BACKGROUND:Mycoplasma hyopneumoniae causes respiratory disease in swine and contributes to the porcine respiratory disease complex, a major disease problem in the swine industry. The M. hyopneumoniae strain 232 genome is one of the smallest and best annotated microbial genomes, containing only 728 annotated genes and 691 known proteins. Standard protein databases for mass spectrometry only allow for the identification of known and predicted proteins, which if incorrect can limit our understanding of the biological processes at work. Proteogenomic mapping is a methodology which allows the entire 6-frame genome translation of an organism to be used as a mass spectrometry database to help identify unknown proteins as well as correct and confirm existing annotations. This methodology will be employed to perform an in-depth analysis of the M. hyopneumoniae proteome.RESULTS:Proteomic analysis indicates 483 of 691 (70%) known M. hyopneumoniae strain 232 proteins are expressed under the culture conditions given in this study. Furthermore, 171 of 328 (52%) hypothetical proteins have been confirmed. Proteogenomic mapping resulted in the identification of previously unannotated genes gatC and rpmF and 5-prime extensions to genes mhp063, mhp073, and mhp451, all conserved and annotated in other M. hyopneumoniae strains and Mycoplasma species. Gene prediction with Prodigal, a prokaryotic gene predicting program, completely supports the new genomic coordinates calculated using proteogenomic mapping.CONCLUSIONS:Proteogenomic mapping showed that the protein coding genes of the M. hyopneumoniae strain 232 identified in this study are well annotated. Only 1.8% of mapped peptides did not correspond to genes defined by the current genome annotation. This study also illustrates how proteogenomic mapping can be an important tool to help confirm, correct and append known gene models when using a genome sequence as search space for peptide mass spectra. Using a gene prediction program which scans for a wide variety of promoters can help ensure genes are accurately predicted or not missed completely. Furthermore, protein extraction using differential detergent fractionation effectively increases the number of membrane and cytoplasmic proteins identifiable my mass spectrometry.

Teladorsagia circumcincta is a major financial burden on the UK sheep farming industry. Disease control is becoming increasingly difficult due to the rapid emergence of anthelmintic resistance. This has prompted the search for alternative, sustainable control measures, including vaccination. Vaccine design would be aided by a thorough knowledge of the mechanisms involved in immunity to T.circumcincta. Most research has focussed on humoral and cellular responses to infection with this nematode. This thesis focuses on the impact of infection with regards to the proteins found locally within the abomasum. Using a well established infection model, proteomic analysis of lymph draining the abomasum was carried out by means of 2-dimensional electrophoresis (2-DE). The identity of many of the proteins in gastric lymph was revealed by means of MALDI-TOF analysis. The relative quantities of the lymph proteins were monitored over time using gel analysis software in both primary infection and immune challenged infection models. This study revealed a number of proteins of interest, including the acute phase proteins serum amyloid A, alpha-1 acid glycoprotein and haptoglobin, as well as the actin depolymerising protein, gelsolin. The effect of infection and immunity to T.circumcincta on these proteins was investigated further by means of biochemical assays, western blotting and real-time PCR. The impact of infection on the permeability of the abomasal mucosa will affect the resultant gastric lymph proteome. This “leak lesion” phenomenon is well documented in T.circumcincta infection but the underlying cause is unknown. Tight junction proteins in the abomasum were studied, using immunofluorescence techniques, in an attempt to define the role of these proteins in this important immunological/pathological event. The aim of this thesis is to contribute to the knowledge of innate immune responses and local pathology occurring within the abomasum during T.circumcincta infection.

