Ph.D. Theses

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Characterization of GPR139 and investigating its possible role in brain development
(Thesis (Ph.D.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 2022., 2022) Aslan, Tolga.; İyison, Necla Birgül.
G protein coupled receptors are a large family of cell surface receptors and are involved in a plethora of physiological functions from vision to various neuronal func tions. They have been a major target in the pharmaceutical industry and more than one third of currently available drugs target a GPCR. Although extensively studied, there are still 140 orphan GPCRs. GPR139 is an orphan GPCR in the rhodopsin fam ily and is primarily expressed in medial habenula. In addition to its cognate ligand, its function is still not known. At the time of the initiation of our study, the literature only suggested a possible role of GPR139 in neurological disorders such as ADHD and Alzheimer’s disease. Objective of this study is to investigate possible the role(s) of GPR139 in brain development via employing a Gpr139 knock-out mice model, and via in vivo ablation of Gpr139 in adult mice, and subjecting the animals to a set of behavioral experiments to measure the impact of deletion and ablation, respectively. In addition to animal experiments, various bioinformatic analyses were also performed to further characterize Gpr139. Our findings showed that Gpr139 is expressed differen tially in the brain tissue during embryonic development and its expression is most likely controlled by histone modifications and not DNA methylation. We also showed that ablation of GPR139 does not have any significant effects on spatial learning, locomotor activity, recognition memory and anxiety.
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.
From genome scan to disease gene identification
(Thesis (Ph.D.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2008., 2008.) Uğur, Sibel Aylin.; Tolun, Aslı.
In the framework of this study, genome wide linkage scans of the following five inherited disorders were evaluated with parametric programs and suggestive loci were subsequently investigated with fine-mapping studies and candidate gene approach. Split-Hand/Foot Malformation (SHFM) affects the central rays of the autopod. We identified a novel SHFM locus at 12q13.11-q13 and a homozygous WNT10b mutation (p.R332W) in all affected individuals plus in an asymptomatic female. We propose that either a second locus contributes to the manifestation of SHFM phenotype or a suppressor locus prevented trait manifestation in the non-penetrant female. This is the first reported WNT10b mutation on the pathogenesis of limb development and recessive mutation in SHFM. Hypomyelination and congenital cataract is a recessive white matter disorder caused by mutations in gene DRCTNNB1A. Here we report a large intragenic deletion that does not lead to congenital cataract in all of the patients in an afflicted family. A novel form of recessive cone rod dystrophy was mapped to chromosome 17p13.2-p13.1, and the disease gene was identified as GUCY2D encoding the retinal guanylyl cyclase gene. The mutation (p.I949T) resided in the catalytic domain of the protein where other mutations had previously been associated with Leber congenital amaurosis, a common cause of childhood blindness. The milder phenotype observed in our patients implicate that either the mutation does not disturb the catalytic activity completely or modifier locus/loci interfere with the phenotype. Lastly, a recessive form of mental retardation and a dominant arthrogryposis syndrome were mapped to chromosomes 7q21.3-q31.1 and 13q31.3-q32.1, respectively.
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.
Genetic and molecular analyses of Turkish patients with pelizaeus - merzbacher disease
(Thesis (Ph.D.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2007., 2007.) Bilir, Birdal.; Battaloğlu, Esra.
