Doktora Tezleri

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Computational establishment of microRNA metabolic networks
(Izmir Institute of Technology, 2017-06) Saçar Demirci, Müşerref Duygu; Allmer, Jens
MicroRNAs (miRNAs) are single-stranded, small, non-coding RNAs, that control gene expression at the post transcriptional level through various mechanisms such as translational inhibition, degradation and destabilisation of their target mRNAs. Despite the fact that thousands of miRNAs have been reported in various species, most still remain unknown. Due to this, the identification of new miRNAs is an essential process for analysing miRNA mediated post transcriptional regulation mechanisms. Moreover, many biological approaches suffer from limitations in their capacity to reveal rare miRNAs, and are further restricted to the state of the organism under examination. Such limitations have resulted in the construction of sophisticated computational tools for identification of possible miRNAs in silico. However, these programs suffer from low sensitivity and/or accuracy and as a result they do not provide enough confidence for validating all their predictions experimentally. In this study, the aim is overcoming these challenges by creating a new and adaptable machine learning based method to predict potential miRNAs in any given sequence. The efficiency of proposed method is shown by comparison with available tools on various data sets. By using this approach, miRNAs from the genomes of various organisms like human (Homo sapiens), fly (Drosophila melanogaster) and tomato (Solanum lycopersicum) are identified. Moreover, networks between the possible miRNAs of virus and human genes as well as the communications among nuclear and organelle genomes of Solanum lycopersicum through miRNAs are investigated.
Detection of the metastatic potential of breast cancer cell lines to specific target tissues
(Izmir Institute of Technology, 2021-03) Fıratlıgil Yıldırır, Burcu; Yalçın Özuysal, Özden; Izmir Institute of Technology
Breast cancer is one of the most frequently diagnosed cancer types and the second leading cause of cancer-associated deaths in women. Breast cancer begins as a local disease which can then metastasize to distant sites specifically to bone, lung and liver. The increasing rate of the metastasis-related deaths asserts the need to develop in vitro diagnostic strategies representing in vivo properties better. In this study, two different lab-on-a-chip (LOC) platforms, IC- and EX-chips, were used to detect the invasion and extravasation potentials, respectively, of breast cancer cells to 3D in vitro generated bone, lung, liver and breast microenvironments. The metastatic MDAMB231, but not non-metastatic MCF7 breast cancer cells showed higher invasion and extravasation potentials towards lung and liver microenvironments than breast microenvironment. Lung-specific but not bone-specific metastatic subclonal cells invaded significantly towards lung microenvironment. On the other hand, an intensive invasion was observed in bone-specific but not lung-specific metastatic subclonal cells towards bone microenvironment demonstrating different in vivo metastatic behaviors of breast cancer cells. Overall, the tissue-specific invasion and extravasation capacities of breast cancer cells were demonstrated with IC- and EX-chips where the physiologically more relevant bone, lung, liver and breast homing target sites were generated by a specific emphasis on ECM components, stromal cells and secreted factors. This study is important in providing a basis for the development of diagnostic tools and precision therapeutics for breast cancer metastasis.
Development of sequence based markers for molecular genetic analysis in sesame (Sesamum indicum L.)
(Izmir Institute of Technology, 2015-06) Uncu, Ayşe Özgür; Frary, Anne
Sesame (Sesamum indicum L.) is an orphan crop with most molecular genetic research work done in the last decade. In this study, pyrosequencing was used for the development of genomic SSR (Simple Sequence Repeat) markers in sesame. The approach proved successful in identifying 19,816 SSRs, 5727 of which were identified in a contig assembly that covers 19.29% of the sesame genome. As a result of this work, 933 experimentally validated sesame specific markers were introduced, 849 of which are applicable in Sesamum mulayanum, the wild progenitor of cultivated sesame. Using a subset of SSR markers, molecular genetic diversity and population structure of a collection of world accessions were analyzed. Results of the analyses revealed a pattern of gene flow among sesame diversity centers. Taken together with the high rate of genomic marker transferability between S. indicum and S. mulayanum, the results provide molecular genetic evidence for designating the two taxa as cultivated and wild forms of the same species.In related work, a Genotyping By Sequencing (GBS) approach was applied on recombinant inbred lines for single nucleotide polymorphism (SNP) identification and mapping in the sesame genome. As a result, 15,521 SNPs were identified and a high-resolution genetic linkage map was constructed using a core set of selected SNPs (781 SNPs) appropriate for use in linkage analysis. The 15,521 putative SNP markers represent a substantial contribution to the existing pool of sesame-specific markers. The genetic linkage map constructed in this work will enable the identification of loci involved in the genetic control of agriculturally important traits in sesame.
