Proteomics is a set of analytical methods which enable qualitative and quantitative characterization of the proteome. Expression proteomics quantitatively compares proteomes of cells, tissues, body fluids or other biological materials to find differencies in protein expression and, based on these differencies, to describe the biological processes occuring in investigated organisms. An initial material for expression proteomic studies are complex mixtures containing thousands of proteins, which are analyzed using separation (electrophoretic and chromatographic) methods, and identified, possibly quantified using mass spectrometry. The aim of this Thesis is to demonstrate the application of the tools of expression proteomics in solving diverse challenges in biomedicine. We employed various proteomic approaches and tools for studying molecular mechanisms of human diseases using pacient biological samples, or a model organism and a cell culture. We were conducting three different research projects, namely: A quest for potencial molecular targets for selective elimination of TRAIL-resistant mantle cell lymphoma cells; Investigation of molecular mechanisms of heart failure using a rat model of the disease induced by volume overload; and Searching for diagnostically usable serum biomarkers of ovarian...

Proteomics is a set of analytical methods which enable qualitative and quantitative characterization of the proteome. Expression proteomics quantitatively compares proteomes of cells, tissues, body fluids or other biological materials to find differencies in protein expression and, based on these differencies, to describe the biological processes occuring in investigated organisms. An initial material for expression proteomic studies are complex mixtures containing thousands of proteins, which are analyzed using separation (electrophoretic and chromatographic) methods, and identified, possibly quantified using mass spectrometry. The aim of this Thesis is to demonstrate the application of the tools of expression proteomics in solving diverse challenges in biomedicine. We employed various proteomic approaches and tools for studying molecular mechanisms of human diseases using pacient biological samples, or a model organism and a cell culture. We were conducting three different research projects, namely: A quest for potencial molecular targets for selective elimination of TRAIL-resistant mantle cell lymphoma cells; Investigation of molecular mechanisms of heart failure using a rat model of the disease induced by volume overload; and Searching for diagnostically usable serum biomarkers of ovarian...

Indiana University-Purdue University Indianapolis (IUPUI) / Winter survival is variable among commercially grown strawberry (Fragaria x ananassa) cultivars. The main objectives of this study were to evaluate the molecular basis that contribute to this difference in strawberry cultivars and to identify potential biomarkers that can be used to facilitate the development of new strawberry cultivars with improved overwintering hardiness. With these goals in mind, the freezing tolerance was examined for four cultivars, ‘Jonsok’, ‘Senga Sengana’, ‘Elsanta’, and ‘Frida’ (listed from most to least freezing tolerant based on survival from physiological freezing experiments) and the protein expression was investigated in the overwintering relevant crown structure of strawberry. Biomarker selection was based on comparing the protein profiles from the most cold-tolerant cultivar, ‘Jonsok’ with the least cold-tolerant cultivar ‘Frida’ in a comprehensive investigation using two label-free global proteomic methods, shotgun and two dimensional electrophoresis, with support from univariate and multivariate analysis. A total of 143 proteins from shotgun and 64 proteins from 2DE analysis were identified as significantly differentially expressed between ‘Jonsok’ and ‘Frida’ at one or more time points during the cold treatment (0, 2, and 42 days at 2 ºC). These proteins included molecular chaperones, antioxidants/detoxifying enzymes, metabolic enzymes, pathogenesis related proteins and flavonoid pathway proteins. The proteins that contributed to the greatest differences between ‘Jonsok’ and ‘Frida’ are candidates for biomarker development. The novel and significant aspects of this work include the first crown proteome 2DE map with general characteristics of the strawberry crown proteome, a list of potential biomarkers to facilitate the development of new strawberry cultivars with improved cold stress tolerance.

Biofilm formation is one of the most common bacterial survival strategies. Majority of bacterial species are able to form these three-dimensional structures, including pathogens like Mycobacterium tuberculosis. Representatives of Mycobacterium genus widely occur in the nature, although they can cause serious problems when they appear in medical equipment and artificial replacements of the human body. Non-pathogenic Mycobacterium smegmatis mc2 155 was used as a model organism in our experiments. We investigated morphology of the three- and six-day-old colonies (in fact biofilms) on agar and agar covered with cellophane using Stereo microscope and Scanning Electron Microscope. We found that a type of surface as well as a carbon source has a great influence on the morphology of the M. smegmatis colonies. We isolated proteomes from the agar and cellophane cultures and from planktonic culture. Two-dimensional electrophoresis was used as the main proteomic method. Proteomic data were analyzed using PDQuest software. Then the sets of proteins detected by qualitative and quantitative analyses were compared using Venn diagrams. As a result, we recognized 7 unique proteins that might be specific for recognition and adhesion of bacteria to the cellophane, no unique protein in agar proteome and 46 unique...

