Células multipotentes mesenquimais estromais (CTM) são células adultas multipotentes que podem ser isoladas a partir de diferentes tecidos e são capazes de atingir sítios danificados, exercer papéis na regeneração tecidual e modular a resposta imune. Estas células demonstraram resultados discrepantes em estudos in vivo dependentes de sua fonte de obtenção. Há na literatura hipóteses de que o mecanismo predominante pelo qual as CTMs atuam no reparo tecidual estaria relacionado à sua atividade parácrina, criando um microambiente com sinais tróficos. Nesse sentido, a avaliação do conteúdo do secretoma destas células é de grande interesse. Portanto, este projeto teve como objetivo analisar o meio condicionado de CTMs obtidas de diferentes fontes (tecido adiposo, músculo esquelético e tubas uterinas) de mesmos indivíduos. A abordagem experimental consistiu em proteômica shotgun (nanocromatografia líquida acoplada a espectrometria de massas em tandem) com o intuito de identificar alvos diferentemente expressos entre as culturas que possam sugerir funções específicas de cada linhagem celular. Os dados espectrais foram obtidos pelo modo de aquisição dependente de dados (Top15). Os dados adquiridos foram processados pelas plataformas MaxQuant e TPP (Trans-Proteomic Pipeline). Foi realizada análise qualitativa de vias enriquecidas por meio do programa Ingenuity utilizando as proteínas em comum nos secretoma de todas as CTMs analisadas. Essa análise permitiu observar vias enriquecidas de proliferação celular, migração celular e desenvolvimento do sistema cardiovascular, demonstrando que as proteínas secretadas por quaisquer das CTMs analisadas podem ser relacionadas a resultados encontrados na literatura utilizando estas células para terapias para patologias. As análises estatísticas para determinar se haveria dependência da composição do secretoma em função do indivíduo doador ou tecido fonte das CTMs revelaram proteínas diferencialmente expressas entre todos os grupos. Estas proteínas diferencialmente expressas são relacionadas à proliferação, sinalização e interação celular, além de modulação do sistema imune e da angiogênese. Neste contexto, podemos concluir que o secretoma das CTMs é muito semelhante, que as CTMs isoladas de quaisquer tecidos ou indivíduos são capazes de secretar moléculas que possivelmente exercem benefícios em determinado tratamento. Entretanto, estes benefícios podem ser exacerbados ou suprimidos pelas moléculas diferencialmente expressas, as quais são dependentes tanto dos tecidos quanto dos indivíduos dos quais as CTMs foram obtidas / Multipotent Mesenchymal Stromal Cells (MSCs) are multipotent adult cells that can be isolated from different tissues and are able to reach damaged sites, play a role in tissue regeneration and modulate immune response. These cells showed conflicting results in studies in vivo depending on their tissue origin. It is hypothesised that the predominant mechanism by which MSCs function could be related to its paracrine activity, creating a microenvironment with trophic signals. Accordingly, the evaluation of the content of the secretome of these cells is of great interest. Towards this end, this project analyzed the proteins of conditioned medium of MSCs obtained from different sources from the same donors (adipose tissue, uterine tubes and skeletal muscle). The MSCs were characterized by flow cytometry for the presence of membrane markers and by differentiation in vitro into adipocytes, chondrocytes, and osteoblasts. The conditioned media were obtained and the protein profile was analysed by liquid nanochromatography coupled to tandem mass spectrometry. Spectral data were obtained by full-acquisition mode MS / dd-MS2 (Top15). The acquired data were processed by MaxQuant software and TPP (Trans-Proteomic Pipeline). Qualitative analysis of enriched pathways through the Ingenuity program using the shared proteins between the cell lineages was performed.It showed enriched pathways related to cell proliferation, cell migration and development of the cardiovascular system. This allows considering that the secreted proteins from the analyzed MSCs might be related to findings in the literature using these cells for therapies. After this, the proteins were analyzed for differential expression by comparing the MSCs into groups of different sources or different donors. In which were observed differentially expressed proteins related to proliferation, cell signaling and interaction, modulation of the immune system and angiogenesis. In this context, we can conclude that MSC\'s secretome is very similar in the analyzed lineages, and that any MSCs are able to secrete molecules which potentially exert for certain treatment benefits. However, these benefits can be exacerbated or annulled by differentially expressed molecules, which are dependent both as the individual and tissues from which MSCs were obtained

Candidate: Nikola Kollárová Title of diploma thesis: Detection of Sap2 in the secretome of Candida albicans cell wall and secretory mutants Charles University, Faculty of Pharmacy in Hradec Králové, Department of Biological and Medicinal Sciences Complutense University of Madrid, Faculty of Pharmacy, Department of Microbiology II Study program: Pharmacy Backgound: The aim of this diploma thesis was to search for C. albicans proteins involved in the secretion of the secreted aspartyl proteinase 2 enzyme (Sap2) evaluating the ability to degrade BSA (bovine serum albumin) as a source of nitrogen in several cell wall and secretory mutants of C. albicans. The work was carried out at the Department of Microbiology II, Faculty of Pharmacy, Complutense University of Madrid. Methods: The supernatant samples of several Candida albicans mutants were tested by SDS-PAGE electrophoresis and stained. Bands corresponding to BSA were observed and compared to controls. The other method was counted with 96-well plate. Results: The correlation between optical density and degradation of BSA was observed. Some mutants with disability to degrade BSA were found in a pilot screening of the ability to degrade BSA using 96-well plate method. That fact was confirmed by SDS-PAGE electrophoresis. C. albicans mutants showing...

