Made available in DSpace on 2014-07-29T15:13:42Z (GMT). No. of bitstreams: 1 tese pos defesa.pdf: 2322225 bytes, checksum: 4027ee4b5a7e38a6c0d20eb192a18551 (MD5) Previous issue date: 2011-03-18 / This study has aimed do evaluate a risk factor for bovine digital dermatitis and the efficacy of 5% aqueous barbatimão extract solution as ancillary in interdigital skin incisional wound repair. One hundred bovine females, 50 Holstein and 50 Girolando, from two different farms were used for the risk factor study. Experimental units were divided into four groups: healthy Holstein cattle group (GHS) and digital dermatitis Holstein cattle group (GHL), healthy Girolando cattle group (GGS) and digital dermatitis Girolando cattle group (GGL). Ten milliliters of blood were collected from all animals and stored in anticoagulant added tubes. Samples were refrigerated and submitted to hemolytic tests for eight bovine blood systems: A, B, C, F, J, L, S and Z. The frequency of blood factors was determined and compared, in each system and each group. Data were analyzed through non parametric tests: Chi Square and Fisher s exact. The results indicated that the blood factors may contribute to the occurrence of digital dermatitis lesions, but further studies are needed for a more accurate conclusion. Thirty confined, 10 months old calves were selected in order to evaluate the efficacy of 5% barbatimão solution. A biopsy punch was introduced in each interdigital space, up to the dermis, to produce a lesion measuring one centimeter in diameter. The lesions were numbered 120 in total and named experimental units, which were divided into two groups of 60 units each: GB treated with 5% aqueous barbatimão bark extract solution and GC treated with water. Tissue repair was evaluated through macroscopic and histological findings in days three, seven, 14 and 21 of treatment. Macroscopic findings were analyzed descriptively, while histological findings were submitted to Mann Whitney test, with p<0.05. For lesions treated, GB group showed less intense polymorphonuclear cells infiltration on the first 14 days of treatment. A marked collagen deposition was also observed at day 14 while collagen alignment was more expressive on day 21. More specific studies may be necessary in order to determine if blood factors can contribute to the occurrence of digital dermatitis. Barbatiman 5% footbath solution for confined cattle helps qualitatively in modulation of interdigital cutaneous incisional wound repair, but does not avoid the extension of inflammatory phase of healing. / Objetivou-se nesta pesquisa estudar um fator de risco para a dermatite digital bovina e avaliar a eficácia do extrato de barbatimão em solução aquosa a 5% como auxiliar no reparo de feridas cutâneas induzidas no espaço interdigital de bovinos. Para pesquisar o fator de risco utilizaram-se 100 fêmeas bovinas, sendo 50 Holandesas e 50 Girolando, de duas propriedades rurais. Dividiram-se as unidades experimentais em quatro grupos: grupo de animais saudáveis da raça Holandesa (GHS) e grupo com lesões de dermatite digital (GHL); grupo de animais saudáveis da raça Girolando (GGS) e grupo com lesões de dermatite digital (GGL). De todos os animais colheram-se 10 ml de sangue, em tubo a vácuo, contendo anticoagulante. As amostras foram refrigeradas e submetidas a testes hemolíticos, para oito sistemas sanguíneos de bovinos: A, B, C, F, J, L, S e Z. A frequência dos fatores sanguíneos foi determinada e comparada, dentro de cada sistema, em cada grupo de animais. Os dados foram analisados através dos testes não paramétricos de Qui-quadrado e Exato de Fisher. Pelos resultados obtidos concluiu-se que os fatores sanguíneos podem contribuir para a ocorrência de lesões de dermatite digital, contudo são necessários estudos mais específicos para uma conclusão mais precisa. Para avaliar a eficácia da solução de barbatimão a 5%, utilizaram-se 30 animais com dez meses de idade, mantidos em sistema de confinamento. Em cada espaço interdigital dos animais, com auxílio de um punch de um centímetro de diâmetro, produziu-se uma lesão cutânea até a derme, totalizando 120 feridas. As feridas foram denominadas unidades experimentais e distribuídas em dois grupos com 60 unidades