Made available in DSpace on 2016-01-26T12:51:34Z (GMT). No. of bitstreams: 1 alessandravidotto_tese.pdf: 5410688 bytes, checksum: e1af0ab5bd616652ebf0225cd33f4c1d (MD5) Previous issue date: 2009-08-27 / The regional lymph nodes play a pivotal role in diagnosis, staging and management of head and neck squamous cell carcinomas (HNSCC). Despite their importance, detailed understanding of the probable mechanisms of lymphatic metastases has not been completely achieved. Subjects and Methods: We analyzed metastatic and normal lymph node tissues, as well as saliva and serum from sixth-two patients with HNSCC, and twenty-nine controls using two-dimensional electrophoresis, MALDI-Q-TOF and western blot. Results: Several proteins were found to be significantly increased in metastatic nodes, such as stratifin, glutathione S-transferase pi, apoliproteín A-I, alpha-1-microglobulin, disulfide isomerase, galectin, citokeratins, immunoglobulins, transtirretin, calciun-binding protein (família S100) and fat-binding protein (FABP). Among the down-regulated proteins in metastatic lymph nodes are calreticulin, tropomiosin 3, triosephosphate isomerase, piruvate quinase, anidrase carbonic, gamma actin, peroxiredoxin 2, profilin 1, gliceraldeyde 3- fosfato desidrogenase and heat shock proteins. These proteins are involved in epidermis development, cell proliferation, migration and adhesion, apoptosis, defense and inflammatory response and xenobiotic metabolism. Our data on the expression of heat shock proteins and enzymes of the glycolytic pathway suggest an effect of the lymph node environment in controlling tumor progression or in metabolic reprogramming of the metastatic cell. In saliva, 13 proteins showed an altered pattern of expression in samples patient, including over-expression of keratins, immunoglobulins, alphaamylase, PLUNC and zinc-alpha-2-glycoprotein and down-regulation of myosin. In serum samples, six proteins were over-expressed (serum albumin, alpha-1- microglobulin/bikunin precursor, apolipoprotein A-I, haptoglobin, serotransferrin, transthyretin) and two were under-expressed (hemoglobin subunit alpha, hemoglobin subunit beta) compared to the control group. Conclusion: New potential markers, such as profilin-1 and E-FABP, were identified and may be proved useful for defining the invasive phenotype of head and neck carcinomas. / O comprometimento de linfonodos regionais por células neoplásicas é atualmente o indicador mais utilizado para prognóstico em pacientes com carcinoma epidermóide de cabeça e pescoço (CECP). Apesar disso, a compreensão detalhada dos mecanismos envolvidos na formação de metástases linfáticas ainda não foi completamente atingida. Casuística e Método: Foi avaliado o perfil protéico de linfonodos metastáticos e não metastáticos, bem como de amostras de saliva e soro de 62 pacientes em diferentes estágios da doença e de 29 controles, utilizando eletroforese bidimensional, espectrometria de massas por MALDI-Q-TOF e experimentos de validação por Western blot. Resultados: Os resultados mostraram várias proteínas com expressão elevada em linfonodos metastáticos em relação aos não metastáticos, como stratifina, glutathiona S-transferase pi, apoliproteína A-I, alfa-1-microglobulina, dissulfeto isomerase, galectinas, citoqueratinas, imunoglobulinas, transtirretina e proteínas de ligação ao cálcio (família S100) e a ácidos graxos (FABP). De forma inversa, as proteínas calrreticulina, tropomiosina 3, triofosfato isomerase, piruvato quinase, anidrase carbônica, gama actina, peroxirredoxina 2, profilina 1, gliceraldeído 3-fosfato desidrogenase e proteínas de choque térmico mostraram níveis reduzidos em linfonodos metastáticos. Essas proteínas estão envolvidas em processos de desenvolvimento epidérmico, proliferação, migração e adesão celular, apoptose, resposta inflamatória e metabolismo de xenobióticos. Os dados relacionados à expressão de proteínas de choque térmico e enzimas da via glicolítica sugerem um efeito do ambiente dos linfonodos e no controle da progressão do tumor ou na reprogramação das células metastáticas. Em saliva, 13 proteínas exibiram um padrão alterado nas amostras de pacientes com câncer, incluindo expressão elevada de queratinas, imunoglobulinas, alfa-amilase, PLUNC e zinc-alfa-2-glicoproteína e expressão reduzida de miosina. Em amostras de soro, seis proteínas apresentaram expressão aumentada (albumina, alfa-1-microglobulina/bikunina precursor, apolipoproteína A-I, haptoglobina, serotransferrina e transtirretina) e duas estavam com expressão diminuída (hemoglobina alfa e hemoglobina beta), quando comparadas com o grupo controle. Conclusão: Os resultados obtidos revelaram novos marcadores potenciais, como profilina 1 e E-FABP, PLUNC e transtirretin que podem ser úteis na definição do fenótipo invasivo e no rastreamento e diagnóstico desse grupo de neoplasias.