Pelizaeus-Merzbacher disease (PMD) is a rare inherited leukodystrophy with Xlinked recessive segregation. About 80 per cent of patients with PMD have been associated with mutations of the PLP1 gene on Xq21.3-Xq22 which encodes two proteins, PLP1 and DM20, expressed abundantly in oligodendrocytes. Mutations of GJA12 gene on 1q41-42 are responsible for some of the PMD cases with autosomal recessive inheritance. In the framework of this study, the molecular basis of PMD was investigated in a cohort of 21 Turkish families with PMD. In total, pathogenic mutations were identified in 57 per cent of the families, 19 per cent of which were due to PLP1 duplications, and nine and 29 per cent were due to mutations in the PLP1 and GJA12 genes, respectively. The distribution of the mutations identified in our cohort of patients was different from those reported in the literature, which may result due to the high frequency of consanguinity and autosomal recessive cases in our population. Absence of mutations in PLP1 or GJA12 genes in 43 per cent of the cases analyzed suggests presence of further genetic heterogeneity in PMD. In vitro immunocytochemical analyses of two PLP1 mutations identified in our cohort revealed that accumulation of mutant proteins in the endoplasmic reticulum, leading to UPR activation and subsequent apoptosis were observed for the mutant proteins. However, one of the mutations showed a different pattern of localization for DM20 isoform. Since patients present similar clinical features, the results implicate that PLP1 and DM20 may have different roles in myelin.|Keywords: Pelizaeus-Merzbacher Disease, Dysmyelination, Proteolipid Protein 1 (PLP1) Gene, Gap Junction Protein Alpha12 (GJA12) Gene, Unfolded Protein Response (UPR)
Effects of extremely low frequency electromagnetic fields on caspase activities
(Thesis (Ph.D.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2006., 2006.) Ozansoy, Mehmet.; Başak, A. Nazlı.; Denizhan, Yağmur.
All life on Earth is bathed in a sea of natural low-frequency electromagnetic fields from conception to death. Since the World Health Organization (WHO) launched its international electromagnetic fields (EMF) project in 1996, it has conducted international reviews of the evidence on whether exposure to static and extremely low frequency (ELF) fields might be harmful to health. ELF fields for WHO’s EMF project are defined as those having frequencies above zero and below 300 Hz. In the framework of this study, the activation of seven different caspases will be investigated systematically, when extremely low frequency electromagnetic fields, which are thought to be an environmental hazard according to WHO, are applied to the HEK 293 cell line. The selected frequency will be 50 Hz, which is the power transmission line frequency in most parts of the world. Two different magnetic field strengths will be applied to HEK 293 cells, and two different exposure durations will be chosen. Caspase activity levels are to be measured at different time points after exposure. The common pattern seen in all of them was the oscillation of enzyme activities from the beginning. At 100 μT, caspases gave four peaks at four, eight, 16 and 34-hour incubation periods. This oscillatory behavior can also be seen when 25 μT magnetic field was applied, but the behaviors of the enzymes were different in a certain extent. The location and the number of the peaks at 25 μT exposure were quite variable, but the activity periods of all caspases seemed to be shorter than those exposed to 100 μT. The data presented here indicate that when ELF-EMF is applied to the HEK 293 cells, all seven caspases investigated are found to be activated, but this activation shows an oscillatory pattern, and in the long run it seems to be damped by some intracellular mechanisms.
Investigation of molecular signals regulating cell proliferation and neurogenesis in the intact and injured Zebrafish (Danio rerio) olfactory epithelium
(Thesis (Ph.D.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 2021., 2021.) Demirler, Mehmet Can.; Fuss, Stefan H.
The zebrafish olfactory epithelium (OE) can recover from the near total loss of olfactory sensory neurons (OSNs) within only a week when the OE is damaged by external causes. This remarkable neurogenic activity depends on the differential activation of horizontal basal cell (HBC) progenitors, which are quiescent in the intact tissue. Under physiological conditions, an independent pool of mitotically active progenitors, the globose basal cells (GBCs), constitutively generate new neurons to replace OSNs that reach the end of their natural life span. Unique to the zebrafish OE, the two progenitor types are spatially segregated, and HBC and GBC activity can easily be discriminated by analyzing the epithelial position of mitotic activity under different tissue conditions. However, detailed knowledge about the diversity of non-neuronal cells and the molecular cues that regulate the rate of maintenance neurogenesis or trigger OE regeneration, is largely missing. This study shows that HBCs, GBCs and sustentacular glial cells (SCs) express Sox2, that they segregate within distinct layers of the basal OE, and that they are selectively sensitive to purine compounds. HBCs occupy the basal OE and respond strongly to ATP and 2- MeSATP, while GBCs and SCs occupy suprabasal strata and show high and low ATP sensitivity, respectively. ATP stimulates GBC activity but does not affect HBC proliferation, indicating that purinergic signaling has a selective function in regulating maintenance neurogenesis. In contrast, exposure to the cytokine IL-6 results in HBC expansion and subsequent OSN generation. Detailed analysis of the tissue dynamics in the intact OE revealed that HBCs are more active than anticipated but only rarely, if at all, contribute to OSN or SC neurogenesis. A preliminary model of maintenance neurogenesis could be developed, which suggests the existence of a novel SC progenitor within the neurogenic niche of the intact OE.
