Moleküler Biyoloji ve Genetik

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A FLY's view of salt inducible kinases: a synergistic tumorigenesis model
(Thesis (M.S.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 2012., 2012.) Sayın, Sercan.; Çelik, Arzu.
The recent explosion of data generated by high-throughput assays on cancer necessitated reliable and fast in vivo models for evaluation new catalogue of candidate genes as tumor suppressors or proto-oncogenes and their subsequent trials in cancer therapeutics. Drosophila melanogaster, an acclaimed model organism for genetics studies, could now be proposed as the new pinnacle for modeling and screening those candidate genes. As an initiative study in Turkey, we tried to establish a multifaceted, synergistic cancer model based on the Drosophila compound eye, reinforced with fly glia and wing models, while using Salt Inducible Kinase (SIK) family as the choice of candidate genes. Metabolic deregulation in cancers is getting increased attention, thus SIKs were an excellent choice since they stand on the crossroad where metabolic and structural information in a cell meet. Using two backgrounds, low grade sensitized and high grade eyeful, we enquired the contributions on growth and metastasis by two existing Drosophila orthologs of human SIKs. Results showed that SIK2 could assume both suppressive and oncogenic roles; as optimal amounts of SIK2 could repress tumor growth while any fluctuations could enhance tumor and glial migration strength. SIK3, on the other hand, was found to act as oncogene when over-expressed in the studies. SIK3 gain could enforce several different outcomes; constitutive over-expression of SIK3 was highly lethal and could alter developmental fate choice of the respective context. Additionally, effects of SIKs on cancer tissues were investigated at cellular level via cell proliferation and death assays, which showed that SIKs could interact with major developmental pathways, such as Notch, TGF-β, FGF.
A molecular investigation of β-thalassemia in the Aegean and Mediterranean coasts of Turkey :|is there a region-dependent specificity?
(Thesis (M.S.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 1994., 1994.) Senga, Edward Bisweck.; Başak, A. Nazlı.
As in many other Mediterranean countries, β-Thalassemia is also a major public health concern in Turkey. The average gene frequency.is estimated to be two per cent, and regions with higher figures are known to exist. Country scale frequencies of β-thalassemia mutations have been established in Turkey by several investigators in the past, but mutational data maps at regional scale are not available yet. The present study was designed to investigate the presence of a possible locus-specific heterogeneity of β-thalassemia mutations at the Aegean and the Mediterranean coasts of Turkey. Blood samples of patients sent to our laboratory from the Medical Schools in Izrnir, Antalya and Adana were chosen as being representative for the Western and Southern parts of the country. The method of choice for screening a large number of chromosomes for point mutations, involves the PCR amplification of the gene under investigation, followed by hybridization of the amplified DNA to Allele Specific Oligonucleotide (ASO) probes. A total of 191 chromosomes were analyzed in the framework of this thesis using 19 oligonucleotide probes specific for the Mediterranean countries. The results obtained do confirm the remarkable molecular heterogeneity of β-thalassemia in all three districts investigated. Although a marked locus-specific heterogeneity of mutations was not observed, different patterns of mutational distribution were obvious, which may help in the elucidation of the molecular heterogeneity of Turkey in certain cases.
A novel post-translational modification on the central CRISPR enzyme :|discovery of Cas9 ubiquitylation
(Thesis (M.S.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 2019., 2019.) Çelen, Arda Baran.; Şahin, Umut.
