Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / IntroduÃÃo: Guanilinas, peptÃdeos natriurÃticos (ANP e urodilatina) e bradicinina estÃo implicados na fisiopatologia, com potencial terapÃutico, do metabolismo do sal e da hipertensÃo. Objetivo: Estudar o mecanismo de aÃÃo e possÃveis interaÃÃes destes peptÃdeos, pelo emprego de inibidores da guanilato ciclase isatina e ODQ. MÃtodo: Foram realizadas experiÃncias no rim isolado e perfundido de rato com ferramentas farmacolÃgicas, isatina (IS; 0,3 ou 10&#956;M) ou com ODQ (37&#956;M), nos estudos com uroguanilina (UGN; 0,3 ou 0,6&#956;M), bradicinina (BK; 0,3 ou 0,9 ou 1,8nM) ou urodilatina (UD; 0,03nM). Investigaram-se ainda possÃveis interaÃÃes entre os referidos petÃdeos. Resultados: Isoladamente, IS (0,3&#956;M), ODQ, UGN (0,3&#956;M), BK (0,9 ou 0,3nM) nÃo interferiram signficativamente na presssÃo de perfusÃo, na diurese ou na reabsorÃÃo tubular fracionada renal de eletrÃlitos (sÃdio - %TNa+; potÃssio - %TK+; cloreto - %TCl- ). Nas condiÃÃes experimentais, ODQ mostrou-se eficaz (p<0,05) em inibir os efeitos de BK (1,8nM) sobre a pressÃo de perfusÃo (ODQ+BK120min: 111+3mmHg; BK120min: 139+5mmHg) e %TNa+ (ODQ+BK120min: 80+1%; BK120min: 76+2%). IS (3&#956;M) mostrou-se parcialmente eficaz em inibir efeitos de UGN (0,6&#956;M; IS+UGN90min: 76+2%; UGN90min: 72+2%) e de BK (1,8nM; IS+BK90min: 81+2%; BK60min: 0,76+2%) sobre %TNa+. UD (0,03nM90min: 86+2%; UGN+UD90min: 73+4%; p<0,05) potencializou a natriurese produzida por UGN (0,3&#956;M90min: 83+2%), cujos efeitos vasculares foram inibidos com BK (0,3nM90min: 104+5mmHg; UGN0,6&#956;M; 90min: 135+4mmHg; UGN+BK90min: 110+2mmHg). ConclusÃes: IS e ODQ comprovaram a participaÃÃo da via de sinalizaÃÃo NO-GMP no mecanismo de aÃÃo dos peptÃdeos estudados. A perfusÃo simultÃnea com mais de um peptÃdio, comprovou que hà interaÃÃes em suas diferentes vias de sinalizaÃÃo. / Introduction: Guanylins, natriuretic peptides (ANP and urodilatina) and bradykinin are involved in the pathophysiology, with therapeutic potential, of salt metabolism and hypertension. Objective: To study the mechanism of action and possible interactions of these peptides, with the employment of guanylate cyclase inhibitors isatina and ODQ. Method: Experiments were performed on isolated perfused rat kidney with pharmacological tools, isatin (IS; 0.3 or 10&#956;M) or with ODQ (37&#956;M), in studies with uroguanylin (UGN, 0.3 or 0.6&#956;M), bradykinin (BK , 0.3 or 0.9 or 1.8nM) or urodilatin (UD, 0.03nM). It was also investigated possible interactions between those peptides. Results: Alone, IS (0.3&#956;M), ODQ, UGN (0.3&#956;M), BK (0.9 or 0.3nM) did not interfere significantly in perfusion perfusion (PP), in diuresis or in fractional renal tubular reabsorption of electrolytes (sodium - %TNa+; potassium - %TK+; chloride - %TCl-). In experimental conditions, ODQ proved to be effective (p<0.05) in inhibiting the effects of BK (1.8nM) on the PP (ODQ+BK120min: 111+3mmHg; BK120min: 139+5mm Hg) and %TNa+ (ODQ+BK120min: 80+1%; BK120min: 76+2%). IS (3&#956;M) proved to be partially effective in inhibiting effects of UGN (0.6&#956;M; IS+UGN90min: 76+2%; UGN90min: 72+2%) and BK (1.8nM; IS+BK90min: 81+2%; BK60min: 0.76+2%) on %TNa+. UD (0.03nM90min: 86+2%; UGN+UD90min: 73+4%, p<0.05) increased the natriuresis produced by UGN (0.3&#956;M90min: 83+2%), whose vascular effects were inhibited with BK (0.3nM90min: 104+5mmHg; UGN0, 6&#956;M; 90min: 135+4mmHg; UGN+BK90min: 110+2mmHg). Conclusions: IS and ODQ confirmed the participation of the NO-GMP signalling pathway in the mechanism of action of peptides studied. The infusion simultaneously over a peptide, proved that there are interactions in their different signalling pathway.

