Ph.D. Theses

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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.
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.
Parkinson's disease in Turkish patients: molecular defects in familial and isolated cases
(Thesis (Ph.D.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2009., 2009.) Pirkevi, Caroline Selma.; Başak, A. Nazlı.
Parkinson's disease (PD) is worldwide the second most common neurodegenerative disorder after Alzheimer's disease. Recently, several genes have been shown to result in PD phenotype. Mutations in these genes may lead to autosomal dominant ( -synuclein, LRRK2), autosomal recessive (Parkin, PINK1, DJ1) or sporadic PD. In the framework of this study, the above five genes were investigated in Turkish patients. For the Parkin gene, we have performed dHPLC analysis prior to sequencing. The high percentage of abnormal dHPLC profiles observed in the patient population revealed only one mutation and 136 polymorphisms. Exon rearrangements were investigated by semi-quantitative multiplex PCR and by MLPA; 12 rearrangements were described in the Parkin gene. PINK1 and DJ1 exons were also sequenced; 75 variations were identified, two being heterozygous mutations. Point mutations in -synuclein are a rare cause of PD and were not found in our patient population. In contrast, triplications and duplications of the whole gene are found more frequently in autosomal dominant forms of the disease. A heterozygous duplication of exons 3 and 4 was described in several patients of a large kindred, and the whole family was subjected to haplotype analysis for chromosome 4q. LRRK2 consists of 51 exons and most mutations are seen in 15 of these. Sequencing of these exons revealed a novel S1508R and a G2019S mutation. G2019S frequency differs among populations and it is associated with three different haplotypes so far. The haplotype of our patient has been shown to have a great homology with the Japanese haplotype. Although an independent origin of the Turkish haplotype cannot be excluded at this point, the huge and centurieslong migration of the Turkic people in Central Asia rather supports a common origin.
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.
Defining novel cancer genes, sage tags and co-regulated regions of the human genome evolving from the search and identification of novel Wnt/TCF/B-catenin targets
(Thesis (Ph.D.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2009., 2009.) Kavak, Erşen.; Koman, Ahmet.; İyison, Necla Birgül.
The Wnt signaling pathway is involved in many differentiation events during embryonic development and can lead to tumor formation after aberrant activation of its components. β-catenin, a cytoplasmic component, plays a major role in the transduction of canonical Wnt signaling. The first part of this study aimed at identifying novel genes that are regulated by active β-catenin/TCF signaling in hepatocellular carcinoma-derived Huh7 cells with high (transfected) and low β-catenin/TCF activities. High TCF activity Huh7 cells led to earlier and larger tumor formation when xenografted into nude mice. SAGE (Serial Analysis of Gene Expression), genome-wide microarray and in silico promoter analysis were performed in parallel, to compare gene expression between low and high β-catenin/TCF activity clones, and also those that had been rescued from the xenograft tumors. SAGE and genome-wide microarray data were compared and contrasted. BRI3 and HSF2 were identified as novel targets of Wnt/β-catenin signaling after combined analysis and confirming experiments including qRT-PCR, ChIP, luciferase assay and lithium treatment. In the course of analyzing SAGE and microarray data of Huh7 cells, we felt the need to overlay this data with different data sources. Starting with this motivation in the second part of the study, we performed the first meta-analysis of SAGE and microarray data together, which we termed common gene expression patterning (COGENT). By using COGENT data, we introduced the “being multi-cancer” concept for genes being differentially expressed in several types of tumors. There are not only multi-cancer genes but also multi-cancer co-regulated regions (RIDGEs) on the genome. “Multi-cancerness” of a gene correlates well with the number publications stating that gene in a cancer context, its rank within a single study and the number of its orthologs. There are many multi-cancer genes which have not been related to tumorigenesis as yet. In addition, there are 81 tumor associated RIDGEs (TA-RIDGEs) on the human genome which change similarly in multiple types of tumors.
Characterization of thermophilic gene expression enzymes
(Thesis (Ph.D.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2010., 2010.) Çağlayan, Melike.; Bilgin, Neşe.
