Phd Degree / Doktora

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Department: search.filters.department.Thesis (Doctoral)--İzmir Institute of Technology, Molecular Biology and GeneticsPublisher: search.filters.publisher.01. Izmir Institute of Technology

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Now showing 1 - 7 of 7
  • Doctoral Thesis
    Therapeutic Efficacy Of Intrathecal Administration Of AAVrh10-mHEXA Vector In A Mouse Model Of Tay-Sachs Disease
    (01. Izmir Institute of Technology, 2025) Seyrantepe, Volkan; Seyrantepe, Volkan; 01. Izmir Institute of Technology; 04. Faculty of Science; 04.03. Department of Molecular Biology and Genetics
    Tay-Sachs hastalığı, bir lizozomal depolama hastalığıdır ve HEXA genindeki mutasyonlar sonucu oluşmaktadrr. HEXA geni, GM2 gangliosidini parçalamaktan sorumlu β-hekzosaminidaz A enziminin α-alt birimini kodlar. Son zamanlarda, Hexa ve Neu3 genlerinin birleşik eksikliklerine sahip Tay-Sachs hastalığı fare modeli (DKO fareleri), şiddetli nöropatolojik semptomlar ve nöroinflamasyon sergileyerek 20 haftaya kadar hayatta kalmıştır. Birçok lizozomal depolama hastalığı için tedavi stratejileri değerlendirilmiş olsa da, Tay-Sachs hastalığı için etkin tedaviler henüz geliştirilememiştir. Bu çalışmada, AAVrh10-mHexa (AAV) vektörünün intratekal uygulanmasının DKO farelerinde terapötik etkinliğini araştırmayı amaçladık. Ayrıca, AAV ilişkili gen tedavisi ile bir anti-inflamatuar ajanı olan istradefylline tedavisinin, DKO farelerinde nöroinflamasyonu hafifletme potansiyelini değerlendirdik. Moleküler biyolojik, immünohistokimyasal ve davranışsal analizler yapıldı. AAV tek başına ve istradefylline ile kombinasyon halinde uygulandıktan sonra DKO farelerinin yaşam süresinin 30 haftaya kadar uzadığını gösterdik. Moleküler biyolojik analizler, AAV tedavisi uygulanan ve istradefylline ile kombinasyon halinde tedavi edilen DKO farelerinin korteks, beyincik ve böbrek, karaciğer ve dalak gibi organlarında Hexa aktivitesinin arttığını ve lizozomal markerlar ile pro-inflamatuar sitokinler olan Ccl2 ve Ccl3 seviyelerinin azaldığını ortaya koydu. İmmünohistokimyasal veriler, AAV ve AAV-istradefylline kombinasyonu ile tedavi edilen farelerin beyinlerinde GM2 birikintisinin temizlendiğini, lizozom sayılarının azaldığını, aktif astrosit düzeylerinin düştüğünü ve nöronlar ile oligodendrositlerde iyileşmeler olduğunu gösterdi. Ayrıca, bu farelerde motor aktivitesi de iyileşti. Bu sonuçlar, AAVrh10 ilişkili intratekal uygulamanın, tek başına veya istradefylline ile kombinasyon halinde, Tay-Sachs hastalığını tedavi etmek için umut verici bir terapötik yaklaşım olduğunu göstermektedir.
  • Doctoral Thesis
    Investigation of the Effects of Ketogenic Diet Therapy in a Mouse Model of Gm2 Gangliosidosis
    (01. Izmir Institute of Technology, 2024) Seyrantepe, Volkan; Seyrantepe, Volkan; 04.03. Department of Molecular Biology and Genetics; 04. Faculty of Science; 01. Izmir Institute of Technology
    GM2 gangliosidosis is an autosomal recessive lysosomal storage disorder in which GM2 ganglioside is accumulated, especially in the brain. GM2 gangliosidosis is divided into three different variants: Tay Sachs (B-variant or type I), Sandhoff (O-variant or type 2), and GM2AP deficiency (AB-variant). Accumulation of the GM2 ganglioside in brain causes disease pathology as neurodegeneration and neuroinflammation. Our lab first displayed the novel GM2 gangliosidosis type-I mouse model (Hexa-/-Neu3-/-). Compared to control, this model could survive a maximum of five-months due to severe pathologies, neurodegeneration, and neuroinflammation. The ketogenic diet is high-fat and low-carbohydrate/protein diet that triggers burning fat instead of carbohydrates. The ketogenic diet is used comprehensively in neurodegenerative disorders such as Sandhoff, Alzheimer’s, and Parkinson’s to regulate inflammation, neurodegeneration, and autophagy. In addition, elevation of CCL2 expression in Hexa-/-Neu3-/- mice resulted in increased amounts of active microglia and astrocytes. Therefore, the CCL2/CCR2 signaling inhibitor of propagermanium was used in addition to ketogenic diet. Thus, reducing neuroinflammation is aimed to be more effective as a combined therapy. In my Ph.D. thesis, expression and protein levels of autophagy and inflammation-associated genes were analyzed in the mouse brain to exhibit whether beneficial effects on autophagic flux and neuroinflammation are found after the ketogenic diet and propagermanium treatment. The pathology of the GM2 gangliosidosis mouse type-I brain was illustrated by thin-layer chromatography and immunofluorescence staining to display whether the ketogenic diet affects the ganglioside metabolism. Briefly, ketogenic diet therapy and its anti-inflammatory and neuroprotective effects were explored in the GM2 gangliosidosis mouse model.
