Submitted by Edineia Teixeira (edineia.teixeira@unioeste.br) on 2017-12-18T18:47:09Z No. of bitstreams: 2 Caroline_Covatti2016.pdf: 1272706 bytes, checksum: 2b328da4ea5385205d047b1e31662783 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-12-18T18:47:09Z (GMT). No. of bitstreams: 2 Caroline_Covatti2016.pdf: 1272706 bytes, checksum: 2b328da4ea5385205d047b1e31662783 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-06-15 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Cerebral palsy (CP) is characterized by movement and postural disorders that limit the individual‘s activity, and is attributed to the occurrence of non-progressive disturbances during the development of the fetal or infant brain. Animal models have been used in an attempt to reproduce the lesions and characteristics of CP. However, there is still no intervention capable of reproducing the characteristics of this pathology in the experimental area. Thus, the objective of this study was to verify the effects of a CP model that combines prenatal exposure to lipopolysaccharide (LPS), perinatal anoxia and sensorimotor restriction on the muscle fibers and neuromuscular junctions (NMJs) of the extensor digitorum longus (EDL) of rats. Male Wistar pups were separated into two groups: Control group (CTL - n = 10) - pups of mothers injected with saline during pregnancy and Cerebral Palsy Group (CP - n = 10) - pups of mothers injected with LPS during pregnancy. submitted to perinatal anoxia and sensorimotor restriction. On the day of birth, the offspring of the CP group were placed in a closed chamber with nitrogen flow (100%) to perform the perinatal anoxia. Additionally, from the first postnatal day (P1) to P30, those pups were also submitted to sensorimotor restriction by immobilizing the hind limbs. Motor performance was assessed in both groups during open field tests on P29 and P45. After euthanasia, EDL muscle samples were processed for morphological and morphometric analysis of the muscle fibers and NMJs. Regarding motor performance, the time of locomotion and the number of rearings were significantly lower in the CP group compared to CTL group at 29 days of age (p <0.001 and p <0.01, respectively). At 45 days of age, the time of locomotion of the animals in the CP group was also lower in relation to the CTL group (p <0.05). The total body weight, weight and length of the EDL muscle were 18%, 17% and 15% lower, respectively, in the CP group in relation to the CTL. The animals in the CP group presented hypertrophy of the type IIB fibers. However, regarding the other fibers there was no difference between groups. There were no differences between groups in the number of muscle fiber types I, IIA and IIB. The nuclei/fiber ratio, and the capillary/fiber ratio, were significantly higher in the CP group (21% and 18%, respectively). Regarding the intrafusal fibers, the animals from the CP group presented atrophy in 26% of the cross-sectional area and a reduction of 26% in the muscle spindle area. Intramuscular collagen increased by 34% in the animals from the CP group. The ultrastructural study of the EDL muscle in the CP group showed myofibrillar disruption and Z-line disorganization and dissolution. The NMJs in the CP group presented an increase of 22% in area and 11% in diameter when compared to the CTL group. In conclusion, the CP animal model that uses injections of LPS, perinatal anoxia and sensorimotor restraint produces motor deficits that are also observed in children with CP. / A paralisia cerebral (PC) caracteriza-se por desordens do movimento e da postura, por isso causam limitação na atividade do indivíduo, as quais são atribuídas por distúrbios não progressivos que ocorreram no desenvolvimento cerebral fetal ou infantil. Modelos animais têm sido utilizados na tentativa de reproduzir as lesões e características da PC. Porém, ainda não existe uma intervenção capaz de reproduzir as características desta patologia no âmbito experimental. Assim, o objetivo deste estudo foi verificar os efeitos de um modelo de PC que associa exposição pré-natal ao lipopolissacarídeo (LPS), anóxia perinatal e restrição sensório-motora sobre as fibras musculares e junções neuromusculares (JNMs) do músculo extensor longo dos dedos (EDL) de ratos. Filhotes Wistar machos foram separados em dois grupos: Grupo controle (CTL - n = 10) - filhotes de mães injetadas com solução salina durante a gestação e Grupo Paralisia Cerebral (PC - n = 10) - filhotes de mães injetadas com LPS durante a gestação, submetidos à anóxia perinatal e à restrição sensório-motora. No dia do nascimento, os filhotes do grupo PC foram colocados em câmara fechada com fluxo de nitrogênio (100%) para a realização da anóxia perinatal. A partir do primeiro dia pós-natal (P1) até o P30, esses filhotes também foram submetidos à restrição sensório-motora por imobilização das patas pélvicas. Avaliações do desempenho motor foram realizadas em um campo aberto no P29 e P45 para os dois grupos. Após a eutanásia, amostras do músculo EDL foram processadas para análise morfológica e morfométrica das fibras musculares e JNMs. Quanto à performance motora, o tempo de locomoção e o número de erguidas (rearing) foi significativamente menor no grupo PC em comparação ao CTL aos 29 dias de idade (p< 0,001 e p< 0,01, respectivamente). Aos 45 dias de idade, o tempo de locomoção dos animais do grupo PC também foi menor em relação ao grupo CTL (p < 0,05). O peso corporal, peso e comprimento do músculo EDL foram 18%, 17% e 15% menores, respectivamente, no grupo PC em relação ao CTL. Os animais do grupo PC apresentaram hipertrofia das fibras do tipo IIB, todavia, não houve diferença entre os grupos nas demais fibras. Não teve diferenças entre os grupos estudados quanto ao número de fibras musculares dos tipos I, IIA e IIB. A relação núcleo/fibra, e a relação capilar/fibra, foi significativamente maior no grupo PC (21% e 18%, respectivamente). Quanto às fibras intrafusais, os animais do grupo PC apresentaram atrofia de 26% na área de secção transversal e redução de 26% na área do fuso muscular. O colágeno intramuscular aumentou 34% nos animais do grupo PC. O estudo ultraestrutural do músculo EDL do grupo PC mostrou desarranjo miofibrilar, desorganização e dissolução da linha Z. As JNMs do grupo PC apresentaram aumento de 22% na área e de 11% no diâmetro maior quando comparado ao grupo CTL. Em conclusão, o modelo animal de PC que utiliza injeções de LPS, anóxia perinatal e restrição sensório-motora produz déficits motores que também são observados em crianças com PC.

