Master Degree / Yüksek Lisans Tezleri

Permanent URI for this collectionhttps://hdl.handle.net/11147/3008

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Subject: search.filters.subject.Autophagy

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Now showing 1 - 4 of 4
  • Master Thesis
    Elucidation of Lysosomal Cathepsin a in the Regulation of Autophagy
    (01. Izmir Institute of Technology, 2023) Yanbul, Selman; Seyrantepe, Volkan
    Lysosomal Cathepsin A (CathA) is a multifunctional enzyme with independent catalytic and protective functions. It has a serine carboxypeptidase activity in acidic pH conditions for the degradation of short bioactive peptides that are vasoactive peptides including endothelin-1, angiotensin-I, bradykinin and neuropeptides including oxytocin and substance P. Lysosomal CathA enzyme also forms a lysosomal multienzyme complex (LMC) with α-neuraminidase (Neu1) and ß-Galactosidase (ß-Gal) enzymes to protect them from hydrolytic degradation in lysosomes and due to its protective function. Genetic defects in the lysosomal CathA enzyme causes a rare lysosomal storage disorder, Galactosialidosis (OMIM #256540), with secondary deficiencies of Neu1 and ß-Gal enzymes. Catalytically inactive Cathepsin A knock-in mouse model, CathAS190A has point mutation in the active catalytic site which serine was replace with alanine amino acid. Accumulation of short bioactive peptides has been reported in previous studies in different tissues of the CathAS190A mouse model. In this thesis study, investigation the role of the lysosomal CathA enzyme in the regulation of autophagic flux in neuroglia and fibroblast cell lines derived from CathAS190A mice was aimed. For this aim; RT-PCR, Western Blot and Immunocytochemical analyses were for performed for autophagy markers. Thesis study results have exhibited that catalytically deficient CathA causes the impairment in autophagic machinery with secondary accumulation of autophagic substrates and alterations in the expression of the autophagy marker genes. Accumulation of the short bioactive peptides due to the catalytically inactive CathA enzyme may be related to to impaired autophagic flux. Autophagy-inducing Rapamycin and Starvation treatment conditions may restore the impaired autophagic flux due to catalytically inactive CathA enzyme with the clearance of accumulation of secondary autophagic substrates.
  • Master Thesis
    Preparation of Some Semi-Synthetic Saponin Analogs and Investigation of Their Mechanism of Action on Necrotic Cell Death
    (Izmir Institute of Technology, 2019) Üner, Göklem; Bedir, Erdal; Ballar, Petek
    Since antitumor potency of saponins is relatively weak, researchers focus on semi-synthetic modification of saponins to obtain highly potent structures. With the same motivation, we prepared cytotoxic sapogenol (AG-08), from cycloastragenol. Our preliminary studies revealed that AG-08 was inducing necrotic cell death together with autophagic inhibition. Furthermore, immunoblotting experiments suggested that AG-08 promoted cleavage of various proteins. A continuation study was performed in this thesis with aims of: i) verifying previous studies; ii) identifying molecular mechanism of AG-08; iii) preparing further analogs of AG-08 and deduce structure activity relationships(SAR). Accordingly, necrotic cell death and autophagic inhibition via AG-08 was verified, and cytotoxicity of AG-08 on 13 cell lines was examined. Furthermore, inhibitors of calpain-1, general caspases, cathepsin B/L/S, and caspase-8 were found to partially alleviate cell death, whereas cathepsin D/E inhibitor were not able to do. Additionally, lysosomal impairment due to loss of acidic nature was demonstrated. Later data and effect of cathepsin inhibitor on AG-08 mediated cell death suggest lysosomal membrane permeabilization. In synthesis part, 15 AG-08 analogs were prepared, three of which were cytotoxic. Additionally, active analogs triggered similar cell death mechanism with AG-08. SAR evaluation reveals that presence of tosyl, and tetrahydrofuran ring are required for activity, while double bond at C-6 is not essential. Consequently, this thesis provides important data on mechanism of necrotic cell death and autophagic inhibition via AG-08 treatment as well as relationship between structure and activity. However, further studies are warranted to clarify complete mechanism of AG-08 and substantiate structure activity relationship deductions.
  • Master Thesis
    Investigation of Autophagy Related Markers in Brain Tissue of Early-Onset Tay-Sachs Disease Mouse Models
    (Izmir Institute of Technology, 2018) Ateş, Nurselin; Seyrantepe, Volkan
    Tay-Sachs disease is an autosomal recessively inherited lysosomal storage disorder that mainly affects the central nervous system. It is caused by mutations on the HEXA gene encoding α-subunit of β-Hexosaminidase A enzyme. The enzyme normally catalyses GM2 to GM3 conversion but when it is absent or dysfunctional the GM2 degradation is interrupted. Progressive accumulation of the undegraded GM2 ganglioside in neurons causes neurodegeneration and eventual death for the patient. The Hexa-/- mice generated as Tay-Sachs model was nearly normal and a bypass mechanism mediated by a sialidase was discovered. Neu3 sialidase involvement in ganglioside degradation in the Tay-Sachs disease pathology was reported and the Hexa-/-Neu3-/- mice was observed to mimic the neuropathologic and clinical phenotype of the disease. Therefore, it can be used as early-onset-Tay-Sachs disease mouse model. Lysosomal storage diseases have been reported as disorders of autophagy as the lysosomal accumulation expected to affects the autophagical-lysosomal pathway in one way or another. In the concept of our study comparative analysis of WT, Hexa -/- ,Neu3 -/- and Hexa -/- Neu3 -/- mice provided the information that early-onset Tay-Sachs disease model exhibit impairment in autophagic flux and secondary accumulation of autophagic components. The effect of abnormal GM2 and this secondary accumulation on apoptotic regulators and trigger factors were also investigated. In the light of our study, impairment in autophagic flux, increased oxidative stress and ER-stress are involved in the disease pathology of early-onset Tay-Sachs disease mouse model.
  • Master Thesis
    Effects of Polyether Antibiotics on Autophagy
    (Izmir Institute of Technology, 2017) Khan, Nasar; Tosun, Çiğdem; Bedir, Erdal; Tosun, Çiğdem; Bedir, Erdal
    Treatment of cancer is one of the crucial enigma for scientific world and that’s why much effort needs to be put in place for the resolution of this challenge in alternative ways. Autophagy is believed to have an important role in tumor development and progression. The natural polyether antibiotics might be important chemotherapeutic agents to cure cancer by modulating autophagy. The primary goal of this study was to investigate the cytotoxic effects and autophagic mechanism of actions of three polyether antibiotics, one of which was a new secondary metabolite isolated from the marine Streptomyces cacaoi. The effects of these polyether antibiotics were investigated along with previously known autophagy modulators from the same group (Monensin). To achieve this goal, cytotoxicities of these polyether type compounds on three different type of cancer cell lines along with two healthy cell lines were investigated followed by a search to reveal the effects of these compounds on autophagy in cancer cell lines. Methodology of this study consists of mammalian cell culturing, cytotoxicity screening, staining and quantification of acidic compartments inside the cells and studying different autophagy markers along with other associated proteins under various conditions by using Western blotting. This study revealed that the tested polyether antibiotics were autophagy inhibitors as well as inducers of apoptosis in cervical, colorectal and prostate cancer cells. The obtained results will be of significance for the field of anticancer drugdevelopment; however, before one places these secondary metabolites as potential drug candidates, further studies including in vivo experiments are warranted.