Master Degree / Yüksek Lisans Tezleri
Permanent URI for this collectionhttps://hdl.handle.net/11147/3008
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Master Thesis Tay-sachs Hastalığı Fare Hücrelerinde MiRNA'ların Biyolojik Rolünün Araştırılması(2025) Kaya, Beyza; Seyrantepe, VolkanTay-Sachs hastalığı, HEXA geminin mutasyonu ve β-N-asetilheksosaminidaz A enzim eksikliğine bağlı bir lizozomal depo hastalığıdır. GM2 gangliozidinin lizozomal birikimi ve bozulmuş otofajik akış daha önce Tay-Sachs hastalığı fare modeli olan Hexa- /-Neu3-/-'de bildirilmiştir. Birkaç tedavi stratejisi araştırılmış olsa da Tay-Sachs hastalığı için küratif bir tedavi yoktur. ML-SA1 bir lizozomal kalsiyum kanalı agonistidir ve otofaji ve ekzositozu indükleme yeteneği çeşitli hastalıklarda bildirilmiştir. miRNA'lar, gen ekspresyonunu transkripsiyon sonrası düzenleyen kodlamayan küçük RNA'lardır. miRNA'ların düzensiz ekspresyonları birkaç lizozomal depo hastalığı için incelenmiştir. Ancak, Tay-Sachs hastalığında kapsamlı miRNA profillendirmesi ve ML-SA1 tedavisi hakkında bir çalışma yoktur. Bu çalışmada ML-SA1 ile tedavi edilmiş ve edilmemiş koşullarda Hexa-/-Neu3-/- fibroblastları ve nöroglialarının kapsamlı miRNA profillerini belirleyerek hastalık patolojisiyle ilişkili spesifik miRNA'ları tanımlamak ve ML-SA1'in lizozomal ekzositoz ve bozulmuş otofaji üzerindeki etkisini değerlendirmek amaçlanmıştır. Buna göre, yeni nesil dizileme ile birlikte enzim aktivite analizi, otofaji ve ekzositoz belirteçlerinin protein ve gen ekspresyon analizleri gerçekleştirilmiştir. Sonuç olarak, ML-SA1 lizozomal ekzositozu artırmış ve otofajiyi indüklemiştir. Ancak otofajik temizlenme gözlemlenmemiştir. ML-SA1 uygulanmamış Hexa-/-Neu3-/- fibroblastlar ve nöroglialarda mmu-miR-672-5p ve mmu-miR-5624-3p, WT kontrollerine kıyasla önemli ölçüde yukarı regüle edilirken, mmu-miR-6940-3p önemli ölçüde aşağı regüle edilmiştir. ML-SA1 tedavisi üzerine, WT hücrelerine kıyasla mmu-miR-375-3p her iki hücre tipinde de önemli ölçüde yukarı regüle edilirken mmu-miR-7091-5p önemli ölçüde aşağı regüle edilmiştir ve mmu-miR-672-5p'nin yukarı regülasyonu etkilenmemiştir. Bu bulgular lizozomal kalsiyum modülasyonunun hastalık koşulları altında miRNA regülasyonunu belirgin şekilde etkilediğini göstermektedir.Master Thesis Identification of Neuroinflammatory Markers in a Mouse Model With a Deficiency of Neu1(01. Izmir Institute of Technology, 2024) Seyrantepe, Volkan; Ada, Ebru; Seyrantepe, VolkanLysosomal neuraminidase 1 is responsible for sialic acid removal from oligosaccharides and glycoconjugates. Neu1 sialidase forms enzyme complex with protective protein cathepsin A, and glycosidase β-galactosidase. Mutations in Neu1 sialidase cause sialidosis, and Neu1-/- mice show symptoms of sialidosis patients. GM3 ganglioside is defined as substrate of lysosomal sialidase in vitro, and marked increase in GM3, GD3, and GM4 ganglioside levels in brain, spleen and liver autopsy tissues of sialidosis patients. Additionally, it was monitored that Neu1 has regulatory roles in immune response; expression of interleukins, activation of Toll-like receptor, and production of NF-kB in immune cells. However, relationship between inflammatory pathways and secondary lipid metabolisms in Neu1 sialidase deficiency remains unclear. Here, we aimed to investigate secondary lipid alterations and inflammatory response in tissues of Neu1-/- mice. In this study, lipidomic, molecular, histological and immunohistochemical analyses were performed in brain, spleen and kidney tissues of 2- and 5-month-old Neu1-/- mice. Decreasing levels of secondary lipids (phosphotidylcholine, phosphatidylethanolamine, and phosphotidylinositol) and elevated levels of pro-inflammatory cytokines, glycoconjugate accumulations, morphological degenerations, oligodendrocyte and neuronal loss, astrogliosis, and microgliosis were observed in brain, spleen and kidney of 2- and 5-month-old Neu1-/- mice. In the light of our findings, reduced levels of glycerophospholipids may be considered as biomarkers of activated inflammatory response in Type II sialidosis mice model. In the future studies, novel therapeutic