Made available in DSpace on 2016-03-15T19:40:50Z (GMT). No. of bitstreams: 1 Adelisandra Silva Santos Castelhano.pdf: 492676 bytes, checksum: 6118cc3994884f74daed04e57878c9e4 (MD5) Previous issue date: 2010-01-26 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The Autism Spectrum Disorders (ASD), conditions which affect the brain development, are characterized by impairments in reciprocal social interactions, communicative use of verbal and nonverbal language, and restricted/repetitive behaviors. Despite a wealth of descriptive data obtained from patient histories, imaging techniques, and genetic and molecular studies, the pathogenesis of ASD remains poorly understood. Progress toward understanding the etiology of an acquired neurological disorder, such as ASD, is largely dependent on the degree to which experimental animal models reflect the human condition. Status epilepticus (SE), the condition of ongoing seizures or repetitive seizure activity, is a clinical emergency more common in children than adults, with almost 40-50% of the case occurring in children younger than 2 years of age. Clinical and experimental studies have been showing that despite of immature brain to be more resistant to structural damage when compared with adult brain, it has been demonstrated that neonatal SE may produce learning deficits, memory impairment, and emotional sequels in adulthood, altered GABAergic intracortical and hipocampal circuitries and reduced dopamine levels in the prefrontal cortex. Taking all information together, the aim of the present study was to evaluate a possible social impairment, learning and exploratory deficits after pilocarpine-induced SE in rats of both genders during development. Wistar rats of both gender at PN9 received intraperitoneal injection of pilocarpine (380 mg/kg). Control animals received saline solution instead pilocarpine. Social and exploratory behaviors were assessed between 30-35 postnatal days using paired-exposure paradigm. Learning performance was assessed, at PN90 using skinner Box apparatus, by quantification of the number of sections to acquire bar pressing conditioning. Our results demonstrated that neonatal SE produced social impairment, learning and exploratory deficits. Furthermore, the social impairment was gender-dependent, affecting predominantly male rats. On the other hand, the exploratory behavior was reduced in both genders, however female rats seems to be more affected, since selfgrooming was enhanced in this specific group. Quite interesting, both behaviors are affected by emotionality and environmental context, suggesting that SE was able to affect more severely the emotionality of the female animals. Moreover, it is also important to note that learning deficits were observed in both genders as well. Based on this evidences, we propose that this animal model can be a valuable tool to investigate the neurobiological basis of ASD. / Os Transtornos do Espectro Autista (TEA) são condições que afetam o desenvolvimento cerebral prejudicando a interação social recíproca, a comunicação verbal e não verbal e são acompanhados por comportamentos repetitivos e padrões anormais de interesses e atividades. Apesar da riqueza dos dados obtidos a partir da história dos pacientes, dos exames de neuroimagem, dos estudos genéticos e moleculares, a patogênese do TEA permanece mal compreendida. Modelos animais têm propiciado a ampliação do conhecimento acerca da neurobiologia dos TEA. O Status epilepticus (SE), uma condição aguda caracterizada por convulsões repetitivas ou prolongada, é uma emergência clínica que ocorre mais frequentemente em crianças que em adultos, e em 40-50% dos casos em crianças com idade inferior a dois anos. Estudos clínicos e experimentais indicam que embora o SE produza menos danos estruturais no cérebro imaturo que no cérebro adulto, SE em neonatos leva a prejuízos na aprendizagem, memória, sequelas emocionais na idade adulta, alteração da circuitaria GABAérgica hipocampal e intracortical, e redução dos níveis de dopamina no córtex pré-frontal. Este trabalho teve como objetivo avaliar se o SE em ratos neonatos de ambos os gêneros produz prejuízos na interação social, na cognição e no comportamento exploratório. Ratos wistar de ambos os gêneros com nove dias pós-natal (PN9) receberam injeção intraperitoneal de pilocarpina (380 mg/kg). Animais controles receberam solução salina. Os comportamentos sociais e exploratórios foram avaliados entre PN30-PN35, empregando-se o paradigma de observação por pares. O desempenho cognitivo foi avaliado empregando-se a caixa de Skinner, quantificando-se o número de sessões para aquisição do comportamento de pressão à barra. Os resultados mostraram que o SE no cérebro em desenvolvimento alterou a sociabilidade, a cognição e o comportamento exploratório. Os prejuízos na interação social foram gênero-dependentes, afetando predominantemente os machos. O comportamento exploratório foi reduzido em ambos os gêneros, no entanto, as fêmeas parecem ter sido mais afetadas, desde que paralelamente observou-se aumentou do selfgrooming. Ambos os comportamentos são afetados pela emocionalidade e pelo contexto ambiental, sugerindo que o SE afetou mais severamente a emocionalidade das fêmeas. Os prejuízos cognitivos foram igualmente observados em ambos os gêneros. Baseando-se nestas evidências, sugerimos que este modelo pode ser utilizado para explorar mecanismos neurobiológicos do TEA.