The DNA polymerase I genes of seven newly identified Geobacillus species within the family Bacillaceae, were cloned, sequenced and overexpressed in Escherichia coli. The polA gene of these species encodes DNA polymerase I of 878 amino acid residue protein with a predicted molecular weight of 99.3 kDa. Similarity analyses suggested that DNA polymerases belong to family A polymerases and lack 3'-5' exonuclease activity. The complete coding sequences of the genes were submitted into the GenBank. The recombinant (His)6-tagged DNA polymerases were purified by Ni2+-affinity chromatography and the homogeneous proteins were obtained after TEV protease digestion. DNA polymerases from Geobacillus anatolicus (Gana DNApolI) and Geobacillus kaue strain NB (Gkaue DNApolI) were further characterized in vitro and optimum conditions with respect to temperature, pH, monovalent and divalent ions were determined. Geobacillus DNA polymerase I fragment (GF DNApolI) was cloned and purified after homology modeling using Bacillus DNA polymerase I fragment (BF) as the model protein structure. The accuracy of GF DNApolI was measured by two M13 based fidelity assays which score errors produced during in vitro DNA synthesis of the lacZα complementation gene in M13mp2 DNA at 37°C, 50°C and 72°C. Base substitution errors increase three-fold when temperature is raised from 37°C to 72°C. DNA sequencing of the phage mutants showed that some of the base substitutions are more temperature sensitive than others. The most common base substitution error is the misincorporation of dGMP opposite to template G. Single nucleotide incorporations for both correct and for incorrect nucleotides were also studied under single-turnover conditions at 22°C, 37°C and 50°C. For both correct and incorrect dNTP insertions, the rate of polymerization, kpol, increased (seven- and four-fold, respectively) when temperature is raised from 22°C to 50°C, whereas only a slight change in Kd was observed. As a result, kinetic efficiency of the enzyme (kpol/Kd) shows five-fold increase over this temperature range.
Gene hunt in four inherited diseases
(Thesis (Ph.D.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2010., 2010.) Çetinkaya, Murat.; Tolun, Aslı.
Genetic linkage analysis is applied to identify loci that harbor gene or genes associated with a disease. It is possible to map disease loci by observing alleles the segregation of microsatellite or SNP markers with the disease in families, it is possible to map disease loci. Availability of densely spaced microsatellite marker maps and genomewide SNP scans have made such studies feasible. In the framework of this thesis, genomewide linkage data were evaluated with parametric linkage analysis software and haplotype segregation studies for four inherited disorders. Furthermore, candidate loci identified were further investigated by fine-mapping and candidate gene approach. Autosomal Recessive Ataxia (ARA) is a subgroup of hereditary ataxias, and characterized by slowly progressive impaired coordination of gait, hands, eye movements and speech. Nine patients from two consanguineous families were included in the study. We found that Aprataxin gene was mutated in six patients. Three patients were homozygous for a known nonsense mutation, and three patients for a novel missense mutation. For the three remaining patients clinical reevaluation revealed that they were afflicted with another disease, but no responsible locus could be identified. Autosomal recessive Larsen Syndrome (LRS) is characterized by congenital largejoint dislocations and craniofacial abnormalities. Seven consanguineous families were analyzed. In four families disease was mapped to 17q25.3 and homozygous mutations were found in Calcium Activated Nucleotidase 1. Three patients had the same known missense mutation. In one family, a novel missense mutation was identified. No common candidate region could be defined for the remaining three families. Juvenile Parkinsonism (JP) is characterized by classical triad of parkinsonism signs, bradykinesia, rigidity and resting tremor and disease onset before the age of 40 years. A missense variant in AK3L1 was identified in homozygous state in all patients but also in a healthy individual in the family. This variant was not detected in any of the 130 control individuals. We propose that an undetermined second locus either contributes to the disease manifestation in patients or protects the homozygous healthy individual. Lastly, autosomal recessive Congenital Cerebellar Hypoplasia (CCLH), characterized by nonprogressive congenital cerebellar ataxia with mental retardation, delayed motor development, disturbed coordination, hypotonia and cerebellar Hypoplasia was studied in two families. In one of the families we mapped the disease to 6q16.1-22.1. No candidate gene could be defined in the region. In the other family, several candidate loci at other chromosomal regions were found.
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.
Mutation profile of hemophilia a patients with inhibitors and association of interleukin and cytokine gene polymorphisms with inhibitor development
(Thesis (Ph.D.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2010., 2010.) Fidancı, İnanç Değer.; Çağlayan, S. Hande.