The CRISPR/Cas9 system offers a simple method for genome engineering by utilizing the ability of the bacterial Cas9 enzyme to cleave any desired genomic region under the guidance of a complementary RNA molecule. Due to its minimalism and versatility, the CRISPR/Cas9 system is increasingly being used as a gene editing platform in higher organisms, with the current applications encompassing diverse fields such as disease therapy, biotechnology and agriculture. Despite such widespread use of this prokaryotic protein, there is a lack of knowledge regarding its behavior and regulation in eukaryotic systems where the majority of these applications are implemented. In this study, we aim to elucidate the mechanisms of Cas9 regulation in eukaryotic systems with the specific goal of investigating the potential post-translational modifications (PTMs) on this protein. As part of this, we show that Cas9 gets ubiquitylated, promoting its proteasomal degradation. In addition, we present a cell culture-based experimental setup where we aim to discover that the Streptococcus pyogenes Cas9 protein undergoes SUMO modification following the engulfment of these bacteria by human immune cells. We expect that these results will lead to a better understanding of the eukaryotic posttranslational regulation of Cas9. Our hope is that uncovering the functional implications of these PTMs will contribute to the development of safer therapies and improve the current CRISPR-based applications, while paving the way for new ones. Moreover, we hope that a potential discovery of in vivo Cas9 sumoylation may create a new frontier for future research focusing on host-pathogen interactions.
A novel tool for the identification of locus-specific chromatin proteins
(Thesis (M.S.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 2019., 2019.) Ogmen, Anna.; Emre, Tolga.
Genomic DNA is maintained and regulated by chromatin proteins. Methods for research of chromatin organization are numerous and established, however, it is still a challenge to identify novel chromatin proteins associated with a speci c locus. In the scope of this project, we work on implementing a novel tool for the identi cation of chromatin proteins associated with a given genomic region of interest. The method we used is based on combining the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and SICAP (Selective Isolation of Chromatin-Associated Proteins) techniques. CRISPR technique is used for targeting the aimed locus, and SICAP technique provides bene ts by employing two tandem pulldowns. First pulldown is dCas9-mediated and locus-speci c, while second is based on streptavidin binding to in vitro-biotinylated DNA of chromatin fragments. Second pulldown enables to get rid of contaminants by stringent denaturing washes and limits contaminant proteins to the chromatin. Resulted pool of chromatin proteins is eluted, and is ultimately aimed to be analyzed with mass spectrometry. In current work, most of the critical components of the method were developed and veri ed. Also, we implemented the model genomic locus for the validation of the method. CRISPR-SICAP method was then probed on di erent genomic regions by CRISPR-SICAP-qPCR and was shown to be more speci c than regular dCas9- ChIP. These results can be extrapolated to future applications to locus-speci c protein research. Both CRISPR-SICAP and model genomic loci are optimized and ready for being applied for obtaining pool of locus-associated chromatin proteins.
A privileged hunter: role of NLRP7 in immune privilege
(Thesis (M.S.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 2012., 2012.) Demiröz, Duygu.; Özören, Nesrin.
NOD-like receptors (NLRs) are a family of cytoplasmic receptors with members regulating apoptosis and inflammation. NLRP7 is a novel PYRIN domain containing NLR family member and its functions are still under investigation. NLRP7 is known to possess an oncogenic role in testicular seminomas and found to be overexpressed in cer-tain cancers. Interestingly, in addition to some other tissues, NLRP7 is expressed in brain, testis and placenta which are immune privilege sites in the body. Furthermore, mutations in NLRP7 can cause recurrent hydatidiform moles leading to stillbirths and abortions. One study suggested that NLRP7 down-regulates IL-1β and hence it is thought that NLRP7 may have an immuno-suppressive function. The aim of this project is to clarify the possible role of NLRP7 in immune privilege and also in inflammation. As a first step to the project, NLRP7 cDNA was cloned into several tagged (FLAG-, HA-, pcDNA3-MYC, RFP and EGFP) mammalian expression vectors for confocal anal-ysis for cellular localization and for detection of its interaction partners by co-immunoprecipitation. The effect of NLRP7 overexpression on NF-κB signaling regula-tion was measured by performing luciferase assay in HEK293FT cells. In order to eluci-date, in order to elucidate the possible role of NLRP7 in immune privilege, shifts in death receptor and HLA expressions were measured using real-time PCR and FACS analysis, respectively, in stable NLRP7 knock-down HEC-1-A cell line. Confocal analy-sis of RFP or EGFP fused NLRP7 reveals that NLRP7 is a cytosolic protein with minor co-localization to mitochondria. Furthermore, confocal co-localization studies show clear overlap between NLRP7 and caspase-1 and/or ASC. We also found that NLRP7 forms an inflammasome that activates IL-1β through binding to ASC and Caspase-1. NLRP7 appears likely to regulate IL-1β expression. Finally, NLRP7 down-regulation decreases the expression of TRAIL-R3, TNF-R2 and TWEAK and also HLA A, B, C and increases Fas expression.
