Ph.D. Theses
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Item From mutations to disease mechanism in Rett Syndrome, breast cancer, and congenital hypothyroidism(Thesis (Ph.D.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2008., 2008.) Barış, İbrahim.; Battaloğlu, Esra.Epidemiological studies provide the correlative data to understand the etiology of human inherited diseases and develop efficient genetic testing assays. Additionally, the accumulated data of genetic and epigenetic findings, expression profiling, and proteomics allows disease diagnosis, to understand the molecular mechanisms leading to the disease pathogenesis, and to develop efficient therapeutic approaches. In the framework of this thesis, we have investigated genetic and epigenetic changes and performed genotypephenotype correlations to unravel the molecular mechanisms that lead to three different diseases, Rett Syndrome, breast cancer, and congenital hypothyroidism. The genetic basis of Rett Syndrome (RTT) was investigated in a total of 71 RTT patients. A heterogeneous spectrum of disease-causing MECP2 mutations was identified in 68.2 per cent of a clinically well defined group of cases whereas in only 12.5 per cent of the patients referred for differential diagnosis suggesting that this gene does not represent a major cause of the disease among patients with Rett-like features. For the first time, we have identified gene duplications as causative mutations in female atypical RTT cases. Consistent with the animal models, our results support the possibility that duplication of MECP2 that leads to increased expression might underlie some cases of X-linked delayedonset neurobehavioral disorders including Rett Syndrome. Our results showed that exon rearrangements that could not be detected by standard techniques contribute to 19.3 per cent of these MECP2 mutations, and should be considered in especially RTT variants in order to determine the actual significance of the gene in the etiology of RTT. Genotype/phenotype correlation was performed based on comparison of severity score of patients with the type and location of the mutation and the XCI pattern. The results did not reveal a statistically significant correlation, but, the patients with exon deletions were found to be more severely affected than patients with all other types of mutations and patients with exon duplications to present with severe eye contact problems. Additionally, we have developed and validate a novel multiplexed amplification refractory mutation system (ARMS) assay for identification of seven common mutations that accounts for almost 65 per cent of all MECP2 gene mutations. The validation studies revealed that our novel assay is an efficient, reliable, and cost-effective screen for molecular genetic testing of patients with RTT. Furthermore, we tested the effect of DNA concentration on reliablity and reproducibility of SYBR green dye-based Real Time PCR analysis to detect the MECP2 exon rearrangments. The results suggested that Real Time PCR analysis is reliable for determination of the exon copy number if the DNA amount is in the range of 1-50 ng. To our knowledge, there are no known reports investigating the role of methylation of hHR23A and hHR23B genes in the tumor tissues. We have characterized the 5' flanking region of the hHR23A and hHR23B genes using web-based analysis and investigated the involvement of methylation status of putative promoter region of hHR23A and hHR23B genes in breast carcinogenesis. The observations of the hypermethylation of hHR23A gene and the presence of methylated conserved motifs and transcription binding sites in hHR23B gene among the analyzed tumor tissues suggested the involvement of methylation of hHR23 genes in the breast carcinogenesis. Investigation of epigenetic changes in tumor samples of breast cancer patients was a pioneering work since available literature implicates its presence only in cell lines. Since our CH patient was the first case with Bamforth Syndrome and suffered the plasma cholinesterase deficiency, the genetic mechanisms leading to congenital hypothyroidism and prolonged paralysis after mivacurium were investigated. In contrast to other reported two patients with TTF2 gene mutation, the presence of thyroid tissue in our patient suggested further phenotypic heterogeneity associated with human TTF-2 mutations. The functional study with a collaborative work also helped to understand the genetic mechanisms and provided original evidence that implicated differential effects of TTF-2 mutations on downstream target genes required for normal human thyroid organogenesis.Item SIK2 expression in retinal cells and its possible involvement along with PKA in FGF9 signal transduction(Thesis (Ph.D.