Dissertation (Ph.D.)--University of South Florida, 2008. / Includes vita. Includes bibliographical references (leaves 102-128).

Dissertation (Ph.D.)--University of South Florida, 2008. / Title from PDF of title page. Document formatted into pages; contains 132 pages. Includes vita. Includes bibliographical references.

De nos jours, la validation des unités de contrôle électronique ECU se fonde généralement sur la simulationHardware-In-the-Loop où les systèmes physiques qui manquent sont modélisés à l’aide deséquations différentielles hybrides. La complexité croissante de ce type de modèles rend le compromisentre le temps de calcul et la précision de la simulation difficile à satisfaire. Cette thèse étudie et proposedes méthodes d’analyse et d’expérimentation destinées à la co-simulation temps-réel ferme de modèlesdynamiques hybrides. Elle vise notamment à définir des solutions afin d’exploiter plus efficacement leparallélisme fourni par les architectures multi-coeurs en utilisant de nouvelles méthodes et paradigmesde l’allocation des ressources. La première phase de la thèse a étudié la possibilité d’utiliser des méthodesd’intégration numérique permettant d’adapter l’ordre comme la taille du pas de temps ainsi quede détecter les événements et ceci dans le contexte de la co-simulation modulaire avec des contraintestemps-réel faiblement dures. De plus, l’ordre d’exécution des différents modèles a été étudié afin dedémontrer l’influence du respect des dépendances de données entre les modèles couplés sur les résultatsde la simulation. Nous avons proposé pour cet objectif, une nouvelle méthode de co-simulationqui permet le parallélisme complet entre les modèles impliquant une accélération supra-linéaire sanspour autant ajouter des erreurs liées à l’ordre d’exécution. Enfin, les erreurs de retard causées par lataille de pas de communication entre les modèles ont été améliorées grâce à une nouvelle méthoded’extrapolation par contexte des signaux d’entrée. Toutes les approches proposées visent de manièreconstructive à améliorer la vitesse de simulation afin de respecter les contraintes temps-réel, tout engardant la qualité et la précision des résultats de simulation sous contrôle. Ces méthodes ont été validéespar plusieurs essais et expériences sur un modèle de moteur à combustion interne et intégrées àun prototype du logiciel xMOD. / Nowadays the validation of Electronic Control Units ECUs generally relies on Hardware-in-The-Loopsimulation where the lacking physical systems are modeled using hybrid differential equations. Theincreasing complexity of this kind of models makes the trade-off between time efficiency and the simulationaccuracy hard to satisfy. This thesis investigates and proposes some analytical and experimentalmethods towards weakly-hard real-time co-simulation of hybrid dynamical models. It seeks in particularto define solutions in order to exploit more efficiently the parallelism provided by multi-core architecturesusing new methods and paradigms of resource allocation. The first phase of the thesis studied the possibilityof using step-size and order control numerical integration methods with events detection in thecontext of real-time modular co-simulation when the time constraints are considered weakly-hard. Moreover,the execution order of the different models was studied to show the influence of keeping or not thedata dependencies between coupled models on the simulation results. We proposed for this aim a newmethod of co-simulation that allows the full parallelism between models implying supra-linear speed-upswithout adding errors related to their execution order. Finally, the delay errors due to the communicationstep-size between the models were improved thanks to a proposed context-based inputs extrapolation.All proposed approaches target constructively to enhance the simulation speed for the compliance toreal-time constraints while keeping the quality and accuracy of simulation results under control and theyare validated through several test and experiments on an internal combustion engine model and integratedto a prototype version of the xMOD software.