A quest for the upstream activators of ochratoxin a (OTA)- induced MAPK/ERK1/2
(Thesis (M.S.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 2016., 2016.) Gül, Gizem.; Yaman, İbrahim .
Ochratoxin-A (OTA) is a mycotoxin, produced as a secondary metabolite by fungi belonging to Aspergillus and Penicillium genera. It has been recognized as a carcinogen in rodents and a possible carcinogen to humans. In our previous study, we demonstrated that OTA induces sustained MAPK/ERK1/2 activation in HK-2 cell line. Upstream activator of ERK1/2 protein activation remained obscure. In this study we screened a number of proteins via Western blot to determine if they cause ERK1/2 activation in OTA-exposed HK-2 cell line. We observed no dramatic increase in the expression levels of neither Integrin and subunits nor Integrin Linked Kinase (ILK). Also there was no discernable increase in the phosphorylation of Focal Adhesion Kinase (FAK) residues that might be involved in OTA mediated ERK1/2 activation under the experimental conditions employed in this study. Although a stable level of PKC phosphorylation was not observed, inhibition of various PKC isoforms reduced OTA-induced ERK1/2 activation. These results suggest PKC as a possible upstream activator in the activation of MAPK/ERK1/2 in OTA-treated HK-2 cells. Moreover, modulation of G protein-coupled receptor (GPCR)-dependent signaling through pertussis and cholera toxins suppressed ERK1/2 activation in response to OTA treatment. In addition, chemical agents that increase intracellular cAMP levels subdued OTA-induced ERK1/2 activation considerably. We also observed that H- 89, a cAMP-dependent kinase (PKA) inhibitor, augments OTA-induced activation of ERK1/2. Taken all together, this study suggests that OTA may cause stimulation of a putative GPCR that results in a decrease in intracellular cAMP levels and ultimately activation of MAPK/ERK1/2 in HK-2 cell line where PKC may be an intermediary in relaying signal from GPCR to ERK1/2.
A study for the detection of known and novel mutations in the cystic fibrosis transmembrane conductance regulator gene
(Thesis (M.S.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 1996., 1996.) Kılınç, Okyay.; Tolun, Aslı.
Cystic fibrosis (CF) is one of the most common and severe autosomal recessive genetic disorders worldwide. It results from mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). The gene has 27 exons which encode a 1480 amino acid transmembrane ion channel protein. The major CF mutation F508 accounts for about 67% of the 30000 CF Caucasian chromosomes screened worldwide. Moreover, more than 600 other mutations have been identified, each with a frequency of at the most a few percent. The mutations responsible for CF in the Turkish population are not yet known. In the Turkish patients, F508 is found at a significantly lower frequency (13%), which indicates that CF is caused predominantly by other mutations. A systematic study was initiated to characterize the CF mutations. First, we screened for four mutations which are frequent in neighboring geographical areas, and found them to be infrequent. It became obvious that, an efficient, quick and reliable mutation screening method which would cover all of the coding region was needed. We applied to 124 CF chromosomes the DGGE technique to analyze all of the gene except for the first and last exons with the purpose of detecting any variants in the gene. These variants would later be subjected to DNA sequence analysis to determine mutations or polymorphisms which they represent. Our previous hypothesis that the profile of CF mutations in the Turkish population shows great heterogeneity is consistent with the data obtained. As expected, several mutations and variants were identified which were distributed randomly throughout 14 exons in the coding regions of the CFTR gene, each of which would account for a relatively small fraction of the CF mutations.