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2006., 2006.) Özmen, Yeşim.; Buğra, Kuyaş.FGF signal transduction pathway activates a cascade of kinase and phosphatase dependent phosphorylation and dephosphorylation events. The pathway is subject to tight control mechanisms through negative feedback exerted by the the activated effector molecules, action of kinases and phosphatases. Besides the cellular context and interplay between the regulators, integration of heterologous signaling is also critical in shaping the cellular responses. In this study, we have shown FGF9 receptors FGFR2, FGFR3 and a putative modulator SIK2 is widely expressed in neuronal cells and Muller glia, FGF9 is detected in neuronal cell layers but not in Muller cells. Our data, when compared with the primary Muller cells, indicate that MIO-M1 cells are equipped with the components of signal transduction. Using these cells as model system we had shown the activation of FGF9 pathway and that the pathway is modulated by PKA activity. We had also shown that SIK2 is modulated both in response to PKA and FGF9 by phosphorylation and nuclear translocation. Regulation of SIK2 translocation correlates with levels of ERK phosphorylation. When both stimuli are present SIK2 tends to act in accordance with FGF9 but not PKA.Item Omics comparison of two common bean genotypes and study of pvSPS4 knockdown in composite plants under saline conditions(Thesis (M.S.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 2021., 2021.) Niron, Harun.; Türet, Müge.Soil salinity is an abiotic stress factor that limits global agricultural output. Common bean is an important protein source in developing countries, however sensitive to salinity. To understand the underlying mechanism of salt stress responses, transcrip tomics, metabolomics, and ionomics analyses were performed on both salt-tolerant and susceptible common bean genotypes under saline conditions. Transcriptomics revealed enhanced photosynthesis together with active carbon and amino acid metabolism in the tolerant genotype. Metabolomics revealed increased carbohydrate and amino acid metabolism in the tolerant genotype. Ion content comparison indicated that the tol erant genotype blocked the accumulation of Na+ in the leaves. The results of this omics study have demonstrated the differences in contrasting genotypes and provided information on the novel mechanisms salt tolerance to pinpoint genes with high po tential for functional analyses. Stress-related carbohydrate metabolism is a dynamic network and disruptions in this system can have negative effects on tolerance. Su crose phosphate synthase (SPS) enzymes operate in the sucrose synthesis pathway and have significant roles in sugar metabolism. This study has focused on the function of SPS homolog, pvSPS4, in the roots of salt-tolerant common bean under salt stress. Composite common bean plants with pvSPS4 knockdown roots exhibited sensitivity to salinity. Disturbed root carbohydrate and ion balance resulted in a reduction in photosynthesis together with osmoregulation and antioxidant capability. These results indicate that pvSPS4 is an important gene for carbohydrate balance regulation in the salt-stress response in the common bean root tissues.Item Neuropeptidome and gpcrome of stick insect: Specific structural and functional aspects of allatostatin receptor(Thesis (Ph.D.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 2018., 2018.) Duan, Burçin.; İyison, Necla Birgül.The arthropods constitute about three-quarters of the world’s animal species. The molecules that regulate many physiological events, from the feeding to develop ment, from locomotion to the social behavior, from the reproductive behavior to the intestinal motility, are the neuropeptides and their cognate receptors. Carausius mo rosus, also known as laboratory rodent, is a species that is studied on locomotion and can easily reproduce via parthenogenesis. On the other hand, among the arthropod neuropeptides, those that regulate Juvenile Hormone, namely allatostatin (AST), is specially important. The subject of this thesis is understanding the interaction be tween allatostatin C receptor (AlstR-C) of C. morosus and AST-C, as well as finding the other neuropeptides and GPCRs. At the beginning of our work, it was found that amino acids were conserved in the ligand binding pocket of AlstR-C and these amino acids were mutated and utilized in atomic force microscopy studies. This IXTPP mo tif, located in the third extracellular loop, together with the N-terminus were found to be important for this interaction. RNA sequencing analysis was then performed to access other AlstR types and AST peptides. As a result,at least 23 different neu ropeptide transcripts and 43 GPCR transcripts were obtained from adult C. morosus. Tissue expression profiles of these GPCRs were found. This information will facilitate future neuropeptide-GPCR studies. In this study, about the Alstr-AST system being homologous to the somatostatin receptor of human, we have also asked whether this neuropeptide may affect the proliferation of cancer cells. However, both XTT and in vivo xenograft experiments showed that the peptide or active receptor does not affect tumor growth.Item Molecular and cellular development of cortical projection neurons: specification, diversity, and directed differentiation from endogenous progenitors for functional circuit repair(Thesis (Ph.D.