The recent increase in availability of next generation sequencing methodologies has led to extensive analysis of the genome of Clostridium difficile. In contrast, protein expression analysis, crucial to the elucidation of mechanisms of disease, has severely lagged behind. In this study, in-depth proteomic analysis of three strains of varying virulence, demonstrated previously in an animal model, has been undertaken against a background of the sequenced genomes. Strain B-1 is a historic, virulent, ribotype 005 clone, strain A represents the emerging hypervirulent 027 ribotype, while strain Tra5/5, ribotype 001, is of low virulence. To undertake a comprehensive overview of the expressed proteome, both 1D and 2D gel electrophoresis were used to separate and display the protein content of each isolate. This was coupled to MALDI-TOF and LC-MS/MS mass spectrometry for protein identification. A total of 888 different proteins were characterised by comparative analysis of isolates grown in parallel for 64 hours on blood agar. Of these, only 38% were shared between all isolates. An additional 350, 243 and 398 proteins were detected from broth cultures, and the use of a hexapeptide bead library, designed to capture low abundance proteins, led to the detection of a further 148, 127, and 171 proteins in strains A, B-1 and Tra5/5 respectively. Relative differential expression was investigated using Differential In Gel Electrophoresis (DIGE), and five proteins were shown to have a statistically higher concentration in strain A, twelve in strain B-1 and eight in strain Tra5/5. A number of these were surface proteins, with selected S-layer proteins found to be up-regulated in each strain, and the flagellar protein, FliC, up-regulated in both A and B-1. Furthermore, differential post-translation modification events were seen in flagellar and S-layer proteins. In-vivo expression of these proteins was mapped using Western blotting. Immunodetection of the majority of these, including FliC and the high molecular weight S-layer protein, were conserved between the three strains, but a notable series of immunoreactive protein spots were present in strains A and Tra5/5 but not B-1, most likely corresponding to an additional S-layer protein present in the genomes these strains, but not that of B-1. Protein expression differences for a number of previously proposed virulence proteins were evident between strains, including toxin B, sporulation, flagella and the S-layer proteins, metabolic enzymes, stress response proteins and ABC transporters. This study strongly supports the view that the virulence of Clostridium difficile is multifactorial, and that a number of related factors, although not directly required for pathogenicity, may serve to modulate the virulence of individual strains.

Internal fertilization occurs in birds and eutherian mammals. Foetal development, however, is either extra- respectively intra-corpore (egg vs uterus). In these animal classes, the female genital tract stores ejaculated spermatozoa into a restricted oviductal segment; the functional pre-ovulatory sperm reservoir, where they survive until ovulation/s occur. Paradoxically, this immunologically foreign sperm suspension in seminal fluid/plasma, often microbiologically contaminated, ought to be promptly eliminated by the female local immune defence which, instead, tolerates its presence. The female immune tolerance is presumably signalled via a biochemical interplay of spermatozoa, as well as the peptides and proteins of the extracellular seminal fluid, with female epithelial and immune cells. Such interplay can result in gene expression shifts in the sperm reservoir in relation to variations in fertility. To further aid our understanding of the underlying mechanisms, this thesis studied the proteome of the seminal fluid (using 2D SDS-PAGE and mass spectrometry) including cytokine content (using Luminex and/or ELISA) of healthy, sexually mature and fertile boars and cocks. As well, gene expression changes (using cDNA microarray) in the oviductal sperm reservoirs of sexually-mature females, mated or artificially infused with homologous sperm-free seminal fluid/plasma were studied. Pigs were of commercial, fertility-selected modern breeds (Landrace), while chicken belonged to the ancestor Red Junglefowl (RJF, low egg laying-capacity), a selected egg-layer White Leghorn (WL) and of their Advanced Intercross Line (AIL). Ejaculates were manually