Uma fração considerável de pacientes com doença de Chagas, decorrente da infecção pelo protozoário Trypanosoma cruzi, desenvolve a cardiopatia crônica, que pode levar a morte. A regulação da resposta imune específica contra o parasita é essencial para controlar a atividade efetora excessiva anti-T. cruzi. Na falta dessa regulação, a resposta imune acaba por induzir lesão dos tecidos. Uma vez que, em hipótese, componentes autoimune participam da cardiopatia chagásica, a regulação da resposta poderia ser necessária para controlar a reatividade contra o próprio. Nesta tese, nós avaliamos o envolvimento das células T reguladoras (TREG) em modelo murino de doença crônica de Chagas. Na fase aguda, período em que ocorre uma forte ativação policlonal de linfócitos, observa-se um pequeno aumento no número de células esplênicas CD4+CD25+FoxP3+ TREG, no entanto, um aumento de maior magnitude ocorre nas células efetoras CD4+CD25+FoxP3-. O tratamento de camundongos na fase aguda da infecção com MoAb anti-CD25 (PC61) resulta em discreta redução no número de células TREG, mas essa não afeta os níveis de parasitemia ou patologia do coração na fase aguda. Na fase crônica, os números de células TREG dos animais crônicos retornam aos mesmos níveis dos observados nos animais não infectados, no entanto, o número de esplenócitos CD4+ está discretamente aumentado. O tratamento de camundongos crônicos com PC61 resulta em uma redução no número de células TCD4+CD25+ e uma tendência à redução no número de células CD4+FoxP3+. Mais importante, a resposta imune anti- T. cruzi, carga parasitária sistêmica e patologia cardíaca não foram consistentemente alteradas nos grupos tratados com PC61 em comparação aos tratados com o controle isotípico ou IgG de rato. O fato do tratamento com MoAb PC61 não modificar a patologia do coração de camundongos crônicos pode ser devido a depleção incompleta das células TREG, ou a um pequeno envolvimento das TREG no controle dos mecanismos efetores anti- T. cruzi. / A large fraction of patients with Chagas disease, an illness due to infection by protozoan Trypanosoma cruzi, develops chronic myocardiopathy that often leads to death. Regulation of parasite-specific immune responses is essential to control excessive anti-T. cruzi effector activity that may result in tissue damage. Moreover, because autoimmunity has been hypothesized to contribute to Chagas myocardiopathy, regulation could be also necessary to control anti-self reactivity. In this paper we evaluated the involvement of TREG in a murine model of chronic T. cruzi infection. In the acute phase, a period when there is strong polyclonal lymphocyte activation, a small increase in the number of CD4+CD25+FoxP3+ spleen cells (TREG) cells is observed, albeit of considerably lower magnitude than that of CD4+CD25+FoxP3- effector cells. Treatment of acutely-infected mice with anti-CD25 (PC61) mAb results in discrete reduction of TREG cell numbers, but it does not affect parasitaemia levels or acute phase heart pathology. At the chronic phase, TREG cells numbers return to numbers of non-infected mice, in spite that the total number of CD4+ splenocytes is discretely increased in chronic mice. Treatment of chronic mice with PC61 results in a reduction in TCD4+CD25+ cell numbers, but in a small, non significant reduction in total CD4+FoxP3+ cells. More important, the anti-T. cruzi immune response, systemic parasite load and heart pathology were not consistently altered in PC61-treated chronic mice compared to mice treated with isotypic control antibodies or normal rat IgG. Failure to modify heart pathology in PC61-treated chronic mice could be due to incomplete TREG depletion or to discrete involvement of TREG in control of the anti-T. cruzi effector mechanisms.

A descoberta de aminopeptidase regulada por insulina (IRAP), a qual hidrolisa peptídeos como ocitocina e vasopressina e é receptora de angiotensina IV, destaca a importância do estudo do envolvimento de peptidases na função endócrina do adipócito. Estudos recentes detectaram alterações das atividades de aminopeptidase neutra e de dipeptidil peptidase IV (DPPIV) no plasma e no hipotálamo e hipocampo na obesidade induzida por glutamato monossódico (MSG). A presente tese propôs (i) a existência de atividades aminopeptidásicas ácida (APA), básica (APB), neutra insensível (APM) e sensível à puromicina (PSA), metionil (MetAP) e DPPIV, além da já conhecida (leucil (LAP)/cistil (CAP)/IRAP), nas frações de membrana plasmática (FM) e de alta (HDM) e baixa (LDM) densidade microssomal de adipócitos isolados do depósito de gordura retroperitoneal; (ii) que suas atividades catalíticas, expressões gênicas e tráfego subcelular seriam diferenciados entre obesos induzidos por MSG, submetidos ou não à privação alimentar e em animais controle sadios, submetidos ou não a privação alimentar; (iii) e influenciadas por insulina, angiotensina II, angiotensina IV e vasopressina. A existência de todas essas aminopeptidases no adipócito foi demonstrada, sendo a APM a que apresenta maior Vmax e maior eficiência catalítica e a APA a maior afinidade. Os animais obesos apresentaram aumento do diâmetro médio dos adipócitos e aumento do lipócrito, caracterizando hipertrofia adipocítica, enquanto os controles privados de alimento tiveram um aumento no número de adipócitos e aumento no lipócrito, caracterizando hiperplasia adipocítica. Pela primeira vez, um perfil variado de atividades aminopeptidásicas distribuídas em diferentes compartimentos subcelulares do adipócito é evidenciado juntamente com a demonstração de diferenças no padrão de distribuição destas atividades entre os ratos obesos e sadios, privados ou não de alimento. Dentre as novas aminopeptidases detectadas no adipócito não há nenhuma com a característica clássica de IRAP. No geral, as alterações das atividades catalíticas nas diferentes situações sob estudo mostram o envolvimento dessas novas aminopeptidases na regulação endócrina