Dept. of Molecular Biology and Genetics - Ph.D. / Sc.D.
Permanent URI for this collection
Browse
Browsing Dept. of Molecular Biology and Genetics - Ph.D. / Sc.D. by Title
Now showing 1 - 20 of 82
Results Per Page
Sort Options
Item Open Access The ability to generate differentiated and senescent progeny is a major determinant of breast cancer heterogeneity(Bilkent University, 2009) Mumcuoğlu, MineBreast cancer displays distinct subtypes, such as luminal A, luminal B, and basallike. The prognosis and therapeutic response of each subtype is different. The mechanisms involved in the generation of these tumor types are poorly understood. Our aim was to test whether the ability to generate senescent progeny contributes to breast cancer heterogeneity. A panel of 12 breast cancer cell lines, 31 isogenic clones, and 12 breast tumors were used. We classified breast cancer cell lines into senescent cell progenitor (SCP) and immortal cell progenitor (ICP) subtypes. All ER+ cell lines tested and some ER-positive (ER+) breast tumors displayed senescence. Acute loss and tamoxifen-mediated inactivation of ER triggered a robust senescence response in SCP type T47D cell line. In contrast, ER-overexpression, estrogen treatment and p21Cip1 knockdown inhibited senescence. Neutralization of reactive oxygen species also abolished senescence. Breast cancer cell subtypes displayed divergent ability to produce differentiated progeny. The SCP subtype cells produced CD24+ or ER+ luminal-like and ASMA+ myoepithelial-like progeny, in addition to CD44+ stem/progenitor-like cells. In contrast, ICP cell lines acted as differentiation-defective stem/progenitor cells. Some cell lines generated only CD44+/CD24-/ ER-/ASMA- progenitor/stem-like cells, and others only CD24+/ERluminal-like, but not ASMA+ myoepithelial-like cells. SCP cell lines were less tumorigenic, and they clustered with luminal A/normal like tumors. In contrast, ICP subtypes were more tumorigenic, and they clustered together with basal/luminal B tumors. Our results show that breast cancer cell lines clustering with luminal A/normal-like and basal/luminal B tumors respectively, differ from each other by the ability to generate differentiated and senescence-arrested progeny.Item Open Access Acquired expression of transcriptionally active p73 in hepatocellular carcinoma cells(Bilkent University, 2002) Sayan, A. EmreP53 gene is the most common mutated tumor suppressor gene during tumorigenesis. From its description till 1997, p53 gene was thought to stand alone in the human genome. In 1997, p73 gene and in 1998, p63 gene was identified which are encoding functional homologues of p53 protein. Unlike p53, the knock out mice for p73 and p63 genes did not yield a tumor prone phenotype and the mutation frequency of these genes is very low compared to p53 gene. There is also extensive alternative splicing and changes in the expression pattern of p73 and p63, unlike p53. Thus the new p53 homologues were considered as non-classical and non-Knudson type tumor suppressor genes. A codon specific, aflatoxin ingestion related p53 mutation was shown to be important in the ethiopathology of HCC so loss of p53 function is a major factor during HCC development. The rate of p53 functional inactivation was determined by lots of studies in HCC but the knowledge for new p53 homologues is scarce. We aimed to define the probable function of the new p53 homologue, p73 in HCC development. For this purpose, we have analyzed the 3’ alternative splicing and expression pattern of p73 in a series of HCC derived cell lines. Our results showed the alteration of splicing and expression in HCC cell lines compared to normal liver. After the completion of human genome project, the contig harboring the p73 gene was entered to the public database. With the hints of the presence of an alternative promoter in the p63 gene and the description of the alternative promoter in mouse p73 gene, we have made an in silico analysis to identify the probable promoter and exon within p73 gene. Our studies revealed the in vivo description of a new human p73 encoded transcript. The proposed protein product of the transcript was lacking the transactivation domain so it was named as Dominant Negative p73 (DN-p73) and the former p73 was renamed as Transactivating p73 (TA-p73). Since the promoters of these two transcripts are different and probably under the regulation of different transcription factors, we studied the expression pattern of them by semi quantitative RT-PCR method. We have shown the presence of only DN-p73 in normal in normal liver. HCC derived cell lines and primary HCC tumors also express DN-p73 together with the acquired expression of TA-p73 in most of the cell lines and some of the primary HCC tumors. The promoter of TA-p73 was shown contain E2F1 transcription factor binding sites. The Retinobastoma protein (pRb) is the most potent inhibitor of the E2F1 transcription factor and the dysrégulation of the Rb pathway components is a common event in HCC development (Rb gene mutations and proteolytic dysrégulation of pRb and mutational and epigenetic inactivation of pi6). We have shown the expression of TA-p73 in some of the HCC derived cell lines and primary HCC tumors so the acquired expression of TA-p73 in HCC cells might be the indicator and the effect of of Rb pathway dysrégulation. We tested this hypothesis by analyzing the expression of pRb and pi6, together with the endogenous E2F1 transcription factor targets such as cyclin E, pi4'^’^ and TA-p73. Our results showed a 75% inactivation of Rb pathway components and a partial correlation of TA-p73 expression in HCC cells. The acquired expression of TA-p73 in HCC cells is unfavorable during tumorigenesis since TA-p73 mimics the pro- apoptotic and cell cycle regulatory, function of wild type p53. Mutant p53 proteins were shown to inhibit the pro-apoptotic fliction of wild-type p53 and TA-p73. We have analyzed the p53 protein status of 15 HCC derived cell lines and defined the