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Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2015-11-19T10:15:55Z No. of bitstreams: 2 Dissertação - Amanda Rafaela Rodrigues - 2014.pdf: 1648839 bytes, checksum: 9a00f301e577d05e94214afe813688c5 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2015-11-19T10:17:31Z (GMT) No. of bitstreams: 2 Dissertação - Amanda Rafaela Rodrigues - 2014.pdf: 1648839 bytes, checksum: 9a00f301e577d05e94214afe813688c5 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-11-19T10:17:31Z (GMT). No. of bitstreams: 2 Dissertação - Amanda Rafaela Rodrigues - 2014.pdf: 1648839 bytes, checksum: 9a00f301e577d05e94214afe813688c5 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2014-10-23 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The genus Trichoderma is represented by non-pathogenic soil fungi that have been studied by act as biological control agents against fungal pathogens, such as Sclerotinia sclerotiorum, the fungus that causes white mold, besides that, reduce environment and human health risks by being able to replace agrochemicals. Proteomic strategies with MS techniques have been important tools in studies of patterns identification for protein expression in different growth conditions. This work aims to development new strategies that enable the detection and identification of proteins secreted by Trichoderma asperellum (TR356) and Sclerotinia sclerotiorum when inoculated together and separated. The results obtained by MALDI / TOF analysis allowed the identification of a β-1.3--glucanosiltransferase and α-1.2-Dmannosidase, demonstrating the possibility of proteins identification for better comprehension about interaction between these organisms. It was therefore possible to identify the proteins through strategies used, but further analysis are required in order to elucidate the function and interaction of proteins secreted by the fungus Trichoderma asperellum TR356 against S. sclerotiorum. / OgêneroTrichodermaérepresentadoporfungosdesolonãopatogênicosquesãoestudad osporsuaaçãocomoagentesdecontrolebiológicocontrafungosfitopatógenos,comoScler otiniasclerotiorum,ofungocausadordomofobranco.Osfungos Trichoderma spp.atuamcomoimportantesagentesdecontrolebiológico,diminuindoosriscosàsaúdehu manaeaomeioambienteporseremcapazesdesubstituirosagroquímicos.Estratégiasprot eômicas juntamente com técnicas de MSsãoimportantesferramentasemestudosdepadrão, identificação e expressãodeproteínasemdiferentescondiçõesdecrescimento.Levando em consideração a importância de tais estudos, o presente trabalho tem por objetivo o desenvolvimento de novas estratégias que possibilitem a detecção e identificação de proteínas secretadas por Trichoderma asperellum (TR356) e Sclerotinia sclerotiorum quando inoculados juntos e/ou separados os dois organismos vivos. Os resultados obtidos através das análises por MALDI/TOF permitiram a identificação dasproteínas, β-1,3-glucanosiltransferase e α-1,2-D-mannosidase,demonstrando a possibilidade de identificaçãopara o entendimento futuro acerca da interação destes organismos. Foi possível, portanto, a identificação das proteínas através das estratégias utilizadas, porém serão necessárias futuras análises afim de elucidar a interação e função das proteínas secretadas através do fungo T. asperellum TR356sobre S. sclerotiorum.