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.
Cellular and molecular analysis of regenerative neurogenesis in the zebrafish (Danio rerio) olfactory epithelium
(Thesis (Ph.D.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 2021., 2021.) Kocagöz, Yiğit.; Fuss, Stefan H.
The olfactory epithelium (OE) has a high regenerative capacity that ensures the lifelong replacement of olfactory sensory neurons (OSNs). Different modes of neurogen esis originate from the selective activity of two distinct stem cell populations in the OE. In mammals, maintenance and regenerative neurogenesis are attributed to the selective activity of globose basal (GBC) and horizontal basal cells (HBC), respectively. How ever, the identity and function of similar progenitor cells in the OE of non-mammalian vertebrates remain elusive. This study demonstrates that a dual progenitor cell sys tem also exists in the zebrafish OE but with distinctive features when compared to mammalian models. HBC-like cells reside within the basal layer along the sensory OE, while GBC-like cells are confined to two isolated regions flanking the sensory OE. The positional and functional differences between these progenitor populations establish a balance between constitutive and regenerative modes of neurogenesis in the OE. Here, OE regeneration in response to global and OSN-specific injuries is examined in detail. The OE launches a rapid regenerative response upon chemical lesion, which results in HBC-like cell activation and OSN regeneration within the sensory OE. However, in response to selective OSN ablation, accelerated OSN regeneration is characteristically seen around the OE margins similar to the neurogenic activity in the intact OE. This crucial finding suggests that different injury conditions may affect HBC- and GBC-like cells selectively. RNA-sequencing analysis of the regenerating OE suggests that the diffusible signaling factor HB-EGF may have a critical role at the onset of regenerative neurogenesis. Exogenous HB-EGF administration induces OSN regeneration, while pharmacological inhibition of HB-EGF activity suppresses the regenerative response.
The effect of nNav1.5 gene expression in breast cancer metastasis
(Thesis (Ph.D.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2006., 2006.) Yamacı, Rezan Fahrioğlu.; Battaloğlu, Esra.
Breast cancer is the most common cancer among women. Its metastasis is lethal that can not be detected at microscopic levels using current techniques. Thus, there is need for reliable early metastasis markers. Neonatal Nav1.5 (nNav1.5) is a voltage-gated sodium channel (VGSC) and one of the potential early markers for breast cancer metastasis. In this study, we determined that nNav1.5 expression was in parallel with breast cancer metastasis and estrogen receptor (ER) expression in a group of patients. To provide data for future drug development, we analyzed the expression pattern of nNav1.5 protein in normal human tissues. The protein was not expressed in skeletal and heart muscle, brain, small intestine, colon, stomach, esophagus, urinary bladder and prostate but expressed in breast at basal level. We also investigated the distribution of VGSC in these non-excitable human tissues. Except urinary bladder, VGSC protein was determined mostly in secretory cells in all of the tissues above that may indicate a role in secretion. Upon identification of VGSC upregulation in tumor regions of different cancers including, colon, stomach, urinary bladder, kidney and lung it is possible that VGSC expression could be a widespread mechanism in cancer metastasis. Within the scope of this thesis, we also investigated the possible role of estrogen on nNav1.5 upregulation and activity in metastatic breast cancer. Estrogen had no effect on proliferation of cells but slightly increased motility through nNav1.5 in highly metastatic cells that express the protein. In weakly metastatic cells that do not posses nNav1.5, estrogen decreased motility slightly. The quantity of nNav1.5 protein was not affected by estrogen but functionally available form on the plasma membrane was increased only in the highly metastatic cells. These results may suggest that estrogen increases motility capacity of breast cancer cells by regulating nNav1.5 activity.
Investigation of novel recessive causative genes and gene / allele frequency for CMT disease in Turkey
(Thesis (Ph.D.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 2021., 2021.) Candayan, Ayşe.; Battaloğlu, Esra.