Made available in DSpace on 2016-06-02T19:51:45Z (GMT). No. of bitstreams: 1 3188.pdf: 2904896 bytes, checksum: 9d486740c6a99b495aeb0b7d3f2aad98 (MD5) Previous issue date: 2010-08-23 / The raising of alligator in captivity began in Brazil as an option to protect the species and the land properties against invasions of alligator hunters. This activity was legalized by the decree 126/1990 of Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis (IBAMA). Increasingly, actions which are linked to the breeding of this animal in captivity are accomplished in the state of Mato Grosso by institutions like Sistema Brasileiro de Apoio às Micro e Pequenas Empresas (SEBRAE), Fundação Estadual do Meio Ambiente (FEMA) e IBAMA. These actions have as an aim to develop the productive chain of Jacaré do Pantanal, that have as the main product the skin of the animal and as sub-products, the meat, craft and animal food. The sub-product meat received detachment in this productive chain in 2008, due to the processing unit which belongs to the Cooperativa de Criadores de Jacaré do Pantanal (COOCRIJAPAN) that obtained the registration of the Serviço de Inspeção Federal (SIF), allowing the trading of alligator meat in national territory and abroad. To meet the quality requests and the security of the food, in general and at this unit, actions and procedures are necessary of the Quality Management. The objective of this study is to present a set of propositions for the Quality Management for the Processing Unit of COOCRIJAPAN, that meets the requests of the Good Manufacturing Practices (GMP), of NBR ISO 22000:2006 and of Hazard Analysis Critical Control Point (HACCP), which are considered specific methods for the Quality Management of Food Industries. With this objective, data were collected at COOCRIJAPAN, through interviews with the members of the unit and observations were made at the local to know the practices of the quality management of the Cooperative during the month of July of 2009. From the bibliographic revision and the field study, proposals was made for the Quality Management and Food Security for the Cooperative as well as a definition of a implementation plan for it. / A criação de jacaré em cativeiro teve início no Brasil como opção para proteção da espécie e das propriedades de terra contra invasões de caçadores de jacaré. Esta atividade recebeu suporte legal através da portaria 126/1990 do Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis (IBAMA). De maneira crescente, ações ligadas à criação deste animal em cativeiro são realizadas no Estado de Mato Grosso por órgãos como o Sistema Brasileiro de Apoio às Micro e Pequenas Empresas (SEBRAE), Fundação Estadual do Meio Ambiente (FEMA) e IBAMA. Essas ações objetivam desenvolver a cadeia produtiva do Jacaré do Pantanal, que tem como principal produto o couro do animal e como subprodutos a carne, artesanatos e ração animal. O subproduto carne recebeu destaque nesta cadeia produtiva em 2008, devido à unidade processadora, pertencente à Cooperativa de Criadores de Jacaré do Pantanal (COOCRIJAPAN), ter obtido o registro no Serviço de Inspeção Federal (SIF), possibilitando a comercialização da carne de jacaré em território nacional e no mercado externo. Para atender os requisitos da qualidade e da segurança do alimento, de modo geral e nesta unidade processadora, são necessárias ações e procedimentos da Gestão da Qualidade. O objetivo deste trabalho é apresentar propostas para a Gestão da Qualidade para a unidade processadora da COOCRIJAPAN, que atenda aos requisitos das Boas Práticas de Fabricação (BPF), da NBR ISO 22000:2006 e do Sistema de Análise de Perigos e Pontos Críticos de Controle (APPCC), que são consideradas abordagens específicas para a Gestão da Qualidade em indústrias alimentícias. Para isso, foram coletadas informações na COOCRIJAPAN, através de entrevistas com membros da unidade e feitas observações in loco para conhecer as práticas de gestão da qualidade da Cooperativa, durante o mês de julho de 2009. A partir da revisão bibliográfica e da pesquisa de campo, elaborou-se a proposta para a Gestão da Qualidade e da Segurança do Alimento para a Cooperativa, bem como um plano para implantação.