Orientadores: Gonçalo Amarante Guimarães Pereira, Johana Rincones Pérez / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-21T12:47:51Z (GMT). No. of bitstreams: 1 Oliveira_BrunoVazde_D.pdf: 5827031 bytes, checksum: c9ff7bf065563150ef6ea14b5630ca52 (MD5) Previous issue date: 2012 / Resumo: O fungo basidiomiceto Moniliophthora perniciosa é o agente causador da vassoura-de-bruxa do cacaueiro, doença que se tornou um dos principais problemas fitopatológicos do Brasil. O fungo possui duas fases de vida, biotrófica e necrotrófica, as quais estão associadas aos sintomas de vassoura-verde e vassoura-seca, respectivamente, durante a progressão da doença. Em vista dos grandes prejuízos econômicos causados pela vassoura-de-bruxa, foi iniciado o Projeto Genoma Vassoura-de-Bruxa, cujo objetivo é a decodificação genética do fungo com o intuito de identificar genes candidatos que possam estar relacionados ao estabelecimento e à progressão da doença para serem caracterizados. M. perniciosa é capaz de utilizar metanol como fonte única de carbono. Na maioria dos organismos estudados, a enzima metanol oxidase (MOX) é a responsável pelo metabolismo de metanol através do metabolismo peroxissomal, à qual também está associada uma catalase (CAT). Em plantas, uma das principais fontes de metanol é a pectina presente na estrutura da parede celular vegetal. Algumas espécies de fungos fitopatogênicos tem a capacidade de secretar oxalato como produto da enzima oxaloacetato acetilhidrolase (OAH), o qual é capaz de quelar íons cálcio da estrutura das pectinas, deixando-as vulneráveis ao ataque de enzimas que degradam a parede celular vegetal. Dentre essas enzimas, encontra-se a pectina metilesterase (PME), a qual é capaz de desmetilar as pectinas, liberando metanol. Como M. perniciosa possui em seu genoma sequências com similaridade a mox, cat, pme e oah, formulamos uma hipótese, segundo a qual M. perniciosa seria capaz de degradar o metanol proveniente das pectinas, através de um metabolismo peroxissomal, durante sua interação com o cacau na vassoura-de-bruxa. De fato, M. perniciosa é capaz de secretar oxalato, formando cristais de oxalato de cálcio. Nossos resultados sugerem fortemente que M. perniciosa utiliza o metanol proveniente das pectinas durante a vassoura-de-bruxa; no entanto, esse processo se daria através de um metabolismo extracelular, visto que M. perniciosa produz uma MOX e CAT extracelulares, e não através do metabolismo peroxissomal como inicialmente proposto. Além disso, M. perniciosa possui outras catalases, as quais se apresentam diferencialmente expressas nas diferentes fases do ciclo de vida do fungo e em diferentes estágios da progressão da vassoura-de-bruxa. Por fim, apresentamos uma análise preliminar do secretoma de M. perniciosa / Abstract: The hemibiotrophic basidiomycete fungus Moniliophthora perniciosa is the causal agent of Witches' broom disease (WBD) in cacao. M. perniciosa is classified as a hemibiotrophic pathogen and presents two morphologically distinct life phases, biotrophic and necrotrophic, that are correlated with the green broom and dry broom symptoms, respectively, during the progression of WBD. The introduction of WBD in Bahia, the main Brazilian cacao-producing state, reduced the cacao production drastically and Brazil has become a net importer of cacao in order to supply the national chocolate industry. Due to the extreme losses in cacao production, the Witches' Broom Genome Project was initiated to decode the M. perniciosa genome and, based on the data acquired, select genes that could be relevant during the progression of WBD for characterization. M. perniciosa is able to grow in methanol as the sole carbon source. In methylotrophic yeasts and other methanol-degrading organisms, a methanol oxidase (MOX) is the key enzyme in methanol metabolism and it is also related to a catalase (CAT); both MOX and CAT characterize a peroxisomal metabolism. In plants, one of the main sources of methanol is the pectin present in the plant cell walls. Many phytopathogenic fungal species produce oxalate, a product of the enzyme oxaloacetate acetyl hydrolase (OAH), which removes calcium ions bound to pectin to produce calcium oxalate crystals, thus exposing the host cell walls to plant cell wall-degrading enzymes (PCWD) of fungal origin. One of the main PCWDs is pectin methylesterase (PME), an enzyme that removes the methyl ester radicals from esterified pectin, releasing methanol. As M. perniciosa possesses mox, cat, oah and pme on its genome, we hypothesized that M. perniciosa would utilize a peroxisomal metabolism to metabolize the methanol released from the pectin demethylation in WBD. Indeed, M. perniciosa secretes oxalate, thus producing calcium oxalate crystals. Our data strongly suggest that M. perniciosa able to utilize the methanol released from pectin demethylation; however, this metabolism is probably extracellular due to the presence of secreted MOX and CAT. Moreover, M. perniciosa possesses other cats, which are differentially expressed in vitro and in planta. Finally, we present a preliminary analysis of M. perniciosa secretome / Doutorado / Genetica de Microorganismos / Doutor em Genetica e Biologia Molecular

Spinal muscular atrophy (SMA) is an inherited neuromuscular disorder, which causes dysfunction/loss of lower motor neurons and muscle weakness as well as atrophy. While SMA is primarily considered as a motor neuron disease, recent data suggests that survival motor neuron (SMN) deficiency in muscle causes intrinsic defects. We systematically profiled secreted proteins from control and SMN deficient muscle cells with two combined metabolic labeling methods and mass spectrometry. From the screening, we found lower levels of C1q/TNF-related protein 3 (CTRP3) in the SMA muscle secretome and confirmed that CTRP3 levels are indeed reduced in muscle tissues and serum of an SMA mouse model. We identified that CTRP3 regulates neuronal protein synthesis including SMN via mTOR pathway. Furthermore, CTRP3 enhances axonal outgrowth and protein synthesis rate, which are well-known impaired processes in SMA motor neurons. Our data revealed a new molecular mechanism by which muscles regulate the physiology of motor neurons via secreted molecules. Dysregulation of this mechanism contributes to the pathophysiology of SMA.