Development of single nucleotide polymorphism markes for fingerprint analysis of Turkish olive (Olea europaea L.) cultivars and detection of adulteration in Turkish olive oil
(Izmir Institute of Technology, 2015-06) Uncu, Ali Tevfik; Doğanlar, Sami
Olive (Olea europaea L.) tree and oil are signature figures of the Mediterranean culture. Because of its high economic value, olive oil is extremely vulnerable to fraud. The aim of this study was to develop molecular tests for authenticating cultivar and botanical origin in olive oils. In order to authenticate the botanical origin and detect adulteration, a plastid DNA region was utilized for standardizing a capillary-electrophoresis barcode assay. The performance of the assay was evaluated on series of olive oil : seed oil admixtures. The assay proved successful in identifying seed oils in olive oil down to a limit of 10%. The molecular assay described in this work enables adulteration detection regardless of compositional similarities between the adulterant and adulterated oil species, thus will complement the shortcomings of analytical chemistry approaches. In order to establish a DNA-based identification key to ascertain the cultivar origin of Turkish monovarietal olive oils, short fragments from five olive genes were sequenced for SNP (Single Nucleotide Polymorphism) identification. CAPS (Cleaved Amplified Polymorphic DNA) assays were designed for SNPs that alter restriction enzyme recognition motifs. When applied on the oils of 17 olive cultivars, a maximum of five CAPS assays were necessary to discriminate the varietal origin of the samples. Admixture detection threshold of the assays was identified as 20% when tested on olive oil admixtures. The SNP-based CAPS assays developed in this work can be used for testing and verification of the authenticity of Turkish monovarietal olive oils, for olive tree certification, and in germplasm characterization and preservation studies.
Development of tomato plants over-expressing cytokinin synthesis gene and characterization by proteomic approach
(Izmir Institute of Technology, 2020-01) Şelale, Hatice; Frary, Anne
Cytokinins (CKs) are plant hormones controlling growth and development including cell division and differentiation, apical dominance and delay of senescence. CKs take part in regulation of the abiotic stress response in plants. In this study, transgenic tomato plants overexpressing the IPT (CK biosynthetic gene) were developed. Homozygous transgenic plants exhibited a phenotype with reduced plant stature and lost apical dominance. Increased shoot biomass and leaf water content with a reduction in fruit yield were observed in all transgenic lines. Proteomics analysis was conducted to understand high CK response in molecular level. Proteins supporting a strong sink phenotype and vasculature development were upregulated in transgenic lines and reflected the phenotypic changes observed in homozygous plants. Proteins related to stress response such as detoxification enzymes and PR proteins were upregulated in a gradual manner in transgenic lines with the strongest up-regulation in T6 homozygous line indicating the metabolic stress induced by high CK levels. The transgenic plants were tested for drought stress and observed to have improved water use efficiency, antioxidant response and delayed senescence compared to nontransgenic plants. Proteomic analyses from leaf total and nuclear enriched extracts were conducted to understand the molecular basis of improved drought tolerance. Proteins related to photosynthesis and oxidative stress response were the most prominent groups of differentially abundant proteins in the transgenic line under drought, which could contribute to tolerance. Eighteen transcription factors were differentially abundant in the nuclear proteome of drought stressed plants. These transcription factors could control the gene expression contributing to tolerance.
