M.S. Theses
Permanent URI for this collection
Browse
Recent Submissions
Item Investigation of the role of NLRP13 in cell death(Thesis (M.S.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 2022., 2022) Çakır, Elif Öykü.; Özören, Nesrin.NLRP13 is an intracellular protein that is included in the NOD- like receptor family. It contains the N-terminal pyrin domain, central NOD domain, and leucine-rich repeats, LRR at C terminus. Stably NLRP13 expressing THP-1 cells induced a higher pro-inflammatory response upon LPS/ATP treatment and P. Aeruginosa infection. Moreover, the activation of procaspase-8 is relatively increased in the stably NLRP13 expressing THP-1 cell. NLRP13 can interact with caspase-8 according to Co-IP results. Besides these, NLRP3 inflammasome components are significantly higher in the stably NLRP13 expressing THP-1 cells upon inflammasome activation. This thesis study aimed to investigate whether NLRP13 has a role in cell death via the caspase-8 activation complex after inflammasome activation. To elucidate this, Annexin V-PI staining and LDH release assay were performed after pyroptosis induction via inflammasome activation.No significant difference was observed between stably NLRP13 expressing THP-1 cells and control cells. Furthermore, Gasdermin D cleavage was shown to be similar for each group while PARP-1 cleavage in stably NLRP13 expressing THP-1 cells was slightly higher than control. The experiments were repeated after caspase-8 was inhibited. There was no significant difference in Annexin V-PI staining, LDH release assay, and gasdermin D cleavage. However, PARP-1 cleavage was slightly decreased in stably NLRP13 expressing THP-1 cells when caspase-8 was inhibited.It was shown that NLRP13 is not involved directly in pyroptosis via induction of the caspase-8 activation complex; however, it could be involved in the molecular switch mechanism between apoptosis and pyroptosis with the caspase-8 activation complex.Item Investigation of interaction partners of NLRP13(Thesis (M.S.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 2022., 2022) Ok, Hilal.; Özören, Nesrin.NOD-like receptors (NLRs) are the cytoplasmic members of pattern recognition receptor family, broadly with functions in inflammasome formation, signal transduction, transcription activation, cell death, reproduction, and embryonic development. NLRP13 is a member of NLRs containing PYRIN domain as an effector domain. Its expression is conserved in primates and various mammals, but not in rodents. It is a novel protein and there is not a single dedicated article in the literature. Its information can only be achieved from a limited number of sources and the studies of our former lab members. Based on its expression in oocytes, it is suggested that NLRP13 is a maternal-effect gene. We have shown that NLRP13 is involved in inflammasome formation. After LPS/ATP treatment or P. aeruginosa infection, NLRP13 is upregulated in THP-1 monocytes, and proinflammatory cytokine secretion increases in stable THP- 1 macrophages. NLRP13 is cleaved by Caspase-8 activated upon Fas/FasL-mediated FADD recruitment, and the cleaved C-terminal of NLRP13 is partly localized to mitochondria. NLRP13 do not directly involve in pyroptosis via Caspase-8, and PARP-1 cleavage, an apoptosis marker, is decreased in NLRP13-stable THP-1 cells after Caspase-8 is inhibited. In this thesis, the main aim of the study is to identify the novel interaction partners of NLRP13 so that to propose a notion about its role in signaling pathways and its molecular function. To achieve this goal, NLRP13- FLAG-stable THP-1 and Tera-2 cell lines were generated. Co-immunoprecipitation of NLRP13 from stable THP-1 monocytes, LPS/ATP-treated stable THP-1 macrophages and stable Tera-2 cells was performed with FLAG antibody followed by mass spectrometry. Due to various glitches, the results of repeated experiments could not be reached. However, the analysis of the obtained immunoprecipitates continues.Item Whole-transcriptome analysis of protein-coding potential in the model plant medicago truncatula(Thesis (M.S.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 2022., 2022) Çakır, Umut.; İyison, Necla Birgül.; Kryvorchko, Igor.How many different proteins can be produced from a single spliced transcript? Genome annotation projects usually do not consider the coding potential of altORFs. However, many altProts have been shown to carry out essential functions in various organisms. In addition to the existence of protein-coding potential in all the three reading frames, spliced eukaryotic transcripts may undergo programmed single or multiple ribosomal frameshifting events. Depending on whether a protein is produced by one or several such events, this novel protein is called either a chimeric protein or a mosaic protein, respectively. Proteins produced via single ribosomal frameshifting events have been known in viruses for a