cada: grupo GB tratado com solução de extrato de barbatimão a 5% e grupo GC tratado com água. A evolução do processo de reparação tecidual das feridas foi acompanhada por meio de avaliações macroscópicas e histológicas nos dias três, sete, 14 e 21 de tratamento. Os achados macroscópicos foram analisados descritivamente e os histológicos submetidos ao teste de Mann Whitney, com p<0,05. Para as feridas tratadas, observou-se que o grupo GB apresentou menor intensidade de infiltrado de células polimorfonucleares nos primeiros 14 dias de tratamento. Nesse grupo observou-se ainda que a deposição de colágeno mostrou-se mais acentuada aos 14 dias e aos 21 dias as fibras colágenas apresentavam-se mais alinhadas. O uso do extrato de barbatimão a 5% em pedilúvio, em bovinos confinados, auxiliou qualitativamente no processo de modulação da reparação de feridas cutâneas interdigitais incisionais, mas não impediu o prolongamento da fase inflamatória do processo cicatricial.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Os terpenos Ãcido centipÃdico e lactona do Ãcido hawtiwaico (LAH), isolados dos capÃtulos florais de Egletes viscosa Less, popularmente conhecida como macela ou macela da terra, foram avaliados em modelos de dermatite de contato irritativa induzida pelo 13-acetato-12-o-tetradecanoil-forbol (TPA) e de dermatite de contato alÃrgica induzida por oxazolona (OXA) em camundongos. O Ãcido centipÃdico e a LAH, por via tÃpica (0,125; 0,25 e 0,5mg/orelha), reduziram significativamente o edema de orelha induzido por TPA (2,5Âg/orelha) em 45,5; 55,5; 61,1% e 33,3; 42,2 e 63,3%, respectivamente. Quando administrados por via oral, (12,5; 25 e 50mg/kg), reduziram o edema em 46,6; 59,3; 67,9% e 58,7; 59,7; 71%, respectivamente quando comparado ao controle veÃculo. A dexametasona na dose tÃpica de 0,05mg/orelha e na dose oral de 1mg/kg reduziu o edema de orelha em 90 e 73,1%, respectivamente. O Ãcido centipÃdico e a LAH, na dose de 0,5mg/orelha, via tÃpica reduziu de a atividade da mieloperoxidase (MPO) em 90,1 e 94,6%, respectivamente, enquanto a dexametasona (0,05mg/orelha) reduziu em 96,2% a atividade da MPO quando comparado ao controle veÃculo. Os terpenos na dose tÃpica de 0,5mg/orelha foram capazes de reduzir os nÃveis teciduais de TNF-&#945;, o mesmo sendo observado com a dexametasona. A aÃÃo antiinflamatÃria dos terpenos no modelo da dermatite pelo TPA foram confirmados pelos achados histolÃgicos. O modelo de dermatite de contato induzida por oxazolona (OXA, 1%/orelha), provocou uma hiperplasia epidÃrmica onde o INF-&#947; teve papel crucial. Os terpenos Ãcido centipÃdico (0,25 e 0,5) e a LAH (0,5mg/orelha), por via tÃpica, diminuÃram, a hiperplasia epidÃrmica induzida por oxazolona (1%/orelha)em todos os perÃodos de observaÃÃo (4Â - 19Â dia). O Ãcido centipÃdico e a LAH na dose de 0,5mg/orelha diminuÃram significativamente os nÃveis teciduais de INF-&#947; (92,2 e 99,4%, respectivamente) quando comparados ao controle veÃculo. Quando administrado por via oral, o Ãcido centipÃdico (12,5; 25 e 50mg/kg) reduziu de forma significativa a hiperplasia epidÃrmica do 7Â ao 19Â dia de observaÃÃo. A dexamentasona na dose tÃpica (0,05/mg) e na dose oral (1mg/kg) reduziu a hiperplasia epidÃrmica em 100 e 75%, respectivamente. Os resultados encontrados, comprovados pelo estudo histolÃgico, demonstram o efeito antiinflamatÃrio desses terpenos em modelo de dermatite de contato alÃrgica crÃnica onde a hiperplasia epidÃrmica e o aumento nos nÃveis teciduais de INF-&#947; sÃo caracterÃsticas importantes, como no caso da psorÃase / The terpens centipedic acid (CA) and lactone of hawtriwaic acid (LAH) isolated from the flowers chapters of Egletes viscosa Less, popularly known as macela or macela da terra, were evaluated on 13-acetate-12-o-tetradecanoil-forbol (TPA) induced irritative contact dermatitis model and on oxazolone induced allergic contact dermatitis in mice. The centipedic acid and LAH on the following dosage of 0,125; 0,25 and 0,5 mg/ear using the skin surface as a via reduced in a significative way the ear oedema induced by application of TPA (2,5 &#956;g/ear). The values of reduction are the following respectively: 45,5; 55,1; 61,1% and 33,3; 42,2 and 63,3%. When the oral administration