No description available.

Traumatic brain injury (TBI) caused by traffic and fall accidents, sports-related injuries and violence commonly results in life-changing disabilities. Cognitive impairments following TBI may be due to disruption of axons, stretched by the acceleration/deceleration forces of the initial impact, and their surrounding myelin in neuronal networks. The primary injury, which also results in death to neuronal and glial cells, is followed by a cascade of secondary injury mechanisms including a complex inflammatory response that will exacerbate the white matter injury. Axons are supported and protected by the ensheathing myelin, ensuring fast conduction velocity. Myelin is produced by oligodendrocytes (OLs), a cell type vulnerable to many of the molecular processes, including several inflammatory mediators, elicited by TBI. Since one OL extends processes to several axons, the protection of OLs is an important therapeutic target post-TBI.  During development, OLs mature from oligodendrocyte progenitor cells (OPCs), also present in the adult brain. The aim of this thesis was to investigate white matter pathology, with a specific focus on the OL population, in experimental and clinical TBI. Since the inflammatory response may contribute to OL cell death and OPC proliferation, neutralization of interleukin-1β (IL-1β) was investigated. The lateral and central fluid percussion injury models were used in mice and rats where memory, learning and complex behaviors were investigated by two functional tests. Brain tissue, surgically resected due to life-threatening brain swelling or hemorrhage, from TBI patients was also investigated. Axonal injury, myelin damage, microglia alterations and OPCs and OL cell death were investigated by immunohistochemical techniques. In focal and diffuse experimental TBI, OL cell death was observed in important white matter tracts. OL cell death was accompanied by myelin damage, axonal injury and presence of microglia as well as an increased number of OPCs in both the experimental and human setting. OPCs were found to proliferate in diffuse TBI in mice where both complex behavioral changes and impaired memory were observed. Neutralization of IL-1β normalized and improved these behavioral alterations and also lead to a preserved number of mature OLs although without influencing OPC proliferation. The results provided in this thesis indicate that white matter pathology is a key component of the pathophysiology of TBI. The OPC proliferation may influence regeneration post-injury and might be an important future therapeutic targets for TBI. The present studies also suggest that treatment strategies targeting neuroinflammation may positively influence behavioral outcome and OL cell death in TBI. / <p>(Faculty of Medicine)</p>