long time, and more recently, they have also been found in higher eukaryotes. In contrast, mosaic proteins so far are elusive, with only one example found in viruses. Detection of altORFs can help identify these unusual proteins because altORFs may act as building blocks for chimeric proteins and mosaic proteins. This way of extracting and combining genetic information from different reading frames may significantly increase proteome diversity, thus promoting organisms' flexibility and adaptability to various environmental conditions. This project aims to identify altProts based on the conservation evidence or detection by mass spectrometry (MS) analysis and to find proteins produced via single and multiple ribosomal frameshifting events to demonstrate the existence of mosaic translation. Our study in Medicago truncatula, a well- established model legume, detected 715 translated altProts and 146 chimeric proteins. Two transcripts support the existence of mosaic proteins and mosaic translation, which has never been detected in non-viral organisms before. In addition, we have found evidence for many thousands of conserved altProts. This work pioneers a new field of proteomics and is of immense value for plant biologists and specialists interested in translation. It also paves a way towards the major shift in current understanding of proteome complexity and diversity.Item Dissecting the crosstalk between inhibitory receptor signaling and the T cell receptor in natural killer cells(Thesis (M.S.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 2022., 2022) Ergün, Ebru Zeynep.; Sütlü, Tolga.Killer-cell immunoglobulin-like receptors (KIRs) are among the most prominent receptors regulating NK cell development and function. Most KIR ligands are the MHC class I molecules found on target cells. KIRs are responsible for NK cell licensing which is required for the NK cell to kill and differentiate self from non-self. Adoptive cell therapy based on T cell transfer relies on T cell receptor (TCR) or chimeric antigen receptor (CAR). However, with the TCR-T cells, the most important challenge is mispairing of transferred TCR chains with the endogenous TCR chains. Recently, TCR modification of NK cells has been applied as a solution to the mispairing of TCR chains in T cells. NK cells, with their similar downstream signaling machinery to T cells, can replace T cells in TCR-T cell therapy. These modifications were performed on NK92 cells that lacks inhibitors. This study tries to elucidate how inhibitory signals interfere with TCR signaling. TCR-NK cells against the melanoma-associated antigen Tyrosinase, have been transduced with KIR2DL1 coding lentiviral particles for stable KIR2DL1 expression. The function of the TCR-NK-KIR2DL1 cells was measured against K562 or melanoma cells A375 and A375Tyr. The degranulation of TCR-NK-KIR2DL1 cells was observed to be slightly higher independent from the antigen. Similarly, real- time cell analysis showed the cytotoxicity induced by KIR2DL1 did not show a significant difference. Cy tokine secretion showed there might be a KIR2DL1-mediated TNF-α decrease against A375Tyr cells, but not the same for IFN-γ secretion. Phospho- protein analysis of TCR-NK-KIR2DL1 showed a decrease in PLCγ1 and Erk1/2 levels against all targets.Item Co-receptor mediated regulation of T cell receptor signalling in natural killer cells(Thesis (M.S.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 2022., 2022) Karahan, Zeynep Sena.; Sütlü, Tolga.Antigen-specific cancer immunotherapy has been developing since the 1990s and showing promising results. T cell receptors (TCR) and Chimeric antigen receptors (CAR) are the most commonly used tools for modifying T cells with antigen-specific cytotoxicity. TCR-T cells have the mispairing problem caused by the endogenously produced TCR chains pairing with the ectopically delivered ones and creating T cells with unknown specificity. While NK cells contain all the necessary molecules for the downstream signalling pathway of TCR-CD3 mediated cytotoxicity they do not have the full TCR-CD3 complex. In order to overcome the mispairing problem in T cells, TCR and CD3 genes are used to modify NK cells instead of T cells. TCR-NK cells have shown promising cytotoxic effects against their target cells in vitro and in vivo, however their signalling mechanisms remain to be studied. CD8 coreceptors are known to increase the strength of the bond between TCR-CD3 and peptide-major histocom patibility complex (pMHC) and also aid the signalling cascade to begin. There are two types of CD8 chains, α and β. While T cells mostly utilise CD8αβ heterodimers, some cells have the CD8αα homodimer. This thesis aims to investigate the possible role of CD8 dimers in TCR-NK cells in order to optimise the TCR-NK cell cytotox icity. Lentiviral constructs containing CD8 genes were delivered to TCR-NK cells. These TCR-NK-CD8 cells’ cytotoxicity and antigen-specificity against the target cells were analysed with degranulation, cytokine secretion and real-time cell analysis assays. Western blot analysis was performed to observe the upregulated signalling pathways if present. Our results provide valuable data on the effect of CD8 coreceptors on TCR- NK cells.Item Investigation of the effects of SUMO modification on two critical proteins : a pathogenic NEK1 mutant that drives ALS pathogenesis and the CRISPR-associated CAS9 protein(Thesis (M.S.