Made available in DSpace on 2016-06-02T19:52:13Z (GMT). No. of bitstreams: 1 DissAC.pdf: 1825602 bytes, checksum: 66578e867a58642d29d296cfb15d5fc3 (MD5) Previous issue date: 2005-12-05 / The steady growth of the fruit juices sector is a significant factor strengthening the Brazilian economy; the richness of the tropical fruits, the quality of juices they are processed into and the luring presentation to consumers expanded the internal market and helped conquer demanding markets abroad. All this happened as a result of meeting stringent and assorted requirements, since food fruit juices included quality and safety are a must today. The purpose of this paper is to present a proposal to integrate management systems involving quality, good manufacturing practices and related legal requirements, as well as HACCP, in a small fruit juices company in the State of São Paulo. The proposal includes integrated cost-effective actions to implement the various standards. The research carried out concludes that the integration of management systems involving quality, good manufacturing practices and related legal requirements, as well as HACCP, is feasible. A relationship matrix of the respective requirements was designed during the research, which allowed conducting an initial joint and revealing assessment to evaluate the degree of fulfillment of those requirements. Bearing in mind the company´s scenario where the research was performed and the first application of this Integrated Management System took place, the proposal was not totally implemented. The necessity was perceived to test the Integrated Management System and monitor its operation in other food sector companies, thus not only evaluating the consistency of the proposal but also its flexibility to meet local specific requirements and detect improvement opportunities. / O crescimento do setor de produção de sucos de frutas vem contribuindo ao fortalecimento da economia brasileira; a rica variedade de frutas tropicais, a diversidade de formas de apresentação e a qualidade dos sucos ampliaram o mercado interno e conquistaram mercados externos, atendendo exigências cada vez mais rígidas. As empresas do setor estão submetidas a crescentes exigências de qualidade e segurança de seus produtos. O objetivo deste trabalho é apresentar uma proposta para integrar os sistemas de gestão da qualidade, das boas práticas de fabricação e requisitos legais correlatos, bem como da APPCC, em uma pequena empresa de sucos de frutas situada no interior do Estado de São Paulo, submetida às crescentes exigências dos clientes. A proposta inclui ações integradas contribuindo para racionalizar os recursos aplicados ao processo de adequação aos diferentes referenciais normativos. O trabalho conclui que é viável a integração dos sistemas de gestão da qualidade, das boas práticas de fabricação e requisitos legais correlatos e da APPCC. A matriz de correspondência entre requisitos respectivos, desenhada durante a pesquisa realizada, permitiu a avaliação conjunta do grau de atendimento dos mesmos, facilitando o processo de implementação a partir dos resultados obtidos. Tendo em vista o cenário da empresa em que foi desenvolvida a pesquisa e realizada a primeira aplicação da proposta do Sistema Integrado de Gestão, não foi possível implementar a proposta integralmente. Foi percebida a necessidade de testar sua implementação e operação em outras empresas do setor de alimentos, para avaliar não só a consistência da mesma, senão também sua flexibilidade para se ajustar às especificidades locais e detectar oportunidades de melhoria. Palavras-chave: qualidade do alimento, segurança do alimento, BPF, ISO 9001 (2000) e APPCC, Resolução ANVISA RDC 275/01, indústria de sucos, sistema integrado de gestão.

A produção de kits para diagnóstico in vitro deve ser feita seguindo-se a legislação vigente de Boas Práticas de Fabricação e Controle (BPF). O objetivo deste trabalho foi elaborar um procedimento para desenvolvimento, produção e validação de um produto para diagnóstico in vitro, de acordo com a legislação vigente. Adotamos como modelo um kit imunoenzimático para Doença de Chagas. Dentro dos requisitos de BPF, a validação é uma etapa importante, pois tem por objetivos, dentre outros: auxiliar no estabelecimento de procedimentos de produção e controle de qualidade, avaliar desvios e dimensionar possíveis erros, avaliar o desempenho quanto à utilidade médica dos resultados obtidos e estabelecer condições ideais de uso. No estabelecimento dos requisitos para validação devem-se considerar as características do método utilizado, a utilidade clínica e diagnóstica dos resultados e as condições de uso do kit. Os parâmetros para validação devem ser definidos considerando a finalidade do uso do produto. Os resultados obtidos em três lotes pilotos demonstraram que o kit pode ser utilizado tanto com soro como com plasma, as amostras podem ser congeladas e descongeladas antes do uso por até 5 ciclos, o índice de concordância com kit comercial é de 0,9 (ótimo) e o kit mantém-se estável por pelo menos 7 dias à 37ºC, o que neste trabalho foi equivalente a pelo menos um ano na sua condição ideal de armazenamento de 2 a 8ºC. Além disso, o kit apresentou 100% de sensibilidade, 99% de especificidade, com coeficiente de variação 15,2% tanto na repetitividade como na reprodutibilidade de amostras positivas. Quanto à análise de interferentes, amostras hemolisadas