strategies can target these altered glycerophospholipids, and their regulation can be crucial for alleviation of pathogenesis in sialidosis patients.Master Thesis Investigation of Biomarkers Using Lipidome-Based Research Analysis in Sialidosis(01. Izmir Institute of Technology, 2024) Gümüş, İlker; Seyrantepe, VolkanNeuraminidase 1, also known as N-acetyl-α-neuraminidase, is an enzyme found in lysosomes and encoded by the NEU1 gene. This enzyme is responsible for eliminating terminal sialic acids from glycoproteins and oligosaccharides. When mutations occur in the NEU1 gene, it leads to a particular lysosomal storage disorder known as sialidosis. Sialidosis is a rare genetic disorder that is inherited in an autosomal recessive manner. Sialidosis is classified into two subtypes: Type I, which has a later onset and Type II, which presents with early onset. In previous studies, an increase in glycolipid levels in visceral organs, and accumulation of sialyloligosaccharides and sialoglycoproteins were reported. However, the effect of NEU1 sialidase on secondary lipid expression levels in sialidosis pathology remains unknown. The relationship between lipid expression levels and inflammation of human and mice sialidosis fibroblasts cell lines was analyzed for the first time in this study. To understand the connection between secondary lipid alterations and inflammation in sialidosis molecular biological approaches and shotgun lipidome analysis were followed. The link between the secondary lipid alterations and its association with sialidosis was determined in our research. We have concluded that the findings not only provide the elucidation of the lipidome characteristics in sialidosis models of mice and patients, but they also have the potential to establish a connection between the lipidome features and the occurrence of neuroinflammation in sialidosis. Grasping the correlation between the changes in secondary lipids and inflammation may offer therapeutic strategies for sialidosis patients by modulating the expression of secondary lipids.Master Thesis Elucidation of Lysosomal Cathepsin a in the Regulation of Autophagy(01. Izmir Institute of Technology, 2023) Yanbul, Selman; Seyrantepe, VolkanLysosomal 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 Investigation of the Biological Role of Mouse Acylneuraminyl Hydrolase Enzymes in the Regulation of Neuroinflammation(Izmir Institute of Technology, 2022) Tabakacılar, Doğa; Seyrantepe, VolkanSialidosis is a lysosomal storage disorder, and it is inherited by autosomal recessive mutations in the Neuraminidase 1 (NEU1) gene. Neuraminidases or sialidases are catalytic enzymes that carry out the desialylation of glycoconjugates. Deficiencies of neuraminidases lead to the accumulation of sialoglycoconjugates in membranes of cells. Neuroinflammation and neurodegeneration are present in some lysosomal storage diseases such as Tay-Sachs. However, in the sialidosis mouse model, neuroinflammation was never studied. In this study, we investigated the effect of neuraminidase 1, neuraminidase 3, and their combined deficiency on neuroinflammation by using Neu1-/-, Neu3-/- knockout, and Neu1-/- Neu3-/- double knockout mouse models. Neu1-/- Neu3-/- knockout mouse model was smaller in comparison to its littermates and showed muscle weakness, tremoring, and 2-3 weeks of a lifetime. Some of the Neu1-/- Neu3-/- mice died prematurely. To unravel the pathology immunohistochemical, biological, and chromatographic techniques were used. The expression of inflammatory cytokines was altered in the Neu1-/-, Neu3-/-, and Neu1-/- Neu3-/- mice with respect to the brain section. Neu1-/- Neu3-/- mice showed generally the highest expression of cytokines in the cerebellum compared to the cortex. Neuronal loss was observed in the Neu1-/- Neu3-/- mice in the cortex, thalamus, and cerebellum. The most remarkable change was in the ganglioside expression pattern in the Neu1-/- Neu3-/- mice cortex. GD3 expression was present in the cortex of Neu1-/- Neu3-/- mice where expression of this ganglioside is related to neuroinflammation, neurodegenerative stimuli, autophagosome remodeling