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 2022., 2022) Ergünay, Tunahan.; Şahin, Umut.Small Ubiquitin-like modifier (SUMO) is an essential eukaryotic post-translational modification. SUMO isoforms attach to specific lysine residues on target substrates to modi-fy their function, activity, solubility and stability. Dysregulation of sumoylation is associated with various pathological conditions ranging from cancer to neurodegeneration. In this study, we focused on a mutant form of NEK1 protein, called truncated NEK1 (or tNEK1), which was recently linked to the pathogenesis of Amyotrophic Lateral Sclerosis (ALS). Previous studies from our lab had established that tNEK1 was prone to aggregation and associated with PML nuclear bodies (NBs). Here, we showed that PML also facilitates tNEK1 sumoylation and ubiquitylation. Furthermore, pharmaceutical agents that induce PML NB biogenesis, such as interferon-alpha (IFN), promote tNEK1 hypersumoylation in a PML-dependent manner. These findings have important implications for the management of tNEK1-linked ALS in the clinic, as IFN may promote the hypersumoylation, degradation, and clearance of this toxic protein in vivo, in a PML-dependent manner. In addition, our lab has created a transgenic mice model that expressed tNEK1 to study the effect of this mutation in vivo. In this study, we have performed motor neuron function assays (specifically, foot-print and walking assays). Our results indicate that tNEK1-expressing mice display a walk-ing disorder, implying that tNEK1 expression in vivo may lead to ALS pathogenesis. Finally, we have also conducted studies on the CRISPR-associated Cas9 protein that we have recently discovered to be a sumoylation target. We have identified the major SUMO conjugation site on this protein and showed that sumoylation impacted on both the stability and DNA-binding ability of this important protein.Item Further characterization of heart - and skeletal muscle-specific mitochondrial aspartyle -tRNA synthetase (DARS2) knockout mouse(Thesis (M.S.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 2022., 2022) Kartal, Özlem.; Doğan, Şükrü Anıl.Mitochondrial respiratory chain defects are the primary cause of mitochondrial disorders. Mitochondria have developed various adaptive responses to counteract the effects of these defects. In this study, the heart- and skeletal muscle-specific mito chondrial aspartyl-tRNA synthetase (DARS2) knockout mouse model (hmKO) was further characterized phenotypically and molecularly by previously unchecked param eters. Phenotypic characterization demonstrated that hmKO mice have lower exercise performance, poorer coordinated activity, and weaker muscles than their wild-type lit termates. Furthermore, respiratory chain deficiency led to mitochondrial dysfunction exemplified by reduced oxygen consumption rate, reactive oxygen species (ROS) flux, and ATP production. In addition, components of the mitochondrial integrated stress response (ISRmt) were upregulated, while the antioxidant response was downregulated in the heart. Although there is no specific cure for mitochondrial diseases, various ap proaches can slow down the disease progress or attenuate the symptoms, one of which is ketogenic diet (KD). Animals were fed with KD to mitigate the effects of the severe phenotype of hmKO mice. Unfortunately, KD could not prolong the shortened lifespan and improve the exercise performance and muscle strength of hmKO animals due to our model’s severe diseased phenotype.Item Investigation of NLRP13's cleavage and its role in macrophage polarization(Thesis (M.S.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 2022., 2022) Çıracı, Sevgi.; Özören, Nesrin.The NOD-like receptors (NLRs) are mainly known for their roles in inflamma tory responses and host defense against microbial pathogens, but they are also asso ciated with metabolic disorders, autophagy regulation, transcription regulation, early embryogenesis, reproduction, and yet unknown functions. Several NLRs have been demonstrated to assemble inflammasomes which are cytosolic multiprotein complexes that can induce pyroptosis. NLRP13 is a PYRIN containing NLR protein that is found in non-rodents. There is little to no information about NLRP13 in the literature. It is determined that NLRP13 is involved in inflammasome formation and cleaved forms of this protein can be found in the cell, by the former students