presence of mutant p53 or no functional p53 protein in 87% of the HCC derived cell lines. As a summary, we have identified the human homologue of mouse DN-p73 and defined the 3’ alternative splicing and 5’ differential promoter initiation products of p73 gene encoded products in normal liver versus a series of HCC derived cell lines and primary tumors. Moreover we have correlated the expression of TA-p73 with Rb pathway inactivation and expression of mutant p53 proteins.Item Open Access Acquired tolerance of hepatocellular carcinoma cells to selenium-deficiency : a selective survival mechanism(Bilkent University, 2003) Irmak, Meliha BurcuSelenium-deficiency causes liver necrosis. Selenium is protective against viral hepatitis and hepatocellular carcinoma (HCC). The underlying molecular mechanisms of selenium effects are ill-known. In this study in vitro response of hepatocellular carcinoma-derived cell lines to selenium-deficiency is examined alone or in conjunction with Vitamin E and Copper/Zinc. Here we show that in vitro selenium-deficiency in a subset HCC-derived ‘hepatocyte-like’ cell lines causes oxidative stress and apoptosis. The oxidative stress and consequent cell death induced by selenium-deficiency on these cells are reverted by the antioxidant effect of Vitamin E. However, ten among thirteen HCC cell lines are tolerant to selenium-deficiency and escape its deadly consequences. Nine of ten tolerant cell lines have integrated hepatitis B Virus (HBV) DNA in their genomes, and some display p53-249 mutation, indicating past exposure to HBV or aflatoxins, established factors for oxidative stress and cancer risk. Thus, as demonstrated by the gain of survival capacity of apoptosis sensitive cell lines with Vitamin E, such malignant cells have acquired a selective survival advantage that is prominent under selenium-deficient and oxidative stress conditions.Item Open Access Analysis of 45S rDNA promoter methylation and expression of rRNA transcripts in breast cancer(Bilkent University, 2015-06) Karahan, GurbetRibosome biogenesis has a central role in cell growth and proliferation that is usually disrupted in tumor cells by the inactivation of tumor suppressor genes and activation of oncogenes. Ribosomal RNA (rRNA) gene expression is one of the most important factors regulating ribosome production, which is controlled by CG rich 45S ribosomal DNA (rDNA) promoter. The effect of DNA methylation at 45S rDNA promoter on rRNA gene expression is a subject of controversy in the literature. In this thesis, a 434 bp region (-380 bp to +54 bp) spanning both upstream control element (UCE) and core promoter located in 45S rDNA promoter containing 54 CpGs was analysed in breast cancer. We also analysed the related rRNA expression levels in the same samples in order to clarify the role of 45S rDNA promoter methylation on rRNA gene expression. 45S rDNA promoter region was highly methylated (74%-96%) in all cell lines including non-tumorigenic breast cell line (MCF10A). Even though 45S rDNA promoter region of breast cancer cell lines are extensively methylated, rRNAs (18S, 28S, 5.8S and 45S ETS) were expressed independent of the heavy methylation. Expression levels of rRNAs are assessed either using housekeeping genes (ACTB, TBP, ACTB&TBP) or geometric mean of rRNAs (GM-rRNAs). We propose GM-rRNA normalization as a new method to identify relative expression differences between rRNA transcripts. Epigenetic drugs 5-Aza-2'-deoxycytidine (5-AZA) and Trichostatin A (TSA) were used to determine the effect of DNA methylation and histone acetylation on rRNA expression. Demethylation with 5-AZA resulted in an unexpected decrease in the expression of all rRNA. TSA treatment did not lead to any significant expression difference in cell lines. To better evaluate the effect of DNA methylation on the expression of rRNA transcripts we analysed the methylation status of 19 breast tumor and matched normal frozen tissue samples. The results showed that majority of the tumors (13/19) have significantly higher methylation levels than their normal pairs. Using the GM-rRNA as reference helped us to determine significant differences in the proportionate expression of rRNAs in these tissue samples. The 5.8S rRNA ratio was significantly lower whereas the 18S rRNA ratio was significantly higher in breast tumor samples. Furthermore, the 45S rDNA promoter methylation levels in normal breast tissue samples were negatively correlated with the18S rRNA ratio but this correlation was disrupted in breast tumors. Similarly, rRNA transcript levels were significantly correlated with each other in normal samples, were lost in tumor samples. It is clear that, there is a dysregulation both in rDNA methylation levels and spliced rRNA transcripts specific to breast tumor samples, which was not observed in normal breast tissues. rRNA gene expression is controlled by mechanisms other than promoter DNA methylation. Tumorigenesis may cause disruption of many control mechanisms that are required for proper rRNA expression, splicing and maturation, resulting in a dysregulation of the correlation between spliced rRNA expression levels, which should be investigated further.Item Open Access Analysis of BRCA1 and BRCA2 genes in Turkish breast cancer patients(Bilkent University, 2000) Özdağ, HilalBreast cancer is the most frequent cancer type and the second cause of death among women. It is estimated that 10 to 15% of breast cancer cases are hereditary. The majority of hereditary breast cancers can be attributed to germ-line mutations in ^Jgeast CAncer susceptibility genes BRCAl and BRCA2. In this study, germ-line BRCAl and/or BRCA2 gene mutations were screened in 50 Turkish breast and/or ovarian cancer patients divided into four groups of hereditary, familial, early onset, and male cancer by heteroduplex analysis and DNA sequencing. Two BRCA2 mutations, one novel (6880insG) and one previously reported (3034delAAAC), were found in the hereditary group. A novel BRCAl (1200insA) mutation was formd in the early onset group. All three mutations cause premature- termination codons. In addition, five BRCAl sequence variants have been identified in 23 patients. K654E (2080 A—>G), D693N (2196 G->A), P871L (2731 C—>T), and K1183R (3667 A—>G) result in a change of amino acids. 