Aberrant global DNA methylation in neurodegeneration: ALS and trinucleotide repeat disorders
(Thesis (M.S.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 2016., 2016.) Hamzeiy, Hamid.; Battaloğlu, Esra.; Başak, A. Nazlı
Amyotrophic lateral sclerosis (ALS) is a late onset neurodegenerative disease of the motor neurons, leading to death within two-three years of onset. Extensive studies have thus far helped identify many genetic causes for this devastating disease, but despite such efforts, more than 80% of ALS still remains unexplained. ALS is generally considered to be a polygenic disease, and many different factors may be involved in its pathogenesis, including a combination of rare mutations and environmental factors which could ultimately lead to epigenetic modifications. Here, we investigated ALS from an epigenetic perspective, focusing on 5-methylcytosine (5-mC), a well-characterized epigenetic modification, aiming to conclude disputes between different studies reporting inconsistent results for global 5-mC levels detected in blood samples of sporadic ALS (sALS) patients. The study was further extended to different subtypes of ALS, including familial ALS (fALS), C9orf72 expansion carrier ALS (C9orf72+ ALS) and ATXN2 intermediate expansion ALS along with spinocerebellar ataxia types 1 and 2 (SCA1 and SCA2), Huntington’s disease, Friedreich’s ataxia and myotonic dystrophy type 1. In order to analyze the global 5-mC levels, in DNA isolated from blood, an enzyme-linked immunosorbent assay (ELISA) kit was selected upon testing of commercially available kits. The results showed that increased global 5-mC levels are not exclusive to sALS (p < 0.001 [F(1, 214) = 11.993, p = 0.000645]) and that this can also be observed in different subtypes of fALS. Interestingly, SCA1 (p < 0.01 [F(1, 32) = 8.778), p = 0.00571]) and SCA2 (p < 0.01 [F(1, 56) = 10.784, p = 0.001768]) patients also showed increased levels of global 5-mC when compared to age- and sex-matched healthy controls. Additionally, direct bisulfite sequencing was utilized to investigate the C9orf72 promoter in C9orf72+ ALS patients and healthy controls. Promoter hypermethylation was observed in patients and was moderately correlated (rs = 0.3902, p < 0.05) with the global levels of 5-mC. We also tested several commercial kits for the quantification of 5-hydroxymethylcytosine and could not find a suitable kit for its detection in blood.
Absence epilepsy in Turkish patients: a novel gene at susceptibility locus 2q36 and the role of GABRG2
(Thesis (Ph.D.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2009., 2009.) Çapan, Özlem Yalçın.; Çağlayan, S. Hande.
Idiopathic absence epilepsies (IAE) are complex disorders mainly caused by genetic factors. Two major whole-genome linkage studies performed on many IAE samples pointed to chromosome 2q36 as a susceptibility locus for absence epilepsies. Whole genome linkage study on absence epilepsy model Wag/Rij rats, also showed the syntenic 2q33-37 region to be linked to the quantative trait of absence seizures. Candidate ion channel genes at 2q36 were screened but causative mutation could not be identified. On the other hand, mutations have been found in the subunits of GABA receptors and Ca channels in a few patients. At present, therefore, the complete picture of pathogenesis of the absence seizures is not known. In this study, to assess the possible role of 2q36 region in absence epilepsy, 205 Turkish absence patients and 219 healthy controls were used in an association analysis. Based on the haplotype block structure of the Turkish population in this region 10 tagSNPs were selected to cover the 160kb region at 2q36. The patients were subgrouped according to the syndrome and seizure types. The results revealed a significant association of two neighboring SNPs (rs7588807 and rs2840128) with JAE syndrome and even higher significant association with GTCS with the same SNP, rs7588807 that resided in the INHA gene. The point mutation and qPCR analysis of the INHA gene revealed mutations/variations in several patients and a large deletion that covered 30-50 kb in at least seven JAE patients supporting the association of INHA with the epilepsy phenotype and its establishment as a novel gene involved in the pathogenesis of JAE. The presence of other candidate genes in the deleted region paves the way for further molecular genetic analysis to reveal the role of each candidate gene in the pathogenesis of epilepsies, if any. The study further supports the role of GABRG2 in the pathogenesis of absence seizures by the identification of novel variations/mutations especially affecting the splice sites in CAE patients.