was used the doses of 12,5; 25 and 50 mg/kg had showed a significative oedema reduction of 46,6; 59,3; 67,9% and 58,7; 59,7; 71%, respectively. Dexametasone through skin surface administration on dosage of 0,05 mg/ear and oral dosage of 1mg/kg have reduced in a significative way the ear oedema in 90 and 73,1%, respectively. The centipedic acid and LAH using the dosage of 0,05 mg/ear through the skin surface had reduced the mieloperoxydase activity in 90,1 and 94,6%, respectively, while dexametasone (0,05 mg/ear) had reduced in 96,2% when compared to the group treated only with the vehicle. The terpens on the topic dosage of 0,05 mg/ear were capable to reduce the tecidual levels of TNF-&#945; as well as dexametasone. The antiinflamatory action of the terpens on TPA induced dermatitis model was confirmed through histological analysis of the tissue. The oxazolone induced contact dermatitis model (OXA 1%/ear) had promoted an epidermic hyperplasia in which INF-&#947; have a central role. The products under evaluation centipedic acid (0,25 and 0,5 mg/ear) and LAH (0,5 mg/ear) through skin surface reduced in a significative way the epidermic hyperplasia Throughout the observation period (4Â- 19Â day). Centipedic acid and LAH on the following dosage 0,5/mg reduced in a significative way the tecidual levels of INF-&#947; (92,2 and 99,4%, respectively) after 19 days of treatment. When using oral administration the centipedic acid (12,5; 25 e 50 mg/kg) reduced the epidermic hyperplasia from seventh through tenth ninth day. Dexametasone through skin surface administration on dosage of 0,05 mg/ear and oral dosage of 1mg/kg have reduced the epidermic hyperplasia in 100 and 75%, respectively The data that were found plus the histological analysis have showed the antiinflammatory effect, mainly through topic administration, of these terpens on allergic contact dermatitis models in which epidermic hyperplasia and increased tecidual levels of INF-&#947; are important features, such as psoriasis

Introdução: A dermatite atópica (DA) é uma doença cutânea inflamatória de caráter crônico, recidivante, em que o prurido intenso e a xerose cutânea são frequentes. A etiopatogenia da DA é multifatorial, envolvendo fatores genéticos, ambientais e imunológicos. Eosinófilos são leucócitos polimorfonucleares multifuncionais que estão implicados na patogênese de diversos processos inflamatórios, incluindo a DA. Além da produção e secreção de diversas proteínas presentes nos grânulos citoplasmáticos, os eosinófilos também apresentam potencial para secretar metaloproteinases, enzimas proteolíticas que degradam vários componentes da matriz extracelular, e estão presentes em diversos processos fisiológicos e patológicos. Objetivo: Avaliar: 1) o perfil fenotípico dos eosinófilos na dermatite atópica do adulto, através da expressão das moléculas CCR3, CD23, CD38, CD69 e CD62L; 2) o perfil funcional, a partir da secreção de metaloproteinases, inibidores teciduais de metaloproteinases e RANTES por eosinófilos purificados. Métodos: Foram incluídos 41 adultos diagnosticados com DA, de acordo com os critérios de Hanifin & Rajka e 45 controles adultos sadios. A gravidade da doença foi mensurada através do escore de gravidade EASI (Eczema Area and Severity Index). Eosinófilos (LIN 1- CCR3+) do sangue periférico foram analisados para os marcadores CCR3, CD38, CD69, CD23 e CD62L através da citometria de fluxo (LSRFortessa, BD Biosciences) a análise foi realizada com o FlowJo 7.5.6 software. Eosinófilos purificados de indivíduos com DA e indivíduos controles foram estimulados com enterotoxina de Staphylococcus aureus B (SEB) e FSL-1 (agonista de receptores Toll-like 2 e 6), e os sobrenadantes foram coletados para dosagem de metaloproteinases (MMPs), inibidores teciduais de metaloproteinases 1 e 2 (TIMP-1 e TIMP-2) e RANTES por ELISA e por Cytometric bead array. Resultados: Indivíduos com DA apresentaram maior frequência de eosinófilos (LIN1- CCR3+), relacionada à gravidade da doença. Observou-se também, que a frequência de CD62L (L-selectina) e de CD23 (receptor de baixa afinidade para IgE) em eosinófilos (LIN1- CCR3+) diminui em pacientes com DA. Os receptores de ativação precoce (CD69) e tardio (CD38) não mostraram