Elucidation of boron stress signaling pathways in yeast
(Izmir Institute of Technology, 2015-12) Uluışık, İrem; Koç, Ahmet
Boron is an essential micronutrient not only for plants but also for many other organisms. The excess of boron causes toxicity and the mechanism of this toxicity is not known. The yeast Saccharomyces cerevisiae was used as a model system in this study. In order to reveal boron metabolism related genes, a genome-wide screen has been conducted. Among the identified mutants, six boron resistant and eight boron sensitive mutants were chosen for further investigation to understand how cells cope with boron stress. Boron resistant mutants were found to have increased levels of boron efflux pump ATR1 and its transcription activator Gcn4. The sensitive mutants were lacking the genes that are involved in different cellular pathways. They were found to accumulate higher amounts of boron inside the cells upon boron treatment. To reveal how boron stress is conducted to Gcn4 transcription factor, the deletion mutants of transcription factors that are known to regulate GCN4 were investigated in terms of their effects on Gcn4 and ATR1 expression. Additionally, signaling cascades that converge on Gcn4 transcription factor such as TOR, PKA, and SNF1 pathways were analyzed for their roles in boron stress response mechanism. We found that the Gcn system is activated by the uncharged tRNA stress in response to boron treatment and that GCN1, which plays a role in transferring uncharged tRNAs to Gcn2, was necessary for the kinase activity of Gcn2. Additionally, boron treatment caused the phosphorylation of eIF2α in mammalian cells, in a similar manner to that of yeast cells, which suggested that boron toxicity and tolerance mechanisms were conserved between yeast and mammals.
Elucidation of boron tolerance mechanisms in Puccinellia distans (Jacp.) Parl. using a transcriptomic approach
(Izmir Institute of Technology, 2017-06) Öztürk, Saniye Elvan; Frary, Anne
The amount of boron in soil is important for agronomic plants. As an abiotic stress condition, boron toxicity causes significant decreases in crop yields. Puccinellia distans (Jacq.) Parl. (P. distans), common alkali grass, is found throughout the world and can survive in soils with boron concentrations that are lethal for other plant species. Indeed, P. distans accumulates very high levels of this element. Despite these interesting features, very little research has been performed to elucidate the boron tolerance mechanism in this species. In this study, P. distans samples were analyzed by RNA sequencing to identify genes and miRNAs related to boron tolerance and hyperaccumulation. The results indicated that the hyperaccumulation mechanism of P. distans involves many transcriptomic changes including: alterations in the malate pathway, changes in cell wall components that may allow sequestration of excess boron without toxic effects, and increased expression of at least one putative boron transporter and two putative aquaporins. MiRNAs are also altered under stress conditions. The presence of miRNAs as stress regulator elements is an example of post-transcriptional regulation of stress related mechanisms. Additionally these small RNAs could affect their target genes by positive or negative regulation. Therefore, changes not only in miRNAs but also in their targets are important to understand their roles in hyperaccumulation. For example, downregulation of miRNA under stress could cause target accumulation. These mechanisms could be key in plant adaptation to new conditions. Elucidation of the boron accumulation mechanism is important in developing approaches for bioremediation of boron contaminated soils.
Enhancement and validation of current human genome annotation via novel proteogenomics algorithms
(Izmir Institute of Technology, 2016-12) Has, Canan; Allmer, Jens
Proteogenomics includes the transfer of knowledge from proteomics to genomics and vice versa. To have high confidence in the information transferred it is essential that it be based on experimental results. Genomics is currently fueled by high throughput techniques involving next generation sequencing. Proteomics is based on mass spectrometry (MS) which is also a high throughput approach. Both fields are generating a wealth of data which needs to be correlated and annotated to generate knowledge. Publicly available human blood plasma mass spectrometric data exist for samples in data repositories such as PeptideAtlas, PRIDE. We acquired high-quality collections from this data and stored it in a custom database developed by us. First, we aimed to amend this data by employing a proteogenomic pipeline PGMiner developed in this study against a custom sequence database which includes all predicted alternative open reading frames as well as the six-frame translation of the human genome and exosome. Then, we correlated the existing annotations with the available mass spectrometric measurements. The human genome in tandem with currently available genome annotations from HAVANA and ENSEMBL enabled us to validate and enhance current gene annotations.