do balanço energético (provavelmente via ação hidrolítica sobre angiotensina II e vasopressina) sob modulação por angiotensina IV (APB, APM, DPPIV, LAP/IRAP, MetAP e PSA em animais controle sadios e APB em animais controle privados de alimento), angiotensina II (APB e PSA nos animais obesos) e vasopressina (APB nos animais obesos privados de alimento; e influenciadas em seu tráfego subcelular por angiotensina II (CAP, DPPIV e LAP/IRAP) e angiotensina IV (LAP/IRAP). O tráfego subcelular da conhecida atividade CAP/IRAP mostrou-se suscetível à insulina e vasopressina / The discovery of insulin-regulated aminopeptidase (IRAP), which hydrolyzes peptides such as oxytocin and vasopressin and is an angiotensin IV receptor, highlights the importance of the involvement of peptidases in the endocrine function of adipocyte. Recent studies have detected changes in aminopeptidase activities of neutral aminopeptidase and dipeptidyl peptidase IV (DPPIV) in the plasma and in the hypothalamus and hippocampus of rats with obesity induced by monosodium glutamate (MSG). The present thesis proposes (i) the existence of acid (APA), basic (APB), neutral insensitive (APM) and puromycin sensitive (PSA) aminopeptidases, methionyl aminopeptidase (MetAP) and dipeptidyl peptidase IV (DPPIV), besides the well-known cystyl (CAP) / leucine aminopeptidase (LAP) / IRAP) in plasma membrane (MF) and in high (HDM) and low (LDM) density microsomes of isolated adipocytes from retroperitoneal fat pad; (ii) that their catalytic activities, gene expression and subcellular trafficking were different among MSG-induced obese and healthy control rats (food deprived or not); (iii) and influenced by insulin, angiotensin II and IV, and vasopressin. The existence of all these adypocite aminopeptidases was demonstrated. APM has a highest Vmax and catalytic efficiency, while APA has a highest substrate affinity. The obese animals have increased mean diameter of adipocytes and lipocrit, characterizing hypertrophy, while the food deprived rats had an increased number of adipocytes and lipocrit, characterizing hyperplasia. For the first time, a profile of varied aminopeptidases activities distributed in different subcellular compartments of adipocyte is evidenced together with the demonstration of differences in the distribution pattern of these activities among the obese and healthy rats, food deprived or not. Among these novel aminopeptidases detected in adipocytes there is no one with the classical characteristic of IRAP. In general, changes on catalytic activities in these different situations show the involvement of these novel aminopeptidases in the endocrine regulation of energy balance (probably via hydrolytic action on angiotensin II and vasopressin) under modulation by angiotensin IV (APB, APM, DPPIV, LAP/IRAP, MetAP and PSA in healthy animals and APB in food deprived healthy animals), by angiotensin II (APB and PSA in obese) and by vasopressin (APB in food deprived obese animais); and under influence of by angiotensin II (CAP, DPPIV, LAP/IRAP) and angiotensin IV (LAP/IRAP) on their subcellular trafficking. Subcellular trafficking of the well-known CAP/IRAP was susceptible to insulin and vasopressin

Os tumores cutâneos e subcutâneos em cães representam um terço das neoplasias nessa espécie. O carcinoma de células escamosas (CCE) é uma neoplasia maligna com origem no epitélio estratificado escamoso da pele e de outras superfícies mucosas. A etiologia do CCE em cães não é bem definida, porém é descrito que a exposição aos raios solares é um importante fator para seu desenvolvimento. O CCE, frequentemente, é precedido pela ceratose actínica (CA). Este trabalho tem como objetivo comparar aspectos clínicos, morfológicos e moleculares da ceratose actínica e carcinoma de células escamosas da pele da região abdominal ventral de cães. Foram coletadas dez amostras de pele apresentando CCE e 9 amostras de pele apresentando CA da região abdominal ventral de caninos domésticos. As amostras foram fixadas em formol a 10%, e rotineiramente processadas para inclusão em parafina. Os cortes histológicos de 5?m foram submetidos à coloração de Hematoxilina e Eosina, para análise histopatológica, e às marcações, por imuno-histoquímica, de E-caderina, p53, Ciclo-oxigenase-2, pancitoqueratina AE1/AE2, vimentina, 5 metilcitosina e Ki67. As expressão de E-caderina, p53, ciclo-oxigenase-2, pancitoqueratina AE1/AE e vimentina foram avaliadas qualititivamente nas 9 amostras de CA e 10 amostras CCE, respectivamente. A metilação global do DNA e a proliferação celular (Ki67) foram quantificadas nos cortes histológicos de CA e CCE por meio da contagem de núcleos de células epiteliais marcados positiva ou negativamente. Os dados foram analisados por meio de testes uni e multivariados. Os cães machos da raça American PitBull Terrier de pelagem branca foram os mais acometidos pela CA e CCE, com média de idade de 8,1 e 8,4 anos, respectivamente. Não houve associação significativa na expressão de p53, COX-2 e vimentina na CA e CCE. Houve aumento no número de células com o núcleo marcado positivamente para Ki-67 e 5 metilcitosina em CCE comparado a CA. Observou-se, também, marcação heterogênea de Ecaderina em CCE. Constatou-se também diminuição na expressão de pancitoqueratina AE1/AE3 quando há invasão linfática e maior expressão de 5 metilcitosina em cães mais idosos, no CCE. Estes achados sugerem que há diferenças entre CA e CCE em relação aos parâmetros estudados, que condizem com a transformação maligna da afecção / Cutaneous and subcutaneous tumors in dogs account for a third of neoplasms in this species. Squamous cell carcinoma (SCC) is a malignant neoplasm that originates in the stratified squamous epithelium of the skin and other mucosal surfaces. The etiology of SCC is not well defined, however it is described that exposure to the sunlight is an important factor for its development. SCC is often preceded by actinic keratosis (AK). This