Hemophilia A (HA) is an X linked recessive bleeding disorder characterized by qualitative and quantitative deficiency in the factor VIII (FVIII) protein, mainly caused by Factor 8 (F8) gene mutations. A severe complication in the replacement therapy of HA patients is the development of allo-antibodies (inhibitors) against FVIII which neutralize the substituted FVIII. Genetic risk factors along with F8 gene mutations influence the development of inhibitors. Interleukins and cytokines such as IL4, IL5, IL10, TGFB1 and IFNG that are involved in the regulation of B lymphocyte development are possible targets as other genetic risk factors. The aim of this dissertation was to reveal the F8 gene mutation profile of severe HA patients who developed inhibitors using various methods to assess the possible associations between 9 selected interleukin and cytokine gene polymorphisms with inhibitor development in HA patients with a null mutation in the F8 gene. The most prevalent mutation in inhibitor patients was intron 22 inversion followed by nonsense mutations and large deletions with major effects on FVIII function. Therefore, severe HA patients were screened for intron 22 inversion to constitute inhibitor (+) and inhibitor (–) patient subgroups to carry out a case-control association study. A significant association with the T-allele of rs2069812 located in IL5 gene promoter and patients with inhibitors was found with a p-value of 0.0251. The TT genotype was also significantly associated with the inhibitor (+) patient group with a p-value of 0.0082 and OR of about 7, suggesting that the T-allele as the recessive susceptibility allele and C-allele was the dominant protective allele. The present findings are highly informative about the role played by the polymorphisms in genes involved in B lymphocyte development as genetic risk factors in antibody development in severe HA patients with null mutations and paves the way for furthe studies in the field.
SIK2: a key player in FGF2-induced proliferation and insulin-induced survivale/hyperglycemia-dependent apoptosis in müller cells
(Thesis (Ph.D.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2011., 2011.) Küser, Gamze.; Bilge, Kuyaş Buğra.
Salt inducible kinase 2 (SIK2), a serine/threonine kinase, is primarily expressed in insulin-responsive tissues. It was suggested that SIK2 might be involved in type 2 diabetes by phosphorylating S789 residue of IRS1. In the initial experiments we observed rapid and transient increase of SIK2 activity upon FGF2 stimulation, confirming that the kinase is part of this pathway. Subsequently it was demonstrated that FGF2 dependent ERK and Akt activation was reduced significantly by SIK2 overexpression. SIK2 silencing, on the other hand, enhanced the intensity and the duration of active ERK and Akt levels, and led to a dramatic increase in FGF2-dependent Müller cell proliferation. Gab1 phosphorylation by SIK2 was verified in vitro, their interaction was revealed by coimmunoprecipitation. pSer of Gab1 was diminished significantly by SIK2 silencing. In the same time frame, pTyr levels and binding partner associations (p85/Gab1, Shp2/Gab1, Grb2/Gab1) were increased. Based on this data we propose that SIK2 involved in the negative feedback mechanisms acting on FGF/Ras/ERK and FGF/PI3K/Akt pathways and Müller cell proliferation at the level of Gab1 serine phosphorylation. In the context of insulin pathway we established enhanced tyrosine phosphorylation of IRS1, followed by Akt activation resulting in increased Müller cell survival. SIK2 knockdown leads to considerably earlier FGF2-dependent Akt activation. Its overexpression, hampers Akt activation and cell survival. Coimmunoprecipitation studies indicated IRS1 as an in vivo SIK2 substrate. We observed lower IRS1 expression and tyrosine phosphorylation, enhanced SIK2 activity/expression and downregulation of both basal and insulin-induced Akt activation in MIO-M1 cells under hyperglycemia. SIK2 gene silencing under hyperglycemia restored pAkt level, under normoglycemia in cells overexpressing SIK2, Akt phosphorylation was decreased and apoptosis increased. Based on these observations, we suggest that SIK2 is a negative modulator of insulin-induced IRS1/Akt mediated Müller glial survival pathway, which may contribute to the glial death observed in diabetes.
Regulation of human BFK
(Thesis (Ph.D.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2014., 2014.) Uğurlu, Serkan.; Özören, Nesrin.