Made available in DSpace on 2016-08-17T18:39:43Z (GMT). No. of bitstreams: 1 4434.pdf: 2956570 bytes, checksum: f54aa91fcb424a121affb3c9674f546e (MD5) Previous issue date: 2012-08-17 / Universidade Federal de Minas Gerais / The aim of this work was to perform differential proteomic analysis of the periplasmic protein profiles of the genome strains A, B and C of Xanthomonas spp, which differ in pathogenicity and host range of citrus. The strain Xanthomonas citri subsp. citri (Xac) is the most virulent and infects all types of citrus, while the strain B (Xanthomonas fuscans subsp. aurantifolii, Xau-B) is less virulent and the strain C (Xanthomonas fuscans subsp. aurantifolii, Xau-C) has a unique citrus-host. The comparative proteomic analysis of Xau-B and Xau-C in relation to Xac can reveal genes and proteins involved in pathogenicity and host range of citrus, respectively. The strains A (Xanthomonas citri subsp. citri, Xac) and C (Xanthomonas fuscans subsp. aurantifolii, Xau-C) were grown in XAM-1, which is known to be a pathogenicity-inducing medium for Xac, and in a non-inducing pathogenicity (Nutrient Broth, NB). Proteins from both types (grown in triplicate using both media), were separated by bidimensional electrophoresis (2DPAGE) and the gels were stained using Coomassie Brilliant Blue R-250 and documented. A comparative analysis of the protein profiles of Xac and Xau-B, and Xac and Xau-C, grown in the same culture medium, was performed using ImageMaster Platinum software (GE Healthcare). Statistical analysis (ANOVA) revealed a large number of periplasmic proteins that presented type-specific or differential expression between the two bacteria. For the comparison between Xac and Xau-B we used the twodimensional Xau-B gels of periplasmic proteins obtained by Carnielli (2011). The differential spots between Xac and Xau-C, Xac and Xau-B that showed a significant differential expression (p <0.05) were isolated from gels and identified by ESI-QUADTOF mass spectrometry (LNBio, Campinas-SP), employing Xac, Xau-B and Xau-C databases. Several differentially expressed proteins between strains were identified for the different conditions studied, showing that some of them were strain-specific (approximately 10) while others were expressed in all strains, differing only in intensity. Those proteins potentially related to pathogenicity and citrus host were quantified using the software Scaffold v. 3.0, for the mass spectrometry data.The results showed that Xac, Xau-B, and Xau-C had remarkable differences between the periplasm protein profiles for both conditions, even under conditions of no induction of pathogenicity. The proteomics approach showed that even though the strains showed a different pattern of protein expression, the sequences of genes related to the differential proteins are present in all strains. Based on the proteomic analysis, proteins that showed remarkable differential expression between the genome strainswere selected, and its expression was evaluated by Western blot in periplasmic fraction of the bacteria. The data obtained in the the comparative proteomic analysis for the superoxide dismutase corroborated most quantitative results generated by the software Scaffold. This work showed that the proteomic analysis, combined with quantitative analysis tools, is an important tool that comes to complement genomic investigations designed to differentiate the species of Xanthomonas spp. / O presente trabalho teve por objetivo a análise proteômica comparativa da fração periplasmática das estirpes-genoma A, B e C de Xanthomonas spp, as quais diferem em patogenicidade e gama de citros hospedeiros. A estirpe A (Xanthomonas citri subsp. citri, Xac) é a mais virulenta e infecta todos os tipos de citros, enquanto que a estirpe B (Xanthomonas fuscans