Expression levels of bioactive sphingolipid genes in newly diagnosed and drug-resistant chronic myeloid leukemia patients and their impact on the clinical progress
(Izmir Institute of Technology, 2015-01) Kartal Yandım, Melis; Baran, Yusuf
Bioactive sphingolipids are a family of lipids including ceramide, glucosylceramide (GC), sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P) that have important functions in cellular processes including proliferation, metastasis, invasion, inflammatory response and apoptosis. Many sphingolipidregulated functions are directly related to cancer initiation, progression, and response or resistance to anti-cancer treatments. Ceramide, the central molecule of the sphingolipid metabolism, functions as a tumor-suppressor inhibiting cell division, and inducing cell differentiation, senescence and apoptosis. De novo synthesis of ceramides is regulated by ceramide synthase gene family (CERS1-6). Although ceramide is known to be a pro-apoptotic molecule, GC and S1P which are converted from ceramides by glucosylceramide synthase (GCS) and sphingosine kinase-1 (SK-1), respectively, are anti-apoptotic. Chronic myeloid leukemia is a hematological disorder arisen from the reciprocal translocation between BCR gene on chromosome 22, and ABL gene on chromosome 9, t(9;22)(q34;q11), resulting in the formation of Philadelphia (Ph) chromosome. Ph chromosome encodes BCR/ABL fusion protein having constitutively active tyrosine kinase activity. In this study, we examined the expression levels of CERS1-6, GCS, SK1, and BCR/ABL genes of 66 patients that are newly diagnosed, tyrosine kinase inhibitor (TKI)-resistant, or -sensitive. Q-PCR results showed that there were higher expression levels of apoptotic CERS1-6 in the patients TKI-treated and have shown minimum hematological response than that of the patients newly diagnosed and TKI-resistant. However, expression levels of antiapoptotic GCS and SK-1 genes were significantly higher in TKI-resistant and blastic phase patients than that of the other patients. Additionally, BCR/ABL expression levels were higher in newly diagnosed and TKIresistant patients.
Identification of doxorubicin drug resistance mechanisms by using genomic techniques
(Izmir Institute of Technology, 2015-01) Demir, Ayşe Banu; Koç, Ahmet
Chemotherapy has been an important contributor for the treatment of cancer patients for a long time. The effectiveness of the therapies is influenced from the toxicity effects of the agents on normal cells and from the drug resistance. Therapeutic resistance is believed to cause the failure of the chemotherapy effectiveness in most cancer cases. Therefore, understanding the molecular mechanisms that underlie the drug resistance may contribute to increase the effectiveness of the chemotherapeutic treatment of cancer. Doxorubicin is a natural product that is widely used in treatment of various cancer types, yet many tumors have resistance against these agents. By using the budding yeast Saccharomyces cerevisiae as a model organism, we performed genome-wide screenings to identify the genes that cause resistance against this agent. Overexpression of CUE5, AKL1, CAN1, YHR177W and PDR5 genes have been identified to cause resistance against Doxorubicin at higher concentrations than the identified toxic level. Among these genes, only PDR5 overexpression was found to have cross-resistance to Cisplatin. Real-time PCR and microarray analysis for these genes were also performed. Upon 80μM Doxorubicin treatment for 2 hours, none of the CUE5, AKL1, CAN1, YHR177W and PDR5 genes showed expression changes compared to their correponding untreated wild-type status. Therefore, overexpression of these genes may not be a physiological response of yeast cells against Doxorubicin. Genome-wide microarray analysis showed changes in several cellular and biological functions upon Doxorubicin treatment. Identified genes mainly function in general stress response related events such as, filamentous growth, protein ubiquitination, autophagy, changes in membrane transportation and metabolic processes.
