The skin provides protection and maintains homeostasis, making it essential for survival. Additionally, skin has the impressive ability to grow, as observed in children as they grow into adults. However, skin functions are compromised in large skin defects, a serious problem that can be fatal. The gold standard treatment is to use an autologous skin graft; however, due to donor site morbidity and limited availability, when full-thickness defects surpass 2% total body surface area (TBSA), skin substitutes are preferred. Unfortunately, current skin substitutes on the market: are slow to revascularize (2+ weeks), have low graft survival rates (<50% take), and lead to significant scarring and contracture. Fortunately, a promising solution is to prevascularize engineered skin substitutes in vitro, which has been shown to facilitate rapid tissue integration upon grafting by providing an intact vascular network that readily connects to the host’s circulation. However, current approaches for prevascularizing tissue constructs require long in vitro culture times or implement low extracellular matrix (ECM) density tissue constructs – both which are problematic in a clinical setting. To address this, we implemented a novel multitissue interface culture model to define the design parameters that were essential for rapid vascularization of soft tissue constructs in vitro. Here, we identified endothelial colony forming cell (ECFC) density and maintenance of cell-matrix tensional forces as important factors for rapid in vitro tissue vascularization (18% vessel volume percentage after 3 days of culture). We then applied these parameters to achieve rapid in vitro vascularization of dense, oligomer tissue constructs (12, 20, and 40 mg/mL). We demonstrated, for the first time, rapid in vitro vascularization at 3 days within dense matrices (ECM concentration > 10 mg/mL). Lastly, a rat full-thickness excisional wound model was developed to determine the acellular densified oligomer’s (20 and 40 mg/mL) ability to resist wound contraction and facilitate a wound healing response (recellularization and vascularization) when grafted into wounds. Future work will implement the vascularized, dense tissue constructs into the developed animal model to assess the vascularized graft’s efficacy on treating wounds to reduce scarring and contracture outcomes.

Large doses of recombinant growth factors formulated in solution form directly injected into the body is usual clinical practice in treating second-degree scald injuries, with promising results, but this approach creates side effects; furthermore, it may not allow appropriate levels of the factor to be sensed by the target injured tissue/organ in the specific time frame, owing to complications arising from regeneration. In this research, two delivery methods (infusion pumping and local topical application) were applied to deliver recombinant human erythropoietin (rHuEPO) for skin regeneration. First, rHuEPO was given in deep second-degree scald injury sites in mice by infusion pump. Vascularization was remarkably higher in the rHuEPO pumping group than in controls. Second, local topical application of rHuEPO gel was given in deep second-degree scald injury sites in rats. Histological analysis showed that epithelialization rate was significantly higher in the rHuEPO gel-treated group than in controls. Immunohistochemical studies showed that the rHuEPO gel-treated group showed remarkably higher expression of skin regeneration makers than the control group. An accurate method for visualization and quantification of blood vessel networks in target areas has still not been developed up to this point, because of technical difficulties in detecting such thin blood vessels. A method which utilizes a series of steps to enhance the image, removes noise from image background, and tracks the vessels edges for vessel segmentation and quantification has been used in this study. Using image analysis methods, we were able to detect the microvascular networks of newly formed blood vessels (less than 500 μm thickness), which participate in the healing process, providing not only nutrition and oxygen to grow tissues but also necessary growth factors to grow tissue cells for complete skin regeneration. The rHuEPO-treated group showed higher expression of stem cell markers (CD 31, CD 90, CD 71, and nestin), which actively contribute to in-wound-healing processes for new hair follicle generation as well as skin regeneration. Collectively, both rHuEPO group pumping into the systemic circulation system, and injection into the local injury area, prompted mice and rats to form new blood vessel networks in scald injury sites, which significantly participate in the scald healing process. These results may lead to the development of novel treatments for scald wounds.