subsp. aurantifolii, Xau-B) é menos virulenta e a estirpe C (Xanthomonas fuscans subsp. aurantifolii, Xau-C) possui um único hospedeiro cítrico. Estas estirpes foram cultivadas em meio XAM-1, conhecido como sendo um meio indutor de patogenicidade para Xac, e em meio não indutor de patogenicidade (Caldo Nutriente, CN). Após a extração de proteínas da fração periplasmática em triplicata, as mesmas foram separadas por eletroforese bidimensional (2D-PAGE) e os géis foram corados com Coomassie Brilliant Blue R-250 e documentados. A análise proteômica comparativa de Xau-B e Xau-C relativamente à Xac pode nos levar a genes e proteínas envolvidas com patogenicidade e espectro de hospedeiro cítrico, respectivamente. Assim, Xac e Xau-C foram cultivadas em triplicata em meio XAM-1, conhecido como sendo um meio indutor de patogenicidade para Xac, e em meio não indutor de patogenicidade (Caldo Nutriente, CN), sendo as células coletadas ao final da fase exponencial de crescimento, segundo curvas previamente realizadas. Após a extração de proteínas periplasmáticas, as mesmas foram separadas por eletroforese bidimensional (2D-PAGE) e os géis foram corados com Coomassie Brilliant Blue R-250 e documentados. Uma análise estatística das diferenças observadas nas intensidades dos spots nos géis 2D foi realizada entre Xau-C e Xac e entre Xau-B e Xac (nos mesmos meios de cultura) utilizando o software Image Master 2D-Platinum (GE Healthcare). Para esta última comparação foram utilizados os géis bidimensionais de proteínas periplasmáticas de Xau-B obtidos por Carnielli (2011). As proteínas diferenciais entre Xac e Xau-C e entre Xac e Xau-B que apresentaram uma expressão diferencial significativa (p<0,05) foram isoladas a partir dos géis e identificadas por espectrometria de massas (LNBio, Campinas-SP) após pesquisa em banco de proteínas anotadas a partir do genoma de cada uma das linhagens. Inúmeras proteínas diferencialmente expressas entre as linhagens foram identificadas para as diferentes condições estudadas, demonstrando que algumas foram estirpe-específicas (10 aproximadamente) enquanto outras foram expressas em todas as linhagens, diferindo na sua intensidade. Aquelas proteínas que indicaram alguma potencialidade em relação à patogenicidade e hospedeiro cítrico foram quantificadas com o uso do software Scaffold v. 3,0, a partir de dados provenientes da espectrometria de massas. Os resultados obtidos demonstram que Xac, Xau-B e Xau-C apresentam perfis proteicos marcadamente diferentes na fração de periplasma, mesmo em condições de não indução da patogenicidade. A abordagem proteômica evidenciou que embora as estirpes apresentem um padrão de expressão protéica muito distinto na fração rica em proteínas periplasmáticas, as sequências dos genes relacionados às proteínas diferenciais (superóxido dismutase e fosfoglicomutase) estão presentes em todas as estirpes. Com base na análise proteômica, foram selecionadas proteínas que apresentaram expressão diferencial acentuada entre as linhagens-genomas, nas condições estudadas, sendo sua expressão avaliada por Western blot contra extrato protéico periplasmático das três linhagens- genomas, após cultivo das mesmas em meio CN e XAM-1 Os dados obtidos para a superoxido dismutase corroboraram a maioria dos resultados quantitativos gerados pelo software Scaffold. Este trabalho evidenciou que a análise proteômica, aliada às ferramentas de análises quantitativas, é um recurso que vem complementar as investigações genômicas destinadas a diferenciar as diferentes espécies de Xanthomonas spp. Sob os aspectos biotecnológicos a busca e identificação de proteínas biomarcadoras, em especial aquelas relacionadas com patogenicidade e especificidade à hospedeiro, são importantes alvos para produção de drogas no combate ao cancro cítrico.