collected as single sample in cocks or as the sperm-rich fraction [SRF] and the post- SRF fraction in boars to harvest seminal fluid/plasma for proteome/cytokine and infusion-studies. Oviducts were retrieved for gene-expression analyses via microarray immediately post-mortem (chicken) or at surgery (pig), 24 h after mating or genital infusion. In pigs, the protein-rich seminal plasma showed the highest amounts of cytokines [interferon-γ, interferon gamma-induced protein 10 (IP-10/CXCL10), macrophage derived chemokine (MDC/CCL22), growth-regulated oncogene (GRO/CXCL1), granulocyte-macrophage colony-stimulating factor (GM-CSF), monocyte chemo-attractant protein-1 (MCP-1/ CCL2), interleukin (IL)-6, IL-8/CXCL8, IL-10, IL-15, IL-17 and transforming growth factor (TGF)-β1-3) in the larger, protein-rich and sperm-poor post-SRF, indicating its main immune signalling influence. Chicken showed also a plethora of seminal fluid proteins with serum albumin and ovotransferrin being conserved through selection/evolution. However, they showed fewer cytokines than pigs, as the anti-inflammatory/immune-modulatory TGF-β2 or the pro-inflammatory CXCL10. The RJF contained fewer immune system process proteins and lacked TGF-β2 compared to WL and AIL, suggesting selection for increased fertility could be associated with higher expression of immune-regulating peptides/proteins. The oviductal sperm reservoir reacted in vivo to semen exposure. In chicken, mating significantly changed the expression of immune-modulatory and pH-regulatory genes in AIL. Moreover, modern fertile pigs (Landrace) and chicken (WL), albeit being taxonomically distant, shared gene functions for preservation of viable sperm in the oviduct. Mating or SP/SF-infusion were able to change the expression of comparable genes involved in pH-regulation (SLC16A2, SLC4A9, SLC13A1, SLC35F1, ATP8B3, ATP13A3) or immune-modulation (IFIT5, IFI16, MMP27, ADAMTS3, MMP3, MMP12). The results of the thesis demonstrate that both mating and components of the sperm-free seminal fluid/plasma elicit gene expression changes in the pre-ovulatory female sperm reservoir of chickens and pigs, some conserved over domestication and fertility-selection.

O valor nutricional do milho é limitado devido ao seu alto conteúdo de proteínas de reserva (zeínas), que são deficientes em aminoácidos essenciais como lisina e triptofano. Por esse motivo, a Embrapa (Empresa Brasileira de Pesquisa Agropecuária) desenvolveu a variedade de milho BR473, que tem um excelente valor energético e um maior conteúdo protéico quando comparado ao milho comum. Tal variedade possui 0,9 e 4,0 g/kg de grão de triptofano e lisina, respectivamente, contra 0,6 e 2,6 do milho comum. O alto conteúdo de lisina em algumas variedades de milho como a opaco-2 (o2), por exemplo, é relacionado à presença de proteínas não-zeínas. Para identificar essas proteínas, o que poderia explicar o alto conteúdo protéico da variedade BR473, foi analisado o proteoma dos corpos protéicos dos grãos desse milho. Para tal, foram usadas e avaliadas as cromatografias uni e bidimensional, acopladas a espectrometria de massas (LC-MS/MS e LC/LC-MS/MS), uma vez que tais técnicas se tornaram ferramentas poderosas para seqüenciamento e identificação de proteínas. Neste trabalho, foram identificadas seis proteínas por LC-MS/MS e dezesseis proteínas por LC/LC-MS/MS. / The nutritional value of maize seed is limited due to its high content of zeins (storage proteins), which are deficient in essential amino acids as lysine and tryptophan. Because of this, Embrapa (Brazilian Agricultural Research Corporation) developed the BR 473 maize variety, which has excellent energetic value and higher proteic content than the normal maize. BR 473 maize variety has 0.9 and 4.0 g/kg of grain of tryptophan and lysine, respectively, against the 0.6 and 2.6 from normal maize. The high lysine content in some maize varieties as opaque-2 (o2), for example, has been related to the presence of non-zein proteins. In order to identify them, which could explain the high proteic content of BR 473 maize variety, the protein bodies proteome of these grains were analyzed. For this purpose, one and two-dimensional liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS and LC/LC-MS/MS) were used and evaluated, since these techniques have become a powerful tool for protein sequencing and identification. We have identified six proteins by LC-MS/MS and sixteen proteins by LC/LC-MS/MS.