and programmed cell death.Master Thesis Investigation of Combined Biological Roles of Neuraminidase 1 and Gd3 Synthase Enzymes in Glycolipid Metabolism(Izmir Institute of Technology, 2020) Dağalp, Berkay; Seyrantepe, VolkanNeuraminidases or sialidases are classified enzymes hydrolases the sialic acid residues from the glycoconjugates. In vertebrates, so far four different neuraminidases have been identified having distinct roles besides degradation of glycoconjugates. Neuraminidases differ in subcellular locations where Neuraminidase 1 is mainly localized in lysosomes having crucial regulatory roles and forms a multienzyme complex with protective protein/cathepsin A and ß-galactosidase. Only Neu1 is recognized when its functions or a component from the complex they together forged are defected, resulting two severe lysosomal storage disorders, sialidosis and galactosialidosis. To shed light on these disorders, Neu1-/- mice model lacking the enzyme was generated. By addition of sialic acid residue to the structure of Glycosphingolipids (GSLs), complex sugars in the membrane surface that provide special properties to cell, gangliosides are generated that further processed into 0-, a-, -b, -c series. Since the function of Neu1 in Glycosphingolipid pathway is unclear, to investigate the role of Neu1in this pathway, Neu1-/- mice crossed with the mice lacking b-and c- series of gangliosides, the GD3S-/- mice are used to generate Neu1-/-GD3S-/- mice. Even though mice showed indifferent ganglioside profile with a thin layer chromatography, they displayed decreased apoptotic signals and ER-stress markers with RT-PCR. However, western blotting and immunohistochemical studies revealed severe cell death in the brain. Moreover severe behavioral deficits were observed with open field and rotarod tests. The effects of b- and c- series of gangliosides on double knock-out mice still require further research that might reveal important roles in terms of cell death mechanismMaster Thesis Investigation of Biological Roles of Neuraminidase 3 and N-Acetylgalactosaminyltransferase Enzymes in Glycolipid Metabolism(Izmir Institute of Technology, 2020) Basırlı, Hatice Hande; Seyrantepe, VolkanGangliosides are sialic acid containing biomolecules and perform important functions by locating on cell membranes. Their catabolism regulated by sialidases while their synthesis was performed by glycosyltransferases. Neu3 reacts with gangliosides on plasma-membrane and acts in cellular processes. Neu3 mice model was studied without signs of altered brain activity and phenotypic alterations. In further studies, significance of Neu3 sialidase in bypass mechanism shown in Tay-Sachs disease mice model named as Hexa-/-Neu3-/- mice. Galgt enzyme produces 0-, a-, b-, and c-series of complex gangliosides. Galgt deficiency led to progressive axonal degeneration, decreased myelin volume, altered axo-glial junction integrity, male infertility and hindpaw-clasping in mice. However, the studies for showing In this study, we investigated altered ganglioside metabolism, ER-stress, oxidative stress, and apoptosis mechanisms in cortex, thalamus, and cerebellum tissues of 3- and 6-month-old Neu3-/-, Galgt-/-, and Neu3-/-Galgt-/- mice compared to age-matched WT control by performing molecular biological techniques (TLC, RT-PCR, and Western Blot analyses). Furthermore, we performed histopathologic and immunohistochemical analyses to examine the alterations in myelination, neuron number, and glyco-conjugate content, morphological abnormalities, and apoptosis in brain sections of single and double deficient mice models. We also performed behavioral assays like rotarod, passive avoidance and open field to show altered brain functions and behavioral abnormalities like anxiety, reduced motor balance, strength, and locomotory activity in addition to problems in memory formation. In line with these studies, we found that Neu3 and Galgt enzymes acts in the regulation of ganglioside metabolism in a region-specific and age-dependent manner.Master Thesis Investigation of Combined Biological Roles of Neuraminidase 1 and Gm3 Synthase Enzymes in Glycolipid