Chez les vertébrés, les muscles squelettiques du corps sont dérivés de la partie dorsale dessomites, le dermomyotome, structure transitoire mésodermique. Une première étape demyogenèse aboutit à la formation d’un muscle primitif, le myotome primaire, à partir desbordures du dermomyotome : ces cellules constituent les premières fibres musculaires, et formentl’architecture de base du futur muscle. Dans un second temps, une population de progéniteursmusculaires émerge de la région centrale du dermomyotome. Cette population est primordialedans la constitution du muscle. Elle prolifère, et une partie d’entre elle fusionne aux fibresexistantes pour donner les fibres multinucléées adultes. Finalement, une partie des progéniteursmusculaires reste indifférenciée jusqu’à l’âge adulte et compose la population de cellules souchesmusculaires, les cellules satellites. Ainsi, les progéniteurs musculaires contribuent audéveloppement musculaire tout au long du développement embryonnaire et foetal, mais égalementà la myogenèse post-natale avec les cellules satellites.Lors de ma thèse, je me suis intéressé à cette population de progéniteurs musculaires. Deux souspopulationsde progéniteurs musculaires ont précédemment été identifiées dans notre laboratoireau cours de l’embryogénèse précoce de poulet, l’une exprimant le facteur de transcription Pax7,l’autre co-exprimant Pax7 et le facteur de différenciation myogénique précoce Myf5. Face àl’absence de données concernant les progéniteurs musculaires, et à l’importance de cettepopulation pour la myogenèse, j’ai réalisé une étude systématique des progéniteurs musculairestout au long du développement embryonnaire et foetal de deux organismes modèles : le poulet etla souris. J’ai pu montrer que ces deux sous-populations coexistent tout au long dudéveloppement, depuis l’émergence des progéniteurs de la partie centrale du dermomyotome,jusqu’au moment où ces cellules deviennent des cellules satellites à la fin du développementfoetal. De manière très intéressante, j’ai pu montrer qu’au sein des progéniteurs musculaires, lapopulation principale co-exprime Pax7 et Myf5, et prolifère activement, alors que la populationPax7 est mineure et prolifère à un taux moins élevé. Cette dernière entre de manière importanteen quiescence à la fin du développement embryonnaire. Ces caractéristiques sont semblablesentre le poulet et la souris, et montrent que des stratégies cellulaires et moléculaires similairessont conservées au sein des amniotes. / Duringembryonicandfetallife,skeletalmusclegrowthisdependentupontheproliferationandthedifferentiationofapopulationofresidentmuscleprogenitors,fromwhichderivethemusclestemcellsof theadult,thesatellitecells.Underpoorlydefinedextrinsicandintrinsicinfluences,muscleprogenitorsproliferate,differentiateorenteraquiescentstatetobecomereservesatellitecells.Despitetheir primordialrole,surprisinglylittleisknownonthehomeostasisofresidentprogenitorsduringembryogenesis.Preliminarystudiesinchickandmousedescribingthekeyprogenitorpopulationscontributingtomusclegrowthduringembryogenesishaveledtodifferingresultsthatcouldbeduetotechnicalissuesortofundamentaldifferencesbetweenanimalmodels.Toaddressthisquestion,we haveundertakenacomprehensiveanalysisofthestateofdifferentiationandproliferationofmuscleprogenitorcellsfromthetimeoftheiremergencewithinthedermomyotomeuntillatefetallife,whenthey adoptasatellitecell-likepositionunderthebasallamina.Thiswasdonebyimmunostainingagainstkeyplayersofmyogenicdifferentiation,inmuscleschosenfromdifferentregionsofthebodyintwo modelorganisms,thechickandmouse.This studyidentifiedtwoco-existingpopulationsofprogenitorsduringembryonicandfetallifeinboth chickandmouse:aminor,slow-cyclingpoolofundifferentiatedresidentprogenitorswhichexpress Pax7,co-existingwithamajorfast-cyclingpopulationthatco-expressPax7andtheearlymyogenicdifferentiationmarkerMyf5.Wefoundthattheoverallproliferationrateofbothprogenitorsdrasticallydecreasedwithembryonicage,asanincreasinglylargeportionofslowandfast-cyclingprogenitorsenteredquiescenceduringdevelopment.Together,thisdatasuggeststhatthecellularstrategiesthatdrivemusclegrowthduringembryonicand fetallifeareremarkablyconservedinamniotesthroughoutevolution.Theyrelyonthetightregulationofproliferation,entryinquiescence,andmodulationofthecellcycle’slengthforbothoftheco-existingpopulationsofmuscleprogenitorstomaintainthehomeostasisofgrowingmusclesduringdevelopment.

L’activation du système complément, qui se produit à la suite d’une infection ou d’un trauma, génère de puissants signaux moléculaires capables d’initier la réaction inflammatoire. Parmi ces signaux, le fragment actif C5a permet de recruter sur le site lésé les cellules qui expriment son récepteur (le C5aR/CD88). Bien que le C5aR/CD88 soit initialement connu pour être exprimé par les cellules inflammatoires, il est établi que de nombreuses cellules non immunitaires expriment ce récepteur indiquant son implication dans d’autres processus. Nos résultats ont permis de démontrer que l’activation du système du complément au niveau de la pulpe dentaire est réalisée non seulement à partir des protéines plasmatiques mais aussi des protéines synthétisées par les fibroblastes pulpaires. Ainsi, l’activation locale du système du complément, produit à la suite d’une infection, d’un trauma ou de l’application de biomatériaux, génère du C5a qui induit la migration des cellules progénitrices. Ce travail démontre pour la toute première fois l’implication du fragment actif C5a dans le recrutement de progénitrices pulpaires, étape clef au processus de régénération pulpo-Dentinaire. Ces travaux pourraient donc constituer une piste sérieuse dans l’établissement de nouvelles thérapies permettant de cibler les cellules progénitrices au cours du processus de régénération. / After tissue injury or infection, Complement activation provides powerful signals initiating the inflammatory reaction. These events are mediated by biologically active fragments such as C5a which attracts cells expressing its receptor (C5aR/CD88) to the injury site. Besides inflammatory cells as the main C5aR-Expressing cells, various tissue cells have been reported to express this receptor suggesting its involvement in other processes. In order to investigate the possible relationship between complement activation and pulp regeneration, we investigated Complement activation in the dental pulp and progenitor cell migration from their perivascular niches to the pulp injury site to initiate the regeneration process.Our results indicate that complement activation in the dental pulp is the result of both plasma and fibroblast secreted complement proteins. Thus upon local complement activation, which can occur after pathological injury or biomaterials application, C5a induces pulp progenitors’ migration which is critical in initiating the regenerative processes. To our knowledge, this is the first work to demonstrate the involvement of C5a biologically active fragment in the recruitment human pulp progenitor cells. This may provide a useful future therapeutic tool in targeting the progenitor cells in a dentin/pulp regeneration process.

