Master Degree / Yüksek Lisans Tezleri
Permanent URI for this collectionhttps://hdl.handle.net/11147/3008
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Master Thesis Expression, Purification and Preliminary Functional Characterization of the Acidianus Two-Tailed Virus Tnpb Endonucleases(01. Izmir Institute of Technology, 2024) Burgeia, Arwa Saleh; Oke, MuseTnpB is an RNA-guided endonuclease encoded in the IS200/605 transposon family. It forms a complex with a self-encoded ωRNA that directs TnpB to an appropriate target DNA sequence for activity. DNA cleavage at the target site promotes homologous recombination thus ensuring propagation of IS200/605 elements. Although IS200/605 elements are abundant in prokaryotic genomes, they are rarely found in viruses. However, the Acidianus two-tailed virus (ATV), which possesses 72 genes, encodes four TnpB proteins (gp10, gp40, gp43 and gp68). In this study, bioinformatics analysis of all ATV tnpB genes demonstrated that they were all solo tnpB genes and therefore could be classified as part of the IS1341 group. TnpB proteins are characterized by having a DED catalytic site motif and a C-terminal C4 zinc finger motif. Although the ATV gp10 protein retains the DED active site motif found in most TnpB proteins, the other ATV TnpB proteins possess a different amino acid instead of the Glu residue. Additionally, the ATV TnpB proteins possess the C4 zinc finger motif except for ATV gp40, which possesses three cysteine residues. All four ATV tnpB genes were cloned and expressed in the heterologous Escherichia coli host. The gp40 and gp68 proteins were purified using Ni2+ -NTA and gel filtration chromatography. Although gp68 appears to bind to DNA, there is insufficient evidence for RNA binding. Cleavage assays revealed that gp68 demonstrated nonspecific DNA nickase and cleavage activities. The significance of this study and the broader implications regarding the possible role of TnpB in virus survival are discussed.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.