<p>Tissue engineering of biomimetic skeletal muscle may lead to development of new therapies for myogenic repair and generation of improved in vitro models for studies of muscle function, regeneration, and disease. For the optimal therapeutic and in vitro results, engineered muscle should recreate the force-generating and regenerative capacities of native muscle, enabled respectively by its two main cellular constituents, the mature myofibers and satellite cells (SCs). Still, after 20 years of research, engineered muscle tissues fall short of mimicking contractile function and self-repair capacity of native skeletal muscle. To overcome this limitation, we set the thesis goals to: 1) generate a highly functional, self-regenerative engineered skeletal muscle and 2) explore mechanisms governing its formation and regeneration in vitro and survival and vascularization in vivo.</p><p>By studying myogenic progenitors isolated from neonatal rats, we first discovered advantages of using an adherent cell fraction for engineering of skeletal muscles with robust structure and function and the formation of a SC pool. Specifically, when synergized with dynamic culture conditions, the use of adherent cells yielded muscle constructs capable of replicating the contractile output of native neonatal muscle, generating >40 mN/mm2 of specific force. Moreover, tissue structure and cellular heterogeneity of engineered muscle constructs closely resembled those of native muscle, consisting of aligned, striated myofibers embedded in a matrix of basal lamina proteins and SCs that resided in native-like niches. Importantly, we identified rapid formation of myofibers early during engineered muscle culture as a critical condition leading to SC homing and conversion to a quiescent, non-proliferative state. The SCs retained natural regenerative capacity and activated, proliferated, and differentiated to rebuild damaged myofibers and recover contractile function within 10 days after the muscle was injured by cardiotoxin (CTX). The resulting regenerative response was directly dependent on the abundance of SCs in the engineered muscle that we varied by expanding starting cell population under different levels of basic fibroblast growth factor (bFGF), an inhibitor of myogenic differentiation. Using a dorsal skinfold window chamber model in nude mice, we further demonstrated that within 2 weeks after implantation, initially avascular engineered muscle underwent robust vascularization and perfusion and exhibited improved structure and contractile function beyond what was achievable in vitro. </p><p>To enhance translational value of our approach, we transitioned to use of adult rat myogenic cells, but found that despite similar function to that of neonatal constructs, adult-derived muscle lacked regenerative capacity. Using a novel platform for live monitoring of calcium transients during construct culture, we rapidly screened for potential enhancers of regeneration to establish that many known pro-regenerative soluble factors were ineffective in stimulating in vitro engineered muscle recovery from CTX injury. This led us to introduce bone marrow-derived macrophages (BMDMs), an established non-myogenic contributor to muscle repair, to the adult-derived constructs and to demonstrate remarkable recovery of force generation (>80%) and muscle mass (>70%) following CTX injury. Mechanistically, while similar patterns of early SC activation and proliferation upon injury were observed in engineered muscles with and without BMDMs, a significant decrease in injury-induced apoptosis occurred only in the presence of BMDMs. The importance of preventing apoptosis was further demonstrated by showing that application of caspase inhibitor (Q-VD-OPh) yielded myofiber regrowth and functional recovery post-injury. Gene expression analysis suggested muscle-secreted tumor necrosis factor-α (TNFα) as a potential inducer of apoptosis as common for muscle degeneration in diseases and aging in vivo. Finally, we showed that BMDM incorporation in engineered muscle enhanced its growth, angiogenesis, and function following implantation in the dorsal window chambers in nude mice.</p><p>In summary, this thesis describes novel strategies to engineer highly contractile and regenerative skeletal muscle tissues starting from neonatal or adult rat myogenic cells. We find that age-dependent differences of myogenic cells distinctly affect the self-repair capacity but not contractile function of engineered muscle. Adult, but not neonatal, myogenic progenitors appear to require co-culture with other cells, such as bone marrow-derived macrophages, to allow robust muscle regeneration in vitro and rapid vascularization in vivo. Regarding the established roles of immune system cells in the repair of various muscle and non-muscle tissues, we expect that our work will stimulate the future applications of immune cells as pro-regenerative or anti-inflammatory constituents of engineered tissue grafts. Furthermore, we expect that rodent studies in this thesis will inspire successful engineering of biomimetic human muscle tissues for use in regenerative therapy and drug discovery applications.</p> / Dissertation