1013 T—^>C and 2201 C—>T are silent mutations. One patient in the early onset group was compound heterozygote for K654E and D693N. These results indicate that BRCAl and BRCA2 genes are involved in some but not all hereditary breast cancers in the Turkish population.Item Open Access Analysis of candidate molecular targets in adult (CML) and childhood (AML, ALL) Leukemias(Bilkent University, 2004) Boylu, Cemaliye AkyerliCandidate molecular targets were investigated in three different types of leukemias, chronic myeloid leukemia (CML), acute myeloid leukemia (AML) and acute lymphocytic leukemia (ALL). The first group of these molecular targets was identified through a cDNA based gene expression profile analysis in sixty-seven CML patients who were classified according to clinical parameters known as new prognostic score (NPS). CML patients can be divided into three groups of low-risk, intermediate-risk, and high-risk, based on NPS. Response of these risk groups to treatment is not uniform and the gene expression profiles associated with each risk group remain unknown. Seven genes were chosen from a cDNA microarray study in which two high versus two low-risk patients were analyzed. Semi-quantitative and real-time reverse transcription polymerase chain reaction (RT-PCR) analysis of these differentially expressed transcripts highly correlated with the microarray data. Expression levels of all genes, except PTGS1, were significantly different between the high (n=9) and low-risk (n=7) CML by semi-quantitative RT-PCR (IFITM1 and CXCL3 p=0.001; CCNH p=0.012; RAB1A p=0.01, PRKAR2B p=0.016; UCP2 p=0.04; and PTGS1 p=0.315). Real-time RT-PCR analysis showed similar results for IFITM1 expression in thirty-four low and eleven high-risk patients (p=9.7976 x 10-11). Higher IFITM1 or lower CXCL3 expression correlated with improved survival (p=0.01 and p=0.059 respectively). Gene expression profiling is a valuable tool to identify candidate risk group indicator genes for the development of a molecular classification system for CML, which may also predict survival. Although the connection between DNA-repair gene mutations and hematological malignancies are now well established, germ-line mutations in the base excision repair (BER) pathway was only recently documented in an inherited cancer syndrome in human homologue of E. coli mut Y (MYH). Interestingly, the cancer associated MYH missense mutations Tyr165Cys and Gly382Asp have been documented with a high frequency (1 percent) in a control group of the British population. Therefore, we screened the above mentioned missense variants in two different childhood leukemias, AML (n=45) and ALL (n=140). Neither mutation was present in any of the patient samples and controls, except for one patient diagnosed with AML/M3. Tyr165Cys mutation in the heterozygous state was present in the sample obtained at the time of initial diagnosis. Further sampling, at remission, and the analysis of parental DNA, showed only the normal allele. Therefore, the mutation was considered to be specific for the leukemic blasts. Based on these results, an association between childhood leukemias and the MYH missense variants Tyr165Cys and Gly382Asp was not observed. Also, these variants appear to be absent -if not at a very low frequency- in the Turkish population, contrary to the British population.Item Open Access Analysis of differentially expressed geExpression of notch signaling pathway recenes in breast cancer : BRCA1- induced gene expression profiles and meta-analysis gene signature(Bilkent University, 2009) Dedeoğlu, Bala GürThe aim of the first part of this study was to find out the expression profiles of the genes, which were selected from the former BRCA1-induced gene list (OVCA1, OVCA2, ERBIN, RAD21, XRN2, RENT2, SMG1 and MAC30) in normal-matched primary breast tumors and to correlate the gene expression profiles of selected candidate genes with BRCA1 and various pathology parameters. Among the target genes, the expression of ERBIN, SMG1 and RAD21 were found to be highly correlated with that of BRCA1 both in BRCA1 up- and down-regulated cells and this result was validated with qRT-PCR expression profiling of the eight genes in 32 normal-matched primary breast tumor samples. These genes were found to be discriminative between ER(-) and ER(+) tumors as well as grade 1 and grade 3 tumors. Target genes were also analyzed in independent microarray datasets to assess their predictive power for breast tumor grade, subtype and patient survival. ERBIN, SMG1 and RAD21 were found to have predictive roles in these datasets. The aim of the second part of the study was to found appropriate reference genes (RGs) for accurate quantification of target gene expressions in breast tumor tissues. The expression patterns of fifteen widely-used endogenous RGs and three candidate genes that were selected through analysis of two independent microarray datasets were determined in 23 primary breast tumors and their matched normal tissues using qRT-PCR. Additionally, 18S rRNA, ACTB, and SDHA were tested using randomly primed cDNAs from 13 breast tumor pairs to assess the rRNA/mRNA ratio. The tumors exhibited significantly lower rRNA/mRNA ratio when compared to their normals. Among the eighteen tested endogenous reference genes, ACTB and SDHA were identified as the most suitable reference genes for the normalization of qRTPCR data in the analysis of normal-matched tumor breast tissue pairs. The aim of the third part of this study was to develop a resampling-based metaanalysis strategy. Two independent microarray datasets that contain normal breast, invasive ductal carcinoma (IDC), and invasive lobular carcinoma (ILC) samples were used for the