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2016., 2016.) Özkan, Abdulkadir.; Battaloğlu, Esra.; Macklis, Jeffery D..Responsible for perception, integration of sensory information, cognitive function, motor control, and consciousness, the complex, yet highly organized, six-layered mammalian neocortex contains distinct classes of neurons. Specific subtypes of cortical projection neurons are selectively vulnerable in distinct neurodegenerative, developmental, and acquired diseases of the central nervous system (CNS), resulting in irreversible functional deficits. Evidence for the existence of progenitors in restricted regions of the adult brain, and integration of new neurons into preexisting neural circuitry, support the feasibility of cellular repair in the CNS. However, functional repair of diseased or injured neuronal circuitry requires detailed understanding of molecular controls over development of neuronal lineages, and manipulation of these controls in progenitors to direct the differentiation of functional neurons with appropriate identity, maturity and circuit connectivity. In this study, I target endogenous cortical progenitors present in postnatal and adult brain to direct their differentiation into corticofugal projection neurons. Application of a select combination of central and complementary transcriptional controls, Ngn2, VP16:Olig2 and Fezf2, in cultured cortical Sox6+/NG2+ progenitors directs acquisition of cardinal morphological, molecular, and electrophysiological features of corticofugal projection neurons. These findings demonstrate the feasibility of achieving subtype-specific differentiation of cortical projection neurons from a widely distributed in vivo neocortical progenitor population. Further, in the framework of this thesis, I describe the ongoing effort to identify key molecular controls over development, diversity and connectivity of corticostriatal projection neurons, which would serve as a solid step toward achieving a holistic view of the establishment of corticostriatal circuitry and its potential dysgenesis in disease.Item SIK2 functions as a novel tumor suppressor in the breast tumorigenesis(Thesis (Ph.D.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2015., 2015.) Zohrap, Neslihan.; Buğra, Kuyaş.Breast cancer has highly malignant phenotype and is the leading cause of cancer death in women. The heterogenous character of the disease points to the importance of novel target identification for the development of effective therapies. Perturbation in receptor tyrosine kinase (RTK) pathway elements are frequently implicated in cancer. Our group has shown that SIK2 functions downstream of FGFR signaling, and control proliferation and survival. Given the importance of strict control of RTK pathways in cancer, in this study, we aimed to investigate the potential role of SIK2 in the development of tumorigenicity. In this context, screening of a cDNA array indicated downregulation of SIK2 transcript levels in breast cancer. A query into Oncomine database validated frequent reduction of SIK2 expression and loss of copy number in dataset covering a large breast cancer patient cohort. Immunohistochemical studies localized SIK2 in ductal epithelia of breast tissue and showed that SIK2 levels decline in all triple negative breast cancer (TNBC) cases and in half of the hormone positive tumors. These studies also indicated that inverse correlation exists between SIK2 and Ki67 indicating a negative effect of SIK2 on mitotic potential. Reduced SIK2 expression in breast tumor cell lines was also evident. Modulation of SIK2 expression in MDA-MB-231 and MCF12A breast lines suggested that SIK2 negatively regulate proliferation and survival by preventing ERK and Akt activation. In this context, its kinase activity appears to be required. In vitro studies also indicate that SIK2 may also be involved in the regulation of cellular adhesion and invasion. Xenografting experiments provided strong evidence that SIK2 hampers tumor growth through downregulation of proliferation and survival. In the light of these findings, we propose SIK2 as a novel tumor suppressor, loss of its expression/activity promote breast tumorigenesis.Item Regulation of human BFK(Thesis (Ph.D.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2014., 2014.) Uğurlu, Serkan.; Özören, Nesrin.Bcl-2 protein family members are critical regulators for apoptosis. Reduced levels of apoptotic Bcl-2 family members are detected in gastrointestinal cancers which are responsible for a considerable part of the deaths from cancer. BFK (Bcl-2 Family Kin) is a novel pro-apoptotic Bcl-2 family member specifically expressed in the human gastrointestinal tract. BFK has the characteristic BH3 domain, which was shown to be essential for the apoptosis inducing activity of pro-apoptotic Bcl-2 family members. In the colon four alternatively spliced isoforms were identified. Human and mouse BFK genes share 70% homology at the DNA level and 68.7% homology at the aminoacid levels. Interestingly, human and mouse BFK genes show distinct expression patterns. To explain these differences, we performed gene evolution analyses on the promoter region as well as coding region of these genes. We found that the human BFK promoter experienced positive selective pressure and acquired a distinct set of repetetive elements and transcription factor binding sites. In this study, we identified several novel transcription factor candidates which may have roles in the transcriptional regulation of human BFK. As transcription factors, PARbZIP family members (especially TEF and NFIL3) regulate BFK upon binding to its promoter region. NFIL3 supresses TEF induced BFK transcription in HCT116 cells. We also studied hormonal regulation of human BFK. Tamoxifen, as a mixed agonist/antagonist of estrogen, upregulates human BFK levels in SW707 cells. We hope this study contributes to a better understanding of Bcl-2 family.Item Characterization of thermophilic gene expression enzymes(Thesis (Ph.D.