of our lab. In my thesis project, NLRP13’s cleavage and its possible involvement in macrophage polarization were investigated. We showed that the C-terminal of NLRP13 protein colocalizes with mitochondria after being cleaved by Caspase-8 in an overexpression model utilizing immunofluorescence and visualization by confocal microscopy. It was indicated that NLRP13 does not participate in intracellular endosomal trafficking. Additionally, it was suspected that NLRP13 might be involved in macrophage polarization. For this, THP-1 monocytes were differentiated into M1 and M2 macrophages to be analyzed by RT-qPCR and flow cytometry. No difference was observed for wild-type, plasmid control, and NLRP13 overexpressing THP-1 macrophages. Overall, the localization of cleaved forms of NLRP13 in vitro was determined and it was shown that NLRP13 has no role in macrophage polarization in THP1.Item Identification of HB-EGF positive and HB-EGF responsive cell populations in zebrafish olfactory epithelium(Thesis (M.S.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 2022., 2022) Güler, Kardelen.; Fuss, Stefan H.The peripheral olfactory epithelium (OE) is an exception to the limited capacity of the adult nervous system to undergo neurogenesis, which supports structural repair in response to traumatic injury. It continuously and lifelong generates new olfactory sensory neurons (OSNs) at high rate to replace dying cells. Additionally, the OE is capable of regenerating efficiently following severe structural damage. Similar to the mammalian OE, maintenance and regenerative neurogenesis in zebrafish are associated with the selective activity of globose (GBC) and horizontal basal cells (HBC), respectively, which have distinctive structural and functional characteristics. Transcriptome analysis of the regenerating OE suggested that the diffusible signaling factor heparin-binding epidermal growth factor-like growth factor (HB-EGF) may play a critical role at the onset of regenerative neurogenesis. In response to tissue damage, expression levels of HB-EGF are transiently and rapidly upregulated as an early damage response. HB-EGF stimulates mitotic activity in HBCs through the epidermal growth factor receptor (EGFR) signaling pathway. However, the tissue expression of both HB-EGF and EGFR in the zebrafish OE were not known. The studies described in this thesis identified HB-EGF-expressing and HB-EGF responsive cells by selective in situ-hybridization against hbegfa and egfra transcripts. Hbegfa is expressed largely by Sox2-positive basal cells, including tp63-positive HBCs, in addition to HuC/D- positive OSNs and additional cells that may comprise sustentacular glial cells. In response to injury, the number of hbegfa-positive cells increases across all cell populations. In contrast, egfra is expressed by two cell types with distinct tissue distribution, which include HBCs occupying basal OE layers and a subpopulation of OSNs that are restricted to the apical surface of the zebrafish OE. The expression analysis complements previous findings on the proposed role of HB-EGF during OE regeneration and suggests a model of signaling that includes paracrine stimulation between basal cells and other resident OE cells onto injury-responsive HBCs.Item The role of MAPK and AKT signaling during zebrafish olfactory epithelium regeneration(Thesis (M.S.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 2022., 2022) Dokuzluoğlu, Zeynep.; Fuss, Stefan H.The zebrafish olfactory epithelium (OE) is a neuroepithelium that is able to re generate neurons. The ability of the OE for neuron regeneration stems from the pres ence of a dual progenitor system that persists to the adulthood and consists of globose basal cells (GBC) and horizontal basal cells (HBC). In this dual system, GBCs com pensate for daily loss of neurons while HBCs are dormant but activated upon injury to regenerate neurons. Transcriptome profiling of the damaged OE revealed upregulation of the HB-EGF in an early phase of regeneration and suggested a regulatory role in OE regeneration. Pharmacological inhibition of HB-EGF signaling and its receptor EGFR in addition to exogenous stimulation of HB-EGF confirmed the regulation of regener ation by HB-EGF:EGFR interaction. However, cell types regulated by the binding of HB-EGF to EGFR, and cellular signaling pathways that mediate its pro- regenerative effect needed further investigation. In this work, roles of MAPK/ERK and PI3K/AKT signaling pathways which are both well-established to regulate cell fates were studied in the context of OE regeneration. For this purpose, loss of function approaches with pharmacological inhibitors of these pathways were followed by investigating their activ ity in regenerating OE through phosphorylated isoforms. These experiments revealed that MAPK/ERK signaling is the downstream effector of HBEGF:EGFR interaction in cells most likely to be HBCs and it is necessary for recovery of neurons. Despite the fact that PI3K/AKT pathway was also active in these cells, PI3K/AKT signaling was not found to be necessary for functional OE regeneration and it is proposed to stimulate cell cycle progression by promoting cellular growth.Item Identification of allosteric binding sites of the allatostatin receptor type-C of pine processionary moth(Thesis (M.S.