meta-analysis. The resampling-based meta-analysis has led to the identification of a highly stable set of genes for classification of normal breast samples and breast tumors encompassing both the ILC and IDC subtypes. A subset of this meta-gene list was shown to predict well-established molecular tumor subtypes, e.g., basal vs luminal or ER+/ER-, with high accuracy and sensitivity based on class prediction analysis of existing breast cancer microarray datasets. Expression of selected genes, tested on 10 independent primary IDC samples and matched nontumor controls by real-time qRT-PCR, supported the meta-analysis results.Item Open Access Analysis of GSTM1, GSTT1, GSTP1, and TP53 polymorphisms as genetic risk factors for bladder cancer in the Turkish population(Bilkent University, 2001) Törüner, Gökçe AltayThe effect of the GSTM1 and GSTT1 null genotypes, the GSTP1 Ile105Val, and TP53 Arg72Pro polymorphism on bladder cancer susceptibility was investigated in a case control study of 121 bladder cancer patients, and 121 age-sex matched controls in the Turkish population. The adjusted odds ratio (for age, sex, and smoking status) for the GSTM1 null genotype is 1.94 (95% CI 1.15- 3.26) and for the GSTP1 105 Ile/Val or Val/Val genotypes is 1.75 (95% CI 1.03- 2.99). GSTT1, and TP53 loci was not shown to be associated with bladder cancer. Combination of the two high risk genotypes, GSTM1 null and GSTP1 105 Ile/Val or Val/Val, revealed that the risk increases by 3.91 times (95% CI 1.88-8.13) when compared with the combination of the low risk genotypes of these loci. In individuals with a combined risk of cigarette smoking and the GSTM1 null genotype, bladder cancer risk is 2.81 (95% CI 1.23-6.35) relative to persons who do not smoke and carry the GSTM1 present genotype. The same risk for the GSTP1 105 Ile/Val or Val/Val genotypes is 2.38 (95% CI 1.12-4.95). These findings support the role for the GSTM1 null and the GSTP1 105 Ile/Val or Val/Val genotypes in the development of bladder cancer. Furthermore, gene-gene (GSTM1- GSTP1) and gene-environment (GSTM1-smoking, GSTP1-smoking) interactions increase this risk substantially.Item Open Access Analysis of members of the SLIT-ROBO pathway as diagnostic and prognostic tools in hepatocellular carcinoma with special focus on ROBO2-associated cellular phenotype(Bilkent University, 2009) Avcı, Mehmet EnderHepatocellular carcinoma is the sixth most common cancer in the world, with an annual incidence exceeding half a million. It is associated mainly with hepatitis B and C viral infections; and is the main cause of death among cirrhotic patients. Aflatoxin B1 exposure, chronic alcohol consumption and virtually all cirrhosis-inducing conditions are of the other etiologies. For early diagnosis of HCC, surveillance of the risk groups is a crucial task requiring the development of novel markers for HCC with stronger sensitivity and specificity. In addition, description of biomarkers specific to hepatocellular carcinoma subtypes could identify novel targets for therapy. In this study, we analyzed members of the SLIT-ROBO gene families as novel diagnostic and prognostic markers in hepatocellular carcinoma. We defined an expression signature for members of the SLIT-ROBO gene families in HCC cell lines and tissues by real-time quantitative RT-PCR analysis. We showed that ROBO1 was overexpressed as stage and differentiation of the HCC proceeds. Furthermore, ROBO4 downregulation and SLIT2 overexpression marked late stage and poorly differentiated HCCs. Our results suggest that the expression of ROBO1 and ROBO4 can be used in early diagnosis of HCC. As another focus, we stably knockdowned ROBO2 expression in a model AFP positive cell line Huh7 and characterized the associated cellular phenotype. ROBO2 downregulation caused a significant decrease in proliferation rate whereas in wound-healing assay no significant difference in migration rate was observed. In addition, we performed a microarray experiment and found the differentially expressed genes between stable ROBO2 knockdown and negative clones. In this analysis, we found an overexpression of CK19, CD44, ABCG2/ BCRP1 hepatic progenitor cell markers and CD133 that is also a putative cancer stem cell marker of HCC, in stable ROBO2 knockdown clones. In addition KLF4 expression was augmented in these ROBO2 knockdown clones. We propose a genetic association between SLIT-ROBO pathway and CD133 at transcriptional level.Item Open Access BO-264: Highly potent TACC3 inhibitor as a novel anticancer drug candidate(Bilkent University, 2021-01) Akbulut, ÖzgeBreast cancer has been consistently ranked to be the most common cancer and the second leading cause of cancer-related death in women worldwide for many years. Despite better understanding of tumor biology and the availability of plethora of anti-cancer therapeutics utilizing different strategies, complete response and/or long-term survival is achieved in only a small fraction of patients with aggressive disease. Since microtubule re-organization is an important step during cell division, drugs that interfere with this process have been a major focus of cancer research. Although anti-microtubule agents are widely used in clinic, cytotoxicity to non-tumorigenic cells and drug resistance are still the main obstacles. Therefore, development of alternative target molecules that selectively and efficiently target cancer cells, but restore normal cells are needed. Transforming acidic coiled-coil containing protein 3 (TACC3) is an important TACC family member, having both mitosis-related roles e.g. regulation of centrosomes and microtubule stability and interphase-related roles e.g. regulation of gene expression and cell migration. Being overexpressed in a broad spectrum of cancers and correlation of its expression with disease progression make TACC3 a highly attractive therapeutic target. Although the oncogenic role of TACC3 has been established albeit mostly in in vitro settings, there is currently no TACC3 inhibitor