Inherited peripheral neuropathies are a group of genetic disorders of the periph eral nervous system. The most common type is called Charcot-Marie-Tooth (CMT) disease that constitutes an interesting research focus due to its clinical and genetic heterogeneity. Mutations in more than 90 genes and loci are associated with CMT that presents with autosomal dominant (AD), autosomal recessive (AR), X-linked, or mitochondrial inheritance. Despite the advances in genetic testing, approximately 35% of all CMT cases worldwide remain without a molecular diagnosis. The diagnostic rate is even lower for ARCMT due to the presence of many individually rare genes. This diagnostic gap points to the presence of yet unidentified causative genes, as well as po tential non-Mendelian features of the disease. In order to identify novel genes/alleles causing ARCMT and determine the frequency for known genes in Turkey, we have analyzed 56 consanguineous families diagnosed with CMT who present with early on set polyneuropathy with additional symptoms. Through the screening of patients for GDAP1 mutations, and subsequent whole-exome sequencing and homozygosity map ping, we have identified 22 recurrent and 13 novel alleles in known CMT genes achieving a potential diagnosis rate of 62,5%. Moreover, we identified FXN as a candidate gene for a novel disease in the spectrum between CMT and Friedreich’s ataxia, ATP8B3 for ARCMT2, and SEPT11 for AR-cerebellar ataxia with axonal neuropathy. This study paints the genetic landscape of the Turkish ARCMT population, reports candidate genes that might enlighten new disease mechanisms, and can serve as a reference for diagnosis strategies specific to populations with similar genetic backgrounds.
Divergent functions of NLRP7 in embryogenesis, inflammation and oncogenesis
(Thesis (Ph.D.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 2019., 2019.) Garipcan, Aybüke.; Özören, Nesrin.
NLRP7 is a novel protein about which we have limited information. To date, mutations in NLRP7 gene have been associated with recurrent Complete Hydatidiform Mole (RCHM) and NLRP7 is accepted to be the first causative gene for RHM. CHM is a gestational disease characterized by hyper trophoblast proliferation with no embryo formation. Furthermore, NLRP7 expression was found to be elevated in testicular seminoma, endometrium cancer and embryonal carcinoma. Yet, the possible mechanisms of action of NLRP7 in these biological conditions or pathways have not been enlightened. In this thesis, we generated patient derived induced Pluripotent Stem Cells (iPSC) to address the role of NLRP7 in HM. We revealed that inadequate NLRP7 levels expedited the di↵erentiation of iPSCs towards the trophoblasts through BMP4. Recovery of NLRP7 expression or BMP pathway inhibition decelerated excessive di↵erentiation of patient iPSCs to trophoblasts. Also, as NLRP7 is an NOD Like Receptor Family member, it is expected to play a role in innate immunity. We have found that infection of human monocytic cells with Pseudomonas aeruginosa activated the NLRP7 inflammasome followed by increased IL-1! secretion. Clearly, stable overexpression of NLRP7 is correlated with increased secretion of pro-inflammatory cytokines such as; TNF-alpha, IL-6, i-309. In addition, to elucidate the possible proto-oncogenic role of NLRP7 in tumorogenesis, we performed xenograft experiments in vivo. We found that stable expression of NLRP7 in the endometrial cancer cell line (Hec1a) resulted in increased tumor growth. Furthermore, potential interaction partners of NLRP7 were identified after co-immunoprecipitation followed by mass spectrometry. Our results shed further light to the overlapping and diverging molecular pathways regulated by NLRP7 in embryogenesis, inflammation and oncogenesis.
Functional and structural insights into a novel insect G protein-coupled receptor, allatostatin receptor type C of pine processionary moth
(Thesis (Ph.D.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 2020., 2020.) Shahraki, Aida.; İyison, Necla Birgül.
