Master Degree / Yüksek Lisans Tezleri
Permanent URI for this collectionhttps://hdl.handle.net/11147/3008
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Master Thesis Investigation of the Proteins of Leishmania Tropica Causing Viscerotropism in Humans Using Mass Spectrometry(Izmir Institute of Technology, 2022) Beyaz, Merve; Yalçın, TalatLeishmaniasis is a neglected tropical disease in 98 countries and five continents worldwide. The most prevalent forms of this parasitic disease are Cutaneous, Mucocutaneous, and Visceral Leishmaniasis. While Cutaneous Leishmaniasis causes disfiguring skin conditions and lesions, Mucocutaneous Leishmaniasis damages the mucosal tissues of the mouth, nose, and throat. The visceral form of Leishmaniasis causes weight loss, fever, diarrhea, lymph nodes, and spleen or liver enlargement. Today, Leishmania tropica, one of the strains of the Leishmania parasite, no longer causes only Cutaneous Leishmaniasis (CL) but also Visceral Leishmaniasis (VL). The reason for this visceralism in L. tropica is not fully understood. Mass spectrometry has a vital place in proteomic analyses; it provides information about expression levels and the identification of proteins. In this study, the proteins of L. tropica causing CL and CL are analyzed using the mass spectrometric shotgun method. Off-line HPLC separation followed by LC-MS/MS analyses are performed, and differential proteins between CL and VL isolates of L. tropica are determined. Results indicate that among the differentially abundant proteins between two sample groups, paraflagellar rod proteins, elongation factor 1-alpha protein, and surface antigen proteins might play a role in avoiding immune recognition. Also, proteins with peroxidoxin function, cytochrome b5, and endoribonuclease might help parasite survival in macrophages. And finally, thiol-specific antioxidant protein may have a role in viscerotropism in L. tropica.Master Thesis Investigation of Protein Profiles of Listeria Monocytogenes in the Existence of Phenolic Acids Using Mass Spectrometry(Izmir Institute of Technology, 2020) Şen, Sener; Yalçın, Talat; Soyer Dönmez, FerdaListeria monocytogenes is one of the foodborne pathogens (FBP), which are a threat to the consumers' health, able to cause listeriosis. L. monoytgenes cells, which can easily adapt and survive stresses, can develop resistance to antibiotics used in standard therapy. Phenolic acids that are a natural defense mechanism against stress conditions in plants, might be used as an antibacterial-candidate in foodborne diseases, so there is a need for a better understanding of the stress-induced responses and mechanisms of bacteria against these substances. Proteomic approaches are an invaluable method for identifying the stress response in pathogenic bacteria. For this purpose, in this study firstly, the antibacterial effects of two phenolic acids (3-HPAA and 4-HBA) on bacteria were investigated by determining the minimum inhibitory concentrations (MIC). Subsequently, target changes in the protein profile due to antimicrobial effects of phenolic acids were evaluated using a soft ionization technology and mass spectrometry-based comparative gel-free proteomic approach (Shotgun proteomics). According to the results, Listeria monocytogenes could not develop resistance to both phenolic acids. This study emphasizes the importance of using of phenolic acids as a novel and natural therapy methods to overcome antibiotic-resistant pathogenic bacteria.Master Thesis Mass Spectrometry-Based Proteome Analysis of Leishmania Major Parasite in Two Clinical Isolates Which Exhibit Different Impact on Virulence(Izmir Institute of Technology, 2018) Güvenç, Nazlı; Yalçın, TalatLeishmaniasis is a disease that covered under the title of neglected tropical diseases caused by protozoan parasites called Leishmania which can classify into three groups as visceral, cutaneous and mucocutaneous leishmaniasis. L. major is a type of parasite that causes cutaneous leishmaniasis and it is endemic in Iran, and Syria. However, cutaneous type leishmaniasis caused by L. major has been begun to detect in Turkey due to its close location to such countries. Moreover, the variety in the infectivity of L. major in a different region of Turkey has detected. Therefore, the uncertainty under the virulence effect of L. major is aimed to investigate. Large-scale protein analysis by mass spectrometry based proteomics has cleared up to proteome mapping for different organism recently. Generally, although two methodologies that involve gel-free and gel-based approaches have widely accepted for proteomic analysis, gel-free LC-MS/MS analysis were applied to characterize the