As interações entre as plantas cultivadas e os outros organismos do agroecossistema podem afetar as características dos alimentos, trazendo implicações que abrangem desde as condições socioeconômicas dos produtores até a qualidade nutricional e sensorial dos produtos agrícolas. No caso de agroecossistemas equilibrados, a conservação da biodiversidade na propriedade contribui para diminuir a dependência de insumos externos, principalmente para o controle de pragas e doenças. Nesse sentido a produção de bananas no Vale do Ribeira constitui um mosaico de agroecossistemas, que pressionam os maiores e mais conservados remanescentes florestais de Mata Atlântica. A banana é um fruto climatérico típico, que mostrou grande potencial para o presente estudo, primeiro por ter boa parte de seus processos bioquímicos parcialmente elucidados, segundo, por apresentar seu genoma sequenciado e, terceiro, por ser um cultivo que permeia as áreas de Mata Atlântica do Vale do Ribeira. A perspectiva de análise do Proteoma label-free do fruto foi escolhida por sua ampla capacidade de compreensão de respostas biológicas, especialmente em delineamentos experimentais inéditos como esse, onde não é possível fazer grandes especulações acerca da resposta esperada. Dessa forma, inserido a um projeto de objetivo mais amplo, o objetivo deste trabalho foi avaliar a influência da biodiversidade atlântica no entorno do agroecossistema sobre a expressão de proteínas das bananas, considerando os aspectos físico-químicos, fisiológicos e bioquímicos, de modo a estimar as vias metabólicas influenciadas por esta condição ambiental. O projeto foi desenvolvido a partir da comparação de duas áreas comerciais de bananicultura que possuem idade e tratos culturais idênticos, sendo que a única diferença é que uma delas encontra-se cercada exclusivamente pelo monocultivo de bananeiras (parcela Controle) e a outra possui 60% do perímetro adjacente a um fragmento de Mata Atlântica (parcela Biodiversidade). Foi utilizada uma estratégia holística, contemplando diversos fatores do ambiente (fertilidade do solo e aspectos climáticos), da fisiologia da bananeira (diagnose foliar e infestação por doenças) e da banana (aspectos de qualidade, comportamento pós-colheita e proteoma da polpa). Os resultados mostram que as plantas da parcela biodiversidade apresentaram menor Índice de Severidade de Sigatoka Negra e produziram frutos com maior vida verde. Em relação ao Proteoma, as vias do Ciclo do ácido cítrico, do Metabolismo do piruvato e da Alanina, aspartato e glutamato foram as mais alteradas entre os frutos das duas parcelas, sinalizando uma maior tendência na síntese de ácidos graxos nos frutos da parcela Biodiversidade, que parece ter sido desviada para a síntese de aminoácidos nos frutos da parcela Controle. Algumas evidências reunidas sugerem que a presença da biodiversidade da Mata Atlântica no entorno do agroecossistema favorece o restabelecimento da homeostase vegetal, trazendo efeitos benéficos para o cultivo e para o fruto. / Food quality is affected by crop and other agroecosystem organism interaction. These are a broad and diverse field of study, with unclear central issues, implying since socioeconomic condition of the producer, up to food quality, in terms of nutritional and sensorial issues. In this sense, banana production on Vale do Ribeira represents an agroecosystem mosaic, among the hugest and most conserved remaining Atlantic forest. Banana is a climacteric fruit with great potential for this study, firstly because its biochemical processes has been partially clarified, secondly, because its genome is already sequenced and, finally, because its cultivation area is surrounded by Atlantic forest areas from Vale do Ribeira. Proteomic label-free has been chosen, because of its great capability to understand biological response, especially in unprecedented experimental approaches, in which expectations cannot be done. Thereby, inserted on a broader project, the aim of this work was to evaluate the influence of Atlantic forest biodiversity surrounding the agroecosystem on protein expression of banana fruit, considering physic-chemical, physiological and biochemical aspects, in order to highlight metabolic pathways influenced by this environmental