  • Doctoral Thesis
    Identification and Functional Characterization of M6a Rna Modifications in Cisplatin-Treated Hela Cells
    (01. Izmir Institute of Technology, 2024) Gelmez, Ayşe Bengisu; Akgül, Bünyamin; Akgül, Bünyamin; 04.03. Department of Molecular Biology and Genetics; 04. Faculty of Science; 01. Izmir Institute of Technology
    N6-metiladenozin (m⁶A), RNA üzerinde en fazla bulunan ve dinamik bir modifikasyondur. Bu çalışmada m⁶A modifikasyonun HeLa hücrelerinin sisplatin (CP) uygulamasına verdiği yatının düzenlenmesindeki fonksiyonu araştırılmıştır. İlk olarak, CP uygulanmış HeLa hücrelerinde temel m⁶A düzenleyicilerinin seviyeleri ve ardından m⁶A metilasyon dağılımı tüm transkriptom çapında incelendi. Analiz sonucunda, m⁶A yazıcılarının, özellikle METTL4'ün ekspresyonun değişikliğinin olası etkisi olarak, m⁶A metilasyon profilinde anlamlı farklar belirlendi. Özellikle, CP uygulamasını takriben global metilasyon seviyesindeki düşüş aynı uygulama sonucunda gözlemlenen METTL14'ün protein seviyesindeki önemli azalma ile ilişkilendirilebilir. METTL14 susturması sonucunda hücrelerin, özellikle transfeksiyonun 72. ve 96. saatlerinde CP'ye daha hassasiyet gösterdiği tespit edildi. Bu hassasiyet hücre ölümü ile ilgili genlerin RNA sevilerinde METTL14 susturulması sonucunda gözlemlenen artıştan kaynaklanıyor olabilir. Bununla birlikte, aday genler arasından, ATF3, ATF5, DUSP6 ve PEA15 gibi genler tutarlı ifade modelleri sergilerken, TP73, BMF, DAPK1 ve DAB2IP, CP RNA-seq verilerinde tanımlanan yönlere zıt mRNA seviyeleri sergiledi. Bu bulgular, METTL14'e bağlı gerçekleşen metilasyonun, bu genlerin mRNA düzenlenmesinde, dolasıyla CP etkisini artırmasında çok önemli bir rol oynadığını göstermektedir. Ayrıca, YTHDF2'nin pro-apoptotik adaylara koşula özgün olarak kontrol hücrelerinde daha fazla bağlanma yatkınlığı gösterdiği belirlenmiştir. Bu durum bu genlerin normal koşullarda YTHDF2 tarafından indüklenen RNA bozulması ile seviyelerinin kontrol edildiğini ancak CP uygulaması ile bağlanma etkinliğinin azalması nedeni ile aynı genlerin sabitliğinin arttığını işaret etmektedir. Bu çalışma, CP yanıtı ile dinamik m⁶A modifikasyonu arasındaki karşılıklı etkileşimi göstermektedir. Özellikle METTL14'ün sisplatin etkinliğini m⁶A metilasyonu ekseninde artırdığının altını çizerek, kanser tedavisinde m⁶A dağılımını düzenleyen yolakları hedef alan terapötik stratejilere yönelik araştırmalar için temel sağlayabilir.
  • Doctoral Thesis
    Investigation of the Effects of Antiinflammatory in the Tissues of Gm2 Gangliosidosis Mouse Model
    (01. Izmir Institute of Technology, 2023) Seyrantepe, Volkan; Ateş, Nurselin; Seyrantepe, Volkan; 04.03. Department of Molecular Biology and Genetics; 04. Faculty of Science; 01. Izmir Institute of Technology
    Tay- Sachs disease is an autosomal recessively inherited lysosomal storage disorder caused by mutations on the HEXA gene encoding α-subunit of β- Hexosaminidase A enzyme. The enzyme catalyzes GM2 to GM3 conversion but when it is deficient the GM2 degradation is interrupted and GM2 ganglioside is progressively accumulated especially in neurons. Progressive accumulation of GM2 causes increasing death of neurons, disruption in mental and motor functions and eventually death at 2-4 years of age. The Hexa-/- Tay-Sachs model was normal thanks to a bypass mechanism mediated by Neurominidase3. It was determined that Hexa-/-Neu3-/- mice mimicked the neuropathologic and clinical phenotype of the Tay-Sachs disease. Previously we showed GM2 accumulation in Hexa-/-Neu3-/- Tay Sachs disease mouse model triggers release of proinflammatory cytokines, microgliosis, astrogliosis consequently activation of inflammatory cascades as well as oxidative stress. These inflammatory events contribute to neurodegeneration observed in the disease pathology. In Sandhoff Disease mouse model it was shown that astrocytes express adenosine A2A receptors which induces ccl2 chemokine overexpression. A2A receptor antagonist istradefylline treatment reduces microglial activation and ccl2 expression in Sandhoff mice. In this study; A2A receptor antagonist istradefylline treatment was applied to Tay Sachs disease mouse model and whether this treatment would alleviate the neuroinflammation and redox imbalance; and prolong the lifespan was investigated by molecular biological and behavioural analyses. Modulation of ccl2 expression by istradefylline was used as potential therapeutic target to slow down Tay Sachs disease mouse model.