being tested in clinics. Therefore, by combining rational drug design and screening, we aimed to identify and characterize a novel TACC3 inhibitor hit molecule with high potency and minimum toxicity in in vitro and in vivo systems that is amenable for future drug development. BO-264 was identified as a novel inhibitor targeting TACC3 by direct binding validated by using several biochemical methods, including drug affinity responsive target stability, cellular thermal shift assay, and isothermal titration calorimetry. Compared to two other available TACC3 inhibitors, it showed superior inhibition of mitotic progression and cell viability, especially in aggressive basal and HER2+ breast cancer cell lines. Notably, BO-264 had remarkable cytotoxicity effect on several cancer cell lines in NCI-60 human cancer cell line panel (≥ 90% have less than 1 µM GI50 value) and inhibited the proliferation of FGFR3-TACC3 fusion protein-harboring cells, an oncogenic driver in several malignancies. Importantly, BO-264 did not cause any cytotoxicity to non-cancerous cell lines. Noteworthy, its oral administration significantly suppressed tumor growth in both breast and colon cancer syngeneic and xenograft models, and prolonged survival with no major toxicity. Finally, TACC3 expression level has been identified as a strong independent prognostic factor in breast cancer. Collectively, our preclinical findings suggest that BO-264 is a potent and non-toxic anti-cancer agent targeting TACC3 in breast and colon cancer and can be developed further to obtain better drug-like properties.Item Open Access Cancer testis gene expression as a biomarker of one-carbon metabolic activity, drug sensitivity and phenotypic heterogeneity in non-small cell lung cancer and malignant melanoma(Bilkent University, 2016-09) Şenses, Kerem MertExpression of cancer-testis (CT) genes on X chromosome (CT-X) is restricted to tumors, with very low or no expression in normal adult tissues. CT-X gene expression is one of the factors contributing to tumor heterogeneity and is variably observed in various types of cancers including non-small cell lung cancer (NSCLC) and malignant melanoma. Regulation of CT-X gene expression has been strongly linked to DNA methylation of promoter regions, however, mechanisms leading to re-expression of these genes in tumors, driven by promoter hypomethylation, is remained unsolved. Although tumors expressing CT-X genes are shown to be associated with higher tumor stage, larger tumors and aggressiveness, differential drug sensitivity of CT-X positive and negative tumors have not been investigated. In this thesis, we aimed to find the association between one-carbon pathway polymorphisms and CT-X gene expression. Moreover, we tested various tools and methods to find effective drugs for tumors in different phenotypic subgroups determined by CT-X gene expression or by other factors.Item Open Access The characterization and potential functional role of wdr81, a novel zebrafish gene, associated with cerebellar ataxia, mental retardation and dysequilibrium syndrome (camrq) in humans(Bilkent University, 2016-04) Doldur Ballı, FüsunCerebellar ataxia, mental retardation and dysequilibrium syndrome (CAMRQ) is a neurodevelopmental disorder. The gene encoding WD repeat containing protein 81 (WDR81) was reported to be associated with CAMRQ2 [MIM 610185]. Human and mouse studies indicated the potential importance of WDR81 in neurodevelopment. The first aim in this study was to characterize the transcript and to reveal the expression profile of wdr81 in zebrafish. The second aim was to perform the initial characterization of wdr81 morphants. In silico analysis indicated that the conserved domains are shared in human, mouse and zebrafish orthologous proteins, implying a conserved function of WDR81 in three species. The characterization of the transcript revealed that wdr81 possessed one ORF and one 5’UTR structure. The predicted sequence for 3’UTR was confirmed along with detection of some variants and an insertion site in samples from ten developmental timepoints and in several adult tissues. This region was not detected in kidney, intestine and gills, which might be pointing out an alternative polyadenylation event. wdr81 appeared to be maternally supplied. 5 hpf and 18 hpf were detected as crucial timepoints regarding wdr81 expression. Expression of wdr81 was found to be increased in the eye and brain regions at 18 hpf and 48 hpf. wdr81 was found to be ubiquitously expressed in the adult zebrafish. The expression of wdr81 in the adult brain and eye was detected in several regions including retinal layers, presumptive Purkinje cells and some proliferative zones. The splice blocking morpholino which targets the exon 2-intron 2 junction of wdr81 worked at 3 tested doses; 2 ng, 4 ng and 8 ng. The effect of the wdr81 morpholino was detected to add the intron, which is downstream of the target exon, to the transcript and introduce a stop codon. Preliminary results indicated a significant reduction in the head sizes at a ratio of 3.88% (p:0.027) in the wdr81 morphant group compared to uninjected group and gbx2 expression was observed to be higher in wdr81 morphants compared to the control groups. In short, findings of this study emphasize the significance of wdr81 in neurodevelopment and suggest a potential role in neuronal proliferation. This study also serves as a basis for future functional studies.Item Open Access Characterization of a novel IRE1 substrate pact and interacting miRNAS(Bilkent University, 2022-06) Doğan, Aslı EkinThe double-stranded RNA-dependent protein kinase activator A (PACT) anchors the RNAinduced silencing complex (RISC) to the endoplasmic reticulum (ER)’s membranous platform where RISC nucleation occurs and thus, PACT plays a key role in microRNA (miR)-mediated translational repression. Previous studies have shown that ER stress leads to PACT phosphorylation while simultaneously inducing changes in the expression of many miRs. Here, we