e a presença de fator reumatóide podem interferir nos resultados e anticorpos anti-Leishmania na amostra podem dar reação cruzada. Conclui-se que o procedimento elaborado e o kit desenvolvido e validado atenderam aos requisitos pré-estabelecidos, de acordo com as regras de BPF vigentes. / The production of an in vitro diagnostic kit should be done following current Good Manufacturing Practices (GMP). The objective of this work was to establish a procedure for the development, production and validation of an in vitro diagnostic product in accordance with current regulations governing Medical Devices. An enzyme-linked immunoassay kit for Chagas\' disease was used as a model. Validation is a very important step contained within GMP requirements. Validation provides documented evidence that processes and product batches are consistent, it aids in the establishment of production and quality control procedures, evaluate deviations and identify possible mistakes, evaluate the performance and medical usefulness of the product based on the obtained results, and establish ideal conditions of use and storage. In order to establish validation requirements for product development, it is necessary to consider the characteristics of the assay method, the clinical and diagnostic usefulness of the results and the conditions of use of the kit. The parameters for validation should be defined considering the purpose of the use of the product. In the case of this Chagas assay, results obtained in three pilot lots demonstrated that the kit could be used with both serum and plasma, samples could be frozen and thawed before use for up to 5 cycles. The agreement index when compared with a commercially licensed kit is 0,9 (optimum correlation). The kit remained stable for at least 7 days at 37ºC, which is equivalent to at least one year stability in its ideal storage condition of 2 to 8ºC. The kit presented 100% sensitivity and 99% specificity, with variation coefficient of 15,2% for both repeatability and reproducibility of the positive samples. Interference analysis indicated that: hemolyzed samples and the presence of reumathoid factor could interfere with test results. Antibodies anti-Leishmania in the test sample can cross react with T. cruzi proteins. In conclusion: the established procedure for development and validation of chagas kit, and the actual developed and validated kit are in accordance with pre-established current GMP requirements.

Multipotent mesenchymal stromal cells (MSC) were first discovered in bone marrow and can be isolated from “virtually all organs”. They could participate in tissue maintenance and self-renewing process. They are able to adhere to plastic surfaces and acquire a fibroblastic shape when isolated. They are characterized by a particular phenotype and are able to differentiate into several cell types if cultivated in a specific induction medium. These characteristics were defined on MSC in culture and do not represent how they may be in situ.MSC present particular properties. They can secrete growth factors and several cytokines that give them a trophic activity on one hand and the ability to modulate the immune system on the other hand. They are also able to differentiate. These different properties make them an attractive candidate for cell therapy.MSC are already the focus of several pre-clinical and clinical studies. Nevertheless, the results of these studies are difficult to interpret due to limited understanding of their basic biology. MSC are poorly defined in situ and are heterogeneous. Their heterogeneity is dictated by their tissue of origin and cell preparation. To date, there is no standard protocol for MSC isolation and culture. This leads to numerous questions regarding patient safety, and these questions require answers.The first part of the work deals with the methods used to optimize the extraction of MSC and purification from adipose tissue, one of the main sources of autologous MSC with bone marrow. Classical methods require an enzymatic digestion step. The enzyme used and the duration of adipose tissue digestion time can induce cellular alterations and modify cell functions. Moreover, the addition of a xenobiotic increases the risk of contamination and complicates the monitoring of good manufacturing practices (GMP). We propose a method that does not require this enzymatic digestion step while being easier, safer, faster, gentler and less expensive. Compared to the classical enzymatic method, our method yields an equivalent number of MSC from adipose tissue while preserving their properties.The second part of this work focuses on the characterization of the MSC subpopulations from adipose tissue and compares them to those from bone marrow, which are the historical gold standard. The study made it possible to deepen the knowledge of MSC surface markers in situ from these 2 sources. It also evaluated the various properties of the isolated subpopulations thanks to the cell surface markers CD271, SUSD2, MSCA-1, CD44 and CD34. We showed that MSC from bone marrow express MSCA-1, CD271 and SUSD2 markers in situ. We also found that a population clearly positive for the CD34 does exist in situ with different properties compared to those of the unselected populations or the negative counterpart. 