condition. The development of this project is based on the comparison of two banana commercial plots, with similar age and cultural practices, being the only difference between plots the presence of an Atlantic forest remanant on 60% of the Biodiversity plot, while the Control plot is exclusively surrounded by banana crop. It has been adopted an holistic approach, including several environmental factors (soil fertility and climatic factors), crop physiology factors (foliar diagnosis and disease severity) and banana fruit (quality attributes, post-harvest behavior and pulp proteome). Results revealed a reduction on disease severity and a longer fruit greenlife, which represents the time available to transport and marketing, for plants of the Biodiversity plot. The Proteome has shown alterations on metabolic pathways, as Citric acid cycle, Piruvate metabolism and Alanine, aspartate and glutamate metabolism, suggesting a greater tendency on fatty acid biosynthesis on fruits from Biodiversity plots, whereas fruits from Control plot seems to enhance amino acid biosynthesis. Some evidence suggest that the Atlantic forest surrounding the agroecosystem can be helpful to plant homeostasis, with benefits to the crop and fruit.

A evolução do veneno, uma das misturas mais complexas da natureza, tem sustentado o sucesso da diversificação de inúmeras linhagens de animais. Serpentes deslizantes ou medusas flutuantes utilizam o veneno, um coquetel de peptídeos farmacologicamente ativos, sais e moléculas orgânicas. Esses animais surpreendentes têm provocado grande fascínio ao longo da história humana. Nesta dissertação propomos um estudo da evolução dos venenos no filo Cnidaria, englobando dados proteômicos e genômicos. Este projeto teve como objetivos: (1) caracterizar e elucidar a evolução da composição do veneno em Cnidaria por meio da comparação de listas de proteínas; (2) testar a hipótese de que a variação na família de toxinas específica de cnidários tem sido o resultado de um regime de seleção positiva; e (3) determinar a extensão em que a duplicação de genes pode ser considerada como a principal razão para a diversificação de toxinas em Cnidaria. O capítulo \"Comparative proteomics reveals common components of a powerful arsenal in the earliest animal venomous lineage, the cnidarians\" propõe o estudo comparado mais completo sobre a composição do veneno de cnidários e uma hipótese sobre a montagem evolutiva do complexo arsenal bioquímico de cnidários e do veneno ancestral desse grupo basal. Vinte e oito famílias de proteínas foram identificadas. Destas, 13 famílias foram registradas pela primeira vez no proteoma de Cnidaria. Pelo menos 15 famílias de toxinas foram recrutadas no proteoma de veneno de cnidários antes da diversificação dos grupos Anthozoa e Medusozoa. Nos capítulos \"Evidence of episodic positive selection in the evolution of jellyfish toxins of the cnidarian venom\" e \"Gene duplications are extensive and contribute significantly to the toxic proteome of nematocysts isolated from Acropora digitifera (Cnidaria: Anthozoa: Scleractinia)\", nossas análises demonstram que as famílias de toxinas nos cnidários se diversificam amplamente mediante a duplicação de genes. Além disso, em contraste com as famílias de toxinas do veneno na maioria das linhagens animais; nós identificamos um padrão diferente na família de toxinas específica de cnidários, em que há uma seleção purificadora por longos períodos seguindo longos tempos de diversificação ou vice-versa / The evolution of venom, nature\'s most complex concoction, has underpinned the success and diversification of numerous animal lineages. Slithering serpents or buoyant jellyfishes employ venom, a cocktail of pharmacologically active peptides, salts, and organic molecules. These astonishing animals have generated a great fascination throughout human history. In this dissertation, we propose a study of the evolution of venoms in the phylum Cnidaria, encompassing proteomic and genomic data. This project aimed: (1) to characterize and elucidate the evolution of venom composition in Cnidaria by comparing protein lists; (2) to test the hypothesis that the variation in specific family of cnidarians toxins has been the result of a positive selection