L’artériopathie oblitérante des membres inférieurs conduit progressivement au rétrécissement des artères qui assurent la vascularisation des membres inférieurs. Il en résulte une ischémie des tissus irrigués par ces artères et à terme, en cas d’occlusion artérielle, une ischémie critique conduisant à une amputation du membre. De nouvelles stratégies de thérapie cellulaire basées sur l’injection de cellules progénitrices capables d’induire une angiogenèse thérapeutique se sont développées ces dernières années. Cependant le faible taux d’incorporation des cellules transplantées dans le tissu ischémique limite le développement de ces nouvelles approches. Dans ce contexte, mon travail de thèse a consisté à étudier deux approches thérapeutiques distinctes pouvant améliorer les thérapies pro-angiogènes. La première étude porte sur le fucoïdane, polysaccharide sulfaté d’origine naturelle, antithrombotique favorisant la formation de nouveaux vaisseaux sanguins dans le modèle murin d’ischémie du membre inférieur. Nous avons montré qu’il induisait le recrutement de monocytes en améliorant leur adhésion à l’endothélium activé en condition dynamique, ainsi que leur adhésion à la matrice et leur transmigration in vitro. Cette action est médiée par l’activation des voies de signalisation ERK et p38 et la sécrétion de métalloprotéinases 9. De plus, le fucoïdane entraine une polarisation des macrophages de type pro-angiogènes in vitro. Il augmente leur recrutement dans le muscle ischémié permettant de réduire ainsi la phase inflammatoire post-ischémique, la nécrose et de favoriser le processus de cicatrisation. La deuxième étude porte sur le rôle des neuropilines (NRP), co-récepteurs du VEGF (facteur de croissance pro-angiogène) exprimés à la surface des ECFC, afin de comprendre leur implication au niveau moléculaire dans le mécanisme d’action pro-angiogène des ECFCs et optimiser l’efficacité de la thérapie cellulaire. A l’aide du système d’extinction par ARN interférent, nous avons découvert un mécanisme de compensation jamais étudié auparavant puisque l’inhibition de NRP1 entraine une augmentation de celle de NRP2 et une diminution de la prolifération et de la migration des ECFCs. En revanche, l’extinction de NRP2 n’a pas d’effet sur l’expression de NRP1, mais induit une augmentation de l’adhésion des ECFCs à la matrice extracellulaire associée à une augmentation de la phosphorylation des ERK1/2. / Vascular diseases such as Peripheral Arterial Disease may evolve towards critical limb ischemia, requiring revascularization or amputation. New strategies of cell therapy based on the injection of progenitor cells able to induce therapeutic angiogenesis have been recently developed. However the low level of incorporation of transplanted cells in the ischemic tissue limits the development of these new approaches. In this context, my thesis was to study two different therapeutic approaches that can improve the pro-angiogenic therapies. The first focuses on fucoidan, a marine sulphated polysaccharide with antithrombotic properties. We have previously shown promising angiogenic properties of fucoidan in vivo. We found that fucoidan increases monocyte recruitment and improves their adhesion to activated endothelium under dynamic condition. It also increases in vitro transmigration. This action is mediated by the activation of ERK and p38 signaling pathways and metalloproteinase 9 secretion. Further, fucoidan can lead macrophage polarization to the pro-angiogenic type in vitro. It increases macrophage recruitment in ischemic muscle that could reduce post-ischemic inflammatory phase and necrosis leading to healing process. The second study focuses on the role of neuropilin (NRP), co-receptors of VEGF (pro-angiogenic growth factor). The aim of this part was to understand their involvement in the pro-angiogenic properties of ECFC at molecular level and optimize the efficiency of cell therapy. Using siRNA, we found a compensation mechanism never studied before. The NRP1 inhibition leads to an increase in the NRP2 expression and a decrease of ECFC proliferation and migration. The NRP2 silencing has no impact on NRP1, but induces ECFC adhesion to the extracellular matrix correlated with an increased level of ERK1 / 2 phosphorylation.

L’artériopathie oblitérante des membres inférieurs conduit progressivement au rétrécissement des artères qui assurent la vascularisation des membres inférieurs. Il en résulte une ischémie des tissus irrigués par ces artères et à terme, en cas d’occlusion artérielle, une ischémie critique conduisant à une amputation du membre. De nouvelles stratégies de thérapie cellulaire basées sur l’injection de cellules progénitrices capables d’induire une angiogenèse thérapeutique se sont développées ces dernières années. Cependant le faible taux d’incorporation des cellules transplantées dans le tissu ischémique limite le développement de ces nouvelles approches. Dans ce contexte, mon travail de thèse a consisté à étudier deux approches thérapeutiques distinctes pouvant améliorer les thérapies pro-angiogènes. La première étude porte sur le fucoïdane, polysaccharide sulfaté d’origine naturelle, antithrombotique favorisant la formation de nouveaux vaisseaux sanguins dans le modèle murin d’ischémie du membre inférieur. Nous avons montré qu’il induisait le recrutement de monocytes en améliorant leur adhésion à l’endothélium activé en condition dynamique, ainsi que leur adhésion à la matrice et leur transmigration in vitro. Cette action est médiée par l’activation des voies de signalisation ERK et p38 et la sécrétion de métalloprotéinases 9. De plus, le fucoïdane entraine