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.
Analysis of positive and negative regulatory sites and their interacting transcription factors in the zebrafish OR101-1 gene promoter
(Thesis (M.S.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 2018., 2018.) Özdemir, Metin.; Fuss, Stefan H.
Each olfactory sensory neuron (OSN) of olfactory system expresses a single allele of an olfactory receptor (OR) gene from a large genomic repertoire. How an OSN selects and activates a single OR allele is not well understood yet. It was shown that epigenetic modifications are a key part of the process as well as proximal promoter region and long range cis-regulatory elements. Previous work on proximal promoter of OR101-1 gene has revealed both positive and negative regulatory sequences, deletion of which altered the expression efficiency in zebrafish embryo olfactory epithelium (OE) of transgenic vectors. It was shown that deletion of the intron from 1.2 kb upstream of OR101-1 transcription start site reduced the expression of transgenic construct, yet it was not clear whether this effect was caused by the absence of a putative O/E binding site within the intron site or by the absence of intron directly. To test this, three separate constructs were prepared. These constructs were injected in zebrafish embryos and expression efficiency was observed. While mutation of putative intronic O/E binding site resulted in 3% reduction, double mutation resulted in 7.5% reduction and swapping of intron with another resulted in 4% increase in expression, and after all, it was concluded that the expression reduction effect of intron deletion was due to the absence of the intron and not absence of the putative O/E binding site. A negative regulatory element with 562 bp length (i562) was also investigated in this study. Different length probes for each candidate site were prepared and electrophoretic mobility shift assay (EMSA) was applied to these probes with various protein sources and with anti-Zbtb7b antibody. Overall, it was seen that previously observed expression inhibition was not due to direct recognition of the candidate sites by any protein present in OE of zebrafish. The pattern of DNA-protein interaction in EMSA analyses suggests that there are cryptic sites within i562 participating in inhibitory function of the element.
Analysis of the Cx32, MPZ and PMP22 mutations in the Turkish Charcot-Marie-Tooth patients
(Thesis (M.S.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2006., 2006.) Maracı, Nazmiye Öncü.; Battaloğlu, Esra.
Charcot-Marie-Tooth disease is the most common hereditary peripheral neuropathy with a prevalence rate of one in 2500. Although the disease is classified as demyelinating and axonal CMT based on clinical investigations, genetic studies revealed more than 28 genes and subtypes for both axonal and demyelinating forms. In a total of 161 unrelated CMT patients were analyzed in this study. The incidence of CMT1A duplication in the Turkish population was found to be significantly lower (41.6 per cent) compared to that of other populations (70 per cent). The frequency of the HNPP deletion was found to be 66.6 per cent. Although this rate is lower than that of other populations (86 per cent), the difference was not statistically significant. In this study, five nucleotide variations were identified; a novel and a previously reported mutation in Cx32, a previously reported polymorphism in MPZ and two novel mutations in PMP22. Phenotype/genotype correlations were performed for the identified mutations and the observed phenotype of the patients was found to be compatible with the mutations they carry. Identification of causative mutations in the patients verifies the clinical diagnosis and leads the clinicians for choosing the therapeutic approach. However, for the patients that could not be genetically diagnosed further research is required to identify the pathogenic mutations in other CMT genes. On the other hand, identification of further mutations and genotype/phenotype correlations are of critical importance and will help elucidation of the pathogenic mechanisms leading to the CMT disease.
Analysis of the vascular endothelial growth factor and angiogenin genes as risk factors for amyotrophic lateral sclerosis in the Turkish population; identification of a possible novel mutation
(Thesis (M.S.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2006., 2006.) Güzel, Refika Mine.; Başak, A. Nazlı.
Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disorder, which is primarily characterized by the death of motor neurons in the cerebral cortex, spinal cord, and brainstem. Degeneration of motor neurons leads to progressive wasting and eventual paralysis of skeletal muscles. Only a small portion of all ALS cases show familial inheritance (FALS), and the remaining majority of patients are sporadic (SALS). Still, a genetic component is thought to contribute to SALS pathogenesis. Despite intensive research, the mechanisms leading to neurodegeneration in ALS have not been fully understood. Identification of genes, either underlying ALS or predisposing to ALS, has been an important part of ALS research. Among several etiologic gene candidates, thought to be associated with SALS, Vascular Endothelial Growth Factor (VEGF) and Angiogenin (ANG) aroused particular interest, because they are well-known factors for their angiogenic activities. In the framework of this thesis, we examined the link between ALS and the reported VEGF and ANG gene variations in the Turkish population, since it is very important to confirm the observed genetic association in various different populations. Screening of 101 ALS patients and 99 healthy controls with restriction enzyme analysis and DNA sequencing did not reveal any statistically significant association of these genes with ALS in our study population. But a possible novel mutation in the ANG gene was identified in a juvenile ALS patient. This is a preliminary result, which has to be verified by further analysis of the patient and his family.
Analysis of Turkish cystic fibrosis chromosomes
(Thesis (M.S.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 1995., 1995.) Akarsubaşı, Alper Tunga.; Tolun, Aslı.
Cystic fibrosis (CF results from mutations in the gene encoding the cystic fibrosis transmembrane regulator (CFTR), a protein that regulates chloride ion transport in exocrine gladns. Since the cloning of the gene, more than 375 disease-causing mutations have been identified. The major mutation in the gene is DF508, a deletion of three bp in exon 10 that removes a phenylalanine residue at position 508 and is found in 68 per cent of CF chromosomes worldwide. In this study, heteroduplex analysis was performed for detection of the mutations DF508 and 1677 delTA. The former was found in 15.5 per cent of CF chromosomes in Turkey. The latter was found in relatively high frequency (4.5 per cent) with respect to other mutations in our population. Further, DGGE analysis was used to detect mutations in exon 10, a mutation hot spot. A rare mutation, S466X, found in one patient, was the first case reported in the Turkish population. The DGGE migration pattern for the polymorphism 1540A/G was identified by DNA sequencing. It was confirmed with Hph I enzyme digestion and found with high frequency (37 per cent). Also, again by using Hph I digestion, the CF chromosomes were screened for an interesting mutation, 3849+10 kb C->T, which is associated with a mild type of disease. None of the patients were found to carry this mutation.
Beta thalassemia in Turkey: |distribution, diversity, evolution and phenotype-genotype correlations
(Thesis (Ph.D.)- Bogazici University. Institute for Graduate Studies in Science and Engineering, 1999., 1999.) Tadmouri, Ghazi Omar.; Başak, A. Nazlı.
The present study illustrates the results of years of research on different aspects of beta-thalassemia in Turkey. Methods to detect the C-T change at position -158 upstream of the Gy-globin gene and the (AT)xTy motif 5' to the beta-globin gene were established and implemented. Analysis of these polymorphisms explained the reason behind the increased levels of fetal hemoglobin in nine out of 31 beta-thalassemia patients analyzed and demonstrated a dominant effect exerted by the XmnI Gy-globin polymorphism. Molecular screening of beta-globin genes in 19 beta-thalassemia individuals by genomic DNA sequencing uncovered the presence of 14 mutations; three of these are seen for the first time in Turkey. Another achievement made during this study is the compilation of beta-globin gene data collected since 1988 in a single repository. This allowed an easy mean to investigate the distribution of beta-globin gene mutations in various regions and towns of Turkey. This also demonstrated that the distribution of beta-thalassemia mutant alleles differed within each geographical area with a decreased gradient of mutation numbers from the East to the West of Anatolia. Analysis of nine polymorphic nucleotides and the (AT)xTy motif 5' to the beta-globin gene in 204 non-related beta-globin genes from Turkey exhibited 12 sequence haplotypes. Samples from the Black Sea region demonstrated a remarkable level of genetic heterogeneity in contrast to the homogeneity in Central Anatolian samples. Of the 22 beta-globin mutations analyzed, 18 were related with single sequence haplotypes and each of the other four were associated with a minimum of two sequence haplotypes. Our results demonstrate that the heterozygote advantage against malaria in Anatolia may have occurred at 6500-2000 BC by the oldest beta-thalassemia allele (i.e.,IVS-I-110 G-A). From that date on, most of the common beta-thalassemia mutations in Turkey were established and by the 13th century AD most of them were brought to frequencies close to what is observed at present.
