is embedded into a nuclear envelope throughout its life cycle, thereby nucleating cytoplasmic and nuclear microtubules. The yeast centrosome size changes throughout the cell cycle. In addition, cell cycle arrest conditions have various effects on SPB size. Recent studies illustrated that SPB undergoes growth and exchange cycles, thus portraying a dynamic yeast centrosome to an extent that was not anticipated previously. In this study, we have identified a novel role for Bud14 in SPB size maintenance. Through microscopy-based quantification of relative and exact amounts of SPB structural proteins, we showed that cells lacking Bud14 had more Spc72, Nud1, Spc97, Spc42, Spc29, and Spc110 at their SPBs. Bud14 is also a regulatory subunit of the Protein Phosphatase 1 Glc7. We indicated that Bud14 function in Spc29, Spc97, Spc110, and Spc42 size maintenance depends on its interaction with Glc7. We elucidated Spc110 and Spc72 dynamics by measuring their molecule numbers throughout the cell cycle. This dynamic nature might be integral to the mitotic spindle formation and presents a mechanism for reacting to changes in cellular conditions. Furthermore, this research established that functional Cdc14 phosphatase is required for Spc72 and Spc110 size maintenance in cells lacking Bud14. Forceful localization of Cdc28 to SPB outer plaque disclosed that phosphorylation of SPB structural proteins causes their enlargement at the SPB. This research determined that Bud14 regulates SPB size primarily via its interaction with Glc7. Cdc14 also functions in the SPB size maintenance network.
Empirical and computational inquest into biosimilar and next-generation insulin analogs manufacturing: Unveiling structural, analytical, and dynamic attributes through recombinant technological approaches
The intricate molecular structure of insulin and its nuanced interactions within diverse tissues have carved out a domain within the scientific community dedicated to comprehending and harnessing the significance of this pivotal hormone. Investigative efforts on insulin encompass a broad spectrum of scientific disciplines, spanning cellular signaling, biochemical interactions, and genetic regulation. From this vantage point, insulin assumes a pivotal position at the nexus of modern biotechnology and fundamental science, constructing substantial contributions to the advancements in health and biological sciences. This thesis introduces a biotechnological product and method through the production and characterization of two new-generation domestic insulins but also offers the most comprehensive theoretical and analytical insights into the empirical and computational dynamics of these insulins and oligomeric commercial insulins. Introducing an atlas review encompassing all academic information about insulin, the thesis thoroughly explains the fundamental structural biology over this model protein. Subsequent chapters delve into the experimental and computational aspects of domestic new-generation rapid/ultra-rapid insulins. Furthermore, it presents revolutionary studies on the allosteric hierarchy of commercial long-acting oligomeric insulins. In this regard, this thesis can be regarded as a benchmark work in protein research
Molecular analysis of cortical malformation in mammalian cortex by using proteomics approaches
The layered structure of the cerebral cortex is formed through a complicated sequence of highly controlled stages during corticogenesis. During this process, the perturbation of neuronal migration and cell division can result in a rare disorder called cortical heterotopia. Heterotopia patients can have recurrent epileptic seizures, developmental delays, and mild intellectual disabilities. Heterotopia has been challenging to study because human mutations associated with the disease often fail to form heterotopia in mouse models. Eml1 is a heterotopia-associated gene where the perturbations cause heterotopia formation in humans and mice. During my Ph.D., I focused on the investigation of the functional role of EML1 in heterotopia formation through comprehensive cellular and biochemical analysis, including imaging, BioID proximity labeling, proteomic profiling via label-free proteomics or dimethyl labeling, phosphoproteomics, and microtubule-pelleting assays. In the first part of my thesis, I showed that heterotopia-associated mutant EML1 reduces the protein-microtubule binding affinity. BioID proximity labeling of wild-type and mutant EML1 revealed that EML1 interacts with many microtubule and cytoskeletal organization proteins, most of which are lost in mutant-expressing cells. Thus, the wild-type protein interactome is impacted by a heterotopia-causing mutation. This work identified several novel interactors, along with known interaction partners of EML1. Proteome profiling in cortices and primary neuronal cells in a conditional Eml1 knockout (cKO) mouse versus control showed dysregulations in microtubule organization and protein metabolic processes upon Eml1 depletion. These large-scale proteomic analyses are essential for studying the underlying mechanism in brain development and heterotopia formation. In the second part of my thesis, I present my contributions to the published article, which focuses on the cell cycle-dependent regulation of an adhesion protein, protocadherin-7. In this project, as my side project, I unveiled the spatiotemporal regulation of the protein and its' palmitoylation-dependent regulation in cell division.
The importance of PI(3,5)P2 in mitotic exit
Phosphoinositides are evolutionarily conserved signaling lipids that are critical for many aspects of cellular biology. Phosphatidylinositol-3,5-bisphosphate (PI(3,5)P2), one of the rarest phosphoinositides, is synthesized on vacuole/lysosome membranes through the phosphorylation of Phosphatidylinositol-3-phosphate (PI3P) by the Fab1 kinase. PI(3,5)P2 plays key roles in various cellular processes, including vacuole/lysosome structure and function, stress response, autophagy, transcriptional regulation, and membrane trafficking. Dysregulation of PI(3,5)P2 production has been implicated in various human diseases such as amyotrophic lateral sclerosis and cancer, highlighting the significance of PI(3,5)P2 for human health. The work presented in this thesis discovers that PI(3,5)P2 is crucial for a timely mitotic exit in budding yeast. Our data shows that reduced levels of PI(3,5)P2 results in delayed mitotic exit and lethality in cells with compromised mitotic exit activity. Conversely, overproduction of PI(3,5)P2 rescues the prolonged anaphase and lethality of mitotic exit mutants. Mechanistically, we find that PI(3,5)P2 regulates the localization of the mitotic exit inhibitor Kin4. Accordingly, high levels of PI(3,5)P2 causes recruitment of Kin4 to the vacuole periphery. We further show that this process is dependent on Atg18, a known effector of PI(3,5)P2. Thus, this study unravels a novel link between PI(3,5)P2 and cell cycle progression in budding yeast.
