Master Degree / Yüksek Lisans Tezleri
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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.