une polarisation des macrophages de type pro-angiogènes in vitro. Il augmente leur recrutement dans le muscle ischémié permettant de réduire ainsi la phase inflammatoire post-ischémique, la nécrose et de favoriser le processus de cicatrisation. La deuxième étude porte sur le rôle des neuropilines (NRP), co-récepteurs du VEGF (facteur de croissance pro-angiogène) exprimés à la surface des ECFC, afin de comprendre leur implication au niveau moléculaire dans le mécanisme d’action pro-angiogène des ECFCs et optimiser l’efficacité de la thérapie cellulaire. A l’aide du système d’extinction par ARN interférent, nous avons découvert un mécanisme de compensation jamais étudié auparavant puisque l’inhibition de NRP1 entraine une augmentation de celle de NRP2 et une diminution de la prolifération et de la migration des ECFCs. En revanche, l’extinction de NRP2 n’a pas d’effet sur l’expression de NRP1, mais induit une augmentation de l’adhésion des ECFCs à la matrice extracellulaire associée à une augmentation de la phosphorylation des ERK1/2. / Vascular diseases such as Peripheral Arterial Disease may evolve towards critical limb ischemia, requiring revascularization or amputation. New strategies of cell therapy based on the injection of progenitor cells able to induce therapeutic angiogenesis have been recently developed. However the low level of incorporation of transplanted cells in the ischemic tissue limits the development of these new approaches. In this context, my thesis was to study two different therapeutic approaches that can improve the pro-angiogenic therapies. The first focuses on fucoidan, a marine sulphated polysaccharide with antithrombotic properties. We have previously shown promising angiogenic properties of fucoidan in vivo. We found that fucoidan increases monocyte recruitment and improves their adhesion to activated endothelium under dynamic condition. It also increases in vitro transmigration. This action is mediated by the activation of ERK and p38 signaling pathways and metalloproteinase 9 secretion. Further, fucoidan can lead macrophage polarization to the pro-angiogenic type in vitro. It increases macrophage recruitment in ischemic muscle that could reduce post-ischemic inflammatory phase and necrosis leading to healing process. The second study focuses on the role of neuropilin (NRP), co-receptors of VEGF (pro-angiogenic growth factor). The aim of this part was to understand their involvement in the pro-angiogenic properties of ECFC at molecular level and optimize the efficiency of cell therapy. Using siRNA, we found a compensation mechanism never studied before. The NRP1 inhibition leads to an increase in the NRP2 expression and a decrease of ECFC proliferation and migration. The NRP2 silencing has no impact on NRP1, but induces ECFC adhesion to the extracellular matrix correlated with an increased level of ERK1 / 2 phosphorylation.

A eletroquimioterapia (EQT) é uma modalidade de tratamento recente que se baseia na associação de quimioterápicos potencializados pela eletroporação. Possui indicação para neoplasias sólidas de origens histológicas distintas, apresentando baixa morbidade e elevada eficiência. A ação da EQT ocorre em múltiplos sítios, tanto envolvendo a quebra da molécula de DNA, quanto exercendo efeito sobre a vasculatura tumoral. No presente estudo buscou-se maior compreensão dos eventos vasculares, avaliando-se qualitativamente e quantitativamente, com auxílio de marcação imunológica, com fator VIII e VEGF-A, a vascularização do tumor de Ehrlich implantado na forma sólida em camundongos, não tratados e submetidos à EQT com bleomicina, após sete dias de tratamento. No intuito de melhor elucidar os fenômenos vasculares, também foi investigado o efeito do resveratrol associado à EQT. O resveratrol, presente em vegetais como as uvas, possui efeitos de inibição do HIF1-&#945;, reconhecida proteina que estimula a angiogênese em condições de hipoxia tumoral. Os animais submetidos à quimioterapia com bleomicina não apresentaram redução de volume tumoral, ao contrário dos que sofreram EQT com o mesmo fármaco. Evidenciou-se maior densidade microvascular tumoral em animais tratados com quimioterapia, quando comparados aos não tratados e aos submetidos à EQT. O tratamento com resveratrol diminuiu a expressão de VEGF-A e obteve efeito mais pronunciado quando associado à EQT com bleomicina. Através dos fenômenos pesquisados pôde-se evidenciar que a EQT com bleomicina foi efetiva na redução do volume do tumor de Ehrlich e que houve redução da atividade proliferativa assim como da densidade microvascular tumoral. Também observou-se que o resveratrol, ainda mais quando associado à EQT com bleomicina, reduz a proliferação tumoral e a expressão de VEGF-A / The electrochemotherapy (EQT) is a new treatment modality based on the association of chemotherapy potentiated by electroporation. Has indication for solid neoplasms of histological distinct origins, presenting low morbidity and high efficiency. The action of the EQT occurs in multiple sites, both involving the breakage of the DNA molecule, as having an effect on the tumor vasculature. The present study aimed at better understanding of vascular events, evaluating qualitatively and quantitatively, using immune labeling, with factor VIII and VEGF-A, the vascularization of the Ehrlich tumor implanted in solid form in mice, untreated and submitted to EQT with bleomycin, after