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 2022., 2022) Kahveci, Kübra.; İyison, Necla Birgül.G-protein coupled receptors (GPCRs) are cell surface receptors that are consisted of seven-transmembrane α helices. Drugs targeting GPCRs account for one-third of clinically approved drugs; since many drugs target the conserved orthosteric site, they result in side effects like chronic administration, drug resistance, and desensitization. Allosteric sites are topographically distinct from the orthosteric site. Allosteric modu lators modulate the binding and signaling properties of the orthosteric site and orthos teric ligands, fine-tune receptor signaling, reduce the risk of overdosing, and increase specificity since they bind to structurally less conserved sites. Insect GPCRs are a potential target for developing pest control agents as many of these receptors regulate different physiological functions in insects. C-type Allatostatin Receptor (AstR-C) is a class A GPCR and regulates a vital pathway, Juvenile Hormone synthesis, presenting a potential pesticide target. Thaumetopoea pityocampa is the main factor that limits the development and survival of the Mediterranean pine forests. The study aims to provide a safer pesticide with efficient functionality by utilizing in silico and in vitro methods to identify allosteric binding pockets and find allosteric modulators of AstR-C. Various allosteric site prediction tools and blind-docking methods were employed to identify allosteric binding sites. Virtual screening was applied to three potential sites, and hit molecules were subjected to MD simulations and MM-GBSA analysis. Two potential allosteric sites were detected in transmembrane (TM) domains 4, 5, and ECL-2. A third pocket was determined between TM 3-4 and ICL-2. A total of 5 molecules, two molecules for each binding site, yielded promising results in the analyses.Item Investigation of the effects of ochratoxin-a on global protein sumoylation(Thesis (M.S.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 2022., 2022) Denizli, İrem.; Yaman, İbrahim.Ochratoxin A (OTA), a secondary metabolite produced by especially Aspergillus and Penicillium species, is found as a contaminant in foods such as daily consumed corn flakes, coffee, meat and dairy products. OTA is a potential carcinogenic toxin whose mode of action is not fully understood, yet. It is known that OTA deregulates MAPK/Erk1-2 and PI3K/Akt signaling pathways responsible for the vital cellular activities. Therefore, identification of the mechanisms underlying OTA's carcinogenic and toxic effects is of great importance. SUMOylation is one of the post-translational modifications playing crucial roles in cellular homeostasis and the regulation of the cell's vital functions. There is no study examining the effects of OTA on the global protein SUMOylation changes and contributions of these changes on OTA-induced activations of MAPK/Erk1-2 and PI3K/Akt signaling pathways. In this Master's project, first we showed that OTA induces oxidative stress in immortalized human proximal tubule epithelial HK-2 cell line. Next, we demonstrated that OTA treatment leads to alterations in global protein SUMOylation and activation of PI3K/Akt and MAPK/Erk1-2 pathways, which might be linked to OTA-induced oxidative stress. Moreover, we showed that OTA-induced MAPK/Erk1-2 pathway activation is not affected when global SUMOylation is inhibited using ML-792. In contrast, our data portrayed that global SUMOylation is essential for phosphorylation of Akt under OTA exposure. Taken all together, our data suggests that SUMOylation may play a key role in cellular survival under OTA exposure. Finally, we constructed stable SUMO-1- and SUMO-2- overexpressing HK-2 cell lines to eliminate low efficiency for immunoprecipitation. By using these cell lines we will be able to detect possible SUMO targets under OTA exposure via Mass Spectrometry Analysis.Item Characterization of CkIIα-i1 -a putative ortholog of intellectual disability candidate gene ZBTB11(Thesis (M.S.