Inherited myopathies and congenital myasthenic syndromes in a cohort from different regions of Turkey
Myopathies include a clinically, genetically, and mechanistically heterogeneous group of diseases characterized by muscle weakness and fatigue. Congenital myasthenic syndromes (CMS) are characterized by symptoms such as easy fatigue, muscle weakness, and ptosis. Definitive diagnosis of the disease is often difficult, but next-generation sequencing technologies, especially whole exome sequencing (WES), have contributed to their diagnostic process. This thesis involves the use of WES, bioinformatic analysis, and wet-lab to understand the genetic basis of Turkish Myopathy and CMS patients. In total, 23 cases were solved, including eight novel variants, in 30 index patients. The number of unsolved cases was seven, including three sporadic cases. With this thesis, in the cohort under study, the genetic causes of myopathies and congenital myasthenia syndrome have been revealed and differential diagnoses guided. WES is the current gold standard for genetic investigation of inherited disorders, but it has also limitations and challenges. The diagnosis rate is positively correlated with gene-disease associations, and it is suggested that the more associations, the higher the diagnostic yield. The discovery of new causal genes through large cohort studies is also important for this reason. This thesis is expected to contribute to a better understanding of the clinical and genetic picture of myopathy and CMS patients in Turkey, and thus to a better comprehension of the complicated phenotype-genotype correlations of muscle disorders. Such studies will help to shape a hopeful future in the genetic era.
Ecological niche modeling and genetic structure of brown bear (Ursus arctos) in Türkiye
Türkiye, with its rich biodiversity and unique climatic/topographic characteristics, serves as a land bridge between Asia and Europe. Brown bear (Ursus arctos) is the largest carnivore species in Anatolia. Unfortunately, due to climate change and landscape transformation, brown bear habitats are fragmenting and becoming increasingly isolated, posing a significant conservation concern for the species' future. This thesis comprises the chapters examining the various ecological facets of brown bears across Türkiye. Considering bioclimatic, topographic, and anthropogenic environmental variables, the distribution of brown bears across the country was estimated using an ensemble modeling approach. Potential habitat suitability under different climate change scenarios was also investigated. The results indicate bears are primarily distributed in the Euro-Siberian biogeographic region, followed by Irano-Turanian, and Mediterranean. Climate change is expected to lead to a substantial loss of brown bear habitats. Additionally, it is projected that established protected areas might become insufficient to safeguard the species in the face of future habitat loss, leading to a conservation gap. I modeled the structural connectivity of brown bear habitats across the country, identified priority linkages and core patches, and discussed the potential impact of climate change on the ecological network of brown bears across Türkiye. Ultimately, the Western Black Sea and Eastern Anatolia core patches exhibited a relatively intact structure, while the Mediterranean region was identified as critical in terms of habitat configuration and fragmentation. Climate change is predicted to have significant impacts on brown bear ecological network. Then, I analyzed the spatiotemporal dynamics of human-brown bear conflict across Türkiye. A conflict risk map was generated, and the role of protected areas in mitigating conflict was evaluated. The findings revealed significant variations in conflict frequency by years, seasons, bear seasons, and regions. Conflict risk was found to be particularly high along the Black Sea coast and Eastern Anatolia. The primary cause of human-bear conflicts was identified as the confinement of bears to areas, with limited resources, leading them to seek anthropogenic food alternatives. Additionally, the peripheries of protected areas represented hotspots for human-bear conflict. In the next chapter, I analyzed the population structure and genetic variation of brown bears across Middle East. Results indicated that the Sarıkamış population exhibited a more isolated pattern and lower genetic diversity compared to the Western, Eastern, and Iranian populations. However, high genetic variation was detected across the region. Furthermore, environmental heterogeneity and geographic distance were identified as significant patterns driving the genetic differentiation of populations. Lastly, considering the findings from all preceding chapters, I discuss pro-active wildlife management approaches that promote coexistence. This comprehensive study aims to provide a holistic conservation approach for brown bears by examining their suitable habitats, ecological connectivity, population structure, strategies to mitigate human-bear conflict, and climate adaptation strategies. My results highlight the urgent need for effective conservation measures to safeguard Türkiye's brown bears in the face of climate change, habitat loss, and human-bear conflict.
Kanser kök hücrelerine özgü mikroRNA'larin larenks kanser hücrelerinde in vitro karakterizasyonu
Larynx cancer isthe most common and most aggressive type of cancer in the head and neck region. However, when appropriate diagnosis and treatment is applied, the treatment of the head and neck region is the best responding tumor which is the most common cancer type in the world. The incidence in men is about 10 times higher than in women. The most important risk factors for the development of cancer are smoking and alcohol use. Squamous cell carcinomas constitute 90-95% of laryngeal carcinomas and early diagnosis helps increase the quality of life significantly, and affects the survival time positivelylike other cancer types. MicroRNAs (miRNAs) are small RNA molecules that do not encode proteins which are actively involved in the regulation of gene expression and in almost all cellular mechanisms. A good understanding of the working mechanisms of miRNAs has been the focus of recent research. These researches have showed that microRNAs to play an important role in the pathogenesis of many cancers such as Larynx Squamous Cell Carsinoma (LSCC) In this study, samples were first isolated from microRNAs which was already identified in previous microarray studies with mir-26b, mir200c-3p, mir-203, mir363-3p, and mir-1825 in Trizol. cDNA synthesis was followed by qRT-PCR assays. For the microRNAs ready for transfection, Hep-2 cells, the larynx squamos cell line, were used for functional analysis. After transfection of microRNAs into Hep-2 cells, proliferation, migration and invasion, soft agar analyzes were followed and performed respectively. Initially, 5 microRNAs were expressed in terms of significance. However, as a result of these analyzes mir-203 and mir3633p played an important role in these experiments. As a result of that mir-203 and mir363-3p act as tumor suppressor on Hep-2 laryngeal cancer cells, indicating that cancer cells seriously reduce their ability to proliferate, migrate and invade and colonize. It was determined that these miRNAs could be used during diagnosis. Further experiments and works need to be donefor better understanding of this cancer mechanism. Keywords: Larynx Cancer , microRNA, mir-203, mir363-3p, Hep-2