work aims to compare clinical, morphological and molecular aspects of actinic keratosis and squamous cell carcinoma of the skin of the ventral abdominal region of dogs. Ten samples of SCC and 9 skin samples showing AK of the ventral abdominal region of domestic canines were collected. As samples were fixed in 10% formaldehyde, and routinely processed for inclusion in paraffin. Histological sections of 5?m were submitted to staining of Hematoxylin and Eosin for histopathological analysis, and to immunohistochemistry of E-cadherin, p53, Cyclooxygenase-2, pancytokeratin AE1/AE2, vimentin, 5 methylcytosine and Ki-67. The expression of E-cadherin, p53, cyclooxygenase-2, pancytokeratin AE1 / AE and vimentin were qualitatively evaluated in the 9 AK samples and 10 SCC samples, respectively. Overall DNA methylation and cell proliferation (Ki67) were quantified in the histological sections of AK and SCC by counting nuclei of positively or negatively labeled epithelial cells. The data were analyzed by means of uni and multivariate tests. Male dogs of the American PitBull Terrier breed were the most affected by AK and SCC, with an average age of 8.1 and 8.4 years, respectively. There was no significant association in the expression of p53, COX-2 and vimentin in CA and CCE. There was an increase in the number of cells with the nucleus positively labeled for Ki-67 and 5 methylcytosine in CCE compared to CA. There was also a heterogeneous labeling of Ecaderin in CCE. It was also observed a decrease in the expression of pancytokeratin AE1/AE3 when there is lymphatic invasion and greater expression of 5 methylcytosine in older dogs in the ECC. These findings suggest that there are differences between CA and SCC in relation to the studied parameters, which are consistent with the malignant transformation of the disease

A ruptura do aneurisma da aorta abdominal está entre as principais causas de óbito. A alta morbi-mortalidade associada à ruptura e tratamento dos aneurismas representa um grande desafio aos médicos e um alto risco aos pacientes. Apesar dos modelos experimentais serem úteis para compreendermos, treinarmos, testar novos métodos diagnósticos e terapêuticos para esta doença, os modelos existentes até o momento ainda não são os ideais. Nos modelos existentes, os animais são muito pequenos e não representam a doença nos humanos, ou o procedimento envolve laparotomia, ou o comportamento do aneurisma criado não é semelhante ao de um aneurisma verdadeiro. Desenvolvemos, a partir de uma abordagem minimamente invasiva, um método eficiente de induzirmos a formação de um aneurisma verdadeiro na aorta abdominal infrarrenal de porcos Large White. Os animais foram submetidos a indução química a partir de uma aplicação por via endovascular de cloreto de cálcio a 25% ou elastase pancreática suína. Os animais controles foram submetidos a tratamento com soro fisiológico (NaCl 0,9%). Todos os animais foram submetidos à mesma técnica operatória, sob anestesia geral. Os animais foram acompanhados com exames ultrassonográficos com Doppler semanalmente, e as aortas colhidas para testes biomecânicos e análise histológica após 4 semanas. Apesar das aortas tratadas com elastase apresentarem apenas dilatação, estudos de imagens, histológicos e biomecânicos mostraram que as aortas tratadas com cloreto de cálcio evoluíram para aneurismas verdadeiros, com comportamento biomecânico semelhante ao dos aneurismas de humanos. Estes resultados/achados indicam que a abordagem endovascular para a indução de aneurisma é factível e não ocasiona uma fibrose retroperitoneal / Abdominal aortic aneurysms rupture are among the main causes of death. The high morbidity and mortality associated with aneurysm rupture and repair represents a challenge for surgeons and high risk for patients. Although experimental models are useful to understand, train, and develop new treatment and diagnostic methods for this disease, animal models developed to date are far from ideal. Animals are either too small and do not represent the pathology of humans, or the procedures employ laparotomy, or the aortic behavior does not resemble that of a true aneurysm. We developed a novel, less invasive and effective method to induce true aortic aneurysms in Large White pigs. Animals were submitted to an endovascular chemical induction using either calcium chloride (25%) or swine pancreatic elastase. Controls were exposed to saline solution. All animals were operated on using the same surgical technique under general anesthesia. They were followed weekly with ultrasound examinations and at 4 weeks the aorta was harvested. Although elastase induced only arterial dilation, imaging, histological, and biomechanical studies of the aorta revealed the formation of true aneurysms in animals exposed to calcium chloride. Aneurysms in the latter group had biomechanical failure properties similar to those of human aneurysms. These findings indicate that the endovascular approach is viable and does not cause retroperitoneal fibrosis

O objetivo deste trabalho foi de avaliar em Modelo animal, coelhos (raça Nova Zelândia) divididos em três grupos randomizados, (jovem com 60 dias (G1), um idoso com aterosclerose e ingestão de colesterol (G2) e um idoso com aterosclerose e sem ingestão do colesterol (G3)) se, a dieta rica em colesterol e a idade, causam lesões de aterosclerose e placas ateroscleróticas nos animais, alterações nos comprimentos dos dentes, aumento ou diminuição dos espaços periapicais dos dentes, perda óssea alveolar na maxila e mandíbula. Através da metodologia descrita e após as análises histológicas e morfológicas, verificou-se diferença estatisticamente significante, nas variáveis dos comprimentos dos dentes 1º prémolares superiores entre os grupos G3 versus G1 p<5%. médias de 1247,88 (p=0,017) e G3 versus G2, com diferença das médias de 1190,85 (p=0,025) ou seja o comprimento dos dentes fora diferente no grupo G2. Com relação à variável, espessura