Master Degree / Yüksek Lisans Tezleri
Permanent URI for this collectionhttps://hdl.handle.net/11147/3008
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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.Master Thesis Investigating the Biological Role of Sialidase Neu4 and Gm3 Synthase Enzymes in a Mouse Model of Tay-Sachs Disease(Izmir Institute of Technology, 2017) Barnar, Talha; Seyrantepe, Volkanβ-Hexaminidase A which has role in GM2 degradation in glycosphingolipid pathway is known to be main enzyme for Tay-Sachs disease. Although recessive mutant phenotype of this enzyme causes disease in human, Hexa gene knockout mice show less accumulation of GM2 ganglioside than human. To avoid excess GM2 accumulation, mice uses neuraminidases convert GM2 into GA2. In addition, among neuraminidases, it has been found that Neu4-/-Hexa-/- mice can be good model for Tay-Sachs diseases (Seyrantepe et al., 2010). On the other hand, to prevent GM2 accumulation, blocking GM3 synthase is the finest method because GM3 synthase plays a big part in ganglioside synthesis pathway by producing GM3 that is later converted into GM2 or GD3 ganglioside. In addition, GM3 synthase deficient mice can live longer than 1 year. In this study, Hexa-/-GM3S-/-Neu4-/- mice with single and double variants were produced and brain regions were analyzed with thin-layer chromatography, immunohistochemistry, and real-time PCR methods. This investigation was conducted to clarify real function of GM3S on Tay-Sachs mice model and to search for possible effects of Neu4 in ganglioside pathway. Although GM2 accumulation are present in Hexa-/- and Neu4-/-Hexa-/-mice, analysis of Hexa-/-GM3S-/-Neu4-/-and Hexa-/-GM3S-/- mice revealed that there is no GM2 accumulation without GM3 synthase enzyme. These results are consistent with known ganglioside synthesis pathway. Hexa-/-GM3S-/-Neu4-/- and double deficient Neu4-/-mice variants disclosed change of Neu3 and Neu2 concentration to the wild type mice. In regard of these results, change in other neuraminidase expression is to compensate Neu4 function.Master Thesis Investigating the Biological Role of Sialidase Neu4 and Galnac-T Enzymes in a Mouse Model of Tay-Sachs Disease(Izmir Institute of Technology, 2016) Ateş, Edanur; Seyrantepe, VolkanTay-Sachs disease is a lysosomal storage disorder that is caused by a mutation in the HexA gene coding for the alpha subunit of lysosomal B-hexosaminidase A. HexA is responsible for the removal of N-acetylglucosamine residue form GM2 ganglioside to convert it into GM3 in the ganglioside degradation pathway. Deficiency of HexA causes neuronal death with progressive neurological degeneration. Neu4 is a sialidese and found in lysosomes. Knock-out mice model of Neu4-/- show different ganglioside pattern than wild type mice and there is increased GD1a and decreased GM1 in brains of mice (Seyrantepe et al. 2008). As previously shown, Neu4 is a modifier gene of HexA. On a previous work (Seyrantepe et al. 2010) Neu4-/-HexA-/- double deficient mouse showed more severe phenotype than HexA-/- deficiency alone. 1,4-N-acetylgalactosaminyltransferase; (Galgt1) is one of the key enzymes in the synthesis of complex gangliosides and it work in reverse direction of HexA. In the deficiency of Galgt1, there is only production of simple gangliosides occurs. Absence of complex gangliosides causes neurological degeneration. Mouse model of Neu4-/-HexA-/-Galgt1-/- was generated as a model of substrate deprivation therapy. That mouse has defects in both ganglioside synthesis and degradation mechanisms, so neither synthesis nor degradation of complex gangliosides will occur. By this mean, effects of Tay-Sachs disease were decreased. On previously shown Neu4 has role in the metabolism of GD1a into GM1. With this study, it was speculated that sialidase Neu4 may play a role in the metabolism of simple gangliosides.Master Thesis Investigating the Regulatory Role of Lysosomal Protein Cathepsin a in Vasoactive Peptide Biology(Izmir Institute of Technology, 2016) Çalhan, Osman Yipkin; Seyrantepe, VolkanThe lysosomal carboxypeptidase A, Cathepsin A (CathA), is a serine protease with distinct functions. CathA protects sialidase Neu1 and β- galactosidase against proteolytic degradation by forming a multi-enzyme complex and was shown to be activating sialidase Neu1. Mutations in the CathA gene cause the lysosomal storage disease galactosialidosis. Patients with galactosialidosis are characterized with a broad range of clinical phenotypes, involving growth retardation, neurological deterioration and accumulation of vasoactive peptide, endothelin-1 in the brain. CathA is also a multi-catalytic enzyme with deamidase and esterase activity at neutral pH, and carboxypeptidase activity at acidic pH. Previous investigations have presented that CathA has specific enzyme activity against vasoactive and neuropeptides, including endothelin-1, oxytocin and substance P. A generated mouse model with inactive CathA enzyme activity (CathAS190A) showed significantly increased level of precursor endothelin-1 vasoactive peptide with increased arterial blood pressure. The aim of this study was to ascertain the regulatory role of CathA against other vasoactive peptide precursors such as oxytocin, endothelin-1 and substance P in vivo. In addition to the regulatory role of the enzyme, CathA’s involvement in learning ability, long term memory and motor cortex functioning was elucidated. Our results showed that CathA has a regulatory role on endothelin-1 and oxytocin peptides’ degradation, which is the reason of the increased accumulation in the brain hippocampus region for CathAS190A. Memory based Morris’ water maze and passive avoidance tests illustrated that CathAS190A animals had lower scores than control littermates, which let us to speculate that CathA may play significant role in memory consolidation and learning ability through its regulatory role on vasoactive peptide biology.Master Thesis Investigation of the Molecular and Genetic Response in Enterocytes of Duodenum During Elevated Intracellular Glucose Level(Izmir Institute of Technology, 2016) Boztepe, Tuğçe; Güleç, Şükrü; Seyrantepe, VolkanGlucose is one of the nutritional factor that involves in developing of obesity and type 2 diabetes in human. The studies indicated that enterocyte cells on intestine might play a role in dietary glucose sensing during obesity. Obese people are consumed high amount of dietary glucose and enterocyte cells consequently are exposed to high glucose. Thus, we aimed to find relevant physiological pathways and genome-wide mRNA expression profiles that can be regulated by glucose in fully differentiated human intestinal epithelial (CaCo-2). The cells were maintained two different glucose levels (5.5mM for control, 25mM for high glucose) at least three passages. The cells were grown on transwell system for 21 days to mimic human intestine system. Transepithelial electrical resistances (TEER) were measured to control monolayer formation and polarization. RNA isolation was performed and whole genome mRNA expression profile were determined following gene ontology analysis to find affected molecular pathways. Compared to control relative glucose level was found high in basolateral side of CaCo-2 cells that were under high glucose condition without effecting TEER. GLUT2, SGLT1, GLUT5 mRNA levels were significantly reduced during elevated glucose levels which is consistent with literature. Significant fold change analysis showed that 351 genes upregulated and 468 genes under high glucose condition. We found high glucose significantly leads changes of molecular pathways (downregulated; glycolysis and gluconeogenesis, adherens junction, fructose/mannose metabolism, pentose phosphate pathway and upregulated; protein export). These results provide us better understanding and open new window for glucose metabolism of enterocytes during obesity.