Ph.D. Thesis abstract: Diabetes mellitus is a chronic disease characterized by a metabolic disorder in which there is a low level or complete lack of the insulin. Diabetes mellitus type 1 (DM1) is caused by an autoimmune reaction leading to the destruction of the insulin producing beta cells in the pancreas. In consequence, low or non-existent insulin production leads to a complete dependence on exogenous insulin supplementation. DM1 causes serious long-term complications. Although strict control of blood sugar could prevent the onset and development of diabetic complications only 5% of diabetic patients are able to achieve such control. Hence it is evident that the current methods of treatment are neither sufficient to treat this disease, nor prevent late complications in most patients. The most promising therapeutic approach in the treatment of diabetes is the restoring of insulin production. One such method is the transplantation of insulin-producing tissue. However, a lack of available insulin- producing tissue limits such therapeutic approach. Therefore an alternative source of insulin producing cells have to be found to obtain a sufficient amount of safe and efficient insulin producing tissue. Pancreatic stem/progenitor cells could represent such an available alternative source. Despite the evidence...

No description available.

A malária é uma infecção causada por plasmódios, cujo controle depende do baço, o responsável pelo clareamento dos eritrócitos parasitos. O aumento da parasitemia induz uma ampliação do baço para resolver a infecção, onde participam células precursoras que apresentam CCD34+ na sua superfície. Estudamos a distribuição e a quantidade de células CD34+ em baços de roedores durante malárias de roedores, para compreender sua participação na ampliação do baço e no controle da infecção. Camundongos C57Bl/6j infectados com as cepas AJ e CR de Plasmodium chabaudi, e com a cepa ANKA de Plasmodium berghei, tiveram seus baços removidos e encaminhados para histologia e citometria de fluxo. A distribuição das células CD34+ mostrou-se mais intensa no 4º dia p.i. e menos intensa no 8º dia p.i.. As células CD34+ livres, por citometria de fluxo, surgem com uma onda no 4º dia p.i.. Sua quantidade é similar entre os modelos de P. chabaudi, mas diferente no P. berghei. Neste trabalho, o influxo de células CD34+ no baço não se relaciona com o controle da infecção. / Malaria is caused by Plasmodium sp., which control depends on the spleen, responsible for parasite clearing. The increase of parasitemia implies in spleen amplification to control the infection, with participation of CD34+ cells. We studied the distribution and amount of CD34+ cells in spleen during rodent malaria, to define the role of those cells in spleen amplification and infection control. C57Bl/6j mice were infected with strains CR and AJ of Plasmodium chabaudi, and ANKA strain of Plasmodium berghei. The spleen was removed and processed for histology and flow cytometry. Spleen CD34+ cells was increased in 4th day, p.i., and decreases in 8th day p.i. in all models. By flow cytometry, free CD34+ cells appears as a wave in the 4th day p.i.. P. chabaudi models presented the same level of those cells, which was larger in the P. berghei mice. In this work, increase of spleen CD34+ cells do not correlate with infection control.