proteome analysis of L. major parasite in two clinical case exhibiting passive and aggressive virulence effect on leishmaniasis. Finally, differential and common proteins that can affect the infectivity of L. major invastigated by shotgun analysis. As a result, samples showed that there are conflict results with the literature about GP63, secreted acid proteases, cysteine proteases and Peroxiredoxin proteins existence and also in the aggressive L. major cystathionine beta-synthase protein which has an role to synthesis of CPs and pyridoxal phosphate binding activity were proposed as a critical protein for L. major infectivity due to its association with SAPs, CPs.Master Thesis Mass Spectrometry-Based Comparative Proteomic Analysis of Drug Resistant and Nonresistant Strains of Parasite Trichomonas Vaginalis(Izmir Institute of Technology, 2018) Özyağcı, Begüm; Yalçın, TalatTrichomonas vaginalis (T. vaginalis) causes sexually transmitted disease, trichomoniasis. Acute infections can result in cervical cancer, pelvic inflammatory disease, HIV-1 infection, preterm births, and low birth weight. Metronidazole, an antibiotic, is the standard treatment of the disease. However, in some cases T. vaginalis shows resistance to metronidazole thus treatment fails. Nevertheless, resistance mechanism of the parasite is not fully understood. Mass spectrometry has become an important tool in proteomic analysis with the emergence of soft ionization techniques instead of traditional protein identification and sequencing methods. In this study, a comparative gel-free proteomic analysis based on mass spectrometry with soft ionization technology was performed in two sets for resistant and non-resistant strains of T. vaginalis parasite isolated from clinical cases. Total proteins were digested by in solution digestion method and separated with high pH reversed-phase liquid chromatography. After fractions were concatenated, each fraction of sample was analyzed by reversed-phase liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) and proteins were identified by database search. Common and differential proteins between resistant and two sensitive trichomoniasis samples were compared according to their molecular function. Results indicate that ferredoxin 5 and hydroxyl amine reductase are differential proteins with iron-sulfur cluster binding activity identified for only resistant strain. Thioredoxin reductase, alcohol dehydrogenase, superoxide dismutase, pyruvate:ferredoxin oxidoreductase are studied proteins in the literature and also identified as common proteins in all strains for this study. These proteins might have a role in drug resistance mechanism of T. vaginalis.Master Thesis Importance of Database Normalization for Reliable Protein Identification in Mass Spectrometry-Based Proteomics(Izmir Institute of Technology, 2016) Mungan, Mehmet Direnç; Allmer, Jens; Yalçın, TalatOne of the revolutionary steps towards proteomics, was introducing mass spectrometry to protein inference analysis. Its powerful aspects such as speed, and accuracy towards identifying and quantifying proteins have made it the first choice to obtain highthroughput data. Due to development of a variety of fragmentation techniques, mass spectrometry-based analysis even made it possible to acquire knowledge about single polymorphisms and modifications of amino acids of a peptide. Although this technology provides enormous amounts of data, identification of the proteins is still a hard challenge to overcome due to the shortcomings of computational methods. Herein a novel methodology is offered to better analyze mass spectrometry data and overcome the deficiency of protein identification algorithms in terms of speed and accuracy. When the spectral data is acquired from an organism by mass spectrometry, database search algorithms are used for protein identification if the protein sequences of the organism are known. These algorithms compare the experimental data from mass spectrometry analysis to theoretical data gathered from known databases of organism to try and find the best match by ranking the PSMs via scoring functions. Since the databases can be too large to search and multiple databases with different sizes can contain the peptides of experimental data, database search algorithms may fail to produce fair, fast or complete results. In this work a methodology is presented to overcome unfair scoring of peptides in different size databases and enable database search algorithms to utilize relatively big sized entries such as human chromosome six frame translations. In terms of speed and accuracy the method is found to be better than some of the existing methods.Master