seven days of treatment. In order to better elucidate the vascular phenomena, was also investigated the effect of resveratrol associated with EQT. The resveratrol present in plants such as grapes, has inhibitory effects of HIF1-&#945;, a protein that is recognized to stimulates angiogenesis in tumor hypoxia. The animals submitted to chemotherapy with bleomycin showed no reduction of tumor volume, unlike those who suffered EQT with the same drug. It was evidenced increased microvessel density tumor in animals treated with chemotherapy, when compared to untreated and those submitted to EQT. The treatment with resveratrol decreased the expression of VEGF-A and obtained effect was more pronounced when associated to the EQT with bleomycin. This research can prove that the EQT with bleomycin was effective in reducing the volume of Ehrlich tumor and that there was a reduction of proliferative activity as well as of microvascular density tumor. Also it was observed that the resveratrol, even more when associated with EQT with bleomycin, reduces the tumor proliferation and the expression of VEGF-A

Sabe-se que o conhecimento da anatomia do sistema estomatognático é de fundamental importância para a obtenção do diagnóstico preciso e execussão do tratamento adequado. Porém, as informações encontradas em literatura odontológica veterinária são esparsas. Conseqüentemente, necessita-se buscar esse tipo de informação literatura anatômica geral veterinária, dificultando a consulta rápida e objetiva. Considera-se, portanto, de fundamental importância preparar uma obra inédita sobre anatomia oral de cães e gatos para a prática da odontologia veterinária amparada na ampla literatura anatômica odontológica humana. Propôs-se então, neste trabalho, estudar a vascularização e a inervação do sistema estomatognático de cães e gatos, ampliando e enriquecendo o conhecimento destes sistemas, visando ilustrar e correlacionar suas estruturas, aplicando este conhecimento à prática clínico-cirúrgica, enfatizando os pontos de maior relevância para o desenvolvimento da especialidade. Foi realizada a comparação do comportamento dos vasos sangüíneos e nervos entre os três tipos de conformação craniana de cães, bem como de gatos. Os resultados obtidos demonstram haver pequenas variações, não cirurgicamente significativas, quanto à distribuição, comportamento e sinuosidade dos vasos sanguíneos e nervos entre os três diferentes tipos de conformação cranial de cães. Diferenças significativas foram observadas apenas na inervação da região de lábios, entre cães braquicefálicos quando comparados a dolicocefálicos, mesocefálicos e gatos. / The knowledge of the anatomy of the stomatognathic system is important to accomplish any diagnosis and treatment appropriately. For that is necessary the knowledge of the tegumentar, bony, muscular, vascular, nervous and dental components. However, any specific and exclusive literature about stomatognathic system of dogs and cats are not existent. Consequently, it is needed to search this information in the books of veterinary anatomy, hindering a fast and objective consultation. It is therefore important to accomplish a work on oral anatomy of dogs and cats for the veterinary dentistry based on the oral human anatomical literature. Therefore, the aim is to detail the vascularization and the enervation of the stomatognathic system of dogs and cats, enlarging and enriching the knowledge of these systems, describing and illustrating these structures, and correlating this knowledge with the clinical-surgical procedures, emphasizing the points of larger relevance for the development of the specialty. The illustrations were made with heads of dogs and cats prepared with injection of latex-neoprene in the sanguine vascular system and intra-muscular injection of formalin 10% solution and had the structures identified according to the surgical necessities.

O estresse térmico em touros é um fator muito importante, pois afeta negativamente o comportamento reprodutivo e diminui a eficiência reprodutiva. Uma das causas deste efeito adverso é a ineficiência da termorregulação testicular, que conduz ao aumento do metabolismo celular, causando estresse oxidativo e apoptose das células germinativas, o que caracteriza a degeneração testicular, que pode levar à infertilidade, ou mesmo à esterilidade do animal. A degeneração testicular pode ser diagnosticada pela palpação do órgão, aferição do perímetro escrotal e pelo espermograma. No entanto, outras ferramentas podem ser empregadas para auxiliar no diagnóstico desta alteração, entre elas estão a termografia e a ultrassonografia. Desta forma, neste trabalho avaliou-se a eficiência do termógrafo e do ultrassom como instrumentos auxiliares para o diagnóstico da degeneração testicular em bovinos. Para o presente estudo utilizaram-se 16 touros da raça Nelore separados em dois grupos experimentais: Controle: animais sem indução à degeneração testicular (CON, n = 08); e Degeneração: animais induzidos à degeneração testicular (INS, n = 08) por meio de bolsas insuladoras mantidas por 96 horas. Os animais foram submetidos semanalmente a avaliações das características clínicas e seminais, realizando-se as análises duas semanas antes da insulação testicular (S-2 e S-1), no dia da retirada da bolsa (D0) e durante quatro semanas após a retirada da mesma (S+1 a S+4). Foram avaliadas frequência cardíaca (FC), frequência respiratória (FR) e temperatura retal (TR); avaliações testiculares: perímetro escrotal (PE), consistência testicular (CT), temperatura média da superfície escrotal (TMSE), ecotextura (ETT), ecogenicidade (EGT) e vascularização do parênquima testicular (EVT), vascularização (EVP) e índice de resistência vascular (RIP) do plexo pampiniforme. O sêmen dos touros foi colhido e avaliado considerando motilidade (MT), vigor (VG), morfologia (defeitos maiores, menores e totais), integridade das membranas plasmática e acrossomal e potencial de membrana mitocondrial (PIAIA). Para a análise estatística foi utilizado o Statistical Analysis Software (SAS 9.3). Os dados das avaliações realizadas em S-2 e S-1 foram submetidos ao procedimento MIXED considerando dois grupos (CON e INS). Os dados das avaliações realizadas após a insulação foram submetidos ao procedimento MIXED e adicionando o fator tempo por meio do comando REPEATED. O nível de significância considerado foi de P&le;0,05, sendo considerada tendência quando este ficou entre 0,051 e 0,1. Foram realizadas correlações de Pearson, utilizando-se o programa StatView (SAS, 1999). Os grupos de touros foram semelhantes nas semanas pré-insulação. Nas semanas pós-insulação, notou-se interação entre tempo e tratamento para FR (p=0,04), CT (p=0,0003) e RIP (p=0,03) e tendência para EVT (p=0,08). Foram observados maiores valores para TMSE, ETT e EVT para INS do que para CON, mas menores valores de PE, CT, EVP e RIP para INS do que para CON. Além disso, encontrou-se interação entre tempo e tratamento para MT (p=0,01), defeitos morfológicos (p&lt;0,001) e alto potencial mitocondrial (AP, p=0,01) e tendência para PIAIA (p=0,07). O grupo INS apresentou queda na MT, PIAIA e AP, associado com aumento nos defeitos morfológicos. Foram verificadas correlações entre as características ambientais (temperatura ambiente e umidade relativa do ar) e os termogramas. Os termogramas apresentaram correlações com parâmetros vitais e achados testiculares. A hemodinâmica apresentou correlações mais fracas com outras características testiculares. Concluiu-se que o estresse térmico testicular provoca um quadro de degeneração testicular, caracterizado por redução na CT e na MT, PIAIA, PI e AP, além de aumento de defeitos morfológicos espermáticos. Este quadro é acompanhado pelo aumento da TMSE somente no dia da retirada das bolsas, provoca heterogeneidade do parênquima testicular, aumenta a vascularização testicular, reduz a vascularização do plexo pampiniforme e o RI dos vasos do plexo pampiniforme. Desta forma, a termografia e a ultrassonografia testiculares contribuem para o diagnóstico da degeneração testicular em touros. / Heat stress in bulls is a very important factor because it adversely affects the reproductive behavior and reduces the reproductive efficiency. One of the causes of this adverse effect is the inefficiency of testicular thermoregulation, which leads to increased cellular metabolism, causing oxidative stress and apoptosis of germ cells, which characterizes testicular degeneration, which can lead to infertility or even to animal sterility. Testicular degeneration can be diagnosed by palpation of the testicles, scrotal perimeter and sperm analyses. However, other tools can be used to aid in the diagnosis of this alteration, such as thermography and ultrasonography. Thus, this study evaluated the effectiveness of thermography and ultrasound as auxiliary tools in the diagnosis of testicular degeneration in cattle. For the present study, 16 Nelore bulls were divided in two experimental groups: Control: animals without induction to testicular degeneration (CON, n = 08); and Degeneration: animals induced to testicular degeneration (INS, n = 08) through insulation bags, maintained for 96 hours. Animals underwent weekly evaluations of clinical and seminal characteristics, performed assessments two weeks prior to testicular insulation (S-2 and S-1), on the day of the removal of the bag (D0) and during four week after removal of the bag (S+1 to S+4). The following were evaluated: heart rate (HR), respiratory rate (RR) and rectal temperature (RT); testicular evaluations: scrotal circumference (SC), testicular consistency (TC), mean temperature of the scrotal surface (MTSS), echotexture (ETT), echogenicity (EGT), vascularization of testicular parenchyma (VTP), vascularization of the pampiniform plexus (VPP) and resistance index (RI). The semen of the bulls was collected and evaluated considering motility (MT), vigor (VG), morphology (major, minor and total defects), plasma and acrosomal membrane integrity and mitochondrial membrane potential (PIAIA). Statistical analysis was performed using Statistical Analysis Software (SAS 9.3). Data from the S-2 and S-1 evaluations were submitted to the MIXED procedure considering two groups (CON and INS). Data from the evaluations performed after the insulation were submitted to the MIXED procedure adding the time factor through the REPEATED command. The significance level considered was P&le;0,05, being considered a trend when it was between 0,051 and 0,1. Pearson correlations were performed using the StatView program (SAS, 1999). The groups were similar in the pre-insulation period. In the post-insulation weeks, it was noted the interaction between time and treatment for RR (p = 0.04), TC (p = 0.0003) and RI (p = 0.03) and a tendency to VTP (p = 0, 08). Greater values were observed for