do osso alveolar, não ocorreu significância estatística, porém tendências de que este esteja sendo alterado. Com relação a variável espessura do espaço periapical fora verificado significância estatística com p=0,017 em relação ao G1, na região dos 1º Pré-molares, apresentando a diferença das médias (403,42) a favor do G2, ou seja aumento do espaço periapical. A variável da área de placa de aterosclerose, correlacionada, com os comprimentos dos dentes e espessura do osso alveolar, também teve resultado significante na região 1º e 2º pré-molares entre G2 e G3. com p=0,025 e r=0,476. Confirmando assim a correlação da aterosclerose, dietas hiperclesterolêmicas e as alterações da cavidade bucal e estrutura de sustentação dos dentes. / The aim of this study is, to evaluate on animal model (New Zealand rabbits), on different and randomized groups (G1 a young group, G2 an older rabbit, with atherosclerosis and ingesting hipercholesterolemic fat diet and another group G3, just an older group) investigated than the hipercholesterolemic fat diet induced atherosclerotic lesions and plaques in ascendant aorta, bone loss in maxillary and jaw, alterations on length of the tooth, alterations on tooth periapical spaces. By the methodology described and aplicated, before the histological and morphological analysis, was verified significant statistical variance in the length of the first premolar tooth and second premolar on maxillary region, the length of this tooth is reduced in G2 (p<5%). The medias are 1247,88 (p=0,017) and 1190,85 (p=0,025). Analyzing another variable on the alveolar bone thickness, was not found any statistical significance, but tendencies that this event may occur. According to another variable of the thickness found on the periapical space, it was verified a significant variable statistic such as p=0,017 related on G1, on first premolar region, suggesting a different statistic, between medias, favoring G2 meaning the increase of periapical space. The variable on the atherosclerosis plaque area, related to the teeth length and alveolar bone thickness, also showed significant results on first and second premolars regions between G2 and G3, with p=0,025 and r=0,476. That confirms the atherosclerosis correlations and hipercholestrolemic fat diet, also alterations in oral cavity and teeth implantation structure.

Dans le contexte actuel d'allongement de l'espérance de vie, la prévalence et le coût de prise en charge des maladies liées à l'âge telles que l'ostéoporose sont de plus en plus importants. Dans le cadre du développement de nouvelles stratégies de prise en charge des pathologies osseuses, la nutrition offre un potentiel évident et représente une excellente solution alternative aux traitements habituels. Bien que trop marginales, les études des activités biologiques de certains aliments ont pourtant d'ores et déjà montré des effets protecteurs sur l'acquisition du capital osseux et sa préservation par le biais de leurs propriétés anti-inflammatoires et antioxydantes. A ce titre, la consommation de grenade est envisagée car elle fait actuellement l'objet d'intérêts scientifiques croissants ayant déjà suggéré ses avantages nutritionnels et pharmacologiques pour la prévention de certaines pathologies chroniques associées au vieillissement. Dans le cadre du projet de thèse, les premiers travaux réalisés, focalisés donc sur la grenade, ont permis de dégager le concept selon lequel les propriétés biologiques de ce fruitexceptionnel seraient expliquées par la composition particulière de ses différentes parties : en polyphénols pour le jus (anthocyanines) et la peau (ellagitannins) et en acide punicique pour les pépins, et surtout au potentiel anti-oxydant et anti-inflammatoire de ces molécules. Cette hypothèse a donné lieu à une première étude à vocation phytochimique qui a permis de déterminer, de standardiser, produire et caractériser deux types d'extraits de grenade titrés en molécules d'intérêt (acide punicique et ellagitannins). Nous avons ensuite étudié l'impact de la consommation du fruit (donné dans sa totalité) ou de ses principales parties (jus et peau) ou encore d'extraits concentrés en ellagitannins et en acide punicique, sur la biologie osseuse, dans un modèle expérimental d'ostéoporose post-ménopausique bien décrit (ostéopénie consécutive à une carence oestrogénique induite par ovariectomie chez des souris C57bl6/j). Cette étude préclinique a permis de montrer que tous les régimessupplémentés en grenade (quelle que soit la partie) permettent de limiter les processus de déminéralisation et l'altération micro-architecturale de l'os, s'expliquant par une réduction des marqueurs de résorption osseuse et une amélioration de ceux de la formation ; et une diminution des paramètres inflammatoires, et oxydants. Ensuite, afin de déterminer les structures médiatrices de ces effets, une approche originale ex-vivo a permis d'étudier plus finement les mécanismes cellulaires ostéoblastiques et ostéoclastiques associés à la consommation des extraits de grenade testés. Cette analyse a été développée de façon à tenir compte du métabolisme particulier de ces micronutriments et donc des modifications systémiques engendrées suite à l'ingestion des extraits. Ce travail a permis de mettre en évidence que les effets observés in-vivo sur l'os,pouvaient être expliqués par une action directe aussi bien sur les ostéoblastes que sur les ostéoclastes, et donc une optimisation du remodelage osseux. De fait, la consommation d'ellagitannins et d'acide punicique augmente l'activité des ostéoblastes (activité alcaline phosphatase et formation de nodules de calcium) et l'expression des principaux acteurs et facteurs de transcription impliqués dans leur différenciation, tout en diminuant l'expression de ceux responsables de la différenciation ostéoclastique. Dans le même temps, l'analyse des transcrits met en évidence que les extraits de grenade favorisent l'activation d'acteurs