demonstrate that PACT is phosphorylated by the ER-resident Inositol-requiring enzyme-1 (IRE1), a bifunctional kinase/endoribonuclease (RNase), both under ER stress and no stress conditions. While the role of IRE1 as a stress-induced RNase driving the unfolded protein response (UPR) is well understood, the function or the target(s) of its kinase activity have remained unexplored. Findings of this thesis show that IRE1- mediated phosphorylation of PACT regulates mature miR-181c levels, which suppresses the expression of key regulators of mitochondrial biogenesis (mitobiogenesis). Phosphorylation by IRE1 causes PACT-mediated suppression of mitobiogenesis and respiration. Partial PACT-deficiency in mice leads to enhanced mitobiogenesis during brown fat activation in cells and mice. Furthermore, cardiopulmonary bypass-induced ischemia/reperfusion injury downregulates PACT protein expression in human hearts while simultaneously inducing mitobiogenesis. Collectively, these findings demonstrate PACTmiR- 181c signaling axis is a key regulator of mitochondrial biogenesis and energetics.Item Open Access Characterization of functional and molecular properties of circulating extracellular vesicles of childhood idiopathic nephrotic syndrome patients(Bilkent University, 2021-10) Eroğlu, Fehime KaraNephrotic syndrome (NS) is one of the most common causes of glomerular disease in children and is characterized by the triad of proteinuria, hypoalbuminemia, and edema. The major molecular event in the pathogenesis of NS is the disruption of the glomerular filtration barrier, which is primarily driven by podocyte injury. The most common clinical presentation of NS in children is steroid-sensitive nephrotic syndrome (SSNS), characterized by complete remission within 4 weeks of steroid therapy and no apparent glomerular change in the light microscopic evaluation of kidney biopsies, thereby named as Minimal Change Disease (MCD). Since previous research suggests a role of a circulating factor in the pathogenesis of steroid-sensitive nephrotic syndrome (SSNS), we speculated that circulating plasma extracellular vesicles (EVs) are a candidate source of such a soluble mediator. Here, we aimed to characterize and try to delineate the effects of these EVs in vitro. Plasma EVs from 20 children with SSNS in relapse and remission, 10 healthy controls and 6 disease controls were obtained by serial ultracentrifugation. Characterization of these EVs was performed by electron microscopy, flow cytometry and western blotting. The major proteins from the plasma EVs were identified via mass spectrometry. A Gene Ontology classification analysis and ingenuity pathway analysis were performed on selectively expressed EV proteins during relapse. Immortalized human podocyte culture was used to detect the effects of EVs on podocytes. The protein content and the particle number of plasma EVs were significantly increased during NS relapse. Relapse NS EVs selectively express proteins which involved actin cytoskeleton rearrangement. Among these, the level of RAC-GTP was significantly increased in relapse EVs compared to remission and disease control EVs. Relapse EVs were efficiently internalized by podocytes and induced significantly enhanced motility and albumin permeability. Moreover, relapse EVs induced significantly higher levels of RAC-GTP and phospho p38 (p-p38) and decreased levels of synaptopodin in podocytes. Circulating relapse EVs are biologically active molecules that carry active RAC1 as cargo and induce recapitulation of the nephrotic syndrome phenotype in podocytes in vitro.Item Open Access Characterization of novel monoclonal antibodies that target proteins differentially expressed in hepatocellular carcinoma : a proteomics approach(Bilkent University, 2011) Öztaş, EminHepatocellular carcinoma (HCC) is the sixth common cancer in the world. Because of the late diagnosis of the disease, survival rates are still poor in the HCC patients. Surveillance strategies have to be developed in populations with high risk groups having premalignant diseases for HCC, such as liver cirrhosis. The usage of serum and histology-based biomarkers assists health professionals to evaluate the patients. Despite of the advances in diagnostic methods, there is still a need to develop novel biomarkers for early detection of HCC. Therefore, we aimed to develop new biomarkers with higher sensitivity and specificity for HCC to improve the surveillance of the patients. Using an apoptotic HCC cell line, HUH7, and SIP1 proteins, we generated novel monoclonal antibodies (mAbs). 6D5, 1C6 and 6E5 hybridoma clones were chosen for characterization studies because of their strong reactivity in cell-ELISA assays. We found differential reactivity pattern for those novel mAbs in a panel of human sections consisting of tumors, benign liver diseases, normal tissues and a variety of cell lines. Using proteomics methods, we identified candidate target proteins for the 6D5 mAb. Better characterization of these target proteins will provide a better understanding of the molecular pathways in the HCC and aid in the research for developing newer therapeutic agents. In conclusion, our candidate biomarker mAbs can be used in the early diagnosis of HCC as well as in drug development studies.Item Open Access Characterization of the coiled-coil domain-containing protein 124 (Ccdc124) as a novel centrosome and midbody component involved in cytokinesis(Bilkent University, 2013) Telkoparan Akıllılar, PelinCytokinetic abscission is the cellular process leading to physical separation of two postmitotic sister cells by severing the intercellular bridge. During cell division several functional complexes accumulate at the bridge connecting the two sister cells. The most noticeable structural component of the intercellular bridge is a transient organelle termed as midbody. This novel organelle is localized at a central region, which marks the site of cytokinetic abscission. Despite its major role in completion