Bioinformatic and molecular analysis of olfactory receptor gene regulation in zebrafish
(Thesis (M.S.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 2015., 2015.) Kaya, Ahmet Burak.; Fuss, Stefan H.
In vertebrates, an individual olfactory sensory neuron (OSN) expresses only one allele of one olfactory receptor (OR) gene from a huge genomic repertoire. The molecular mechanism governing “one neuron-one receptor rule” are not fully understood and may be regulated by a combination of a variety of cellular mechanisms. Two OR locus-related regulatory mechanisms previously identified are the interaction of transcription factors with specific DNA-binding motifs as a short-range control and the activity of Locus Control Regions as long-range control. Curiously, several regulatory motifs such as Olf1/Ebf1, homeodomain (Lhx2/Emx2) and BPTF motifs were identified in promoter and LCR regions of mice. Here, RNA-Sequencing and bioinformatic analysis were performed in order to detect conserved regulatory motifs within zebrafish OR promoters. Transcript structures and expression levels of OR repertoire were obtained by analysis of transcriptome data. Also, TSS of 161 OR genes were resolved and promoter regions directly upstream of TSSs were identified. De novo motif search with motif-finding bioinformatic tools resulted in a zebrafish-specific motif which is highly similar to Ebf1. Investigation of known regulatory motifs Ebf1, TBP, Lhx2, Emx2 and BPTF indicated presence and distribution of these motifs in OR promoters. Next, a member of highly expressed OR gene family, OR132-5, was investigated with in situ hybridization and a correlation between FPKM levels and number of OSNs that express a given OR was observed. Furthermore, a candidate regulatory motif was identified in this highly expressed OR gene family.
Cardiovascular disease in the Turkish population: molecular analysis of the renin-angiotensin system and its associated genes with premature myocardial infarction
(Thesis (M.S.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2006., 2006.) Gencer, Pınar.; Başak, A. Nazlı.
Cardiovascular disease (CVD), which is a complex multifactorial disease with many environmental and genetic factors, is the leading cause of death worldwide. Extensive research is ongoing for the identification of the genetic risk factors with association studies between patients and controls. As a result, several polymorphisms in several genes were identified as risk factors for CVD, but the results are mostly not reproducible within different study groups. In the framework of this thesis, selected polymorphisms in angiotensin 1 receptor (AT1R), angiotensin converting enzyme (ACE), angiotensinogen (AGT), bradykinin B2 receptor (BKB2), endothelial nitric oxide synthase (eNOS) and G-protein beta-3 subunit (GNB3) genes were investigated to study the significance of the polymorphisms between myocardial infarction (MI) patients and healthy controls. Moreover, the LightCycler system was optimized for genotyping of the ACE and eNOS genes and the genotyping results wer compared with the conventional PCR and restriction enzyme (RE) analysis. Screening of 84 premature MI patients and 60 age-matched healthy controls with RE analysis, DNA sequencing and LightCycler did not demonstrate any significant association between the studied polymorphims in the six genes mentioned above and premature MI. However, this is the first study of its kind in Turkey, and we hope that it paves the way for more extensive studies with larger patient cohorts and healthy controls to unravel the complex interaction between genes and myocardial infarction.