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Background: The beneficial impact of mesenchymal stem cells (MSC) on both acute and chronic liver diseases has been confirmed, although the molecular mechanisms behind it remain elusive. We aim to identify factors secreted by undifferentiated and hepatocytic differentiated MSC in vitro in order to delineate liver repair pathways potentially targeted by MSC. Methods: Secreted factors were determined by protein arrays and related pathways identified by biomathematical analyses. Results: MSC from adipose tissue and bone marrow expressed a similar pattern of surface markers. After hepatocytic differentiation, CD54 (intercellular adhesion molecule 1, ICAM-1) increased and CD166 (activated leukocyte cell adhesion molecule, ALCAM) decreased. MSC secreted different factors before and after differentiation. These comprised cytokines involved in innate immunity and growth factors regulating liver regeneration. Pathway analysis revealed cytokine-cytokine receptor interactions, chemokine signalling pathways, the complement and coagulation cascades as well as the Januskinase-signal transducers and activators of transcription (JAK-STAT) and nucleotide-binding oligomerization domain-like receptor (NOD-like receptor) signalling pathways as relevant networks. Relationships to transforming growth factor beta(TGF-beta) and hypoxia-inducible factor 1-alpha (HIF1-alpha) signalling seemed also relevant. Conclusion: MSC secreted proteins, which differed depending on cell source and degree of differentiation. The factors might address inflammatory and growth factor pathways as well as chemo-attraction and innate immunity. Since these are prone to dysregulation in most liver diseases, MSC release hepatotropic factors, potentially supporting liver regeneration.

Cette étude a pour objectif la mise en évidence, chez les basidiomycetes du genre Pycnoporus, de nouvelles oxydo-réductases impliquées dasn la dégradation de la biomasse végétale: de l'expression des gènes aux applications biotechnologiques. Les champs d'application visés concernent essentiellement le domaine de la chimie verte, dans le cadre du projet européen BIORENEW. Le travail s'est articulé autour de trois axes principaux. Le premier a concerné l'exploration de la biodiversité naturelle en particulier tropicale, pour la sélection de souches productrices de nouvelles laccases de haut potentiel d'oxydo-réduction. Le gène codant pour la laccase Lac1 chez Pycnoporus a été utilisé comme marqueur moléculaire d'identification et de relation phylogénie-fonction, mettant en évidence une distribution des souches fortement corrélée avec leur écozone. Le deuxième axe a porté sur l'isolement de trois nouvelles laccases issues de P.sanguineus et P. coccineus qui exhibent des caractéristiques biochimiques complémentaires: haute thermostabilité, résistance aux solvants, au pH, constantes catalytiques et potentiels rédox élevés. Ces enzymes constituent de bons modèles pour des applications en biotechnologies blanches:décoloration de colorants polyphénoliques, oxydation de composés modèles de type lignine non-phénolique, oligomérisation de flavonoides naturels adaptés aux applications cosmétiques et pharmaceutiques. Enfin, dans le cadre de l'annotation du génome des souches monocaryotiques P. cinnabarinus BRFM 137 et P; sanguineus BRFM 1264, dont le séquençage a été réalisé par notre Unité, un regard tout à fait nouveau est porté sur le système lignolytique du genre Pycnoporus, longtemps décrit comme produisant que de la laccase comme enzyme du système lignolytique. Pour la première fois, nous avons montré la présence de gènes codant pour tout l'arsenal enzymatique de dégradation des lignines, c'est à dire plusieurs laccases mais surtout de nombreuses peroxydases et des enzymes auxilliaires génératrices d'H2O2 comme les glyoxal oxydases. Ces nouvelles enzymes ont été caractérisées in silico. Pour la première fois également, la sécrétion effective de peroxydases, de glyoxal oxydases et d'autres FOLymes dans nos conditions de culture a également pu être démontrée par analyse protéomique. / The purpose of this work was to prospect, in the genus Pycnoporus, for new oxido-reductases involved in the degradation of lignocellulosic biomass: from gene expression to biotechnological applications. This research was conducted in the framework of green chemistry applications according to BIORENEW European Project. The study was divided in three main research axes. Firstly, the exploration of natural biodiversity, especially tropical biodiversity, for the selection of new high redox