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2010., 2010.) Çağlayan, Melike.; Bilgin, Neşe.The DNA polymerase I genes of seven newly identified Geobacillus species within the family Bacillaceae, were cloned, sequenced and overexpressed in Escherichia coli. The polA gene of these species encodes DNA polymerase I of 878 amino acid residue protein with a predicted molecular weight of 99.3 kDa. Similarity analyses suggested that DNA polymerases belong to family A polymerases and lack 3'-5' exonuclease activity. The complete coding sequences of the genes were submitted into the GenBank. The recombinant (His)6-tagged DNA polymerases were purified by Ni2+-affinity chromatography and the homogeneous proteins were obtained after TEV protease digestion. DNA polymerases from Geobacillus anatolicus (Gana DNApolI) and Geobacillus kaue strain NB (Gkaue DNApolI) were further characterized in vitro and optimum conditions with respect to temperature, pH, monovalent and divalent ions were determined. Geobacillus DNA polymerase I fragment (GF DNApolI) was cloned and purified after homology modeling using Bacillus DNA polymerase I fragment (BF) as the model protein structure. The accuracy of GF DNApolI was measured by two M13 based fidelity assays which score errors produced during in vitro DNA synthesis of the lacZα complementation gene in M13mp2 DNA at 37°C, 50°C and 72°C. Base substitution errors increase three-fold when temperature is raised from 37°C to 72°C. DNA sequencing of the phage mutants showed that some of the base substitutions are more temperature sensitive than others. The most common base substitution error is the misincorporation of dGMP opposite to template G. Single nucleotide incorporations for both correct and for incorrect nucleotides were also studied under single-turnover conditions at 22°C, 37°C and 50°C. For both correct and incorrect dNTP insertions, the rate of polymerization, kpol, increased (seven- and four-fold, respectively) when temperature is raised from 22°C to 50°C, whereas only a slight change in Kd was observed. As a result, kinetic efficiency of the enzyme (kpol/Kd) shows five-fold increase over this temperature range.Item Amyotrphic lateral sclerosis in Turkey : Studies on Familial and sporadic ALS using high-throughput genomic technologies(Thesis (Ph.D.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2010., 2010.) Özoğuz, Aslıhan.; Başak, A. Nazlı.Amyotrophic Lateral Sclerosis (ALS) is a late-onset neurodegenerative disease, characterized by death of motor neurons in cortex, brainstem and spinal cord. It is a multifactorial disease with interacting pathogenic mechanisms. Most incidences are sporadic (SALS), while 10 per cent of cases have a family history (FALS). The genetics of ALS is complex; eight genes and six loci with autosomal dominant (AD), autosomal recessive and X-linked patterns of inheritance have been identified thus far. In the framework of this study, 198 Turkish ALS cases were investigated for possible mutations in the SOD1 gene. Five FALS cases were shown to carry disease-causing SOD1 mutations, while six were carriers of a rare polymorphism. In the next step, AD, nonconsanguineous FALS and juvenile cases were analyzed for the TDP-43, FUS and ANG genes via DNA sequencing. One homozygous D90A case, represented as recessive, was investigated by haplotype analysis and was compared to 21 Scandinavian ALS cases in search of a common ancestry. Additionally, 15 FALS and 13 juvenile cases, who were negative for the tested genes, were analyzed by whole genome genotyping for identification of new ALS genes. While no significant regions were obtained, a recessive family was preselected for the identification of homozygosity regions. Five candidate genes located within homozosity regions were examined in one of the family member; no mutations were identified. The same individual was also assessed by whole exome resequencing. Furthermore, this study also contributed to a collaborative genome-wide association study in SALS, where 14 month-survival advantage was shown for homozygous CC allele at rs1541160 SNP in the gene coding KIFAP3. This is the most comprehensive study performed in Turkey on the molecular genetics of ALS. The high-throughput methodologies used and the findings presented in this thesis are expected to shed light to the complex pathogenesis of amyotrophic lateral sclerosis.