Bcl-2 protein family members are critical regulators for apoptosis. Reduced levels of apoptotic Bcl-2 family members are detected in gastrointestinal cancers which are responsible for a considerable part of the deaths from cancer. BFK (Bcl-2 Family Kin) is a novel pro-apoptotic Bcl-2 family member specifically expressed in the human gastrointestinal tract. BFK has the characteristic BH3 domain, which was shown to be essential for the apoptosis inducing activity of pro-apoptotic Bcl-2 family members. In the colon four alternatively spliced isoforms were identified. Human and mouse BFK genes share 70% homology at the DNA level and 68.7% homology at the aminoacid levels. Interestingly, human and mouse BFK genes show distinct expression patterns. To explain these differences, we performed gene evolution analyses on the promoter region as well as coding region of these genes. We found that the human BFK promoter experienced positive selective pressure and acquired a distinct set of repetetive elements and transcription factor binding sites. In this study, we identified several novel transcription factor candidates which may have roles in the transcriptional regulation of human BFK. As transcription factors, PARbZIP family members (especially TEF and NFIL3) regulate BFK upon binding to its promoter region. NFIL3 supresses TEF induced BFK transcription in HCT116 cells. We also studied hormonal regulation of human BFK. Tamoxifen, as a mixed agonist/antagonist of estrogen, upregulates human BFK levels in SW707 cells. We hope this study contributes to a better understanding of Bcl-2 family.
Parkinson's disease in a large Turkish pedigree with SNCA duplication (PARK4): complexin-1 as a potent biomarker for predictive diagnosis
(Thesis (Ph.D.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2015., 2015.) Lahut, Suna.; Başak, A. Nazlı.; Auburger, Georg.
Accumulation and aggregation of alpha-synuclein (SNCA) is a hallmark of Parkinson’s disease (PD). Considering the roles of SNCA in both idiopathic and familial PD, we studied blood samples from a large pedigree with SNCA duplication (PARK4), to identify effects of SNCA gain-of-function on expression levels of downstream genes as potential disease biomarkers. Downregulation of complexin-1 (CPLX1) expression level was found to be correlated with PARK4 and also the further investigated cohort with REM sleep behavior disorder (RBD), which resembles presymptomatic PD. In global RNAseq profiling of blood from presymptomatic PARK4, significant upregulations for immune system, lysosome, lipid and platelet activation pathways were detected. The representative genes of upregulated pathways, SPP1, GZMH, and PLTP, were validated in PARK4. However, unlike CPLX1, they failed to distinguish presymptomatic PD from healthy individuals. The longest size variant (allele 2) of the Rep1 repeat region of the SNCA promoter is known to be associated with PD risk. This region was analyzed in idiopathic PD and restless leg syndrome (RLS). Rep1 allele 2 frequency was found significantly decreased in RLS, suggesting reduced SNCA levels contributing to disease. The WW Domain Containing E3 Ubiquitin Protein Ligase (WWP2) mRNA expression level was tested in PARK4 and RBD and was observed to mimic SNCA expression profiles in both cohorts. Western blot analyses of double transfected cells suggested that in the presence of WWP2, SNCA-wildtype, but not SNCA-A53T-mutant, to be degraded.
SIK2 functions as a novel tumor suppressor in the breast tumorigenesis
(Thesis (Ph.D.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2015., 2015.) Zohrap, Neslihan.; Buğra, Kuyaş.
Breast cancer has highly malignant phenotype and is the leading cause of cancer death in women. The heterogenous character of the disease points to the importance of novel target identification for the development of effective therapies. Perturbation in receptor tyrosine kinase (RTK) pathway elements are frequently implicated in cancer. Our group has shown that SIK2 functions downstream of FGFR signaling, and control proliferation and survival. Given the importance of strict control of RTK pathways in cancer, in this study, we aimed to investigate the potential role of SIK2 in the development of tumorigenicity. In this context, screening of a cDNA array indicated downregulation of SIK2 transcript levels in breast cancer. A query into Oncomine database validated frequent reduction of SIK2 expression and loss of copy number in dataset covering a large breast cancer patient cohort. Immunohistochemical studies localized SIK2 in ductal epithelia of breast tissue and showed that SIK2 levels decline in all triple negative breast cancer (TNBC) cases and in half of the hormone positive tumors. These studies also indicated that inverse correlation exists between SIK2 and Ki67 indicating a negative effect of SIK2 on mitotic potential. Reduced SIK2 expression in breast tumor cell lines was also evident. Modulation of SIK2 expression in MDA-MB-231 and MCF12A breast lines suggested that SIK2 negatively regulate proliferation and survival by preventing ERK and Akt activation. In this context, its kinase activity appears to be required. In vitro studies also indicate that SIK2 may also be involved in the regulation of cellular adhesion and invasion. Xenografting experiments provided strong evidence that SIK2 hampers tumor growth through downregulation of proliferation and survival. In the light of these findings, we propose SIK2 as a novel tumor suppressor, loss of its expression/activity promote breast tumorigenesis.