Identification of molecular resistance mechanisms against systemically used antifungal drugs, amphotericin B and caspofungin
(Izmir Institute of Technology, 2018-06) Balkan, Çiğdem; Arslanoğlu, Alper
The invasive fungal infections have been evolving for the last few decades and become a crucial factor that negatively affects the survival ability of the patients. Moreover, developing new resistance mechanisms of infectious organisms to the antifungal drugs make this problem more critical. In this study, we identified four different genes PDR16 and PMP3, RMD9 and SWH1which are resistant to amphotericin B and caspofungin, respectively, in Saccharomyces cerevisiae. We also screened whole yeast genome at transcriptional level via microarray analysis and identified metabolic pathways affected by these antifungal drugs. In addition, we applied several characterization methods including physiological and morphological analyses to understand the resistance mechanisms. We found that rmd9 mutants were extremely hyperpolarized and pdr16 and pmp3 mutants were depolarized when compared to control group. In contrast, overexpression of SWH1 increased the membrane potential of wild type cells. Further analyses showed that the absence of RMD9 disrupts the cellular morphology. To show that these genes are clinically important, Candida albicans orthologs of PDR16 and PMP3 were cloned and their roles in amphotericin B drug resistance were confirmed. RMD9 and SWH1 have no known C. albicans orthologs, so the confirmation with pathogenic yeast genome could not be performed against caspofungin.
Immunohistochemical, biochemical and imaging-mass spectrometric analysis of brain tissues of mice with combined deficiencies of ß-hexosaminidase a, sialidase Neu4 and GM2-ap
(Izmir Institute of Technology, 2017-12) Timur, Zehra Kevser; Seyrantepe, Volkan
Gangliosides are complex glycosphingolipids derived from glucosylceramide or galactosylceramide and contain sialic acid in their carbohydrate chain. Sialidases, known also as neurominidases, are a family of glycohydrolytic enzymes functioning in the catabolism of sialoglycoconjugates by removing α-glycosidically linked sialic acid residues. Sialidase Neu4 is the lysosomal sialidase and GM2 activator protein is the cofactor of β-Hexosaminidase a enzyme to degrade the GM2 ganglioside. The activity of sialidase Neu4 activity against GD1a and GM2 gangliosides were significantly increased by the co-transfection with GM2 activator protein to the cells transfected with sialidase Neu4. This in vitro study revealed that lipid-binding proteins can facilitate the glycolipid degradation rendered by sialidase Neu4 in the lysosomes. In the concept of this study, a systematic comparison of the storage levels of gangliosides, gene expression ratios and behavioral features of new double (Hexa-/-GM2AP-/- and GM2AP-/-Neu4-/-) and triple (Hexa-/-GM2AP-/-Neu4-/-) knockout mice models with the existing double (Neu4-/-Hexa-/-) and single (Hexa-/-) knockout models revealed the possible involvement of the GM2 activator protein as a cofactor of sialidase Neu4 in the bypass mechanism in the Tay-Sachs mice, in vivo. Based on the increased GM2 ganglioside level in brain and cerebellum detected by the immunohistochemical and imaging mass spectrometric analysis, we speculate that the sialidase Neu4 functions on the degradation of GM2 ganglioside with GM2 activator protein, in vivo.
Investigating molecular mechanisms underlying resistance to notch inhibitors in breast and ovarian cancer
(2022-11) Telli, Kübra; Yalçın Özuysal, Özden
Breast and ovarian cancers remain highly malignant among women with more than 11% overall of incidence rates worldwide. Traditional treatment strategies including chemotherapy, radiotherapy and hormone therapies continues to be successful yet for the long-term, cancer recurrence and drug resistance remains to be the main issue. In addition to the altering common cell fate regulations, cancer cells modify signaling pathways to overcome cytotoxicity. Notch signalling pathway is a conserved ligand-receptor pathway that necessarily plays role in survival homeostasis, yet it is dysregulated in various cancers. Currently, novel treatment strategies are targeting this pathway through Gamma Secretase Inhibitors (GSI) DAPT, R04929097 and MK0752 that are use both as a single agent and in combinations with Docetaxel or Cisplatin. The clinical success of these inhibitors requires further examination of potential intrinsic or acquired resistance profiles. In this study, we generated breast cancer cells (MDA-MB-231 and MCF-7) resistant to DAPT or R04929097 and ovarian cancer cells (IGROV-1, BG-1, SKOV-3 and A2780) resistant to MK0752 by gradual treatments of increasing doses based on drugs’ IC50 values. Morphological changes, growth rates, migration alterations, mRNA expressions of Notch pathway components and epithelial mesenchymal transition markers, 3D setups for acidosis responses and protein expressions for c-myc and oxidative stress response markers were analyzed. Furthermore, proteomic analysis was carried out with the ovarian cancer cell line IGROV-1. The response of the cells to different drug treatments and dysregulated protein families exposed in resistance mechanisms behind DAPT, R04929097 and MK0752 for both breast and ovarian cancer cells are reported. Overall, this study reveals possible resistance mechanisms against GSIs and emphasizes potential targets through well-known hallmarks of cancer drug resistance.