Prostat kanserinde kallikrein (KLK) gen ailesinin ekspresyon seviyelerinin belirlenmesi
Prostate cancer is one of the deadliest cancer types affecting the male population that results with loss of life. Usually begin to appear after the age of 50, and the incidence increases with advanced age. Diagnosis of prostate cancer at the early stage is important in reducing mortality and morbidity. However, after initial treatment, cancer growth may be slow and recur in 30-90% of male patients with increased prostate cancer risk. The specificity and positive predictive value of prostate specific antigen (PSA), the most widely used biomarker for prostate cancer, is low and the absolute lower limit value for cancer diagnosis has not yet been identified. Biochemical recurrence is defined as an increase in PSA value >0.2 ng/mL, measured twice after radical prostatectomy and nadir PSA value ≥2 ng/mL, after radiotherapy. The Kallikrein (KLK) gene family consist of 15 genes encoding secreted serine proteases that have relevant homologies at both the DNA and amino acid level. Two members of the human kallikrein gene family, prostate specific antigen (PSA) and human kallikrein 2 (hK2), are used clinically as prostate cancer biomarkers. In our study, changes in 15 KLK gene expression levels in 34 recurring 36 non-recurring prostate cancers and 19 normal tissue specimens were investigated. This noninvasive method makes a strong candidate for PCa diagnosis. In conclusion, the difference in expression levels of the KLK gene family in recurrent PCa has been shown for the first time by us in the literature. Easy application of PSA in routine examination and the relationship between prostate cancer and other KLKs have been examined. Our results have shown that KLKs, which are differently expressed in PCa, can be used as recurrence markers. Key words: Prostate Cancer, KLK gene family, gene expression
Investigation of specific oncomir MicroRNAs that modify methylation patterns onto effection of prostate cancer
Prostate Cancer (PCa) is the second most common cause of cancer-related deaths in men. Although the etiopathogenesis of PCa has not been clearly elucidated, the evidences has been obtained that epigenetic factors have been implicated in the onset and progression of cancer for the last 10 years.Hypermethylation of genes has been shown to be associated with PCa. MicroRNA (miRNA) is a single-stranded, small non-coding molecules containing about 22 nucleotides. miRNA plays a critical role in the genetic pathogenesis of many types of cancer and mostly human cancer types. DNA methylation is known to be involved in cancer formation, progression and metastasis. Thus, DNA hypermethylation in CpG islands is a marker in the early detection of cancer that can be used as an indicator. Epigenetic mechanisms can be used as a marker in the diagnosis of the disease as well as in the treatment process. It is suggested that epigenetic changes such as DNA methylation and histone modification may be an effective strategy in the treatment of cancer by targeting. In current thesis, promoter methylation statuses and expression changes ofhsa-mir-192, hsa-mir-512-5p, hsa-miR-513a-2 andhsa-mir-572 were analyzed by methylation specific PCR and real time PCR respectively in prostate cancer tumor tissues. Subsequently, here was shown the gene-specific promoter methylation changes that was generated intumor tissues ofmiRNAs, which were shown to be significant. In this work, the possible tumor suppressor potentials ofmiRNAs have been shown.
Effects of diatery fiber in patients with type 2 diabetes mellitus a meta analysis
DEVAM ETMEKTE
Real-time imaging of nitric oxide (NO) signals derived from differentially targeted endothelial nitric oxide synthases (eNOS) using genetically encoded biosensors
Nitric oxide (NO) is a gaseous molecule produced in many different cell types, including macrophages, neurons, and endothelial cells. In endothelial cells, NO is produced by the calcium-dependent endothelial nitric oxide synthase (eNOS), which is known to regulate smooth muscle cell relaxation and vascular tone. Under physiological conditions, eNOS is localized to the caveolae in the cell membrane and is generally inactive due to protein-protein interactions. It has also been shown that translocation between different compartments of eNOS inside the cell can affect NO production. However, in most of these studies the local eNOS derived NO bioavailability could not be studied due to the lack of proper tools. In this diploma, issue by exploiting novel multispectral imaging techniques for NO and Ca2+ was revisited. For this purpose, the novel genetically encoded NO sensor (geNOps), which provides NO detection in living cells with high-spatial and temporal resolution and high selectivity, was used. To visualize local Ca2+signals the well-established red-shifted and differentially targeted variants of the GECOs was exploited. To study eNOS activity in different cellular locales, HEK293 cells was used as a model system and compared wild type-eNOS versus nuclear-targeted eNOS. In both compartments, Ca2+ signals required for eNOS activation was detected upon stimulation. This approach clearly showed that eNOS activity in the nucleus is significantly reduced but still active compared to the wild type-eNOS under the same experimental conditions. In this master thesis, multispectral imaging techniques was used to unveil differences in eNOS activity in different subcellular locales. This master thesis provided for the first-time direct evidence that the local Ca2+ environment does not dictate differences in endothelial nitric oxide synthase activity.
Klk gene editing using crispr technology in prostate cancer cell lines for cancer therapy
Prostate cancer (PC) has been accounted for as the second leading cause of death by cancer in men. Internal and external factors, such as genetic mutation, ethnicity, age, diet, etc., are observed to have been the causative elements for cancer to occur. Serine proteases, that are encoded by Kallikrein (KLK) gene family, show a remarkable impact on PC and some are introduced as PC biomarkers, such as KLK3 and KLK2, as both androgen-dependent are detected in the bloodstream when PC occurs. However, due to androgen dependence and low specificity, they are not quite reliable. In this study, we focused on KLK5 gene mutation in different PC androgen-independent cell lines, PC3, and DU145 cell lines to observe the effect of mutation of PC tumorigenesis. To monitor the effect of the mutation, the KLK5 gene's detected mutation point was knocked out using a genome-editing tool. Gene-editing technology has opened a new path to the scientists' investigation for designing the method that can benefit cancer treatment with the lowest side effects in the long term. In this study, CRISPR/Cas9 gene-editing technology was used to knock out c.245C>A (p.A82D) mutation in the KLK gene which was detected and confirmed by Sanger sequencing. To evaluate the performed gene editing, various cytotoxicity assays, such as cell viability with trypan blue, MTT assay, and colony formation was carried using Doxorubicin and PARP inhibitor which showed a promising result of CRISPR system as a future prostate cancer treatment.