Insect neuropeptides regulate di erent aspects of insect physiology. They exert their function via binding to their cognate receptor belonging to Class A G protein coupled receptors (GPCRs). Neuropeptide receptors are potential targets for nextgeneration pesticides. Allatostatin (AST) neuropeptides regulate the development of insects through the inhibition of juvenile hormone (JH) secretion. Here, Allatostatin receptor type C (AstR-C) of pine processionary moth, a pest in Mediterranean countries, was extracted from the whole genome sequencing (WGS) data. The receptor was cloned and characterized combining via di erent approaches. Using resonance energy transfer (RET)-based techniques, kinetics of G protein coupling and -arrestin recruitment were investigated. Homology modeling, docking and molecular dynamics (MD) simulation approaches were conducted to predict the orthosteric pocket of the receptor which was validated by in silico and in vitro methods. The binding pocket was subjected to virtual screening studies to nd agonists. As a result, it was found that binding of the native ligand at sab-nanomolar and nanomolar ranges to the receptor induces Gi/o protein coupling and -arrestin recruitment, respectively. Kinetics studies revealed that a brief stimulation of the receptor at nanomolar range is enough to obtain a long-lasting response. The accuracy of the predicted orthosteric pocket was validated via G protein activation assay. Q2716:55 (Ballesteros-Weinstein numbering) was found to be critical for G protein-dependent activation of AstR-C. Virtual screening studies resulted in obtaining a small molecule capable of activating the receptor.
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.
Functional enrichment methodology for analyzing omics data to study the aetiology of rare diseases
(Thesis (Ph.D.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2018., 2018.) Saygı, Ceren.; Özören, Nesrin.; Sezerman, Uğur.
Rare diseases (RDs) are a large and diverse group of disorders and defined by low prevalence, in other words, it is any disease that affects a small percentage of the population. According to OMIM and Orphanet, ~7000 different RDs have been estimated, but the number of phenotypes that remain to be defined could be considerably higher. The difficulty in obtaining the correct diagnosis is the most dramatic problem to be solved for the patients, about 30% still lack a diagnostic definition. The patients living with rare diseases visit an average of 7.3 physicians before receiving an accurate diagnosis and the mean length of time from symptom onset to accurate diagnosis is 4.8 years. Late diagnoses delay specific treatments and may have severe and life-threatening consequences. Molecular diagnosis is the most prominent way to facilitate earlier and accurate diagnosis, and hence an effective treatment for rare undiagnosed cases. In this dissertation project, a novel bioinformatics workflow is constructed for whole-exome/genome sequencing data analysis, variant prioritization and pathogenicity prediction from a cascade of different tools shading light into different aspects of the diagnostic process. The pathogenicity mechanisms of mutations are elucidated via molecular dynamics (MD) simulations. The newly developed pipeline is planned to be used for diagnosis of undiagnosed patients with a suspected genetic disorder, where other testing modalities have been inconclusive or noninformative. The workflow was tested on several undiagnosed clinical cases with their family members and achieved high success rates by identifying the causative variant. For two of these families, the pathogenicity mechanisms of mutations were described via MD simulations, and these findings have been submitted to two different SCI journals and passed the editorial approval. The diagnosis of one of these families was Periventricular Nodular Heterotopia, while the other was Nail Dysplasia-10. Both of the diseases are extremely rare that is seen in one in a million cases. In conclusion, we developed a unique workflow for molecular diagnosis of rare undiagnosed diseases. Our pipeline contributes to the already existing knowledge through the combination of population frequency, pathogenicity prediction tools, gene intolerance scores, and MD simulations for the first time.
The molecular landscape of ALS in Turkey: A multifaceted approach to the complex genetics of ALS
(Thesis (Ph.D.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2018., 2018.) İskender, Ceren.; Çağlayan, S. Hande.; Başak, A. Nazlı.