2 populations that are negative and positive for CD44 also exist with similar properties.In contrast to bone marrow MSC, only one selection was able to effectively isolate MSC from adipose tissue by a positive selection based on the expression of CD34. We also isolated a CD271+ population but only from lipoaspirate samples and not from abdominoplasty samples. Collectively, our results suggest that MSCA-1 seems to be the best marker through which to isolate MSC from bone marrow and that CD34 is the only marker able to positively isolate cells from adipose tissue. Thus, we show that the MSC from the different sources share similar properties although they have specific characteristics. The choice of the source and of the marker with which to isolate a particular subpopulation is important depending on their intended clinical use. / Les cellules stromales mésenchymateuses multipotentes (CSM) ont été mises en évidence dans la moelle osseuse et peuvent être isolées de « virtuellement tous les organes ». Elles participeraient à la maintenance et au renouvellement des tissus. Une fois isolées, elles sont capables d’adhérer à des surfaces en plastique en prenant une forme fibroblastique. Elles sont caractérisées par un phénotype particulier et peuvent se différencier en divers types cellulaires lorsque cultivées dans un milieu d’induction spécifique. Ces caractéristiques ont été définies sur les CSM en culture et ne reflètent pas forcément ce qui se passe in situ.Les CSM présentent des propriétés particulières. Elles peuvent sécréter des facteurs de croissance ainsi que de nombreuses cytokines qui leur permettent d’une part d’avoir une activité trophique et d’autre part de moduler le système immunitaire. Elles sont aussi capables de se différencier. Ces différentes propriétés les rendent particulièrement attractives pour la thérapie cellulaire.Les CSM font déjà l’objet de nombreuses études pré-cliniques et cliniques dont les résultats sont difficilement interprétables car nous n’avons à l’heure actuelle qu’une compréhension limitée de leur biologie de base. Les CSM sont encore mal définies in situ et sont hétérogènes. Cette hétérogénéité provient de leur différence d’origine et de leur préparation cellulaire :il n’existe aucune standardisation des protocoles d’isolation et de culture. Cette hétérogénéité entraine de nombreuses questions relatives à la sécurité du patient qui doivent être élucidées.La première partie de ce travail cherche à optimiser les méthodes d’extraction et de purification des CSM du tissu adipeux humain, la principale source de CSM autologues avec la moelle osseuse. Les méthodes classiques requièrent une étape de digestion enzymatique dont l’enzyme utilisée et le temps de digestion du tissu adipeux peuvent induire des altérations cellulaires et modifier leurs fonctions. De plus, l’adjonction de xénobiotiques augmente le risque de contamination et complique le suivi des bonnes pratiques de fabrication (BPF). Nous proposons une méthode qui s’affranchit de cette étape de digestion enzymatique tout en étant plus facile, plus sûre, plus rapide, moins chère et moins traumatisante pour les cellules. Elle permet d’obtenir un nombre tout aussi important de CSM du tissu adipeux que la méthode enzymatique classique en préservant leurs propriétés.La deuxième partie de ce travail vise à caractériser les sous populations de CSM du tissu adipeux humain en les comparant à celles de la moelle osseuse, source de référence historique. Cette étude a permis d’approfondir la connaissance des marqueurs de surface des CSM de ces 2 sources in situ, tout en évaluant les différentes propriétés des sous-populations isolées grâce aux marqueurs de surface CD271, SUSD2, MSCA-1, CD44 et CD34. Nous avons montré que les CSM de la moelle osseuse expriment les marqueurs MSCA-1, CD271 et SUSD2 in situ et qu’il existait une sous-population clairement positive pour le CD34 avec des propriétés différentes de celles de la population non sélectionnée ou négative pour ce marqueur. Il existe aussi 2 sous-populations positive et négative pour le CD44 avec des propriétés similaires.Contrairement aux CSM de la moelle osseuse, une seule sélection a permis d’isoler efficacement les CSM du tissu adipeux par une sélection positive sur base de l’expression du CD34. Nous avons pu aussi isoler une population CD271+ mais seulement des prélèvements de lipoaspirations et non des abdominoplasties.Au vu de nos résultats, MSCA-1 semble le meilleur marqueur pour isoler les CSM de la moelle osseuse tandis que le CD34 est le seul marqueur capable d’isoler positivement celles du tissu adipeux. Ainsi, nous montrons que les CSM issues de différentes sources partagent des propriétés similaires avec cependant des caractéristiques propres. Le choix de la source et du marqueur pour isoler une sous-population sont donc importants en fonction de leur utilité clinique envisagée. / Doctorat en Sciences biomédicales et pharmaceutiques (Médecine) / info:eu-repo/semantics/nonPublished