Investigating the role of oncostatin M in cachexia-associated muscle wasting
Cachexia is a devastating syndrome responsible for the death of more than 20% of cancer patients. Cancer-associated cachexia, marked by significant weight loss and fatigue, leads to reduced quality of life and poor survival. Although inflammatory cytokines are identified as the causative agents of muscle atrophy, current anti-cytokine therapies have not successfully attenuated muscle wasting in cancer patients. Through our examination of muscle gene expression, we identified Oncostatin M (OSM) as a strong inducer of muscle atrophy. OSM causes atrophy in primary myotubes through the JAK/STAT3 pathway. RNA sequencing shows that OSM potently induces the expression of atrophy markers and multiple potential atrophy-related genes. Overexpression of OSM by intramuscular adenovirus injection leads to muscle wasting in mice. Muscle-specific deletion of the OSM receptor (OSMR) in tumor-bearing mice rescues the cachexia-driven loss of muscle mass and function. OSM is upregulated in the plasma of cachectic mice; neutralizing circulating OSM helps preserve muscle mass and strength in tumor-bearing mice. OSMR transcripts are elevated in many muscle disorders including Facioscapulohumeral Dystrophy (FSHD) and Duchenne muscular dystrophy (DMD). Taken together, these results suggest that the OSM/OSMR signaling plays a crucial role in inducing muscle atrophy and targeting this pathway may help prevent muscle wasting.
Antiproliferative and apoptotic effects of proton pumpinhibitor lansoprazole on DU-145 prostate cancer cells
Cancer has been a major threat on human health since 20th century and is one of the most significant causes of death among people. Prostate cancer is the second most cause of death among elder people. In spite of early detection and new treatments, there is a need for improvements for detection, diagnosis and therapy of prostate cancer. Ion channel blockers are promising cancer drugs not only modulate tumor microenvironment and pH but also overcome chemotherapy resistance and slow or even prevent invasion or metastasis by different mechanisms. Lansoprazole is a proton pump inhibitor that blocks V-ATPase and H+/K+ ATPase channels on cells. In this study, we aim to discover antiproliferative and apoptotic effects of lansoprazole on Du-145 prostate cancer cell lines. To explore these features, MTT assay and trypan blue counting for cytotoxicity were performed. To evaluate Caspase-9 and PARP expression, Western Blotting was done. Apoptosis was analyzed by Annexin/PI flow cytometry. In conclusion, it could be stated that lansoprazole promotes a nonapoptotic caspase-independent cell death on Du-145 castration-resistant prostate cancer cells and suppresses cell proliferation and growth dynamics at 6 hours, at 10 and 100 micromolar doses. Keywords: Prostate Cancer, Ion Channels, Lansoprazole, Du-145
The anti-proliferative effect of sericin-coated silver nanoparticles on cancer cell lines
The wide use of silver nanoparticles (AgNP) in many biomedical applications including diagnosis, treatment, drug release and medical devices, pharmacology and biotechnology makes it the most remarkable among other nanomaterials. The main use of silver nanoparticles in medicine includes diagnostic and therapeutic applications. Most therapeutic applications are directed towards investigating the anticancer property of these particles. In this study, the cytotoxic effects of sericin-coated silver nanoparticles extracted from Bombyx mori silk cocoon on HeLa cervical carcinoma, Saos-2 osteosarcoma and hFOB bone cell lines were investigated. It was observed that the anti-proliferative effect on cells increased as AgNP concentrations increased. After AgNP treatment of Saos-2 and hFOB cells, a direct proportional decrease in cell viability was observed from 0.5 µg/mL to 50 µg/mL. IC50 values for Saos-2 and hFOB cell lines were determined as 1.880 and 1.522 µg/mL, respectively. Considering the effects of AgNP in the concentration range of 0.5-8 µg/mL in HeLa, Saos-2 and hFOB cell lines, a significant decrease in cell viability was observed at 2 µg/mL and increasing concentrations in three cell lines. While no significant effect was observed at 0.5-1 µg/mL AgNP concentrations in HeLa and Saos-2 cancer cell lines, hFOB affected cell viability at these low doses in osteoblast cells. It has been observed that AgNPs at different concentrations applied on HeLa, Saos-2 and hFOB cells cause accumulation in the cell cytoplasm and cause cell damage. This study observed structural changes of the actin cytoskeleton by actin staining of three cell types, and the results showed that AgNPs damage the structure of the actin cytoskeleton of HeLa, Saos-2 and hFOB cells.
Structural investigations of the kelch-like-ECH-associated protein 1 at near-physiological conditions and computational analysis for new therapeutics discovery
Kelch-like-ECH-associated protein 1 (Keap1) has a vital role in an important signal transduction pathway in mammalian cells, where it functions to segregate the nuclear factor erythroid 2-related factor 2 (Nrf2) protein transcription factor in the cytosol, targeting it for degradation by the 26S proteasome. Keap1 consists of an N-terminal region (NTR, residues 1–49), a Broad complex, Tramtrack, and Bric-a-Brac domain (BTB, residues 50–179) domain, an intervening region (IVR, residues 180–314), a Kelch domain/double glycine repeats (DGR, residues 315-599), and C-terminal region (CTR, residues 599-624). Keap1 homodimerized through its BTB domain, and homodimerization is critical for Keap1 functionality. As a part of the E3-ubiquitin ligase complex, Keap1 interacts with Cullin3 (Cul3) via the BTB domain. The Kelch domain of Keap1 contains six repeated Kelch motifs, each about 45–55 residues in length, forming a β-propeller structure essential for dual binding Nrf2. Understanding Keap1-Nrf2 interaction is vital because Nrf2 protein regulation leads to a coordinated antioxidant and anti-inflammatory response in the cell. Dimethyl fumarate (DMF), one of the approved drugs for treating Multiple Sclerosis, surrounded Keap1 through its Kelchand BTB domain, releasing Nrf2 and facilitating its activation. Conducting advanced structure determination technique 'X-ray crystallography' at Turkish Light Source, "Turkish Delight," the first ambient and dimeric Kelch domain structure, was solved. The conformational differences in the Nrf2 and DMF binding site within the Kelch domain, resulting from temperature shifts from cryogenic to ambient, were investigated. Furthermore, serial crystallography studies were performed further to elucidate the dynamic behavior of Keap1 under different conditions. Additionally, molecular docking studies were conducted using this new structure to investigate various electrophilic irreversible inhibitors of Keap1/Nrf2, including DMF, as well as potential direct non-covalent inhibitors. In conclusion, the comprehensive structural analysis of Keap1 releaved insights into its dynamics and interactions with associated proteins and drugs. The molecular docking studies reveal the potential of the novel compound, a hybrid of L-carnosine and L-histidyl hydrazide (CNN),as a Keap1/Nrf2 inhibitor. These findings open new avenues for research into targeted therapies that could modulate the Keap1-Nrf2 pathway, potentially offering benefits in treating oxidative stress and inflammationconditions.