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 2022., 2022) Mandacı, Barış Can.; Çelik, Arzu.Intellectual Disability (ID) is a heterogenic neurodevelopmental disorder seen in different spectrums in affected individuals. Resulting problems in the diagnosis of ID and limitations in its treatment constitutes a great emotional and financial burden to society. Therefore, identification of ID-causative genes and their functional characterization are important for the improvement of the current conditions. Two pathogenic variants of one of the ID-candidate genes ZBTB11 was identified and its early characterization was performed. In addition, knockdown of CkIIα-i1, a Drosophila ortholog of ZBTB11 showed deficiency in learning and memory center of Drosophila. The main aim of this study is the further investigate CkIIα-i1 and ZBTB11 in Drosophila in the pursuit of identifying their orthology. Since both CkIIα-i1 and ZBTB11 were understudied genes, transgenic and KO fly lines were generated and utilized in this study. In parallel, the expression of CkIIα-i1 was characterized in mushroom bodies (MB) and clock cells, which are learning- and circadian rhythm- related brain compartments, respectively. In addition, the role of CkIIα-i1 in MB development was analyzed by performing morphological analyses of the MB in CkIIα-i1 knockdown flies. For loss-of-function analysis, two RNA interference lines for the knockdown of CkIIα-i1 were utilized. Furthermore, two CkIIα-i1 KO lines were generated using CRISPR/Cas and used for morphological analysis. Both knockdown and KO of CkIIα-i1 resulted in shrinkage in the α lobe of the MB, with higher frequencies in KO lines. In contrast, the over-expression of CkIIα-i1 using the Gal4/UAS system did not result in any changes in MB morphology. Results of morphological analyses indicated that CkIIα-i1 could have a role in axonal guidance of α lobe axons. In order to investigate orthology between CkIIα-i1 and ZBTB11, wt and two identified variant ZBTB11 constructs were transgenically expressed in Drosophila and their expression was validated in the fly brain.Item The role of EGFR and JAK(Thesis (M.S.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 2022., 2022) Alkiraz, Aysu Şevval.; Fuss, Stefan H.The zebrafish olfactory epithelium (OE) has an exceptional neurogenic capacity, which supports the continuous turnover olfactory sensory neurons (OSNs) and efficient regeneration after injury. These processes depend on the presence of a dual stem cell system that is comprised of globose (GBCs) and horizontal basal cells (HBCs), which selectively contribute to maintenance of the intact and repair of the injured OE, re spectively. Transcriptome profiling of the injured OE revealed strong upregulation of the HB-EGF. Exogenous stimulation and pharmacological inhibition of HB-EGF sig naling show a selective effect on HBCs, suggesting that HB-EGF is a key signal for OE regeneration. Among the known cell surface receptors for HB-EGF, only the tran script levels of ErbB1/EGFR are upregulated in response to injury. EGFR expression localizes to HBCs, suggesting that HB-EGF signals through EGFR to activate injury responsive HBCs. To investigate the roles of EGFR and JAK/STAT signaling on OSN neurogenesis, the effect of pharmacological inhibition of the pathways on maintenance and repair neurogenesis were evaluated. Inhibition of EGFR signaling resulted in the suppression of basal proliferative activity, which, however, only slightly reduced the rate of OSNs turnover in the intact OE. In the injured tissue, on the other hand, EGFR inhibition resulted in a dramatic impairment of OSN regeneration by prevent ing the induction of HBC proliferation. A similar impairment in OSN regeneration could also be observed when JAK/STAT signaling was inhibited. Tissue analysis of JAK/STAT activation pointed to an injury-responsive population that is distinct from HBCs. These results suggest that both EGFR and JAK/STAT signaling are necessary for repair neurogenesis but that they are active in distinct populations of the OSN lineage.Item Preliminary characterization of a novel mitochondrial myopathy mouse model(Thesis (M.S.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 2022., 2022) Yılmaz, Mehmet.; Doğan, Şükrü Anıl.Mitochondria provide a significant portion of the ATP to cells and govern cellu lar energy economics on ever-changing cellular energy requirements. Cells evolved to monitor mitochondrial stress and developed counteracting stress response mechanisms to counteract mitochondrial dysfunction. Mitochondrial diseases are rare but complex diseases; involvement of both nuclear and mitochondrial genomes, threshold effect, and tissue specific manifestations of the disease increase the conundrum. Neuromuscular involvement in mitochondrial diseases is usually associated with mitochondrial my opathies, which are progressive in nature and may cause premature death. For this study, we generated a novel mitochondrial myopathy mouse model by disrupting the function of the mitochondrial aspartyl-tRNA synthetase gene (Dars2 ) in the skele tal muscle. Mitochondrial aminoacyl-tRNA synthetases are vital for mitochondrial protein translation since they aminoacylate uncharged tRNAs, and their dysfunction hampers mitochondrial translation. In skeletal muscle-specific Dars2 deleted mice, we observed severe and progressive mitochondrial