MTSS, ETT and VTP for INS than for CON, but lower values of SC, TC, VPP and RI for INS than for CON. In addition, there was interaction between time and treatment for MT (p = 0.01), morphological defects (p &lt;0.001) and high mitochondrial potential (HMP, p = 0.01) and tendency for PIAIA (p = 0, 07). The INS group presented decrease in MT, PIAIA and HMP, associated with increase in morphological defects. Correlations were found between the environmental characteristics (temperature and relative humidity) and thermograms. The thermograms presented correlations with vital parameters and testicular findings. Hemodynamics showed weaker correlations with other testicular characteristics. In conclusion, testicular heat stress causes a testicular degeneration, characterized by reduction in TC and MT, PIAIA, PI and HMP, in addition to an increase in spermatic morphological defects. This situation, accompanied by the increase in MTSS only on the day of the removal of the bags, causes testicular parenchyma heterogeneity, increases testicular vascularization, reduces pampiniform plexus vascularization and RI of pampiniform plexus vessels. Thus, testicular thermography and ultrasonography contribute to the diagnosis of testicular degeneration in bulls.

O sistema linfoide é composto de órgãos linfoides primários e secundários. O timo é um órgão linfoide primário responsável pela maturação, diferenciação e seleção da linhagem linfocitária do tipo T que é responsavel pela imunidade celular do individuo. Para cumprir estas funções, o timo possui uma disposição peculiar das suas células epiteliais morfologicamente distintas e de suas estruturas vasculares. Seus vasos sanguíneos possuem um papel na oxigenação tecidual e no processo de migração das células precursoras de linfócitos T para o interior do parênquima tímico e por isso apresentam uma arquitetura típica caracterizada por vasos de grande calibre, localizados na junção cortico-medular e uma fina rede de ramos e anastomoses que se estendem para o córtex. Este processo de estruturação e arquitetura vascular ainda possui sua base molecular desconhecida, assim como os mecanismos que provocam a involução do órgão. O VEGF é um fator angiogênico que atua na formação vascular e na modulação de funções relacionadas à vascularização, sendo um importante marcador da angiogênese. A fim de se melhor compreender o comportamento vascular na formação e involução tímica, propôs-se avaliar a expressão gênica e proteica deste fator durante fases de desenvolvimento e involução do órgão, além da quantificação da vascularização do timo pela técnica estereológica, análise do parênquima tímico pela técnica de microscopia eletrônica de varredura e análise dos tipos celulares presentes em cada estágio etário. Para tal utilizou-se amostras de timo de gato em quatro estágios de desenvolvimento fetal (35, 45, 55, 65), e dois estágios pós-natal (6 meses e 1 ano) para a realização da imuno-histoquímica, PCR em tempo real e MEV,e para a técnica estereológica 2 estágios pós-natal (6 meses e 1 ano). Na microscopia eletrônica de varredura foram observados os timócitos de diferentes tamanhos, em estágios de maturação distintos. As proteinas do VEGF-A e dos receptores Fit-1 e KDR foram identificadas no timo de gatos em todas as fases do desenvolvimento foram localizadas no citoplasma de células epiteliais e no interior dos corpúsculos tímicos. A expressão do mRNA no período de 1 ano de idade a expressão do mRNA do VEGF e seus receptores tem um aumento significativo, coincidindo com a diminuição do Nvasc e do Nv(vasc) podendo causar um estado de hipóxia no órgão levando a um aumento compensatório de sistema VEGF. A curva de crescimento vascular obedece a um padrão de desenvolvimento e involuçãio do órgão. / The lymphoid system is composed by primary and secondary lymphoid organs. The thymus is a primary lymphoid organ responsible for maturation, differentiation and selection of the lymphoid T cell lineage that is responsible for cellular immunity. To accomplish these functions has a peculiar arrangement with morphologically distinct epithelial cells and vascular structures. The blood vessels have a role in tissue oxygentation and the migration of T cells into the thymic parenchyma, therefore they presents large vessels in cortico-medullary junction and a fine network branches to the cortex. This process has its molecular basis unknown as well as the involution process of the thymus. VEGF is an angiogenic factor that plays a role in the formation and modulation of vascular functions, being an important marker of angiogenesis. We proposed to evaluate the gene and protein of VEGF during the thymus development and involution, stereological quantification and scanning electronic microscopy. Samples of cat´s thymus from 35, 45, 55, 65 days of development and 6 months and 1 year of age. In scanning electronic microscopy different stages maturation thymocytes were observed. Protein expression of VEGF and its receptors were identified in all development stages in epithelial cells, endothelial cells and thymic corpuscles. The VEGF mRNA expression and its receptors in 1 year old animals was significantly increased, coinciding with the decreasing Nvasc and the Nv(vasc) causing a hypoxic condition in the thymus resulting in a compensatory increase of VEGF system. The vascular growth curve follows a pattern of development and involution of the organ.