regime; and (3) to determine the extent to which the genes duplication may be regarded as the main reason for the diversity of toxins in Cnidaria. The chapter \"Comparative proteomics reveals common components of a powerful arsenal in the earliest animal venomous lineage, the cnidarians\" presents the most comprehensive comparative study on the cnidarians venom composition and a hypothesis about the evolutionary assembly of the complex biochemical arsenal of cnidarians and of the ancestral venom of this basal group. Twenty eight protein families were identified. Of these, 13 families were described for the first time in the proteome of Cnidaria. At least 15 types of toxin families were recruited in cnidarians venom proteome before the diversification of Anthozoa and Medusozoa groups. In the chapters \"Evidence of episodic positive selection in the evolution of jellyfish toxins of the cnidarian venom\" and \"Gene duplications are extensive and contribute significantly to the toxic proteome of nematocysts isolated from Acropora digitifera (Cnidaria: Anthozoa: Scleractinia)\", our analyses indicate that the families of toxins in cnidarians diversify broadly through gene duplication. Besides, in contrast to the families of venom toxins in most animals lineages, we identified a different pattern in the specific family of cnidarians toxins, where there is a purifying selection for periods long, followed by long periods of diversification or vice versa

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2016-08-09T13:39:32Z No. of bitstreams: 2 Dissertação - André Luís Elias Moreira - 2014.pdf: 2397469 bytes, checksum: 4a631b1ca63986a489d7e4bec3dfe90c (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-08-09T13:41:32Z (GMT) No. of bitstreams: 2 Dissertação - André Luís Elias Moreira - 2014.pdf: 2397469 bytes, checksum: 4a631b1ca63986a489d7e4bec3dfe90c (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-08-09T13:41:32Z (GMT). No. of bitstreams: 2 Dissertação - André Luís Elias Moreira - 2014.pdf: 2397469 bytes, checksum: 4a631b1ca63986a489d7e4bec3dfe90c (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2014-08-22 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The paracoccidioidomycosis (PCM) is a systemic disease caused by the thermo dimorphic fungus Paracoccidioides spp.. The route of infection of the PCM occurs by the inhalation of conidia or mycelia fragments. Until now, no proteomic studies were performed with conidia of Paracoccidioides sp. In this sense, characterize the proteome of the conidia, may contribute to the detailed knowledge of the proteins expressed during the propagation phase and their potential roles in virulence and pathogenicity, providing possible targets for antifungal strategies. For the conidia production, the mycelia of isolate Pb01 (ATCC MYA-826) were cultured in potato agar medium during 90 days at 18 ºC. After production, the conidias were collected and purified. The proteins were extracted and subjected to tryptic digestion with subsequent identification by NanoUPLC-MSE. We identified a total of 242 proteins of conidia of Paracoccidioides. The in silico analysis were used to characterize the proteins present in Paracoccidioides conidia. Proteins as GAPDH, enolase, triosephosphate isomerase, and some glycoproteins like ECM33 and β-1,3-glucanosyltransferase gel2 were identified during analysis. Some of these have been described as adhesins in Paracoccidioides and in other pathogenic fungi. Were also identified proteins related to signal transduction pathways, such as: Ras GTPase, RhoA GTPase and calmodulin, described in other fungi involved in morphologic changes. Proteins related to evasion, defense and virulence of the fungus, such as HSP90, catalase, mitochondrial peroxiredoxin Prx1, have been described with functions related to temperature shifts or oxidative stress provided by the host environment, were also identified. These results highlight that Paracoccidioides conidia contain proteins that can contribute to its maintenance in the environment and have molecules related to important processes necessary for the initial steps of infection in the host. / A paracoccidioidomicose (PCM) é uma micose sistêmica causada pelo fungo termodimórfico Paracoccidioides spp.. A