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease (NDD), characterized by degeneration of both upper and lower motor neurons leading to muscle wasting. With an incidence of two and a prevalence of four in 100.000/year, it is the third most common NDD after Alzheimer’s and Parkinson’s diseases. The last decade has proven that the ‘condition’ called ALS is both clinically and genetically heterogenous, and that the genetic component in 90% of the cases that are considered as sporadic, might be stronger than expected. In this thesis, we investigated the complex genetics of ALS in the ethnically heterogeneous yet inbred Turkish population. Familial ALS account for 22% of our cases (159/722) and 563 cases were classified as sporadic (78%). Consanguinity is calculated as 25% among familial and 17% in sporadic cases. Conventional screening of the most common ALS genes (C9orf72, SOD1, TARDBP and FUS) in patients with a family history of disease, explained the disease cause in only 33%, pointing towards marked locus heterogeneity within the population. C9orf72 hexanucleotide repeat expansion was further detected in 3% of ‘apparently sporadic’ patients. Application of whole exome sequencing (WES) in dominant and recessive Turkish pedigrees presenting with ALS or non-ALS motor neuron diseases (MNDs), revealed distinct rare or novel mutations in 20 out of 39 families and enabled differential diagnosis in cases with atypical ALS features. Common current themes in ALS, like oligogenic inheritance and possible genetic modifiers are addressed in families with incomplete penetrance and in C9orf72 expansion carriers. Oligogenic inheritance of known ALS genes was not prominent in the Turkish C9orf72-positive cases. Hypermethylation in the promoter region of C9orf72 was confirmed in Turkish patients, however it did not seem to modify age of onset. Finally, analyses of whole genome sequencing data of 625 Turkish ALS patients and 152 healthy controls in the framework of Project MinE showed population-specific aspects and once more substantiated the concept of low penetrance of rare ALS genes among sporadic patients.
Identification on new loci, genes, and mutations responsible for hereditary spastic paraplegia
(Thesis (Ph.D.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2018., 2018.) Özeş, Burçak.; Battaloğlu, Esra.
Hereditary Spastic Paraplegia (HSP), characterized by lower limb spasticity and progressive weakness, is a group of inherited neurodegenerative disorders. 47 of the 63 genes responsible for HSP, have been associated with autosomal recessive (AR) HSP. In this study, whole exome sequencing analysis was performed for one patient from each 27 ARHSP families to identify causative genes. When WES data was not informative, homozygosity mapping was performed by using WES and/or whole genome SNP genotyping data. After segregation analyses of candidate variants, the causative variants were identified in fifteen families. SPG11 gene was causative in four families. Single families had mutations in CYP7B1 (SPG5A), SPG7, SPG15, and ALS2 genes. SACS and CYP27A1 genes that are associated with Charlevoix-Saguenay Spastic Ataxia and CTX, respectively were determined as the causative genes in two families providing differential diagnosis to these families. KIF1C (SPG58) (Caballero-Oteyza et al., 2014) and PLA2G6 genes (Ozes et al., 2017) identified in two families were reported as novel HSP genes. SAMHD1, ATAD1, and SEMA3C were identified as strong HSP gene candidates. Immortalized B-lymphocytes derived from family members were analyzed to unravel the involvement of candidate HSP genes on disease pathogenesis. ATAD1, SAMHD1 and CYP27A1 protein levels were shown to be reduced in immortalized cell lines of patients. This study contributed to understanding of the genetic heterogeneity of HSP by identifying five novel HSP genes, one of which locates to SPG27. We also underlined the importance of genetic analysis for differential diagnosis, and the necessity of primary genetic screening of SPG11 in AR-HSP-TCC and CYP7B1 in pure AR-HSP cases highlighting the effect of correct genotype-phenotype correlation.
Disease gene discovery by linkage mapping and exome analysis
(Thesis (Ph.D.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2018., 2018.) Bölükbaşı, Esra Yıldız.; Özören, Nesrin.; Tolun, Aslı.