A hematopoese tem sido bem descrita em modelos murinos nas últimas décadas, contudo, trabalhos demonstrando os mecanismos da hematopoese em humanos ainda são escassos. A derivação da primeira linhagem de células-tronco embrionárias humanas (CTEhs) em 1998, gerou novas perspectivas tanto para o estudo da hematopoese na tentativa de mimetizar o que ocorre naturalmente durante o desenvolvimento embrionário, quanto para a aplicação clínica das células hematopoéticas obtidas a partir da diferenciação dessas células. Contudo, apesar de inúmeros trabalhos terem demonstradoa obtenção de células hematopoéticas a partir de CTEhs, os protocolos têm gerado quantidades variáveis de células, com baixa eficiência e com propriedades funcionais de células primitivas. Desse modo, este trabalho procurou estabelecer um modelo próprio de diferenciação de CTEhs-H1 em células progenitoras hematopoéticas para que estas pudessem ser melhor caracterizadas e obtidas de forma mais eficiente. Para isto, foi desenvolvido um sistema de diferenciação baseado no co-cultivo da linhagem de CTEh-H1 com fibroblastos de embrião de camundongo (MEFs), em meio de diferenciação suplementado soro fetal bovino (SFB) e citocinas e fatores de crescimento hematopoéticos em baixas concentrações. Como resultado, o desenvolvimento do presente trabalho permitiu o estabelecimento de um método para geração de populações mistas de células enriquecidas em CPHs positivas para o marcador CD45, o qual mostrou ser coexpresso com outros marcadores hematopoéticos (CD31, CD43, CD71 e CD38), e células hematopoéticas maduras positivas para marcadores mielóide-específicos (235a, CD14, CD15, CD16) e com características morfológicas típicas. Foi demonstrado que as células obtidas expressavam genes relativos ao sistema hematopoético (CD45, CD31, runx1, tal1, lmo2, prom1, CD34 e notch1), e possuíam potencial clonogênico in vitro da ordem de 1/574 células plaqueadas. Em adição, corroboramos os achados de que as células hematopoéticas apresentam duas origens distintas: a partir do endotelio hemogênico e a partir de células com propriedades hemangioblásticas independentes do endotélio hemogênico. / Hematopoiesis has been well described in murine models in recent decades, however, studies demonstrating the mechanisms of hematopoiesis in humans are still scarce. The first human embryonic stem cells line (hESCs) derived in 1998, has generated new perspectives about the study of hematopoiesis as in attempting to mimic what naturally occurs during embryonic development, as for clinical application of hematopoietic cells obtained from the differentiation of these cells. However, although numerous studies have shown the production of hematopoietic cells derived from hESCs, the protocols have generated varying quantities of cells with low efficiency and functional properties of primitive stem cells. Thus, this study sought to establish our own model for hESC-H1 differentiation in hematopoietic progenitor cells so that they could be better characterized and obtained more efficiently. For this way, we developed a differentiation system based on co-culture of hESC-H1 line with inactivated mouse embryonic fibroblasts (MEFs) in differentiation medium supplemented with fetal calf serum (FCS) and cytokines and hematopoietic growth factors in low concentrations. As a result, the development of this study allowed the establishment of a method for generation of mixed population of cells enriched in hematopoietic progenitor cells positive for the marker CD45, which proved to be co-expressed with other hematopoietic markers (CD31, CD43, CD71 and CD38), and mature hematopoietic cells positive for myeloid-specific markers (235a, CD14, CD15, CD16) and morphological characteristics typical. It was shown that these cells expressed genes related to the hematopoietic system (CD45, CD31, runx1, TAL1, LMO2, prom1, CD34 and NOTCH1), and had clonogenic potential in vitro of 1/574 plated cells. In addition, we corroborate the findings that hematopoietic cells have two distinct origins: they can arise as from an hemogenic endothelium as from cells with hemangioblastic properties by an hemogenic endothelium-independent way.