Kistik fibrozis teşhis ve tedavisinde kullanılacak RNA sekansında ileri veri analizleri
KF çok çeşitli klinik ve genetik varyantları olan, yaşamı sınırlayan, OR geçişli bir genetik hastalıktır. Epitelyal hücre zarı boyunca anormal klorür ve sodyum taşınmasına katkıda bulunan kromozom 7q'da'ki KF transmembran iletkenlik düzenleyicisindeki gen mutasyonlarından kaynaklanmaktadır. YND platformu tabanlı RNASeq tekniklerinin geliştirilmesi sonucunda son on yılda büyük ilerlemelerin kaydedilmesiyle bu teknikler sayesinde birçok biyolojik alanda araştırmalar hızlanmıştır. Bu çalışmada, RNASeq transkriptomik analizi yapılan, 2 ayrı veri kaynağından elde edilen toplamda 44 KF örneklerinde kendi belirlediğimiz pipeline ile birlikte RNASeq datasında mutasyon taraması ve ayrıntılı bir transkriptom yanıtın varlığının ortaya çıkarılması amaçlanmıştır. Birinci veri kaynağımızda epitelyal disfonksiyonu incelemek için birkaç in vitro KF modeli geliştirilerek stromal değişikliklere odaklanılarak morfolojik ve transkriptomik özellikler araştırılmıştır. İkinci veri kaynağımızda 'modifiye edici genlerin' olası etkisi nedeniyle, benzer KFTR mutasyonlarına sahip hastalar arasında bile hastalık şiddeti değişken olduğu için bu genleri belirlemek için, orta ve ağır akciğer fenotipi olan KF hastalarında analizler uygulanmıştır. Çalışmamızda kulladığımız 2 veri kaynağından varyant arama için kullandığımız GATK aracı ile daha fazla varyant elde ettik. Bu da literatür ile uyumludur. Her iki veri kaynağımızda yaptığımız varyant çağırma sonrasında, KF ile RNASeq datalarda anlamlı bir sonuç bulunamamıştır. Transkriptom analizimizde ise ilk veri kaynağnda toplamda 426 transkript 2D, 352 transkript de 3D durumunda, 2. veri kaynağımızda da 140 transkriptin yeniden düzenlendiği gösterilmiştir. İlk veri kaynağıyla uyumlu olarak WNT2 ve TBX4 genlerinin up regüle olduğu bulunmuştur. Diğer veri kaynağıyla uyumlu olarak çalışmada EGR1 ve CXCL8 genlerinin down regüle olduğu saptanmıştır. Literatür ile uyumlu olarak ZNF683 geninin ağır KF örneklerinde aşırı ekspresyona uğradığını gösterdik.
The investigation of plasma membrane localization and molecular function of PCDH7 and CLIC4 during cell division
The regulation of the plasma membrane during cell division is essential for maintaining membrane dynamics and cortical stability, both of which are critical for the fidelity of cell division. This thesis investigates the underlying mechanism of two mitosis specific plasma membrane-enriched proteins, PCDH7 and CLIC4, and uncovers their importance in coordinating membrane-cytoskeleton interactions. PCDH7 is identified as a regulator of the actomyosin network during cytokinesis, where it facilitates proper cleavage furrow ingression and completion of cytokinesis. Palmitoylation emerges as an essential mechanism in the translocation of PCDH7 to the plasma membrane at the onset of mitosis through its interaction with a palmitoyl acyltransferase, ZDHHC5. Dynamic plasma membrane localization of PCDH7 promotes the activation of essential cytokinetic regulators, such as RhoA and myosin II, which facilitate actomyosin ring constriction and ensure successful cell division. In contrast, CLIC4 plays a dual role in stabilizing membrane dynamics and cortical tension during cytokinesis. My results suggest that CLIC4 may localize to the plasma membrane in a palmitoylation-dependent manner and bind phosphoinositols, such as PI(3,5)P2 and PI(4,5)P2, to regulate membrane-cytoskeleton interactions. Live-cell imaging reveals its sequential recruitment during the bleb life cycle, where it supports membrane stabilization in the expansion phase and maintains cortical integrity. These findings highlight CLIC4's distinct role in coordinating membrane tension and bleb regulation, processes critical for cell shape maintenance during mitotic progression. Together, the work on PCDH7 and CLIC4 advances our understanding of how protein-membrane interactions contribute to cytokinesis and provides a framework for exploring their broader relevance to cellular architecture and function.