principal rota de infecção da PCM ocorre por inalação de conídios ou fragmentos de micélio. Até o momento, estudos proteômicos não foram realizados com conídios de Paracoccidoides sp. Neste sentido, caracterizar o proteoma do conídio poderá contribuir para o conhecimento detalhado das proteínas expressas durante a fase de propagação e seus potenciais papéis na virulência e patogenicidade, fornecendo prováveis alvos para estratégias antifúngicas. Para a produção de conídios, o micélio do isolado Pb01 (ATCC MYA-826) foi cultivado em meio ágar batata durante 90 dias a 18 ºC. Após a produção, os conídios foram coletados e purificados. As proteínas foram extraídas e submetidas a digestão tríptica com subsequente identificação por NanoUPLC-MSE. Foram identificados um total de 242 proteínas de conídios de Paracoccidioides. As análises in silico foram utilizadas para caracterizar as proteínas presentes em conídios de Paracoccidioides. Proteínas como a GAPDH, enolase, triosefosfato isomerase e algumas glicoproteínas como ECM33 e β-1,3-glicanosiltransferase Gel2 foram identificadas durante as análises. Algumas destas já foram descritas como adesinas em Paracoccidioides e em outros fungos patogênicos. Também foram identificadas proteínas relacionadas as vias de transdução de sinais, tais como: Ras GTPase, RhoA GTPase e calmodulina, descritas em outros fungos envolvidas em alterações morfológicas. Proteínas relacionadas à evasão, defesa e virulência do fungo, tais como HSP90, catalase B, peroxiredoxina mitocondrial Prx1, foram descritos com funções relacionadas a mudanças de temperatura ou estresse oxidativo proporcionado pelo ambiente hospedeiro, também foram identificados. Esses resultados demonstram que o conídio de Paracoccidioides contem proteínas que podem contribuir para sua manutenção no meio ambiente e possuem moléculas relacionadas a processos importantes necessários para os passos iniciais da infecção no hospedeiro.

As toxinas de veneno de serpentes têm como principal função alterar a homeostase das presas para fins de alimentação ou defesa. O estudo aprofundado da composição do veneno de serpentes é importante para a produção de soros antiofídicos mais eficientes, para a descoberta de novos fármacos e na compreensão de processos biológicos, ecológicos e evolutivos. As pesquisas com toxinas têm mostrado uma versatilidade natural, refinada pela evolução, na diversificação de funções em famílias de proteínas recrutadas de suas funções endógenas, por meio de sucessivas duplicações e acumulo de mutações levando a uma evolução acelerada. A miríade de toxinas disponíveis e sua diversidade de funções ainda não foram completamente descritas. A combinação das análises em larga escala do transcriptoma de novo da glândula de veneno e do proteoma do veneno permite elaborar um perfil mais completo do toxinoma do veneno, permitindo inclusive um aumento na sensibilidade da detecção de toxinas pouco representadas e inesperadas nos venenos. O objetivo geral deste estudo foi analisar o toxinoma do veneno de uma das mais perigosas espécies australianas, a Pseudonaja textilis (Elapidae). Foi possível identificar no veneno as toxinas: fatores de coagulação de veneno do complexo protrombinase, subunidades de fosfolipases A2 (PLA2) da neurotoxina textilotoxin e PLA2 de atividade procoagulante, neurotoxinas tipo three-finger toxin (3FTx), inibidores de protease do tipo-kunitz textilinin, e pela primeira vez, uma nova variante de 3FTx, lectinas tipo C, CRiSP além de indícios de toxinas de lagarto Heloderma e outras proteínas candidatas a toxinas como calreticulin e dipeptidase 2. Metaloproteinases, pouco estudadas em Elapidae, foram clonadas e detectadas no veneno por ensaios de fracionamento e imunoreatividade. A análise do transcriptoma identificou novas isoformas e variantes de toxinas, principalmente das 3FTx e dos inibidores de serinoproteases, assim como transcritos de toxinas que não foram detectadas no veneno e que merecem mais investigações. O quadro de sintomas com acidentes em humanos é bem explicado pelas toxinas identificadas, porém, em seu habitat natural, as toxinas pouco conhecidas e até então não descritas devem ter funções importantes e específicas na predação. Identificar esta diversidade de variantes é