All cells are equipped to sense changes in their environment and make adaptive responses according to the stimuli. Signal recognition usually occurs at the cell membrane (with the exception of steroid signalling) where the ligand, which can be a small molecule, a peptide or a protein, binds its cognate receptor. This results in a change in the conformation of the receptor which in turn can regulate the production of second messengers. Second messengers can now modulate specific pathways which control gene expression and modify various aspects of cell behaviour. The signalling cascade is terminated by the removal of second messenger and/or by desensitisation of the receptor to the extracellular signal. Cyclic guanosine monophosphate (cGMP) was first identified in the rat urine and since then has emerged as an important second messenger regulating diverse cell processes. Subsequent to its discovery in mammalian cells, enzymes responsible for its synthesis (guanylyl cyclases), hydrolysis (phosphodiesterases) and its most common effectors (cGMP-dependent protein kinases) were identified. Guanylyl cyclases exist in two forms, cytosolic and membrane bound. Both have a conserved guanylyl cyclase domain, but differ in their choice of ligands, overall structure and tissue localization. It is now known that cytosolic and the membrane-bound forms are involved in eliciting distinct cellular responses. Receptor guanylyl cyclase C (GC-C) was identified as the target for a family of heat-stable enterotoxin toxins (ST) produced by enterotoxigenic E.coli. Stable toxin-mediated diarrhoeas are observed frequently in infants and contribute significantly to the incidence of Travellers’ Diarrhea. Early studies demonstrated that the effects of ST were mediated by an increase in intracellular cGMP levels in intestinal cells, and the receptor for ST was almost exclusively expressed in the apical microvilli of the intestinal brush-border epithelia. Effectors of cGMP in intestinal cells include protein kinase G (PKG), cyclic nucleotide gated ion channel 3 (CNG), and the cystic fibrosis transmembrane conductance regulator (CFTR). ST is an exogenous ligand which serves as a hyperagonist for GC-C, in comparison with the endogenous ligands guanylin and uroguanylin, which maintain fluid-ion homeostasis in the intestinal epithelia. The GC-C/cGMP signal transduction pathway also modulates intestinal cell proliferation along the crypt-villus axis by exerting a cytostatic effect on the epithelial cells, thereby regulating their turnover and neoplastic transformation. The current study describes in molecular detail two signalling pathways, one impinging on and one emerging from GC-C, which regulate colonic cell proliferation. The first part identifies the cross-talk and cross-regulation of GC-C and c-src. The second part delves into the molecular basis of GC-C/cGMP-mediated cytostasis and its effect on colonic tumorigenesis. Cross-talk between signalling pathways is believed to play a key role in regulating cell physiology. Phosphorylation of signalling molecules by protein kinases is frequently used as a means of achieving this cross-regulation. Aberrant hyperactivation of the c-src tyrosine kinase is an early event in the progression of colorectal cancer, and activated c-src specifically phosphorylates a number of proteins in the cell. It was found that c-src can phosphorylate GC-C in T84 colorectal carcinoma cells, as well as in the rat intestinal epithelia. Tyrosine phosphorylation of GC-C resulted in attenuation of ligand-mediated cGMP production; an effect which was reversed by chemical or transcriptional knockdown of c-src. These effects were found to be cell line-independent and relied only on the extent of c-src expression and activation in the cell. Mutational analysis revealed GC-C to be phosphorylated on a conserved tyrosine residue (Y820) in the guanylyl cyclase domain. The sequence of GC-C around Y820 allowed for efficient phosphorylation by c-src, and indeed, kinase assays indicated that the affinity of c-src for the GC-C Y820 peptide was one of the highest reported till date. A phospho-mimetic mutation at this site, which mimics a constitutively phosphorylated receptor, resulted in a sharp reduction of guanylyl cyclase activity of the receptor, reiterating the inhibitory role of Y820 phosphorylation on GC-C activity. Phosphorylation of GC-C at Y820 generated a docking site for the SH2 domain of c-src which could interact and thereby co-localize with GC-C on the cell membrane. Intriguingly, this interaction resulted in activation of c-src, setting-up a feed-forward loop of inhibitory GC-C phosphorylation and c-src activation. Treatment of colorectal carcinoma cells with ligands for GC-C reduces cell proliferation and inhibits tumorigenesis. It was observed that this cytostatic effect can be modulated by the status of c-src activation, and consequently, the fraction of tyrosine phosphorylated GC-C in these cells. Since activation of c-src is a frequent event in intestinal neoplasia, phosphorylation of GC-C by active c-src may be one of the means by which the cytostatic effects of GC-C agonists (guanylin and uroguanylin) in the intestine are bypassed, thereby leading to cancer progression. Colonisation of the gut with enteropathogenic microorganisms induces secretion of IFNγ from the host mucosal immune system, which subsequently activates c-src in intestinal epithelial cells. Ligand-stimulated activity of GC-C was found to be reduced in IFNγ treated cells. This could be one of the host defence mechanisms initiated in response to enterotoxigenic E. coli infection. These results provide the first evidence of cross-talk between a receptor guanylyl cyclase and a tyrosine kinase that results in heterologous desensitisation of the receptor. Populations with a higher incidence of enterotoxigenic E.coli infections appear to be protected from intestinal neoplasia. It was found that mice lacking GC-C, and therefore unable to