O saco vitelino é o único anexo embrionário presente em todas as espécies dos embriões vertebrados, répteis, aves e mamíferos. Em mamíferos domésticos o saco vitelino é inicialmente grande, pois nestas espécies ele é transitório. Após a implantação, surge no mesênquima lateral à notocorda agrupamentos de células, denominados ilhotas sanguíneas, que representam os progenitores dos sistemas vascular e hematopoético: os hemangioblastos. Os hemangioblastos centrais das ilhas sanguíneas formam as primeiras células-tronco hematopoéticas, enquanto os hemangioblastos periféricos se diferenciam em angioblastos, os precursores dos vasos sanguíneos. O desenvolvimento inicial da atividade hematopoética no saco vitelino conduz a hipótese de que esse tecido é o local primário de desenvolvimento hematopoético e que as células-tronco derivadas dele semeiam os outros sítios intraembriônicos. Foi possível observar nas análises microscópicas que realmente existe uma relação entre ambas linhagens. Nas análises de expressão gênica, alguns genes expressos pelo hemangioblasto apresentaram alta expressão nas análises D+0 e outros genes também específicos do hemangioblasto, porém em estágios secundários de diferenciação como os encontrados na região aórtica, a nível de endotélio hemogênico apresentaram altos níveis de expressão após 3 dias em cultivo. Concluímos portanto, que o saco vitelino por ser o local primário de formação das células sanguíneas e endoteliais nos estágios iniciais da embriogênese, por serem primitivas e, portanto não expressarem marcadores de células maduras na sua superfície, tornam estas células uma importante fonte de células-tronco relevante para a Terapia Celular para hemofilia e muitas outras doenças humanas. / The yolk sac is the single attachment embryo present in all species of vertebrate embryos, reptiles, birds and mammals. In domestic mammals the yolk sac is initially large, since these species it is transient. After implantation, appears in the lateral mesenchyme to the notochord cell clusters, called \"blood islands\" that represent the progenitors of vascular and hematopoietic systems: the hemangioblasts. The central islands hemangioblasts form the first blood hematopoietic stem cells, while peripheral hemangioblasts, the angioblastic differentiate into the precursors of blood vessels. The initial development of the yolk sac hematopoietic activity leads to the hypothesis that this tissue is the primary site of development and that hematopoietic stem cells derived from them sow other intraembryos sites. It was observed in the microscopic analysis that there is indeed a relationship between the two lineages. In the analysis of gene expression, some genes expressed by hemangioblasts showed high expression in D+0 and other specific genes also hemangioblasts, but in secondary stages of differentiation as found in the aortic region, the level of hemogenic endothelium showed high levels of expression after 3 days in culture. We therefore conclude that the yolk sac to be the primary site of formation of blood and endothelial cells in the early stages of embryogenesis, for its cells be primitive and therefore do not express markers of mature cells on the surface, these cells become an important source of cells relevant to stem cell therapy for hemophilia and many other human diseases.

Introduction: Vascular injury is the crucial initiating event in atherosclerosis and is universal following percutaneous coronary intervention. The cellular response to this injury largely determines vessel outcome. Endothelial progenitor cells (EPCs) and their progeny, late outgrowth endothelial cells (EOCs) are thought to play an important role in this process and characterising this role would be valuable in better understanding vascular injury and repair. Methods: The radial artery in the context of transradial cardiac catheterisation was examined as a model of vascular injury with characterisation of structural injury, longitudinal function and EPC populations. To examine the role of late outgrowth endothelial cells a method for GMP-compliant cell culture and labelling with F18Fluorodeoxyglucose was developed with a view to conducting a cell-tracking study of human administration. Results: Radial artery function was reduced following transradial cardiac catheterisation with recovery over a period of three months. There was no correlation between recovery of arterial function and EPC populations as defined by conventional surface markers. A research grade protocol for EOC culture was successfully translated to a GMP-compliant process producing a viable, phenotypically homogeneous EOC product. Cells were successfully labelled with F18Fluorodeoxyglucose and whilst proliferation was reduced, acute viability and function were not compromised. Conclusion: The radial artery in the context of transradial cardiac catheterisation is a useful model of vascular injury and repair although recovery of vascular function does not appear to be influenced by EPC populations. GMP-compliant culture and labelling of EOCs is feasible and will allow examination of the physiology of these cells in vivo in man.