Master Degree / Yüksek Lisans Tezleri
Permanent URI for this collectionhttps://hdl.handle.net/11147/3008
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Master Thesis Direct of Determination of Surface Proteins of Leishmania Parasite by Proteomic Approach(01. Izmir Institute of Technology, 2023) Akıllı, Ayşe Necla; Yalçın, TalatLeishmaniasis is a neglected tropical disease caused by the Leishmania parasite, primarily seen in developing and underdeveloped countries. Due to immigration to our country, the effective population of the disease has increased recently. The lesion, which has visceral and cutaneous forms, can be lethal when it acts on internal organs. Surface proteins are the most crucial part of the parasite and host interaction. The parasite attaches to the host cell through surface proteins, enters the cell, multiplies, suppresses the immune system, and allows many other biological functions. It is the most critical research part in vaccine and biomarker discovery. Generally, cell surface biotinylation and cationic colloidal silica beads with which the surface is coated are used to analyze surface proteins. These methods break down the cell and are more open to contaminant proteins from the cytoplasm and nucleus. In addition, since the surface proteins are rich in hydrophobic amino acids, they are difficult to dissolve in polar solvents. The shaving method, uses a faster and minimal experimental workflow to cut only cell surface proteins without lysing the cell, has been tried in four Leishmania species (L.Tropica, L.Infantum, L.Major, L.Donovani). The shaving method aims to digest cell surface proteins by treating the cell surface with a proteolytic enzyme for a short time. Thus, it is expected to contain fewer contaminants and unwanted proteins. As a result of shaving method analysis with the Fusion Orbitrap Mass Spectrometer, the rate of surface protein defined in 4 different species was 9.34% in L.Tropica, 7.55% in L.Major, 7.9% in L.Infantum, 7.52% in L.Donovani. Consistent with the literature and candidates for biomarker ISCL, KMP-11, Leishmanolysin, PSA-2, ABC transporter, and lanosterol 14α demethylase, proteins were identified. Familiar and different proteins were tabulated.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 Investigation of Gas Phase Fragmentation Mechanism of Doubly Charged a Ions by Mass Spectrometry(Izmir Institute of Technology, 2019) Kızılkoca, Doğacan; Yalçın, Talat; Arslanoğlu, AlperThis dissertation presents studies of gas-phase fragmentation mechanism of doubly-charged a7 ions from basic amino acid containing model peptides under low-energy collision-induced dissociation (CID). The study includes three sets of C-terminal amidated model peptides which are alanine series containing basic amino acids (His – Arg – Lys). Position of His, Arg and Lys residue is varied from N-to-C terminal. Both positional effect and peptide sequence effect were examined for the fragmentation reactions of doubly-protonated a7 ions for these heptapeptides. The CID-MS4 mass spectra of doubly-protonated a7 ions have internal amino acid losses which provide an evidence for macrocyclization reaction. The proposed reaction mechanism involves production of doubly-charge a ions and charge-separation reaction of doubly-protonated a ions in the gas-phase which generates a protonated direct and non-direct a ion. All model peptides were also studied to understand behavior of doubly-protonated a ions better. Direct and non-direct sequence fragmentations which are singly or doubly protonated were observed for all studies. The reactions mechanisms were adjusted according to the results. In conclusions, the results presented in this dissertation can be used to elucidate the correct and reliable peptide sequences, and this improve protein identification strategies which is required for high-throughput proteomic studies.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 Investigation of Gas Phase Fragmentation Reaction Mechanisms of Doubly-Protonated Bn (n=7-9) Ions by Ion Trap Mass Spectrometry(Izmir Institute of Technology, 2014) Görgün, Özge; Yalçın, TalatIn this dissertation, the gas-phase fragmentation mechanisms of doubly-charged bn (n=7, 8, and 9) ions from basic amino acid containing model peptides were investigated by means of collision-induced dissociation (CID) coupled tandem mass spectrometry (MS/MS). The study utilized two sets of C-terminal amidated model peptides that HYAGFLV-NH2, YHAGFLV-NH2, YAHGFLV-NH2, YAGHFLV-NH2, YAGFHLV-NH2, YAGFLHV-NH2, YAGFLVH-NH2 and HAAAAAA-NH2, AHAAAAA-NH2, AAHAAAA-NH2, AAAHAAA-NH2, AAAAHAA-NH2, AAAAAHA-NH2, AAAAAAH-NH2 where the position of the histidine (His) residue is varied from N-to-C-terminal. Both positional effect and peptide sequence effect were examined for the fragmentation reactions of doubly-protonated b7 ions for these heptapeptides. The CID-MS3 mass spectra of doubly-protonated b7 ions have internal amino acid losses which provide an evidence for macrocyclization reaction. The proposed reaction