  • Doctoral Thesis
    Biochemical and Functional Characterization of Circular Rnas Differentially Expressed in Cisplatin-Treated Hela Cells
    (01. Izmir Institute of Technology, 2023) Akgül, Bünyamin; Akgül, Bünyamin; 04.03. Department of Molecular Biology and Genetics; 04. Faculty of Science; 01. Izmir Institute of Technology
    Circular RNAs (CircRNAs) are a novel class of single-stranded, covalently-closed RNA molecules. Functional investigations of the circRNAs provide insight into the mechanisms underlying gene regulation and cellular responses, which could ultimately lead to the development of new therapies for a wide range of diseases. In this thesis, four cisplatin (cis-diamminedichloroplatinum II, CP)-responsive circRNAs, circGALNT2, circBNC2, circBIRC6, and circCLASP1, were validated. The reverse genetics approaches, such as knockdown and overexpression strategies, showed that circCLASP1 is required for the proliferation of HeLa cells. The knockdown of circCLASP1 disrupts proliferation in HeLa, and its overexpression restores impaired proliferation. Further analyses revealed that circCLASP1 knockdown sensitizes HeLa cells against 20 μM and 40 μM cisplatin treatments. Interestingly, an IC50 dose of cisplatin causes Annexin V-/7AAD + cell death rather than apoptosis when combined with circCLASP1 knockdown. In light of these findings, five circRNA/miRNA/mRNA regulatory networks were constructed using computational approaches. Additionally, a transcriptomics analysis after circCLASP1 knockdown has supported all of these findings in that muscle cell proliferation genes were significantly altered upon circCLASP1 knockdown in HeLa cells. In conclusion, the findings suggest that the knockdown of circCLASP1 represses proliferation and sensitizes HeLa cells against cisplatin. CircCLASP1-knockdown mediated differential gene expression indicates proliferation, ROS response, iron metabolism, lipid peroxidation, and cell death. Further studies are needed to elucidate the precise mechanism of circCLASP1-mediated cell death and proliferation in muscle cells or liver cells and ROS-related diseases.
  • Doctoral Thesis
    Molecular genetic analyses in Origanum (Lamiaceae) taxa in Türkiye
    (01. Izmir Institute of Technology, 2022) Frary, Anne; Frary, Anne; Frary, Anne; 04.03. Department of Molecular Biology and Genetics; 04. Faculty of Science; 01. Izmir Institute of Technology
    Medicinal and aromatic plants (MAPs) belonging to the genus, Origanum L. (Lamiaceae), are called “oregano”. They are economically important and beneficial for trade, medicine, food, cosmetics, and ornamental purposes with their bioactive compound diversity and richness. Although Türkiye is the gene center for generation, speciation, and diversification of oregano throughout the world, their uncontrolled consumption and other factors threaten their status. According to Ietswaart (1980), there are ten morphological sections in the genus. Of these, 25 taxa (including 13 endemics) and 13 hybrids from eight sections grow naturally in Türkiye. The cross-pollinating and gynodioecious nature of oreganos makes their taxonomic classification difficult. In this dissertation, molecular markers (EST-SSRs and SRAPs) were used to assess the complex evolutionary relationships in a herbarium and the Turkish national AARI Gene Bank collection. Cross hybridization due to high gene flow was found to be the main source of genetic diversity within both collections. In both collections, the highest gene flow was observed between two sections, ANA and BRE, with diecious flowers which supports their frequent hybridization when compared to gynodioecious oreganos in nature. The Aegean and Mediterranean regions had the highest gene flow among all regions, while five province pairs had the highest gene flow among all provinces. In conclusion, molecular markers were shown to be a useful tool for examinations of genetic diversity and evolution in oregano.
  • Doctoral Thesis
    Detection of the Metastatic Potential of Breast Cancer Cell Lines To Specific Target Tissues
    (01. Izmir Institute of Technology, 2021) Fıratlıgil Yıldırır, Burcu; Yalçın Özuysal, Özden; Yalçın Özuysal, Özden; 04.03. Department of Molecular Biology and Genetics; 04. Faculty of Science; 01. 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.