Investigating the role of connexin 32 in breast cancer
(Izmir Institute of Technology, 2020-07) Uğur, Deniz; Meşe Özçivici, Gülistan; Izmir Institute of Technology
Connexins (Cx) are primary components of gap junctions, selectively allowing molecules to be exchanged between adjacent cells. Along with their channel forming functions, connexins play variety of roles in different stages in tumorigenesis, both dependent and independent of gap junctions in connexin and cancer dependent manner. Cytoplasmic accumulation of Cx32 was shown in some breast cancers; and compared to the primary tumors Cx32 is further upregulated in metastasis. However, the complete picture for the role of Cx32 in breast cancer remains to be elusive. Through overexpressing Cx32, its functions in breast cancer cells were investigated in Hs578T and MCF7 breast cancer cells. Cx32 overexpression increased cellular proliferation with significant increase in S phase in Hs578T cells with no significant change on MCF7 cells. Cx32 overexpression did not induce hemichannel activity in neither cell; it reduced gap junctional functions in Hs578T cells. Cx32 in both cells localized in cytoplasm did not form intercellular plaques, and decreased Cx43 expression. Cx32 overexpression reduced the migration and invasion capacity in both cells and in Hs578T cells showed reduction of mesenchymal and increase of epithelial marker expressions. In conclusion, Cx32 increases proliferation and decreases communication in Hs578T cells while not affecting MCF7 cells. It decreases aggressiveness and metastatic potential for both cell lines. Due to changes in gap junctional functions, Cx32 might be acting in relation to GJIC in Hs578T cells and outside of it in MCF7 cells. All in all, presence of Cx32 made Hs578T cells act similar to endogenously Cx32 expressing MCF7 cells.
Investigation of molecular effect of phenolic acids on methicillin-resistant and methicillin-susceptible Staphylococcus aureus in comparison to their phenolic acid resistant mutants
(Izmir Institute of Technology, 2018-12) Keman, Deniz; Soyer, Ferda
Staphylococcus aureus is a Gram-positive bacterium whose acquisition through an open wound results in various infections. Methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) strains are responsible for diseases ranging from soft tissue infections to fatal pneumonia which cannot be treated due to multiple drug-resistances of these strains. This situation increases the importance of searching for alternative antimicrobials worldwide. Having all these in mind, the capacity of phenolic acids cannot be denied to be used against pathogenic bacteria. Phenolic acids produced as plant secondary metabolites show antibacterial effects besides many beneficial properties for human health. The aim of this study was to investigate the antibacterial action mechanisms of vanillic acid and 2-hydroxycinnamic acid on MRSA and MSSA. To achieve this, firstly the antibacterial effects of phenolic acids on both bacteria were investigated by determination of minimum inhibitory concentrations. Then, the resistance development ability of bacteria against phenolic acids was tested by continuous exposure to subinhibitory concentrations. Finally, the action mechanisms of phenolic acids on bacteria were elucidated using two different proteomic approaches. According to the results, bacteria were not able to develop resistance against phenolic acids. Proteomic studies displayed alterations in the protein profiles of phenolic acid treated bacteria and provided potential targets in the battle with pathogenic bacteria. By showing the inability of MRSA and MSSA to develop resistance to phenolic acids and the important proteomic alterations that are induced by phenolic acid treatment, this study highlights the significance of phenolic acids to be used against antibiotic-resistant bacteria.