L’artériopathie oblitérante des membres inférieurs conduit progressivement au rétrécissement des artères qui assurent la vascularisation des membres inférieurs. Il en résulte une ischémie des tissus irrigués par ces artères et à terme, en cas d’occlusion artérielle, une ischémie critique conduisant à une amputation du membre. De nouvelles stratégies de thérapie cellulaire basées sur l’injection de cellules progénitrices capables d’induire une angiogenèse thérapeutique se sont développées ces dernières années. Cependant le faible taux d’incorporation des cellules transplantées dans le tissu ischémique limite le développement de ces nouvelles approches. Dans ce contexte, mon travail de thèse a consisté à étudier deux approches thérapeutiques distinctes pouvant améliorer les thérapies pro-angiogènes. La première étude porte sur le fucoïdane, polysaccharide sulfaté d’origine naturelle, antithrombotique favorisant la formation de nouveaux vaisseaux sanguins dans le modèle murin d’ischémie du membre inférieur. Nous avons montré qu’il induisait le recrutement de monocytes en améliorant leur adhésion à l’endothélium activé en condition dynamique, ainsi que leur adhésion à la matrice et leur transmigration in vitro. Cette action est médiée par l’activation des voies de signalisation ERK et p38 et la sécrétion de métalloprotéinases 9. De plus, le fucoïdane entraine une polarisation des macrophages de type pro-angiogènes in vitro. Il augmente leur recrutement dans le muscle ischémié permettant de réduire ainsi la phase inflammatoire post-ischémique, la nécrose et de favoriser le processus de cicatrisation. La deuxième étude porte sur le rôle des neuropilines (NRP), co-récepteurs du VEGF (facteur de croissance pro-angiogène) exprimés à la surface des ECFC, afin de comprendre leur implication au niveau moléculaire dans le mécanisme d’action pro-angiogène des ECFCs et optimiser l’efficacité de la thérapie cellulaire. A l’aide du système d’extinction par ARN interférent, nous avons découvert un mécanisme de compensation jamais étudié auparavant puisque l’inhibition de NRP1 entraine une augmentation de celle de NRP2 et une diminution de la prolifération et de la migration des ECFCs. En revanche, l’extinction de NRP2 n’a pas d’effet sur l’expression de NRP1, mais induit une augmentation de l’adhésion des ECFCs à la matrice extracellulaire associée à une augmentation de la phosphorylation des ERK1/2. / Vascular diseases such as Peripheral Arterial Disease may evolve towards critical limb ischemia, requiring revascularization or amputation. New strategies of cell therapy based on the injection of progenitor cells able to induce therapeutic angiogenesis have been recently developed. However the low level of incorporation of transplanted cells in the ischemic tissue limits the development of these new approaches. In this context, my thesis was to study two different therapeutic approaches that can improve the pro-angiogenic therapies. The first focuses on fucoidan, a marine sulphated polysaccharide with antithrombotic properties. We have previously shown promising angiogenic properties of fucoidan in vivo. We found that fucoidan increases monocyte recruitment and improves their adhesion to activated endothelium under dynamic condition. It also increases in vitro transmigration. This action is mediated by the activation of ERK and p38 signaling pathways and metalloproteinase 9 secretion. Further, fucoidan can lead macrophage polarization to the pro-angiogenic type in vitro. It increases macrophage recruitment in ischemic muscle that could reduce post-ischemic inflammatory phase and necrosis leading to healing process. The second study focuses on the role of neuropilin (NRP), co-receptors of VEGF (pro-angiogenic growth factor). The aim of this part was to understand their involvement in the pro-angiogenic properties of ECFC at molecular level and optimize the efficiency of cell therapy. Using siRNA, we found a compensation mechanism never studied before. The NRP1 inhibition leads to an increase in the NRP2 expression and a decrease of ECFC proliferation and migration. The NRP2 silencing has no impact on NRP1, but induces ECFC adhesion to the extracellular matrix correlated with an increased level of ERK1 / 2 phosphorylation.