Metabolism(Izmir Institute of Technology, 2020) Can, Melike; Seyrantepe, VolkanGangliosides are sialic acid-containing glycosphingolipids, and commonly expressed in nervous system. GM3 Synthase is responsible for production of GM3 ganglioside known as precursor of a- and b- series gangliosides. Sialidases catalyze removing of sialic acid residues from sialoglycoconjugates and classified based on subcellular localization. Lysosomal Neu1 sialidase is responsible for catabolism of glycolipids, glycoproteins and oligosaccharides. Mutations of lysosomal Neu1 sialidase cause sialidosis and Neu1-/- mice mimic symptoms seen in patients. Glycosphingolipid accumulation in visceral organs of sialidosis patients was notified previously, and it was also reported the GM3 ganglioside as substrate of lysosomal sialidase in vitro. However, effect of Neu1 sialidase in the case of complex ganglioside deficiency in brain remains unclear. In the concept of research, we aimed to understand biological role of lysosomal Neu1 sialidase alone and combined with GM3S in ganglioside metabolism in vivo. In accordance with this purpose, cortex, cerebellum and thalamus tissues of 2- and 5-month old Neu1-/-GM3S-/-, Neu1-/- and GM3S-/- mice were compared with age-matched control group using molecular biological, histological, immunohistochemistry and behavioral analyses. Alterations in ganglioside metabolism, oligosaccharide pattern and cellular processes (ER-oxidative stress, apoptosis), structural abnormalities, glycoconjugate accumulation, loss of neurons and oligodendrocytes in addition to age dependent behavioral impairments in motor function, memory and muscle strength were demonstrated in single and double knock-out mice. In regard of these results, we have concluded that altered glycosphingolipid metabolism with accumulated secondary metabolites like oligosaccharides affect cellular processes and brain pathology resulting in behavioral abnormalities in age dependent and region specific manner.Master Thesis Investigation of Combined Biological Roles of Neuraminidase 1 and N-Acetylgalactosaminyltransferase Enzymes in Glycolipid Metabolism(Izmir Institute of Technology, 2020) Şengül, Tuğçe; Seyrantepe, VolkanGangliosides, sialic acid-containing glycosphingolipids, are responsible for neurogenesis and synaptogenesis which are essential for vertebrate nervous system. N-Acetylgalactosaminyltransferase (Galgt) is glycosyltransferase that plays essential role during complex gangliosides biosynthesis. Expression of Galgt increases at the late stage of development which can be evidence for complex gangliosides role in nervous system development and differentiation.Sialidases are responsible enzymes for sialic acid removal from glycoproteins, glycolipids or oligosaccharides. Neu1 is one of the mammalian sialidase which catabolizes sialoglycoconjugates in lysosomes. Neu1 gene mutations in human result in lysosomal storage disease called sialidosis. Created Neu1 knockout mice model have demonstrated similar symptoms with sialidosis type II. In sialidosis patients, increased ganglioside levels are detected in visceral organs but not in brain. In vitro studies have demonstrated that GM3 is a substrate of Neu1. Until this research, role of Neu1 enzyme on glycosphingolipid metabolism in the absence of complex gangliosides has not been investigated. Therefore this study have been provided comparision of 2-and 4-month-old Neu1-/-, Galgt-/- and Neu1-/-Galgt-/- mice to WT mice and each other by molecular biological, biochemical, histological, immunohistochemical and behavioral analysis in cortex, cerebellum and thalamus regions. In the concept of this thesis, we found effect of single and double deficienct of Neu1 and Galgt enzymes on the distinct cellular events (apoptosis, ER stress, oxidative stress), altered glycolipid and oligosaccharide metabolism, nerve cell death,oligodendrocyte intensity decrease, impairment in locomotor activity, motor coordination, memory capability as age dependent and region specific manner.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, VolkanTay-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.