Investigation of the effects of the HIV-1 Tat gene on the expression of secretory leucocyte protease inhibitor in primate cell lines
(Izmir Institute of Technology, 2015-12) Özdemir, Selçuk; Arslanoğlu, Alper
Old world monkey species including African green monkey (AGM) are resistant to HIV-1. Although the virus can gain entry into the susceptible cells of these monkeys, virus replication is blocked prior to viral genome integration by the restriction factors. HIV-1 Tat are the first viral protein that are produced after viral genome integration and they are essential for the production of other viral proteins. According to our preliminary results, AGM CV-1 cells that are known to be resistant to HIV-1 were transfected with HIV-1 regulatory tat. Then, protein profiles of Tat and empty vector expressing AGM CV-1 were compared by 2D-PAGE and that way observed to be induced in cells expressivity tat was identified to be SLPI by mass spectrometry analysis. Based on our literature reviews, SLPI is found to be an anti-bacterial, anti-fungal and anti-inflammatory protein. SLPI also has an extracellular anti-HIV-1 effect. Our study aims to measure SLPI expression level in AGM and human cells in presence of HIV-1 tat and to research effect of SLPI on NF-kB and HIV LTR promoter. In our research, expression level of SLPI gene is measured by QRT-PCR method. SLPI protein was screened by Western Blot method. SLPI’s effect on NF-kB and HIV-LTR promoter was researched through the luciferase experiment. While SLPI gene shows increase in AGM cells in presence of HIV-1 tat, no such increase was observed in human cells. Furthermore, it has been shown that SLPI gene decreases luciferase expression dependent on NF-kB promoter and HIV-LTR. In other words, SLPI suppresses NF-kB promoter and HIV-LTR.
Metabolic and genetic profiling for primary and secondary metabolites in tomato
(Izmir Institute of Technology, 2017-01) Gürbüz, Nergiz; Doğanlar, Sami
Understanding the metabolic content of plants and its genetic basis is important to determine the most appropriate breeding strategies for traits such as yield, fruit quality, nutritional content, tolerance to disease factors, adaptation to various environmental conditions, and tolerance to abiotic and biotic stresses. Recently, post-genomic studies such as metabolomics, proteomics and transcriptomics are attracting attention and being used in conjunction with genomic studies. These studies help to speed research progress with crops as well as model plants. Tomato (Solanum lycopersicum) is an important crop which is cultivated widely in the world and in our country, and is also important for our daily diet. Although there is growing attention to studies on tomato day by day, there are limited studies that identify the quantitative trait locus (QTL) regions responsible for important agronomic, economic, nutritional and health related traits by correlation of metabolomics and genomics data. In this study, an interspecific IBL (inbred backcross line) population derived from the cross S. lycopersicum cv. Tueza x S. pimpinellifolium (LA1589) was both genotyped via genotyping by sequencing (GBS) and quantified for primary and secondary metabolites affecting yield, quality and nutritional value of the fruit to determine QTL regions for the targeted metabolites. In total, 187 QTLs were identified for 143 important traits. The data obtained from this study will help to shed light on the genetic control of plant metabolism and to develop high yield and nutrient-rich cultivars with improved agronomic traits by breeding strategies.
Molecular and genetic investigation of aging: The role of mitochondrial metabolism genes on life span determination
(Izmir Institute of Technology, 2016-09) Khandaker, Asfaqul Muid; Karakaya, Hüseyin Çağlar; Koç, Ahmet
Molecular mechanism of aging and longevity is still a complex phenomenon. In the course of time, an organism or tissue or a post mitotic cell ages, becomes weak, starts losing energy and ultimately falls in death; that implies that mitochondria has a central role of in the aging process. So in this study it is subjected whether manipulations to mitochondrial metabolism genes can extend life span in yeast. 144 strains derived from the yeast (S.cerevisiae) open reading frame (ORF) deletion collection were screened to identify single deleted mitochondrial genes that increase life span. This has resulted in the isolation of three long-lived mutants’ Δppa2 (28% extended), Δdss1(20% extended) and Δafg3 (40% extended) that are chosen for the current study. These long lived cells comprised relatively less amount of mtDNA at the young stage with effective proliferation rate while mtDNA content was highly decreased in old compared to wild type. Relatively less amount of ATP and absence of endogenous reactive oxygen species (ROS) level was observed both in long lived young and old cells. Long lived cell’s mitochondria was viewed as aggregated. In addition, the elevation of the mitochondrial membrane potential (ΔΨmito ) was found to predominate the relative degree of longevity. All long lived cells comprised similar pleiotropic mitochondrial phenotype and whole genome microarray published the sets of genes that were commonly upregulated and downregulated. The induction of peroxisomal glyoxylate cycle along with TCA cycle is suggested upon CIT2 higher expression. Thus this investigation reveals the regulatory properties of these genes through the remodeling of mitochondrial morphology and function.