respond to ST, displayed an increased cell proliferation in colonic crypts and enhanced carcinogen-induced aberrant crypt foci formation, which is a surrogate marker for colorectal carcinogenesis. However, pharmacological elevation of cGMP was able to efficiently induce cytostasis even in GC-C knockout mice, indicating a key role for cGMP in regulating colonic cell proliferation. Through microarray analyses, genes regulated by ST-induced GC-C activation in T84 colorectal carcinoma cells were identified. Genes involved in a number of cellular pathways were differentially expressed, including those involved in signal transduction, protein and solute secretion, transcriptional regulation and extracellular matrix formation. One of the genes found to be significantly up-regulated was the cell-cycle inhibitor, p21. The increase in p21 expression was validated at both the transcript and protein level. This p53-independent up-regulation of p21 was coupled to the activation of the cGMP-responsive kinase, PKGII, since knockdown of PKGII using specific siRNAs abolished ST-induced p21 induction. Activation of PKGII led to phosphorylation and activation of the stress responsive p38 MAPK. Similar to what was seen following knockdown of PKGII, inhibition of p38 MAPK activity attenuated the up-regulation of p21 in response to cGMP, indicating that PKGII and p38 MAPK could be a part of a pathway regulating p21 expression. It was found that active p38 MAPK phosphorylated the ubiquitous transcription factor SP1, enhancing its occupancy at the proximal p21 promoter. Therefore, SP1 could be one of the factors linking cGMP to transcription of the p21 mRNA. Chronic activation of GC-C led to nuclear accumulation of p21 in colonic cells, which entered a quiescent state. These cells arrested in the G1 phase of the cell cycle, consequent to p21-dependent inhibition of the G1 cyclin-CDK complexes. A fraction of these quiescent cells stochastically initiated a cGMP-dependent senescence programme and displayed all the hallmarks of senescent cells, including flattened cell morphology, expression of SA- galactosidase and formation of senescence-associated heterochromatic foci. Activation of senescence and loss of tumorigenicity in these cells was crucially dependent on the up-regulation of p21. This irreversible exit from the cell cycle due to cGMP-mediated activation of the PKGII/p38/p21 axis was well correlated with reduced colonic polyp formation in mice exposed to ST. In summary, these observations may provide a possible explanation for the low incidence of colorectal carcinoma seen in countries with a high incidence of ST-mediated diarrhoea. Interestingly, c-src mediated tyrosine phosphorylation of GC-C prevented p21 accumulation following ligand application. The findings described in this thesis may have important implications in understanding the molecular mechanisms involved in the progression and treatment of colorectal cancer.

Masters of Science / The aim of this study was to develop a Stock Control Assessment Tool for use in the public health care sector and then to apply it to identify problems in the stock control system. This would help authorities to optimize the system. The advantages experienced with such a dynamic Assessment Tool were many: The Tool was quick and easy to apply; it was user friendly; it provided an immediate SWOT analysis of a particular facility; it is in line with the SA NDP directives and it provides an indication of which structures are in place and whether they are functioning properly. Furthermore; it could determine the increase or decrease in performance of a facility (therefore identify trends within the functional status of a system) when data is collected over a period of time; and finally, it could also be used to prioritize drug policy directives. The following steps were established in the development of the Tool: A literature review of pharmaceutical stock control and Drug Supply Management was addressed to provide the background information for the motivation of this study and to identify the various elements that could influence stock control at a facility level. Observational studies were applied at selected private and public facilities to observe the impact of these identified stock control elements. An 'ideal' stock control system was then generated from the literature review and observational assessment. A structured questionnaire was developed and surveyed at these facilities to generate key areas of concern of a stock control system. A study and adaptation of the indicator methods used by the World Health Organization (WHO) to monitor drug use in health facilities resulted in the formulation of a practical Stock Control Assessment Tool based on 11 key indicators and a number of sub-indicators, all of which were objectively defined. The Tool was then applied at selected public facilities and the results were analyzed quantitatively, qualitatively and subjectively. Each of the indicators was then applied and results examined closely with a view to possible refinements of the indicator. The refinements were made and the Tool was re-applied at two selected facilities. These two facilities were randomly selected from the original six facilities included for the testing of the Tool. Final conclusions and specific recommendations were generated to improve the stock control systems at the selected public health care facilities.