impliqués dans les mécanismes de défenses contre le stress oxydant et l'inflammation, au niveau du microenvironnement osseux. Ces résultats révèlent pour la première fois le potentiel de la grenade au regard de la physiologie osseuse en proposant ses possibles mécanismes d'actions, via une approche nutritionnelle complète et intégrée respectant la physiologie. / In the current context of increased life expectancy, prevalence and socioeconomic consequences of age-related diseases such as osteoporosis represent a major public health problem worldwide. This is why development of new strategies of prevention is highly suitable to provide a wide array of options (alternatives to usual therapies) for health professionals. So far, studies targeting nutrient biological activities have been mainly focused on both calcium and vitamin D. Nevertheless, other nutrients have shown a protective effect on bone mass acquisition and preservation through their anti-inflammatory and antioxidant properties. In this light, pomegranate is endowed with such a potential. Indeed there is an increasing scientific interest that has already suggested its nutritional and pharmacological benefits on prevention of some chronic age-associated diseases. As part of this project, the initial work focused on pomegranate has highlighted the link between its health benefit potential and the exceptional composition of its main parts: polyphenols from the juice (anthocyanins) and the peel (ellagitannins) and punicic acid from seed, those micronutrients being able to elicit antioxidant and anti-inflammatory capacities. Our work hypothesis resulted from a first phytochemical study leading to identify, standardize, characterize and produce two types of pomegranate extracts titrated on 2 molecules of interest (punicic acid and ellagitannins). We thus investigated the outcome of consumption, first of the whole fruit or its principal parts (peel and juice) and in a second time of concentrated ellagitannins and punicic acid extracts on bone biology in a well described experimental model of postmenopausal osteoporosis (osteopenia induced by estrogen deficiency after ovariectomy in C57bl6/j mice). In this preclinical study, wedemonstrated that all the diets supplemented with pomegranate significantly prevented bone loss and micro-architecture impairment. Those findings are associated with transcriptional changes in bone tissue, suggesting involvement of both osteoclastogenesis inhibition and osteoblastogenesis improvement, and reduced inflammatory and oxidative parameters, as well. Then, to determine more accurately the molecules and the signaling pathways involved in those effects, an original ex-vivo study was set up on both osteoblasts and osteoclasts, with respect to physiological conditions (i.e., the aim being to mimic systemic changes and generation of specific circulating metabolites associated with the extractsingestion). This work allowed clarifying the bone sparing effects observed in-vivo. Indeed, pomegranate extracts had the ability to elicit a significant increase in alkaline phosphatase activity, matrix mineralization and transcriptional levels of major osteoblast lineage markersinvolving key signaling pathways, while the expression of specific osteoclast differentiation markers and RANK-RANKL downstream signaling targets were down-regulated. In addition, transcripts analysis revealed that pomegranate extracts were able to induce defense mechanisms against oxidative stress and inflammation in the bone microenvironment. Our results show for the first time the pomegranate potential regarding bone physiology, underlying its possible mechanisms on bone remodeling through a complete and integrated nutritional approach respecting the physiology.

Le développement d’un lymphome gastrique du MALT (LGM) émane d’un processus inflammatoire chronique initié par Helicobacter pylori.A partir du matériel issu d’un modèle animal de LGM, préalablement développé au laboratoire, basé sur des infections chez des souris thymectomisées à la naissance, la réponse inflammatoire gastrique favorable à l’émergence de LGM a été étudiée. Une dérégulation de cytokines et chimiokines au stade LGM a été identifiée permettant de recruter, faire proliférer et faire émerger des infiltrats lymphoïdes. La susceptibilité des souris thymectomisées à développer des lymphomes n’est pas liée à un déficit en lymphocytes T régulateurs. Cinq microARNs ont été retrouvés dérégulés au stade lymphome agissant probablement en synergie pour favoriser la prolifération lymphocytaire en particulier via un mécanisme anti-apoptotique. Enfin, nous décrivons un modèle original de LGM basé sur l’utilisation de souris C57BL6 exprimant la chimiokine APRIL humaine au niveau des lymphocytes T infectées par des espèces du genre Helicobacter. Ce modèle est prometteur pour une meilleure compréhension de la lymphomagénèse gastrique. / The development of gastric MALT lymphoma (GML) originates from a chronic inflammatory process initiated by Helicobacter pylori.The gastric inflammatory response was investigated in a mouse model of GML previously described by the laboratory using BALB/c mice thymectomized at day 3 post-birth and infected by H. pylori. A deregulation of numerous cytokines and chemokines at GML stage was identified which explained the recruitment, proliferation and emergence of lymphoid infiltrates. The susceptibility of thymectomized mice to develop lymphoma was not linked to a deficiency in regulatory T cells. A deregulation of 5 microRNAs was observed at lymphoma stage. These microRNAs may be involved in cell survival and lymphocyte proliferation and act in synergy to promote the development of GML. Finally, we described an original model of GML based on infection by Helicobacter species of transgenic C57BL6 mice expressing the human form of the cytokine APRIL in T cells. This model is promising for a better understanding of gastric lymphomagenesis.