Disease gene identification is an important area in genetics. Consanguineous marriages are very common in some populations and lead to emergence of rare diseases. To uncover the functions of our genes and the molecular bases of diseases, novel disease genes need to be identified. Later the effect of the mutation on the protein could be investigated by various analyses. Also, discovery of a new disease gene can be a glimmer of hope for the patients who are hoping for definite diagnosis and development of therapies. The steps of the disease gene identification is mapping the locus by linkage analysis or homozygosity mapping, identifying the causative mutation by exome sequence analysis and relating the mutation to the disease pathology, thus uncovering the molecular pathogenesis. Possible or known effect of the mutation can be investigated via computational algorithms and in the light of the information in published studies and databases. In this thesis study, causative genes were searched in eight consanguineous families afflicted with six different recessive diseases. The two identified genes and the identified mutations are PDIA3 (p.Cys57Tyr) in Syndromic Intellectual Disability family and CEP19 (p.Tyr65*) in Bardet-Biedl Syndrome family. Besides, in BBS family possible modifiers GLI1 p.Gly274Arg, CCDC28B p.Phe110Phe, MKKS/BBS6 p.Ile339Val, C8ORF37 p.Ala178Val and TMEM67 p.Asp799Asp were identified/detected. In Isolated Intellectual Disability family, missense PTRHD1 p.Cys52Tyr mutation confirmed that the gene is responsible for ID. Gene expression assay revealed wide expression in brain. In Intellectual Disability and Hypothyroidism family missense TPO p.Asp240Gly was identified. Intronic splicing c.6375-1G>C in SPTBN2 in Spinocerebellar Ataxia family unraveled the basis of dominant vs recessive effects of the variants in this gene. Synonymous p.Ile382Ile in TNKS2 in one of the Pleuroparenchymal Fibroelastosis families was identified as the strongest candidate. No candidate variant could be identified in the other two families, indicating genetic heterogeneity for this disease.
Proteomics based search for novel SIK2 substrates involved in endoplasmic reticulum homeostasis
(Thesis (Ph.D.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2017., 2017.) Şeker, Tuncay.; İyison, Necla Birgül.; Özcan, Ferruh.
As a result of high calorie diet and low physical activity, obesity has become widespread worldwide in the last two decades. Obesity, which is a risk factor for many diseases including diabetes, hypertension, and fatty liver, threatens human health considerably. Molecular studies aiming the treatment of obesity and related metabolic diseases are mostly concerned with the regulation of the feeding behaviours and metabolic pathways. SIK2, having a central role in metabolic pathways, is considered as a pharmacological target in the development of therapeutic drugs against metabolic diseases. Recent studies have shown that Endoplasmic Reticulum (ER) stress is involved in the pathological development of metabolic diseases and SIK2 plays a key role in maintaining protein homeostasis in response to ER stress. However, the molecular details have not yet been fully understood. In this study, to elucidate the role of SIK2 in ER stress response we searched for the novel ER-resident substrates of SIK2 by LCMS/ MS-based proteomic analysis. IRS4 and CHIP, considered as candidate proteins after proteomic analysis, were found to interact with SIK2 by co-immunoprecipitation experiments. In the subsequent co-IP experiments, it was also found that IRS4 interacts with the ER stress sensor protein IRE1. A series of experiments have been carried to further investigate the possible roles of IRS4 on ER-stress response and it has been concluded that IRS4 has a regulatory role on the XBP1-splicing activity of IRE1 and the degradation of the ERAD substrate CD3 . To sum up, results of this study reveal novel protein interactions which will help better understanding the molecular function of SIK2 in maintaining proteostasis in response to ER stress.
Molecular analysis of the factor IX gene in the Turkish population
(Thesis (Ph.D.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 1999., 1999.) Onay, Ü. Venüs.; Çağlayan, S. Hande.
Hemophilia B, the third common hereditary coagulopathy, is very heterogeneous at both phenotypic and genotypic levels. This X-linked recessive disease is caused by the production of either reduced amounts or functionally defective forms of the coagulation factor IX. In order to undertake a comprehensive molecular analysis of the FIX gene in the Turkish population 41 hemophilia B patients were screened for mutations by ddF and direct DNA sequencing to contribute to the knowledge of genotype-phenotype correlations in hemophilia B and to construct a Turkish mutation profile. A hypervariable polymorphic site within the FIX gene have also been analyzed. Thirty-two mutations were identified in Turkish hemophilia B patients, including 4 novel single base changes and 2 novel gross rearrangements. The mutation profile of the Turkish population was found to be similar to the general profile observed worldwide. Haplotype analysis revealed the independent origin of 4 recurrent mutations. All patient data was compiled in a national database, which efficiently shows the mutation/phenotype/haplotype relations. The analysis of the hypervariable region in intron 1 in 85 Turkish individuals revealed the presence of a novel allele and showed that the Turkish population carries the Caucasian specific allele as the most frequent one.

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