Hemofili A gen tedavisi için transgenik kök hücre platformunun geliştirilmesi ve in vitro etkinliği
Hemofili hastalarının yaklaşık %80' ini etkileyen Hemofili A, Faktör VIII (FVIII) eksikliği sonucu gelişen X' e bağlı resesif kalıtımsal bir kanama bozukluğudur. Hastalar eklem içi ve kas içi kanamalarla ile karakterize olurken, faktör eksikliğinin derecesi, kanama sıklığını ve şiddetini belirlemektedir. Kanamanın kontrolü ve tedavisinde plazma türevli tedaviler ve rekombinant faktör konsantreleri kullanılmaktadır. Araştırmamızda Hemofili A hastalığından sorumlu olan FVIII eksikliğine yönelik gen tedavisi amaçlanmıştır. Bu doğrultuda promotörü EF-1 alpha olan, B domaini silinmiş FVIII gen sekansı ile CD45 R0 truncated yüzey belirteci taşıyan (EF1α - FVIII.CC - CD45R0t) lentiviral vektör dizayn edilmiştir. Tasarladığımız lentiviral vektörün mezenkimal kök hücreleri transdükte etmesi sağlanarak immün cevaptan korunabilen, sahip olduğu yüzey belirteci (CD45R0t) sayesinde in vivo uygulamalarda takip edilebilen, toksik olmayan ve fonksiyonel FVIII sekresyonu sağlayabilen bir gen tedavisi hedeflenmiştir. Faktör VIII kodlayan lentivirüsün karakterizasyon ve kalite kontrol testlerinin ardından mezenkimal kök hücreler ile transdüksiyon verimliliği, FVIII kalıcılık ve ekspresyon analizleri gerçekleştirilmiştir. Faktör VIII proteinin varlığı Western Blot yöntemiyle de doğrulanmıştır. Geliştirilen lentivirüsün (LTR sekansı) polimeraz zincir reaksiyonu (PCR) analizleri ile replikatif olmadığı, güvenliliği test edilmiştir. FVIII sekresyonunun zamana bağlı değişimi Elisa yöntemi kullanılarak değerlendirilmiştir. Faktör VIII proteinin fonksiyonelitesi ise karıştırma testi (mixing test) ile aktive parsiyel tromboplastin zamanı (aPTT) ve FVIII düzeyleri üzerinden gösterilmiştir. Zamana bağlı gerçekleştirilen FVIII sekresyon analizlerinde istatistiksel olarak anlamlı bir azalma olmadığı, stabil seviyelerde FVIII varlığı saptanmıştır (p > 0.05). Karıştırma testi (mixing test) kullanılarak gerçekleştirilen analizlerde ise gen tedavisi uygulanmış ve gen tedavisi uygulanmamış FVIII içermeyen insan plazma ürünleri aPTT ve FVIII seviyesi açısından karşılaştırılmıştır. aPTT seviyeleri değerlendirildiğinde; gen tedavisi uygulanmamış gruba ait ortalamanın 92.69 ± 11.38 sn olduğu ancak gen tedavisi uygulanan grupta ise bu ortalamanın 38.60 ± 13.38 sn' ye düştüğü görülmüştür (p < 0.001). Faktör VIII seviyeleri değerlendirildiğinde ise gen tedavisi uygulanmamış grubun FVIII seviye ortalamasının 0.41 ± 0.03 IU/dL olduğu, gen tedavisi uygulanmış grubun ise FVIII seviye ortalamasının %25.41 ± 33.38 IU/dL' ye yükseldiği saptanmıştır (p < 0.01). Bu araştırma ile literatürdeki çalışmalara alternatif olarak Hemofili A gen tedavisinde yeni bir yöntemin geliştirilmesi sağlanmış, in vivo hayvan modelleri ve klinik denemelere katkı sağlayacak bir çalışmanın ilk adımları atılmıştır.
Advanced parental age association with the development of autism spectrum disorders in children: Meta-analysis
Autism Spectrum Disorder (ASD) is one of the most frequent neurological conditions in infancy, afflicting about 1 in 36 children, with a male-to-female ratio of 4:5:1. Children with autism spectrum disorder often struggle with communication, social relationships, and adjusting to rapid changes. In addition to confined repetitive modes of behavior, pursuits, or activities. This systematic review and meta-analysis explore the link between advanced parental age risk factors and the development of autism spectrum disorders (ASDs) in youngsters. Methods The search cross-referenced multiple databases including Web of Science, PubMed, EMBASE, Medline, Scopus, CINAHL and Cochrane Library to find relevant studies to incorporate in this review and meta-analysis until June 2022. Data was pulled focusing on (i) bibliographic information (ii) research design (iii) sample size, (iv) exposure and outcomes analytic technique and definitions, (v) risk of bias assessments, (vi) effect estimates (adjusted and unadjusted), (vii) confounding variables. Statistical analysis was performed on Review Manager Version 5.4 to compute the effect sizes of the risk in the form of an odds ratio at a 95% confidence interval. Results The search yielded twenty-one studies that were included in the meta-analysis. Advanced parental age compared to the reference points was associated with a 59% surge in the development of risk of autism spectrum disorders in children OR (odds ratio) = 1.59 with 95% CI (1.45-1.74). Meta-regression analysis demonstrated that an increase of 10 years in maternal and paternal age was associated with a 35% and 29% higher risk of autism respectively. Interpretation Advanced parental age was associated with the development of autism spectrum disorder in children. More mechanistic studies are required to further clarify this positive association.
Genetically design and synthesis of multifunctional fusion proteins for biosensor applications
The key to the development of biosensors, which have gained importance in recent years, is to create a biocompatible biosensor interface by preserving the biological activity of the molecules required for proper sensor performance. The biomolecules used to form the interface are difficult to immobilize on the surface of electrodes or other inorganic materials, especially due to the sensitive nature of enzymes, which are functional proteins, and their tendency to denature quickly. The immobilization of the protein provides superior stability, separation and reusability compared to the use of free protein. However, classical methods for protein immobilization have adverse effects on the function of proteins. In this study, a one-step, easy, controlled, oriented and directed immobilization was performed using the peptide that fully adheres to the silica surface to eliminate the undesirable effects of protein immobilization. Multifunctional protein (MBP-SiBP) designed by molecular cloning methods was synthesized in bacteria by expression vector containing Maltose Binding Protein (MBP) and purified by affinity chromatography. The designed protein was characterized by advanced analysis methods such as protein determination methods, electrophoresis techniques and mass spectrophotometer. Functional comparison of MBP-SiBP multifunctional protein with each other was performed using peptide-free MBP (wt-MBP) as a control. Due to the self-assembly feature of the silica-binding peptide, it has been proven by Micro-contact printing method that MBP-SiBP protein provides controlled immobilization without the need for any chemical or physical modification. It was observed that MBP-SiBP multifunctional protein did not lose its functions during immobilization as it interacted actively with fluorescent bound antibody and maltose.
