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Molecular and Genetic Investigation of Aging: the Role of Mitochondrial Metabolism Genes on Life Span Determination
(Izmir Institute of Technology, 2016) 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.
Elucidation of Boron Stress Signaling Pathways in Yeast
(Izmir Institute of Technology, 2015) 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.
Identification of Doxorubicin Drug Resistance Mechanisms by Using Genomic Techniques
(Izmir Institute of Technology, 2015) 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.

Phd Degree / Doktora

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