Molecular characterization of long non coding RNAs that mediate apoptosis in human
(Izmir Institute of Technology, 2019-12) Sweef, Osama Abdel Hady Biaomy; Akgül, Bünyamin
Apoptosis is an evolutionarily form of programmed cell death for development and tissue homeostasis. Apoptosis is regulated by protein-coding genes and plays an important role in a wide range of biological processes. We aimed to identify and characterize differentially expressed lncRNAs in apoptosis. HeLa cells were used as a model system to identify the lncRNAs. The total RNAs was subjected to deep sequencing by next-generation sequencing. OmicsBOX Bioinformatics tools were used for differential expression analysis of lncRNAs that are apoptosis-induced. Gene set enrichment analysis (GSEA) was used to profile the miRNAs targeting lncRNAs. Cytoscape software was used to reconstruct lncRNA-miRNA targeting networks. RT-qPCR was used to validate miRNAs and their targets of lncRNAs and it was found that the overexpression of miR-519d-3p causes downregulation of lncRNAs RAB22A-202, PARD3-211, and AC027237.1-210. Also, the overexpression of miR-124-3p down-regulates the expression level of APEX2-202 and CD59-209. GTF2A1-AS, TNFRSF10B-AS, and CAMTA1-DT were detected in the nucleus and have no poly (A) tail and they belong to TATA-less promoter genes. TNFRSF10B-AS has a coding probability of 0.99 and alignment to High-scoring Segment Pair (HSP) clarifies one hit to Q9UBN6 protein. ChIRP clarifies that TNFRSF10B-AS binds to a protein (25 kDa). miR-519d-3p and miR-124-3p interact with lncRNA targets by miRNA-mediated lncRNA degradation pattern under apoptosis conditions. TNFRSF10B-AS has a putative regulatory function in the nucleus during apoptosis via binding specifically to the ribonucleoprotein partner.
Molecular genetic analyis in faba bean (Vicia faba L.)
(Izmir Institute of Technology, 2019-07) Abuzayed, Mazen Ali; Doğanlar, Sami
Faba bean (Vicia faba L.) is an important food legume crop with a huge genome. In this study, we used Next Generation Sequencing (NGS) technology for development of genomic simple sequence repeat (SSR) markers. A total of 14,027,500 sequence reads were obtained comprising 4,208 Mb. From these reads, 56,063 contigs were assembled (16,367 Mb) and 2138 SSRs were identified. Mono and dinucleotides were the most abundant, accounting for 57.5% and 20.9% of all SSR repeats, respectively. A total of 430 primer pairs were designed from contigs larger than 350 nucleotides and 50 primers pairs were tested for validation of SSR locus amplification. Nearly all (96%) of the markers were found to produce clear amplicons and to be reproducible. Thirty-nine SSR markers were then applied to 46 faba bean accessions from worldwide origins, resulting in 161 alleles with 87.5% polymorphism, and an average of 4.1 alleles per marker. Gene diversity (GD) of the markers ranged from 0.00 to 0.48 with an average of 0.27. Testing of the markers showed that they were useful in determining genetic relationships and population structure in faba bean accessions. In addition, 26 morphological and seven biochemical (phenolics content, flavonoids, protein, L-DOPA, tannins, vicine and convicine) characters of 61 landraces and 53 faba bean cultivars were analyzed. There was high diversity for the studied characters among the accessions. Association mapping for these morphological and biochemical characters with 59 SSR markers (442 fragments) was conducted using a general linear model based on the Q matrix. As a result, 48 significant loci were detected for 22 morphological characters, and 26 loci were detected for six biochemical traits. The range of LD (r2) was from 0.09 to 0.18, and from 0.06 to 0.13 for morphological and biochemical associations, respectively. This study can help breeding programs in selection and improvement of faba bean production.

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