potential-laccase producing strains. These strains were repositionned in a context of phylogenomic/function through the lac1 gene. Molecular clustering based on lac1 sequences enabled the distribution of P. sanguineus and P. coccineus through four distinct, well supported clades and subclades. This distribution was highly correlated with ecozones. The second part of the work deals with the biochemical and molecular characterization of three novel laccases from P. coccineus and P. sanguineus, and their applicability on natural or model phenolic substrates. The three laccases showed complementary biochemical features: high thermo- and pH stability, high catalytic efficiency and resistance to organic solvents. The three novel laccases proved to be suitable models for white biotechnology processes: polyphenolic dye decolourization, non-phenolic lignin model compound oxidation, and synthesis of new oligomers from natural flavonoids suitable for cosmetic or pharmaceutical applications. Finally, annotation of genomic data from the monocaryotic strains P. cinnabarinus BRFM 137 and P. sanguineus BRFM 1264 (genomes sequenced by the UMR1163 BCF ) was performed for lignolytic enzymes. For the first time, new oxidases (peroxidases, glyoxal oxidases and other FOLymes) were evidenced in Pycnoporus and in silico characterized. Moreover, the active secretion of several of these enzymes has been demonstrated in our culture conditions by 1D-proteomic analysis

Orientador: Rui Seabra Ferreira Junior / Resumo: Atualmente são muitos casos de pacientes que perdem estrutura óssea em acidentes ou reabsorção patológica. A bioengenharia óssea é um tratamento promissor que visa reconstruir estas estruturas sem a morbidade do enxerto autógeno. O tecido ósseo é um conjuntivo especializado com a função principal de proteção e sustentação dos tecidos moles, mas também é responsável pela produção de tipos celulares e homeostase de minerais. Sua reparação é complexa com diferentes tipos celulares e agentes quimiotáticos que funcionam de forma orquestrada até a reparação. As terapias celulares vêm sendo estudadas para promover a reparação de defeitos que o organismo por si não consegue resolver. Células menos especializadas como as células-tronco embrionárias (ESCs) possuem grande potencial terapêutico, mas são complicadas eticamente. Já as células-tronco mesenquimais (MSC) podem ser autólogas, o que minimiza o risco de imunogenicidade mas necessitam área doadora do paciente. Atualmente ainda não há consenso quanto ao uso de células tronco na terapia regenerativa pois há grandes variáveis como a melhor forma de aplicação, a quantidade correta e o melhor tipo celular para a regeneração óssea. As células produzem mediadores químicos no local enxertado, que segundo pesquisas recentes é o principal mecanismo de reparação tecidual. Estes mediadores são depositados em abundância no meio de cultura durante a cultura celular e usados na bioengenharia com a ajuda de scaffolds. Os biopolímeros de fibrina ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Chapter I present a review about bone repair and its biological events, bioengineering, cells, fibrin biopolymer as scaffold and the secretoma derived from cell culture. Many patients nowadays lose bone structure in accidents or pathological reabsorption. Bone bioengineering is a promising treatment that aims to reconstruct these structures without autogenous graft morbidity. Bone tissue is a specialized connective tissue specialized in protecting and supporting soft tissues, but it is also responsible for the production of cell types and mineral homeostasis. The bone healing is a complex process where different cell types and chemotactic agents work in an orchestrated way. The cell therapies can promote the repair of defects that the body cannot solve. Less specialized cells like embryonic stem cells (ESCs) have great therapeutic potential, but are ethically complicated. In contrast, mesenchymal stem cells (MSCs) may be autologous, which minimizes the risk of immunogenicity but requires a patient's donor area. Currently there is still no consensus regarding the use of stem cells in regenerative therapy, studies uses different methods, cells and biomaterials for bone regeneration. Recent researches advocate that paracrine secretions by cells are main mechanism of tissue repair. These mediators are deposited in abundance in the culture medium during cell culture. Fibrin biopolymers (BF) are natural biomaterials to the body and can function as drug delivery of growth factors, c... (Complete abstract click electronic access below) / Doutor