Transcriptome analysis of common bean under salt stress and functional study of salt responsive genes
(Thesis (Ph.D.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2015., 2015.) Hız, Mahmut Can.; Türet, Müge.
Salinity is a major abiotic stress that impairs physiology and productivity of economically important crops such as common bean. It is an important grain legume and provides 30% of the protein diet in developing countries. Several common bean genotypes have been phenotypically classified according to its diverse responses to salinity. In this study, a comprehensive high-throughput transcriptome analysis in root and leaf tissues of common bean genotypes with contrasting salt tolerance was performed under stress in hydroponic conditions. Prior to transcriptome analysis, the stress onset time was determined as the end of 3rd day of 125mM NaCl application for tolerant Ispir and susceptible TR43477 varieties following several physiological measurements. Transcriptome analysis have generated 255 million high quality reads which were assembled into 73762 all-unigenes with a mean length of 930 bp. Among the all-unigenes, 75% (55433) were assigned with Nr annotations. A total of 12001 differentially expressed genes (DEGs) were identified. Validation of the RNA-seq quantifications (RPKM values) were performed by qRT-PCR analysis. Within the selected candidate gene pool with contrasting expression levels between the two genotypes and two tissues, five genes (Unc2, Unc4, TT12, CPRD2 and β-Glu) were cloned and transformed to Arabidopsis Col-0 ecotype to generate homozygous overexpression transgenic lines. Functional studies on these lines by both physiological assays and their impact on the expression of salt responsive genes as well as the transcription factors (TFs) regulating them, suggested a highly significant regulatory role for TT12 and CPRD2 genes in improvement of salt tolerance. Major contributions of these genes were implied for better management of osmotic and ionic imbalance most probably by imposing synthesis, accumulation and sequestration of osmolytes and reinforcement of the cell wall structure while minimizing damage through ROS scavenging. To do so, regulation by interacting ABA -independent and ABA-dependent expression of DREB2A, MYB2 TFs and downstream P5CS1 and RD29B genes were also implicated.
Elucidating the role of unzipped in the visual system of drosophila
(Thesis (Ph.D.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2015., 2015.) Terzioğlu Kara, Ece.; Çelik, Arzu.
The Drosophila eye is composed of 800 ommatidial units, each containing eight photoreceptors (PR). The outer PRs R1-R6 are responsible for motion detection, while the inner PRs R7 and R8 and are responsible for colour vision. The outer PRs express Rhodopsin1 (Rh), while the inner PRs express Rh3-Rh6. The pale subtype is formed when the R7 cells express Rh3 and the coupled R8 cells express Rh5, while in the yellow subtype R7 cells express Rh4 and the coupled R8 cells express Rh6. This final state of differentiation is reached after a sequential recruitment process of PRs at the 3rd instar larval stage. At the initial stage, the PR cells start differentiating and their axons target to their temporal layers. At the terminal stage the inner and outer PRs are distinguished from each other, each PR expresses a specicific Rh molecule, and the axons target to their final layers in the optic lobe. The aim of this study was to identify new molecules involved in inner PR differentiation, mainly focusing on three genes expressed in R8 cells that have been identified in a previously performed enhancer-trap screen. One of these genes, unzipped was chosen for further analysis because of its function as cell adhesion molecule (CAM). By immunostainings the expression of Uzip was revealed to be in R8 cells and a subgroup of glial cells at the 3rd instar larval stage, R8 cells and glial cells at the 48 hour pupa stage, and glial cells in the adult stage where some of them project to the medulla. Immunostainings showed that Uzip does not have a direct effect on PR formation and glia expression at the 3rd instar larvae and pupae stages; or on subtype specification at the adult stage. For axonal targeting both the downregulation of Uzip in the target area and the misexpression in neighboring PRs and glia, which are not in the target region, lead to an increase in axonal misprojection phenotype. According to our suggested model, Uzip expressing R8 cells follow the adhesive signal from the Uzip expressing glial cells at the target region of projection. This study identifies Unzipped and describes its expression and involvement in axonal targeting the visual system.