importante para entender o modo de ação das toxinas. / Snake venom toxins alter prey homeostasis for feeding or defense. In depth studies of venom composition are important for better antivenom production, for new drugs lead and discovery and for better understanding of biological, ecological and evolutionary processes. Research on toxins have shown the natures way of innovating, refined by evolution, diversifying functions of protein families recruited from their endogenous function to the venom gland by successive gene duplication and mutation accumulation, leading to an accelerated evolution. A myriad of available toxins and diversity of functions is still available for discovery. Combining high throughput techniques such as venom gland de novo transcriptomics and venom proteomics, one can assess and observe a more complete profile of the snake toxinome, additionally allowing an upscale in low represented and unexpected toxin detection. The aim of this project was to investigate the venom toxinome of one of the most dangerous Australian species, Pseudonaja textilis (Elapidae). The toxins identified in it venom was: protrombinase complex coagulation factors, neurotoxic textilotoxin phospholipase A2 (PLA2) subunits and procoagulant PLA2, neurotoxic three-finger toxins (3FTx), Kunitz-type protease inhibitor textilinin, and for the first time, a new long 3FTx, C-type lectins, CRiSPs, as well as evidences of lizard toxins from Heloderma genus and other toxin candidates calreticulin and dipeptidase 2. Metalloproteinases, little investigated in Elapidae, was cloned and detected in the venom after fractionation and immunoassay. The transcriptome revealed new toxin variants and isoforms, specially 3FTx and serine protease inhibitors, as well as transcripts from toxins not detected in the venom that deserves further investigation. Human accident symptoms are well explained by the identified toxins, however, in its natural environment, little known and undescribed toxins must have specific and important role in predation. Identifying this diversity is important to better understand toxins ways of action.

When cells divide, a series of events must proceed in a timely and co-ordinated manner to ensure that all DNA is replicated and partitioned equally between the two daughter cells. A central component of this process is the kinetochore, a large proteinaceous complex (>100 proteins) found within the centromere of all chromosomes. During the dynamic process of cell division, this machinery must be able to capture microtubules, promote chromosome movements towards the spindle midzone and ensure that segregration only occurs once this alignment has been successfully completed. This requires intricate mechanical and regulatory co-ordination between components and it is therefore no surprise that the structures responsible are structurally and functionally varied. It has, however, become clear that many kinetochore proteins assemble into distinct sub-complexes and despite the fact that their specific contributions are well studied, the way the many unique sub-assemblies come together to form a fully operational kinetochore is still poorly understood. Here, chromosome isolation techniques from chicken DT40 cells combined with mass spectrometry employing Stable Isotope Labeling by Amino acids in Cell culture (SILAC), is used to compare the proteome of mitotic chromosomes from different conditional kinetochore knockout (KO) cell lines. This includes components of the inner kinetochore; CENP-C, CENP-T and CENP-W, and a sub-unit of the Ndc80 complex that is important for microtubule attachment. With these large data sets I have focused on the impact these depletions have on the architecture of the holo-kinetochore by measuring the SILAC ratios of individual proteins. From these measurements I can define whether specific components are decreased, increased or unchanged in terms of their abundance on chromosomes in response to the various deletions. I have found that proteins within the same complex typically behave in a similar manner across the different KO conditions. By integrating all of the data sets, dependency networks are revealed, as well as highlighting potential novel kinetochore proteins worthy of further study.