mechanism involves charge-separation reaction of doubly-protonated b ions in the gas-phase which generates a protonated iminium ion of the N-terminal residue and protonated internal b ion with C-terminal oxazolone group. In addition to these model peptides, the C-terminal acid free forms of peptides such as SVEHAGVIL, SHIGDAVVI, EHAGVISVL, and GRIDKPILK were also used for to understand the extent of macrocyclization of doubly-charged b8 ion and very similar results were obtained. Moreover, the C-terminal acid free forms of peptide KRNGVIIAGY were investigated for the behavior of doubly-protonated larger b9 ion and doubly-protonated internal eliminations were obtained meanwhile singly-protonated amino acid eliminations and the mechanism was adjusted according to the results.Master Thesis A Proteomic Approach for Indentifying Boron-Stress Tolerant Proteins in Barley Genotpes(Izmir Institute of Technology, 2008) Atik, Ahmet Emin; Yalçın, TalatBoron is an essential micro-nutrient for plants. However, when boron is present at high concentrations in the soil or ground water, healthy plant growth and development can be affected by boron toxicity. Turkey constitutes about 72 % of the total boron reserves in the world. In Turkey, barley (Hordeum vulgare) is the second widely grown cereal after wheat. Boron toxicity is one of the major factors limiting the yield of barley in Central Anatolia of Turkey. In the present study, proteomic approach was used to investigate the boronstress tolerant proteins in Anadolu barley genotype (boron-tolerant). Eight-day-old barley plants were treated with 10 mM H3BO3 for seven days. Control plants received no boron treatment during this period. Total proteins of leaves were extracted and separated by two-dimensional polyacrylamide gel electrophoresis. Control and borontreated Anadolu genotype.s proteome maps were compared and the novel proteins were fragmented into peptides using in-gel digestion technique. Liquid chromatographytandem mass spectrometry (LC-MS/MS) analysis and database searching gave way to identify ten spots representing seven different proteins. Two spots were identified as the same protein and one protein could not identified. The identified seven proteins are namely, ribulose 1,5-bisphosphate carboxylase/oxygenase large chain (RuBisCo largechain), thaumatin-like protein TLP5, basic pathogenesis-related protein PR5, RNase Slikeprotein, vacuolar proton-translocating ATPase subunit E, PSI type III chlorophyll a/b-binding protein, and light-harvesting complex I; LHC I. Among the identified seven proteins, vacuolar proton-translocating ATPase (VATPase) subunit E is the important one for boron tolerance in tolerant barley genotype. It is shown that the accumulation of excess boron in the vacuolar compartment of the plant cell by the help of V-ATPase subunit E protein. This is the known as internal tolerance mechanisms for Anadolu genotype of barley to survive under boron stress. It was proposed that, this might be the defense mechanism in boron-tolerant barley genotype under toxic boron concentrations.Master Thesis Investigation of Heat Stress-Induced Proteins of Cold-Adapted Pseudomonas Marginals Using Proteomic Approach(Izmir Institute of Technology, 2008) Taşoğlu, Çağdaş; Yalçın, TalatTemperature alteration is known as a common environmental stress condition which all living organisms encounter and response by producing evolutionary wellconserved specific proteins called heat stress or heat shock proteins in the cell in order to adapt and survive. In the current study, the induction of heat stress proteins in a coldadapted bacterial strain of Pseudomonas marginalis cells grown under heat stress was investigated by proteomic approach. Five different temperatures, 5, 10, 15, 24, and 30C, were examined for the purpose of determining the optimum growth temperature for the bacterium. Consequently, 15°C was observed as optimum temperature for growth while 30C was established as heat stress temperature. Total proteins from Pseudomonas marginalis cells in the late exponential phase of growth at these two temperatures were extracted and separated by two-dimensional polyacrylamide gel electrophoresis. Totally 1391 protein spots were visualized for 15C and 1384 protein spots for 30C. After comparing with 15C, 13 protein spots that were differentially expressed in the cells exposed to heat stress (30C) were cut from the gel and fragmented into their peptides by in-gel digestion method. Finally, these proteins were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and database searching. Among them, ribosome recycling factor, universal stress protein family and chaperonin GroEL were established as direct sensors of heat stress. As a result, the genes encoding these two heat stress proteins can be isolated and cloned into any other useful microorganism such as bacteria used for detoxification of industrial waste or used in bioremediation but not capable of surviving at high temperatures so that they can be efficient at those temperatures, too.