myopathy that causes muscle atrophy and significant reduction in lifespan and body weight. Knockout mice exhibited exer cise intolerance, decreased locomotor activity, and muscle strength compared to their littermates. We detected hypoglycemia and an overall decrease in electron transport chain complexes at the molecular level. On the other hand, various stress responses were evoked in the knockout mice. Terminal stage mice displayed increased mitochon drial biogenesis and mitochondrial integrated stress response in both cell-autonomous and non-autonomous manner. Moreover, an impairment in autophagy and an advanced antioxidant response were observed. Our findings may create windows of opportunity for additional interventions in mitochondrial diseases.Item Characterization of the E3 ubiquitin ligase RNFT2 ortholog CG13605 in drosophila melanogaster(Thesis (M.S.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 2022., 2022) Kahraman, Ayşe.; Çelik, Arzu.Characterized by deficits in both intellectual ability and adaptive behavior, intellectual disabilities (IDs) are one of the major neurodevelopmental disorders with a prevalence of 1- 3% worldwide. Development of Next Generation Sequencing techniques provided an important step for the study of ID and identification of ID-related genes. A whole exome sequencing study of 404 consanguineous families with ID led to the identification of a novel ID-related missense variant (cT1150C; p.C384R) in the RNFT2 gene. RNFT2 (RING finger protein, transmembrane 2) encodes a RING finger E3 ubiquitin ligase. Its fly ortholog Dmel/CG13605 has the same type of RING finger domain (C3HC4) and is predicted to be a ubiquitin E3 ligase. The aim of this study is to characterize the fly orthologue of RNFT2, CG13605, and investigate the role of RNFT2 in ID and the orthology between two proteins. For this aim, first I investigated the expression pattern of CG13605 and observed expression in the mushroom body (MB) Kenyon cells throughout development. Then I conducted loss of function experiments by RNAi knockdown and generating and investigating CRISPR knockout mutants. Knockdown experiments resulted in misguidance defects in the MB lobes. Similarly, knockout experiments also led to various MB phenotypes in α/β lobes. To investigate the orthology between RNFT2 and CG13605, I expressed transgenic wild type and mutant RNFT2 in flies in the CG13605 mutant background. Rescue experiments with wild type RNFT2 resulted in rescue of observed phenotypes, indicating a function- oriented orthology between RNFT2 and CG13605.Item Investigation of the impact of human immunodeficiency virus on cellular sumoylation(Thesis (M.S.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 2022., 2022) Pekbilir, Emre.; Şahin, Umut.The manifestation of Human Immunodeficiency Virus type 1 (HIV-1) infection is acquired immunodeficiency syndrome (AIDS), one of the major health problems worldwide. From the first step of infection to the end, HIV-1 customizes numerous mechanisms of the host cell to optimize its replication, thus invading the immune sys tem. Sumoylation is an essential regulator of the immune system through modulating different immune signaling pathways, including interferon signaling. SUMO proteins are the downstream effectors of interferon to antagonize bacterial and viral infections. Consecutively, various pathogens oppose sumoylation to neutralize immune responses. In this study, we investigated the interplay between host sumoylation and HIV-1. Our study demonstrates that HIV-1 diminishes cellular sumoylation by antagonizing the UBA2 protein, a subunit of the E1 SUMO-activating enzyme. HEK293 and Jurkat cells display abrogated sumoylation profiles by SUMO1 and SUMO2/3 when the HIV 1 genome is expressed. HIV-1 expression in HEK293 and Jurkat cells suppresses UBA2 protein levels as well. Therefore, HIV-1 targets cellular sumoylation by most probably antagonizing UBA2. Altogether, we demonstrated that HIV-1 impairs sumoylation, a cellular mechanism vital for immunity.Item The role of unzipped axon guidance and targeting in the olfactory system of drosophila melanogaster(Thesis (M.S.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 2014., 2014.) Mika, Kaan.; Çelik, Arzu.The olfactory system of Drosophila is a great model to investigate neural wiring complexity. The fly has two sensory appendages named as antenna and maxillary palp. Hair-like structures called as sensilla cover the appendages and house olfactory sensory neurons (OSN) together with supporting cells. Each OSN expresses one specific type of OR out of a total of 62, and project to a single unit of antennal lobe, called a glomerulus. In each glomerulus, specific synapse formation between the dendrites of projection neurons and OSNs occur. Then, the olfactory stimulus is transmitted to higher brain centers such as the mushroom body and lateral horn, in which olfactory information is