Thesis Salt Stress Responsive Proteins Identification in Wild Sugar Beet (beta Maritima) by Mass Spectrometry(Izmir Institute of Technology, 2008) Baydara, Emine Pınar; Yalçın, TalatSalt stress is one of the major abiotic stresses in agriculture worldwide. Seven percent of the land.s surface and five percent of cultivated lands are affected by salinity.Turkey is the fourth in the world and third in Europe in producing sugar beet. It is observed that salt stress affects the sugar beet negatively especially at germination and seedling stages, it limits the productivity of crop plants and affects the quality of plants.In the present study, proteomic approach was used to investigate the salt-stress responsive proteins in wild salt-tolerant beet, Beta maritima. Sugar beet were grown approximately two months. After growing, they were treated with 250 mM NaCl for seven days. Control plants received no salt treatment during this period. Total proteins of leaves and root were extracted. The proteins were fragmented into peptides using insolution digestion technique and liquid chromatography-tandem mass spectrometry (LC-MS/MS) used for identified the proteins. Totally 288 proteins were identified in leave samples and totally 259 proteins were identified in the root samples.Identified protein results were shown that unique of salt leave proteins and upregulated proteins of leave samples were the related to the antioxidant enzymes. On the other hand, active transporter protein of vacular ATP synthase subunit A was identified in the salt responsive of root samples.Master Thesis Investigation of Dichlorvos (ddvp) and Trifluralin Pesticide Levels in Tahtalı Dam Water(Izmir Institute of Technology, 2002) Erdoğan, Murat; Özgen, TamerkanIn this study, dichlorvos (Dimethyl 2,2-dichlorovinyl phosphate - DDVP) and trifluralin (.,.,.-trifluoro-2,6-dinitro-N,N-dipropyl-p-toluidin) pesticide concentration levels in Tahtalı Dam Water were investigated. Dichlorvos is an organophosphorus pesticide, whereas Trifluralin is a dinitroaniline pesticide.Both of these pesticides are widely used for agricultural purposes in Tahtalı Dam Basin. These pesticides could be carried to the Tahtalı Dam Water, and therefore their concentrations should be controlled.Another reason why these pesticides were selected was that, their method of determination was not straightforward and special determination technique has to be used. That is why these pesticides were not studied extensively for İzmir area.For the determination of the above-mentioned pesticides, gas chromatographymass spectrometry (GC-MS) was generally preferred as reported in most papers [1,2,3].The GC-MS instrument in our laboratory has an Ion Trap (IT) mass detector. Operating in Selected Ion Storage (SIS) or Tandem mass (MS-MS) modes can increase the sensitivity and selectivity of this instrument. The matrix effect coming from the aqueous solution was eliminated by GC-SIS-MS and GC-MS-MS. The detection limits of the instrument are 0.8 .g/L for trifluralin and 10.5 .g/L for dichlorvos, therefore a preconcentration process was required because the studied concentrations are in 1-3 .g/L levels for surface water and 0.1 .g/L levels for drinking water.Liquid-Liquid Extraction (LLE) and Solid Phase Extraction (SPE) methods were used for sample preconcentration. Gas chromatography (GC) - Mass spectrometry (MS) and Tandem mass spectrometry (MS.MS) were employed for the identification and quantification of Trifluralin and Dichlorvos (DDVP) pesticides. For Solid Phase Extraction procedure ENVI-18 Disk was used, optimizing the extraction volume, pH and the salt concentration. Liquid-Liquid extraction procedure was also used, optimizing the extraction volume. In GC.MS.MS, the lowest detectable concentrations for the Trifluralin and Dichlorvos were found as 0.8 ng/L and 10.5 ng/L, respectively.Recovery of Dichlorvos for Liquid-Liquid Extraction and Solid Phase Extraction were 86.0 (±5.4) % and 63.0 (±5.7) % in water samples spiked with 200 ng/L pesticides.Recovery of the Trifluralin for Liquid-Liquid Extraction and Solid Phase Extraction were 90.8 (±9.4) % and 107.5 (±4.5) % in water samples spiked with 200 ng/L pesticides.Water samples, which were collected between 01 June 2002 to 30 September 2002 by İZSU (İzmir Büyükşehir Belediyesi Su ve Kanalizasyon Genel Müdürlüğü), were analyzed using GC-MS system with tandem mass (MS-MS) mode after preconcentration process. Analysis of samples showed no detectable Trifluralin and Dichlorvos levels in Tahtalı Dam Water.Master Thesis Identification of Cytosolic Sialidase Neu2 Associated Proteins Bt Mass Spectrometric Analysis(Izmir Institute of Technology, 2013) Akyıldız