Comparing neutral and adaptive genetic variation in shaping phenotypic diversity of the polymorphic bush cricket Isophya rizeensis along an altitudinal gradient
The accelerating impacts of global environmental and climatic change present significant threats to biodiversity, emphasizing the need to understand how natural populations adapt to environmental gradients. Isophya rizeensis, a univoltine bush cricket endemic to Turkey's Fırtına Valley, offers a compelling model for studying local adaptation, owing to its striking altitudinal color polymorphism in males. Lower-altitude populations predominantly exhibit black morphs, while higher altitudes are characterized by pale-green individuals, a pattern suggesting adaptation to microclimatic conditions rather than classical thermal melanism. Using RAD-seq data from 71 individuals across 10 populations, we identified 92,048 high-confidence polymorphic loci, including 1,113 putatively adaptive loci. Genetic analyses revealed significant differentiation driven by altitude, with Mantel tests confirming isolation by distance and selection. Mid-altitude populations exhibited higher genetic diversity, while extreme-altitude populations showed reduced diversity and fixation of adaptive loci, highlighting the dual influence of gene flow and selection. Genome-wide association studies identified 42 loci significantly associated with altitude, supporting the role of disruptive selection in shaping genetic variation and maintaining polymorphism along the gradient. Discriminant analysis of principal components (DAPC) revealed strong genetic differentiation between color morphs, with three major loci contributing to this separation. These loci exhibited bimodal genotype distributions, indicating the existence of distinct genetic groups linked to color morphs and their altitudinal distribution. This study underscores how environmental pressures shape adaptive and neutral genetic variation, maintaining diversity despite high connectivity. Our findings highlight the interplay between gene flow, selection, and local adaptation, providing valuable insights into the mechanisms driving biodiversity in montane ecosystems and informing conservation strategies in the face of climate change.
Allosteric regulation and functional dynamics of CRY1 in the mammalian circadian clock: Insights from mutational and small molecule interactions
Cryptochromes (CRYs) are essential components of the molecular clock that governs circadian rhythms in mammals, functioning as inhibitors of BMAL1/CLOCK-driven transcription. While mammals possess two CRYs with distinct roles in the circadian clock, the mechanisms underlying their differential functions remain unclear. This study elucidates how a specific mutation in CRY1 allosterically alters its dynamic behavior. Molecular dynamics simulations were conducted on eight CRY1 mutants experimentally shown to exhibit reduced repressor activity. My findings reveal that mutations in CRY1 affect the dynamic behavior of the serine loop and the accessibility of the secondary pocket, changes not observed in CRY2. Further analysis highlights that the flexibility of the serine loop influences secondary pocket volume, with S44 and S45 playing crucial roles through their interactions with E382. Additionally, I explored the potential for small molecules to modulate CRY1 allosterically, demonstrating both in silico and in vitro that this regulation impacts CRY1's affinity to CLOCK. This discovery provides a promising avenue for developing therapeutics targeting CRY1, though it also raises concerns about unforeseen side effects of existing small molecules. To address this, virtual screening was performed to identify compounds targeting the CRY1 secondary pocket. Subsequent phenotypic analysis revealed five distinct effects on circadian rhythm, underscoring the complexity of this regulatory mechanism. This study offers critical insights into CRY1 dynamics, its interaction with CLOCK, and the potential for small-molecule modulation, paving the way for advancements in circadian biology and drug design.
Kanser hücre bölünmesinde keratin 8'in düzenlenmesi
Keratins are cytoskeletal proteins classified as Type I and Type II intermediate filaments. These filamentous structures provide mechanical strength and stability to the cells, but it is not known well how these robust structures are regulated and function during dynamic processes, such as cell division. To better understand the biochemistry of cell division and the regulation of keratins, I focused on keratin 8 in cancer cell division. Keratin 8 is a good candidate with a maintained expression during tumorigenesis and has diverse expression patterns in different cell types. During cell division, these filamentous structures split into non-filamentous clusters with mechanisms that are not well understood. To uncover the phosphorylation-dependent disassembly, I analyzed the cell cycle-dependent phosphoproteome of keratin 8 in different cell lines and investigated its commonly regulated phosphorylation sites. I took label-free and dimethyl labeling-based quantitative approaches combined with TiO2 phosphopeptide enrichment methodology. I applied DDA, DIA, and PRM-based mass spectrometry methods and identified and quantified commonly regulated or cell-type specific phosphorylation sites of keratin 8 during cell division. To understand the functional role of keratins, interaction partners of keratin 8 were investigated by immunoprecipitation. I focused on the relation of keratin 8 with other cytoskeletal elements and further searched for its role in mitosis by analyzing its relation with microtubules. I performed microtubule pelleting assays to determine keratin 8-dependent microtubule interactors. I focused on the NDC80 complex during metaphase and further investigated the role of keratin 8 at the kinetochore-microtubule attachment by super-resolution imaging. Our study brought a better understanding of the regulation and functioning of keratins during cell division.
Structural insights into the electrostatic interactions in human serum albumin dimerization patterns and dipyridamole interaction.
Human serum albumin (HSA) is a ubiquitous, multifunctional protein responsible for the body-wide distribution of both endogenous metabolites' nutrients and exogenous pharmaceuticals. Its inherent properties, mainly its ability to transcytosis and leak into the tissue lumens and having multiple ligand-binding sites, have rendered HSA an exploitable building block in nanoparticle-based drug delivery systems, particularly in cancer targeting. In this study, we present high-resolution crystallographic data (PDB ID: 9V61) supported structure revealing two distinct dimerization patterns of HSA, obtained under high-concentration crystallization conditions, in addition to results from dipyridamole dockings. Both dimer types demonstrate extensive interface areas and a significant number of electrostatic interactions. Comparative analysis with previously reported dimer structure (3JQZ) and other high-interface-area structures, (5Z0B, 8CKS) indicate similarities in contact regions, but unique residue-level differences in biophysical electrostatic bonding interactions. Interface surface area distribution and space group histograms further support the rarity and potential therapeutic relevance of the identified dimer forms. Importantly, these dimer configurations do not disrupt Sudlow's drug-binding sites, which is important as the dipyridamole docking analysis presents a strong affinity to Sudlow site I, not affecting their utility in engineered drug delivery. Our findings open new avenues for structure-based mutagenesis and nanoparticle design strategies centered on HSA dimerization dynamics.