converted into a behavioral response. Interaction of neurons and glia is essential for establishment of a proper olfactory system. Glial cells are known to act as guidepost cells in the nervous system, navigating neurons both by secreting molecules and mediating cell-to-cell contacts with cell surface molecules. Several cell surface molecules and transcription factors play an essential role in the establishment of this complex network. Additionally, a subtype of glia in the interhemispheric region of the pupal brain forms a ring-like structure called Transient Interhemispheric Fibrous Ring (TIFR), which is crucial for the formation of the midline commisure. Uzip is a cell adhesion molecule, expressed by glia and neurons (Ding et al., 2011). Uzip mutants show midline crossing and mistargeting phenotypes of OSNs (Zülbahar, 2012). In this study, the endogenous expression of Uzip was analyzed using a mCherry-tagged version of Uzip that was generated by BAC engineering and Uzip enhancer trap line. Additionally, the function of Uzip was analyzed in detail using cell-type specific gain-of-function and loss-of-function analyses. These analyses revealed that Unzip function is required in glial cells and especially in TIFR glia for a proper development of the olfactory commissure.Item Translational regulation of Kidney Injury Molecule-1 MRNA: presence of an internal ribosomal entry site(Thesis (M.S.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 2014., 2014.) Banaz, Nehir.; Yaman, İbrahim .Kidney Injury Molecule-1 (KIM-1) is a type I transmembrane glycoprotein that is highly upregulated on regenerating dedi erentiated renal proximal tubular epithelial cells as a consequence of toxic or ischemic injury. KIM-1 is a recognized biomarker of nephrotoxicity by United States Food and Drug Administration (FDA) and Europe Medicines Agency; therefore, understanding its transcriptional and translational regulation under chemotoxic stress is of the utmost importance. As chemotoxic stress inducers we used three xenobiotics, Ochratoxin A (OTA), Gentamicin (GM) and Cisplatin (CP) which are known to cause damage to kidney cells. The e ects of these three xenobiotics on viability, proliferation, necrosis and apoptosis are examined using the immortilized human proximal tubule epithelial HK-2 cell line. The transcriptional start region of Kim-1 gene is determined by Rapid Ampli cation of cDNA Ends (RACE). The changes in KIM-1 mRNA and protein levels are measured by Quantitative Real Time PCR and Western Blotting, respectively. Although there was a signi cant decrease in global protein synthesis upon xenobiotic treatment, KIM-1 protein levels increased. Furthermore, no signi cant change in KIM-1 mRNA levels can be detected which led us to suspect the existence of a possible post-transcriptional regulatory mechanism by which KIM-1 mRNA is translated via an Internal Ribosome Entry Site (IRES). This hypothesis is tested in vitro using bicistronic reporter vector pRF containing the 5'UTR of Kim-1 gene. Our results con rm our hypothesis of a possible IRES localized around 300 bp upstream of the translation initiation codon.Item Functional analysis of a splice site mutation in BABRG2 gene and its effect on GABAA receptor(Thesis (M.S.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 2014., 2014.) Kaplan, Öyküm.; Çağlayan, S. Hande.Epilepsy is a group of diseases produced by non-controlled discharge of neurons affecting more than 2% of the population and some forms include genetic determinants. The principal group of inherited epilepsy is Genetic Generalized Epilepsies (GGE) and Childhood Absence Epilepsy (CAE) is a common subtype of GGE. Gamma-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the mammalian brain. It acts through two classes of receptors: GABAA and GABAB receptors. Impairment of GABAergic transmission by genetic mutations cause epileptic seizures. GABAA receptor, gamma 2 which is located on 5q34, is encoded by GABRG gene. Besides mutations which are in coding region in GABRG2 gene, mutations in intronic sites of the gene are also observed. It has been shown that a T-G transversion (IVS6+2T-G) mutation altered the GABRG2 intron 6 splice donor site sequence, activated a cryptic splice site and generated partial intron retention. The aim of this study was to investigate the effect of an other splice site variant of GABRG2 gene detected in a CAE patient on GABAA receptor. Scores of splice site tools showed that this mutation could create cryptic splice site. By site directed mutagenesis, the known mutation was created and cloned with the GABRG2 variant in the patient by creating minigenes. After transfection, expressions were checked by RT- PCR. Under similar experimental conditions while the splicing defect of the known mutation was observed, GABRG2 splice site variant in the CAE patient had no effect on splicing suggesting that it was a silent variant of the gene.