Demir, Seçil; Seyrantepe, VolkanSialidases (Neuraminidases) are the enzymes which remove sialic acids from glycoproteins, oligosacharides and glycolipids. Four mammalian sialidases have been identified which are lysosomal sialidase (Neu1), cytosolic sialidase (Neu2), plasma membrane sialidase (Neu3), and mitochondrial/lysosomal sialidase (Neu4). These enzymes differ in their subcellular localizations, expression levels in different cells and tissues, substrate specificities and optimum pH levels. Cytosolic Neu2 enzyme has an active role on a wide range of subtances including oligosaccharides, glycopeptides and gangliosides. Studies on the Neu2 enzyme also showed that this enzyme is involved in different cellular events including cancer metabolism, neuronal differentiation and myoblast differentiation, proliferation and hypertrophy. However, it has not been shown whether Neu2 interacts with other proteins within the cell. Therefore, in this study we aimed to identify Neu2 associated proteins by using InterPlay Mammalian TAP System and ESI-LC-MS/MS analysis. Proteins in the Neu2 protein complex were identified by three different database search software such as PGLS, Mascot and X!Tandem. As a result of experiment Actin proteins (Alpha Actin, Gamma Actin and Beta Actin), and Calsyntenin-2 protein were found as a candidate protein for Neu2 association. The interaction between Neu2 and β-Actin proteins was confirmed by western blot analysis.Master Thesis Analysis of Lysosomal Neu4 Sialidase Associated Proteins by Using Mass Spectrometry (ms/Ms)(Izmir Institute of Technology, 2012) Öztürk, Süleyman Can; Seyrantepe, Volkan; Seyrantepe, VolkanSialidases are glycohydrolytic enzymes which remove sialic acid residues from glycoproteins, oligosaccharides and glycolipids. There are 4 different sialidases known in mammalians. These are Neu1 (lysosomal), Neu2 (cytoplasmic), Neu3 (cell membrane) and Neu4 (lysosomal/mitochondrial) sialidase. Sialidases are involved in many metabolic and cellular processes interactioning with another proteins or work together in multiprotein complexes. For example, Neu1 is only active with betagalactosidase and cathepsin A enzyme in lysosome. Interactions of sialidases Neu2, Neu3 and Neu4 enzyme with other proteins remain unknown In our study, we aimed to identify proteins which have interaction with sialidase Neu4 as well as Neu1 by using mass spectrometry analysis to find new possible roles of sialidases. Our bait protein's cDNA was tagged with calmodulin binding protein as well as streptavidin binding protein. After transfection and expression of vectors to mammalian cells, proteins were purified using tandem affinity purification (TAP). We identified some associated proteins with sialidase Neu1 and Neu4 by using MS/MS analysis and bioinformatics.Master Thesis Method Development for Protein Identification With Maldi-tof/Tof by Using On-Surface Digestion(Izmir Institute of Technology, 2012) Dinç, Melike; Yalçın, TalatProtein identification is predominantly carried out by searching tandem mass spectrometric data of peptides in a protein database. For this reason, proteins are converted to peptides through a digestion process by using some certain endoproteinases. Trypsin is mostly preferred in this sample preparation step due to its high activity and products having appropriate mass range. Whereas in-solution digestion method is applied for the proteins in solution, proteins trapped in the gel can be digested by using in-gel digestion technique. Alternative to these traditional digestion methods, it has been reported that proteins can be digested too while they were adsorbed onto solid surfaces. In this study, digestion process of the adsorbed proteins, namely on-surface digestion is examined widely by using both hydrophobic and ionic adsorbents on different proteins. Results of the on-surface digestion were compared with in-solution digestion and in-gel digestion methods. As a conclusion, on-surface digestion is applicable for the protein identification by mass spectrometry; however, its yield may change from one experiment to another, depending on two separate but related processes: protein adsorption before the digestion and peptide recovery after the digestion. Nevertheless on-surface digestion has the advantages of protein enrichment and protein purification prior to mass spectrometry. These processes are necessary and significant especially for the samples containing minute amounts of protein and an effective enzymatic activity. Last but not least, this method may be performed complementarily to other digestion methods since new and different peptides may be acquired from the same sample source.