Le neuropaludisme (NP) est la forme la plus mortelle du paludisme. C'est une complication neurologique observée uniquement dans les cas d'infection par Plasmodium falciparum, principalement chez les enfants de moins de 5 ans vivant en Afrique Sub-saharienne, et les adultes non-immuns, notamment les femmes enceintes et les touristes visitant les zones d'endémie. Les signes cliniques sont à présent bien décrits (prostration, convulsions répétées, difficultés respiratoires, coma,...), mais les mécanismes physiopathologiques conduisant au NP sont encore mal définis. Leur élucidation est rendue difficile par la localisation cérébrale de la pathologie du vivant des patients et la faible disponibilité des données nécropsiques. Bien que l'accès aux tissus humains soit limité en nombre, les résultats d'autopsie ont permis d'établir que le NP résulte d'une séquestration des globules parasités au niveau de l'endothélium intra-vasculaire, associée à une forte réaction immunitaire. La stratégie de prise en charge du NP combine un traitement étiologique à base de dérivés d'artémisinine, ou de quinine et un traitement adjuvant symptomatique destiné à pallier à la défaillance multiorganique qui est à l'origine de l'issue fatale souvent observée. Le modèle de NP expérimental actuellement le plus utilisé est le modèle souris infecté par P. berghei ANKA. La pertinence de ce modèle est toutefois remise en cause en raison notamment des différences histo-pathologiques observées par rapport à la forme humaine. En effet, les souris manifestant les symptômes du NP ne présentent que très rarement le phénomène de séquestration, caractéristique majeure du NP chez l'Homme. Par ailleurs, comparativement à la réponse immunitaire de la souris, le modèle rat s'est également révélé plus proche de la réaction de l'Homme, dans le cas d'une autre parasitose, la schistosomose. L'objectif de la première partie du projet thèse a donc été la mise en place et l'évaluation d'un modèle alternatif de NP chez le rat. Ainsi un modèle de NP chez le rat Sprague Dawley infecté par la souche murine P. berghei K173 a été caractérisé sur les plans clinique, biologique (paramètres hématologiques et biochimiques),histopathologique et du profil cytokinique (cytokines cérébrales et sériques). La forte similarité des symptômes et des lésions associées au NP du rat Sprague Dawley infecté par P. berghei K173 par rapport au NP humain permet de valider la pertinence de ce modèle pour l'étude de la physiopathologie du NP. L'objectif de la deuxième partie de mon projet de thèse a été d'évaluer les effets pharmacologiques d'un candidat-médicament, l'isoflurane, pour le traitement adjuvant du NP. Ce composé présente l'avantage d'être déjà utilisé chez l'Homme à d'autres fins thérapeutiques et dispose donc d'une autorisation de mise sur le marché. Les résultats obtenus montrent une efficacité nette de l'isoflurane avec une rémission totale des signes de paralysie pour 47.8% des rats traités ainsi qu'un gain de survie des rats NP traités de 2 à 10 jours par rapport aux rats NP non traités. Ce gain de survie des animaux traités pourrait permettre un allongement de la fenêtre du temps de traitement étiologique, améliorant ainsi sensiblement le pronostic du NP. L'isoflurane, dont le mécanisme d'action semble être la réversion de la séquestration des globules parasités, limite les complications neurologiques souvent responsables de séquelles liées au NP. Des études ultérieures permettront d'optimiser ce nouveau protocole de traitement adjuvant du NP. / Cerebral malaria (CM) is the most deadly form of malaria. It is a neurological complication observed only in cases of infection with Plasmodium falciparum that affects mainly children under five years living in Sub-Saharan Africa and non-immune adults including pregnant women and tourists visiting endemic areas. Although clinical signs are well described (prostration, respiratory distress convulsions, coma), the pathophysiological mechanisms leading to CM are still unclear. Their elucidation in vivo is made difficult by the cerebral location and the low availability of autopsy data. Instead of limited access to human tissues, autopsy results have shown that CM results from a strong immune response linked to sequestration of infected red blood cells in the intravascular endothelium. Cerebral malaria management combines an etiological treatment with artemisinin derivatives or quinine and adjunct treatment of the multi-visceral failures, responsible of fatal outcome. P. berghei ANKA-infected mouse is widely used as experimental murine model of CM. However the relevance of this model is still questioned because of the histopathologic differences from the human form. Indeed, CM mice rarely exhibit the red blood cell sequestration that is a major feature of human CM. Furthermore, compared to mouse, the rat displays a closer immune response to human in Schistosoma infection. This PhD research project first aimed to implement and assess an alternative rat model of CM. The clinical, biological, histo-pathological features as well as the cytokine profiling of an experimental model of CM were characterized in Sprague Dawley rats infected with P. berghei strain K173. The strong similarity of the symptoms and lesions observed in this model with those reported in human CM confirms its high relevance. The second objective of this thesis project was to assess the pharmacological effects of a drug-candidate in adjunct treatment of CM. Results demonstrated a strong efficacy of the molecule tested with 47.8% of the treated CM rats showing total remission. Moreover we observed a 2- to 10-day survival gain in the treated CM rats group compared to the non-treated CM rat group. Preliminary data suggest that this drug-candidate may reverse the endothelial sequestration of parasitized red blood cells and so limit the neurological sequels related to CM. It is anticipated that the gain in survival associated with this drug-candidate use will extend the window of the etiological treatment time, thus significantly improving the global prognosis of CM. Further studies are needed to optimize this adjunct CM treatment protocol.