GCRIS

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Proteomic Studies and Its Application To Biological Samples Using Mass Spectrometry
(Izmir Institute of Technology, 2017) Güray, Melda Zeynep; Yalçın, Talat; Karakaya, Hüseyin Çağlar
Mass spectrometry (MS) is a powerful analytical tool with its application in the field of biological sciences for identification of proteins, defining post-translational modifications, studying protein expression and protein interactions. This thesis presents MS analyses of proteins for defining modifications observed during sample preparation and identification of proteins isolated from clinical samples and microorganisms. The first part of the thesis includes proteomic analysis of antimony resistant L. tropica. The results clearly indicated that the generation of antimony resistance by parasites, either in host organism or in vitro, causes alteration of protein expression levels, and the mechanism of antimony resistance in host organism and in vitro conditions follow different strategies. In the second part of the study, proteomic analysis of Bence Jones proteins isolated from urine of multiple myeloma patients was performed. Gel electrophoresis and MS analysis revealed that the proteins from different patients with different nephrotoxicity have different tendencies to form multimeric structures and contained different type of light chain. In the third part, it was shown that precipitation of proteins in acetone causes +98 u mass artifacts on proteins when analyzed by MS. The parameters affecting the formation of modification was studied and it was revealed that this modification is dependent on solution pH, incubation time and temperature. In the last part, aspartic acid and glutamic acid containing synthetic peptides were shown to be methylated upon incubation in acidified methanol solution. MS analysis revealed that the reaction is dependent on temperature and time and is affected by the type of acid included in methanol solution. All in all, this thesis provides a comprehensive study of proteins by mass spectrometry for identification of proteins from different sources, as well as defining protein modifications observed as artifacts during sample handling in proteomic workflows.
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.

Phd Degree / Doktora

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