Molecular and cellular development of cortical projection neurons: specification, diversity, and directed differentiation from endogenous progenitors for functional circuit repair
(Thesis (Ph.D.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2016., 2016.) Özkan, Abdulkadir.; Battaloğlu, Esra.; Macklis, Jeffery D..
Responsible for perception, integration of sensory information, cognitive function, motor control, and consciousness, the complex, yet highly organized, six-layered mammalian neocortex contains distinct classes of neurons. Specific subtypes of cortical projection neurons are selectively vulnerable in distinct neurodegenerative, developmental, and acquired diseases of the central nervous system (CNS), resulting in irreversible functional deficits. Evidence for the existence of progenitors in restricted regions of the adult brain, and integration of new neurons into preexisting neural circuitry, support the feasibility of cellular repair in the CNS. However, functional repair of diseased or injured neuronal circuitry requires detailed understanding of molecular controls over development of neuronal lineages, and manipulation of these controls in progenitors to direct the differentiation of functional neurons with appropriate identity, maturity and circuit connectivity. In this study, I target endogenous cortical progenitors present in postnatal and adult brain to direct their differentiation into corticofugal projection neurons. Application of a select combination of central and complementary transcriptional controls, Ngn2, VP16:Olig2 and Fezf2, in cultured cortical Sox6+/NG2+ progenitors directs acquisition of cardinal morphological, molecular, and electrophysiological features of corticofugal projection neurons. These findings demonstrate the feasibility of achieving subtype-specific differentiation of cortical projection neurons from a widely distributed in vivo neocortical progenitor population. Further, in the framework of this thesis, I describe the ongoing effort to identify key molecular controls over development, diversity and connectivity of corticostriatal projection neurons, which would serve as a solid step toward achieving a holistic view of the establishment of corticostriatal circuitry and its potential dysgenesis in disease.
Neurogenesis and migration of specified chemosensory neurons in the adult zebrafish olfactory epithelium
(Thesis (Ph.D.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2016., 2016.) Bayramlı, Khalid.; Fuss, Stefan H.
In the framework of this study, a systematic and quantitative analysis of the neurogenic process and the integration of adult-born olfactory sensory neurons (OSNs) into the zebrafish olfactory epithelium (OE) was performed. In the postlarval zebrafish OE OSNs are generated from two distinct proliferation zones at the central and peripheral margin of the sensory tissue. Temporal birthdating, using the proliferation marker BrdU reveals centrifugal migration of newly generated cells from the inner proliferation zones located at the interlamellar curves (ILCs) and centripetal migration of cells from the peripheral neurogenic zone at the sensory / non-sensory (S/NS) border. In addition to radial migration a basal to apical ascend was observed over time. Neuronal progenitor cells expressing proneuronal genes indicative of different stages of cellular differentiation and neuronal maturation could be visualized at or close to mitotically active zones at the ILC and S/NS. Fate tracking of new born OSNs at different time points following BrdU incubation showed that OSNs are rather immobile during early differentiation steps and complete neuronal maturation before completing migration. Analysis of newly generated OSNs expressing different chemosensory receptors demonstrated that both proliferation zones at the ILC and S/NS contribute to the generation of different OSN subpopulations albeit with a quantitative biases. A model is proposed in which adult born OSNs turn on chemoreceptor expression close to their site of birth, migrate radially across the sensory OE and over time undergo cell death, creating the impression of defined ring-like expression domains. A computational model based on quantitative parameters extracted from the analysis of the temporal behaviour of new born OSNs was developed to test whether physical and ontogenetic parameters such as speed of OSN migration, birth rate of the progenitors in the proliferation zones, maturating timing and the lifespan of the OSNs are sufficient to mimic the expression profiles of OR genes.
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.
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.

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