Vagococcus lutrae kaynaklı L-alanine dehydrogenase: yapısal analizi ve endüstriyel potensiyeli
L-Alanine dehydrogenase from Vagococcus lutrae (VlAlaDH) catalyzes the reversible oxidation-reduction reaction between L-alanine and pyruvate. The enzyme's ability to generate a valuable product in a single enzymatic step has increased interest in bacterial AlaDHs. In this study, a novel wild-type apo structure of AlaDH from under characterized organism is presented which could serve as a candidate for L-alanine production with minimal pyruvate byproduct. The discovery lays the groundwork for future studies for protein engineering has been laid. The structure was determined using highly complex experimental methodology of X- ray crystallography with the "Turkish Delight" diffractometer in the ambient temperature in near neutral pH. This approach enabled the determination of the apo-structure of VlAlaDH at 3.2 Å resolution revealing a homo-hexamer oligomerization with conserved Rossmann fold motifs. The main takeaway from the study was the enzyme retained the key motif architecture observed in each homolog structure. The retained motifs being one NAD+ -binding Rossmann fold connected with conserved 𝛼-helices linker to a second Rossmann fold which carried the catalytic residues for pyruvate and L-alanine. Notable differences were observed in the NAD+-binding Rossmann fold where it carried some similarities in residue level to a thermostable bacterial AlaDHs called Geobacillus kaustophilus and Thermus thermophilus. Specifically, residue 270 substituted from Asp to Gln, residue 240 also showed change from Val to Ile between Geobacillus kaustophilus, and between Thermus thermophilus and Vagococcus lutrae both carrying Ile at the residue number 198 but not the homologs. These residues participate in NAD+ binding into the cleft through hydrogen bonds and hydrophobic interactions. As the primary binding substrate being NAD+, these substitutions may be targeted for future studies. The phylogenetic tree further supports that even the evolutionary distance of the species shared common adaptations at coenzymelevel.
Room-temperature apo structure of human SIRT2 solved by X-ray crystallography and its application in targeted cancer drug development
Sirtuins are a family of NAD⁺-dependent deacetylases that regulate gene expression through histone modification and play crucial roles in cellular processes such as stress response, metabolism, aging, and cancer. Among the seven mammalian isoforms (SIRT1–SIRT7), SIRT2 is unique in its ability to function in both the nucleus and cytoplasm, modulating the acetylation status of key regulatory proteins involved in cell cycle control. This regulation implicates SIRT2 in tumorigenesis and the development of drug resistance. In this study, we report the ambient-temperature apo structure of human SIRT2 determined by X-ray crystallography, providing a physiologically relevant model that captures the enzyme's intrinsic conformational flexibility under near-native conditions. Compared to traditional cryogenic structures, the room-temperature data reveal a narrower, likely inactive conformation of the inhibitor binding pocket and active site which undergoes expansion upon ligand binding. Comparative structural analyses identify key residues critical for ligand interactions and highlight the dynamic interplay between protein flexibility, temperature, and ligand presence. These insights significantly deepen our understanding of SIRT2's functional mechanism and are vital for the rational design of selective inhibitors. By integrating ambient-temperature structural data, this work establishes a foundation for structure-based drug discovery targeting SIRT2, advancing efforts in the development of targeted cancer therapeutics. Moreover, these findings emphasize the importance of considering physiological flexibility in proteins to improve the effectiveness and specificity of novel drugs.
Expression, purification, and crystallization of recombinant human ABL1 kinase domain for x-ray crystallography-based structural studies
The Abelson1 (ABL1) is a member of the Abelson (ABL) non-receptor tyrosine kinase family and is central to the regulation of several cellular processes, such as proliferation, survival, and differentiation. ABL1 is composed of several functionally distinct and structurally specialized domains; including an N-terminal cap region, SH2 and SH3 domains, a bilobed kinase domain, and a C-terminal tail, each playing a role in both regulating kinase activity and participating in complex signaling cascades. Under normal cellular conditions, the ABL1 kinase is found in an inactive state and becomes activated according to cellular needs. The aberrant ABL1 activity is associated with cellular abnormalities and disease pathogenesis including Chronic Myeloid Leukemia (CML). CML is a blood cancer type that is characterized by the constitutively active BCR-ABL1 protein arising from the fusion of ABL1 with the Breakpoint Cluster Region (BCR) gene. The deletion of the regulatory sequences of ABL1 and the insertion of additional BCR fragments leads to the constitutive activation of the BCR-ABL1 protein and oncogenic effects. The central role of ABL1 in the progression of CML, makes it the main target for the treatment approaches. Tyrosine kinase inhibitors (TKIs) targeting ABL1 provide an effective strategy for CML treatment by blocking the oncogenic, constitutive activity arising from the BCR-ABL1 fusion. Despite the success of the TKIs in the management of CML, there is a need for new strategies and treatment approaches to improve the treatment efficiency and outcomes. The structural studies that focus on the enlightening structural features of the ABL1 kinase are crucial for the development of novel TKIs and therapeutic approaches for the effective management of CML. This study focuses on the expression, purification and crystallization of ABL1 kinase for its structural investigations that could contribute to the development of novel therapeutics and drug screening platforms.
Dissecting centriolar satellite function using inducible genetic tools
Centriolar satellites are membraneless granules that facilitate protein trafficking to the centrosome and primary cilium, but the principles governing their assembly, dynamics, and functional roles remain poorly defined. Here, we introduce two chemogenetic tools to manipulate satellite organization. First, chemically inducible Kin-14 motor protein-based trafficking system acutely concentrates satellite granules at the centrosome, altering client protein organization. This centrosomal foci created by PCM1 and other clients, behave like a solid compartment and does not dissolve during mitosis. Functionally, forced satellite accumulation disrupts mitotic progression, proper spindle formation, and chromosome congression. In our second system, we are utilizing a chemically inducible centriolar satellite dimerization assay to regulate the multimerization state of PCM1. Ligand-induced dimerization changes satellite organization by enlarging the granules and reducing their numbers. Notably, satellite multimerization prevents their mitotic dissolution and disrupts Hedgehog signaling but does not affect cilium assembly. Finally, we applied a high-throughput image-analysis pipeline to patient derived fibroblasts bearing mutations in ciliopathy related genes and showed that there are no significant differences in satellite number, intensity, or spatial distribution compared to control cells. Together, this thesis establishes various chemical assays for acute perturbation of centriolar satellites, demonstrate that dynamicity and multivalency are critical for proper centriolar satellite function, and indicate that satellite mislocalization and content is not a universal feature of ciliopathies.