of cell divison, our understanding of spatiotemporal regulation of midbody assembly is incomplete. In this thesis work, we first characterizated the coiled-coil domain-containing protein-124 (Ccdc124), a eukaryotic protein conserved from fungi-to-man, at the molecular level. We identified that at the sub-cellular level Ccdc124 is localized at centrosomes and the midbody depending on stages of the cell cycle. In interphase cells, as well as in mitosis, the protein is localized to centrosomes. However at later stages of cytokinesis (lateanaphase/ telophase) Ccdc124 translocates to the midbody. Knockdown of Ccdc124 in human HeLa cells leads to accumulation of enlarged and multinucleated cells; however, centrosome maturation was not affected. Similarly, in preliminary in vivo assays involving down-regulation of Ccdc124-homologue in zebra fish early embryos, we observed multinuclear embryonic cells. Furthermore, we have validated a previously observed in vitro interaction in our laboratory between Ccdc124 and the Ras guanine nucleotide exchange factor 1B (RasGEF1B) by co-immunoprecipitation assays. As RasGEF1B is strictly a Rap2 GTP-binding protein specific nucleotide exchange factor, this result has suggested a possible involvement of Rap2 in cytokinesis related events. Thus, subsequently, we assessed the sub-cellular localization of Rap2 in synchronized cells during cytokinesis. We found that even though it does not play a role in cell division, Rap2 is localized to the midbody. This result establishes a functional link between cytokinesis and activation of localized Rap2 signaling at the midbody. Data presented in this thesis work indicate that Ccdc124 is a novel factor operating both for proper progression of late cytokinetic stages in eukaryotes, and for establishment of Rap2 signaling dependent cellular functions proximal to the abscission site.Item Open Access Characterization of the fine-scale genetic structure of the Turkish population(Bilkent University, 2022-01) Kars, Meltem EceThe construction of population-based genetic resources plays a pivotal role in the study of human biology and disease. In this study, the fine-scale genetic structure of the Turkish (TR) population was characterized using the whole-exome (WES, n =2,589)andwhole-genome(WGS, n =773)sequencesof3,362unrelatedin-dividuals from Turkey. Significant levels of admixture from Balkan, Caucasus, Middle East, and Europe were detected in the TR subregions, consistent with the history of Anatolia. Results of the population structure analyses showed that the TR and European populations have a closer genetic relationship than previously appreciated. Inbreeding coefficient calculations and runs of homozygosity analysis reflected the unique effects of the high rate of consanguineous marriage on the TR genome. A TR Variome comprising over 40 million variants was constructed using the data generated in this study. Derived allele frequency (DAF) calculations revealed that 28% of TR-WES and 49% of TR-WGS variants in the very rare frequency bins (DAF < 0.005) were not listed in the Genome Aggregation Database. The lists of clinically-relevant variants and human gene knockouts in the TR Variome were also listed in this study, presenting the potential of the TR Variome being an invaluable resource for future disease gene identification studies. Additionally, a reference panel for genotype imputation was generated using TR-WGS data. Since this panel significantly increased imputation accuracy in both TR and neighboring populations, it will probably facilitate genome-wide association studies in these populations. In the second part of the study, the sequencing data of a total of 3,599 unrelated TR individuals were assessed for previously reported pathogenic (RP) variants and predicted pathogenic (PP) variants in Online Inheritance in Men (OMIM) genes associated with a pheno-type. Analyses revealed that no less than 70% of TR people have at least 1 RP variant, and all individuals possess at least one RP and/or PP variant in their genome. Moreover, 25% of individuals carried at least one RP variant in the newborn screening genes. Each individual in the study also had at least a 1 in 17 chance of carrying an RP variant in one of the 73 American College of Medical Genetics recommended actionable genes. MEFV, ABCA4, CYP21A2, PAH,and CFTR displayed the highest cumulative carrier frequencies (CF), consistent with the high prevalence of the phenotypes they are responsible for. By estimating the CF and genetic prevalence in 3,251 OMIM genes using RP and PP variants, this study presents the most comprehensive data so far demonstrating the landscape of genetic disease in the TR population.Item Open Access Contribution of mesenchymal stem cells in cell based therapies(Bilkent University, 2010) Tokcaer Keskin, Zeyneptem cell research evolved as a new hope and has gained tremendous interest in the last two decades to develop new strategies for many of debilitating diseases. Mesenchymal Stem Cells (MSCs) are multipotent cells capable of self-renewal and differentiating into multiple lineages such as osteocytes, adipocytes, chondrocytes, myoblasts, and hepatocytes. MSCs can migrate to the injured tissue and have immunomodulatory effects. Due to these features, MSCs have high therapeutic value in tissue engineering and regenerative medicine. In this thesis, our aim was to investigate the further contribution of the MSCs in different cellular therapies. We used two approaches to accomplish our aim. First, we investigated the possibility of obtaining functional cardiomyocytes from rat MSC within a shorter time period by determining the induction timing of cardiomyocyte differentiation of MSCs. Our data revealed that it is possible to get functional cardiomyocytes from in vitro MSC culture in a shorter time period than previously achieved. This reduction in time may provide emergency cases with access to cell-based therapies that may have previously been unavailable. In the second