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.
Characterization and regulation of human Kim-1 gene promoter the role of activator protein 1 (AP1) transchiption factor
(Thesis (M.S.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 2014., 2014.) Bergin, Tijen.; Yaman, İbrahim .
Kidney Injury Molecule-1 (KIM-1) is an immunoglobulin superfamily cell surface protein highly upregulated on the surface of dedifferentiated renal proximal tubule epithelial cells regenerating after toxic or ischemic injury. KIM-1 is known to be the most sensitive biomarker of kidney injury approved by FDA for preclinical safety studies in 2008. Therefore, characterization of the human Kim-1 gene promoter and the associated transcription factors regulating its transcription under toxic injury is very crucial. For this purpose, a human-derived proximal tubule epithelial cell line HK2 was utilized. Ochratoxin A (OTA), Gentamicin (GM) and Cisplatin (CP), which are known to induce nephrotoxicity, were used in in vitro HK2 cell culture system as chemotoxic stress inducers. Significant changes were observed in KIM-1 protein and mRNA amounts under chemotoxic stress as shown by Western Blot Analysis and Quantitative Real Time PCR, respectively. The minimal promoter region of Kim-1 gene was characterized by deletional mutation analysis of Kim-1 upstream region. AP1 protein, a well-known stress response transcription factor, was demonstrated to bind to the promoter region of Kim-1 gene by employing Electrophoretic Mobility Shift Assay (EMSA) and Chromatin Immunoprecipitation (ChIP) assay. However, luciferase reporter assays did not reveal any significant change between the wild type and the mutant AP1 binding site at -1010 position bearing plasmids in transient transfections. In addition, KIM-1 open reading frame (ORF) was cloned into bacterial and eukaryotic recombinant protein expression vector systems in order to produce KIM-1 antibody and reveal the functional role(s) of KIM-1 in human cells, respectively. Our results suggest that, the 700 bp upstream region of Kim-1 gene contains the minimal promoter region and Kim-1 gene might be regulated by the cooperative activity of AP1 with other trans-acting or cis-regulatory elements.
Characterization of a novel R7-specific gene in the drosophila visual system
(Thesis (M.S.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 2013., 2013.) Kaçmaz, Güner.; Çelik, Arzu.
The proper functioning of the nervous system depends on the correct specification of many different cell types and the establishment of proper connections between neurons during development. The visual system of Drosophila represents a good model to study these mechanisms. The Drosophila eye consists of 800 small eye-like structures called ommatidia each containing eight photoreceptors (PRs). Photoreceptors are grouped into two; outer and inner, and each expresses sensory receptor molecule called Rhodopsin (Rh). In an attempt to identify novel genes playing a role in photoreceptor specification, the enhancer trap line for CG7985 showing an R7-specific expression was selected. The aim of this thesis was to characterize this gene functionally to understand its role in visual system development. CG7985 was shown to encode a hexosaminidase enzyme that is conserved in evolution. Hexosaminidases are involved in the hydrolysis of GM2 gangliosides. The human homolog of CG7985, HexDC, has not been investigated extensively, while other well-studied homologs are represented by HexA and HexB, which give rise to diseases like Tay - Sachs and Sandhoff that are characterized by symptoms that include blindness, loss of motor function, paralysis and psychosis. As CG7985 has not been studied previously, we generated tools necessary for its functional analysis. Using Flp/FRT recombination a mutant has been generated. In addition, a transgenic line carrying a targeting construct for homologous recombination was generated. A UAS-line for ever-expression analyses was generated. To identify the endogenous expression of CG7985 a genomic BAC construct encoding a fusion protein CG7985::GFP has been engineered and transgenic flies carrying this construct have been analyzed. Initial analyses of the mutant did not reveal any phenotypes affecting Rh choice in the R7 cell. Western blot analysis of R7-specific and glycosylated proteins did not reveal any changes in the glycosylation pattern of these proteins. More studies are needed to elucidate the function of CG7985 protein.

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