part of this thesis, we examined in vivo and in vitro effects of a telomerase antagonist, imetelstat (GRN163L) on MSCs. Telomerase activity is essential for the continued growth and survival of malignant cells, therefore inhibition of this activity presents an attractive target for anti-cancer therapy. MSCs also show telomerase activity in maintaining their self-renewal; therefore the effects of telomerase inhibitors on MSCs may be an issue of concern. Our results showed that inhibiting the telomerase activity does not interfere with the self-renewal and differentiation of MSCs under short term in vitro culture conditions.Item Open Access Development and preclinical characterization of meningococcal outer membrane vesicle vaccine(Bilkent University, 2024-03) Özsürekci, YaseminInvasive meningococcal disease (IMD) is caused by Neisseria meningitidis, with the main serogroups responsible for the disease being A, B, C, W, X and Y. To date, several vaccines targeting N.meningitidis have been developed albeit with a short-lived protection. Given that MenW and MenB are the most common causes of IMD in Europe, Turkey, and Middle East, we aimed to develop an outer membrane vesicle (OMV) based bivalent vaccine as the heterologous antigen source. Herein, we compared the immunogenicity, and breadth of serum bactericidal assays (SBA) based protective coverage of OMV vaccine to X serotype with existing commercial meningococcal conjugate and polysaccharide (PS) vaccines in a murine model. BALB/c mice were immunized with preclinical batches of the W+B OMV vaccine, either adjuvanted with Alum, CpG ODN or their combinations and compared with a MenACYW conjugate vaccine (NimenrixTM, Pfizer) and a MenB OMV-based vaccine (Bexsero®, GSK). The immune responses were assessed through ELISA and SBA. Antibody responses and SBA titers were significantly higher in the W+B OMV vaccine when adjuvanted with Alum or CpG ODN, as compared to the control groups. Moreover, the SBA titers were not only significantly higher than those achieved with available conjugated ACYW vaccines but also on par with the 4CMenB vaccines. In conclusion, the W+B OMV vaccine demonstrated the capacity to elicit robust antibody responses, surpassing or matching the levels induced by licensed meningococcal vaccines. Consequently, the W+B OMV vaccine could potentially serve as a viable alternative or supplement to existing meningococcal vaccines.Item Open Access Development of a specialized zebrafish xenotransplantation database and establishment of ALU-based tumor DNA quantification method in zebrafish: focus on models of overexpression and microenvironment(Bilkent University, 2020-09) Targen, SeniyeSuccessful xenotransplantation of human cancer cells into zebrafish host marked a new era in cancer research enabling high throughput in vivo screens. Zebrafish xenotransplantation literature continues to rapidly accumulate, and this necessitates the development of an interactive database for accommodating the collective data for fined-tune search, visualization and statistical representation purposes. Herein, I have introduced an interactive database, ZenoFishDb v1.1 (https://konulab.shinyapps.io/zenofishdb), housing manually curated details on molecularly-modified cell transplantations, PDXs, stem cell and cancer stem cell transplantation studies as well as transplantation studies bearing modified host details. The database projects collected data in a table format via various attributes and provides graphical representation of the curated details as well as statistical analyses yielding information on incorporated numbers and frequencies of selected attributes; hence can be used for reviews and designing new experiments. Zebrafish PDX studies are separately conceptualized and displayed through ZenoFishDb v1.1. Development of the ZenoFishDb v1.1 leads to a better understanding of tumor analysis methods such as assessment of proliferation and/or tumor growth in xenotransplantation studies and further marks the need for development of novel methods for precise quantification of tumor size. In the light of these findings, I have helped establish a novel qRT-PCRbased proliferation assessment method for xenografts in zebrafish, adapted from previous mouse xenotransplantation studies. Herein, the use and precision of ALU repeat-based quantification of transplanted liver cancer cells in genotyped zebrafish ache mutants and wildtype siblings was shown exemplifying microenvironment as an important factor for tumor growth. I further demonstrated the power of ALU repeatbased quantification in Mineralocorticoid Receptor (MR) overexpressing breast cancer cells (MCF7) injected to the transparent casper zebrafish as a case study. First, I demonstrated that MR expression and signaling was important in breast cancer biology and prognosis based on in silico TCGA and custom RNA sequencing as well as other in vitro and ex vivo assays. I further showed that results obtained from ALU repeatbased quantification of tumor growth in MR-overexpressing MCF7 cells paralleled fluorescent image-based intensity measurements while the former being relatively less time-consuming and more high-throughput. In this study, accurate quantification of MR overexpression in xenografts was also successfully performed by a cDNA-specific primer pair; and the rate of tumor growth based on image analysis, did not correlate with the amount of MR DNA in casper fish xenografts. However, MCF7 cells overexpressing MR exhibited lower cell viability in vitro although some of these effects were due to empty vector (EV) integration. Accordingly, tumor size in xenografts of naïve, EV- and MR-transfected MCF7 cells injected into pigmented AB larvae were quantified for ALU-repeats yet no significant difference was observed due to high within-group variability in vivo. Future studies are needed to assess the role of varying the volume and placement of injected cells along with the amount of MR gene transfected on tumor growth in vivo.