diferença estatística entre os grupos analisados. Os níveis séricos de MMPs e de TIMPs foram similares entre os controles e pacientes. Ao analisarmos a secreção de MMPs e de (TIMPs), a partir de eosinófilos purificados de pacientes com dermatite atópica, observamos diminuição dos níveis basais de TIMP-1 e TIMP-2 e de RANTES. Conclusões: Na DA do adulto, o perfil fenotípico e funcional dos eosinófilos mostrou: perfil de ativação da fase aguda, com expressão aumentada de CCR3; potencial de migração elevado, em decorrência da diminuição da expressão de CD62L; falhas no processo de ativação dos eosinófilos via CD23, bem como, no remodelamento tecidual mediado por TIMP-1 e TIMP-2 e na quimotaxia mediada por RANTES / Introduction: Atopic dermatitis (AD) is an inflammatory, chronic and recurrent skin disease characterized by intense pruritus and xerosis. AD has a complex etiopathogenesis, which involves the influence of genetics, environment, and immunological disorders, among others. Eosinophils are multifunctional polymorphonuclear leukocytes that contribute to the pathogenesis of several inflammatory processes, such as AD. In addition to the production and secretion of diverse proteins of the cytoplasmic granules, eosinophils have also the potential to secrete metalloproteinases (MMPs), proteolytic enzymes with a primary role for degrading several extracellular matrix components, present in distinct physiological and pathological processes. Objective: To evaluate:1) the phenotypic profile of eosinophils in adults with atopic dermatitis through the expression of CCR3, CD23, CD38, CD69 and CD62L molecules; 2) the functional profile through secretion of MMPs, tissue inhibitors of metalloproteinases 1 and 2 ( TIMP-1 and TIMP-2) and RANTES by purified eosinophils. Methods: This work enrolled 41 patients with AD, diagnosed according to Hanifin & Rajka\'s criteria) and 45 healthy controls. Severity of the disease was established utilizing EASI (Eczema Area and Severity Index). Eosinophils (Lineage cocktail 1- CCR3+) from peripheral blood were analyzed for CCR3, CD38, CD69, CD23 and CD62L by flow cytometry (LSRFortessa, BD Biosciences), and analysis was performed using the FlowJo 7.5.6 software. Purified eosinophils were stimulated with Staphylococcus aureus enterotoxin B (SEB) FSL-1 (Toll-like receptor 2/6 agonist), and supernatants were collected for MMPs, TIMPs and RANTES secretion, evaluated by ELISA and cytometric bead array (CBA). Results: Patients with AD have a higher frequency of eosinophils (LIN1- CCR3+), related to disease severity. Moreover, the frequency of CD62L (L-selectin) and CD23 (low-affinity receptor for IgE) in (LIN1- CCR3+) eosinophils was reduced in individuals with AD. CD69 and CD38 (early and late activation receptors) did not show significant difference in the studied groups. Serum levels of MMPs and of TIMP-1 and TIMP-2 were similar in healthy controls and AD patients. When analyzing secretion of MMPs and TIMPs by purified eosinophils from AD individuals, we detected a decrease in baseline levels of TIMP-1, TIMP-2, and reduced RANTES-mediated chemotaxis. Conclusions: Eosinophils in AD exhibit an activation profile of acute phase, with enhanced CCR3 expression, high potential for migration due to reduced expression of CD62, defective activation mechanisms via CD23, altered tissue remodeling process mediated by TIMP-1 and TIMP-2 and reduced RANTES-mediated chemotaxis

Introdução: A dermatite atópica (DA) é uma enfermidade cutânea inflamatória de caráter crônico, na qual o prurido é constante, e com marcada xerose. Dermatose que geralmente se inicia na infância, e pode surgir em indivíduos com história pessoal ou familiar de asma, rinite alérgica e/ou DA. A pele com DA apresenta colonização por Staphylococcus aureus (S. aureus) em 80-100% dos casos, sendo responsável pela produção enterotoxinas, capazes de exacerbar a resposta inflamatória na DA. Nesta enfermidade, existem distintos subtipos de células apresentadoras de antígeno ou dendríticas (DC), tanto na pele quanto circulantes. As DC exercem papel relevante na inflamação da DA, em especial um subgrupo de células dendríticas mieloides (mDC), as chamadas células dendríticas inflamatórias epidérmicas (IDEC). Objetivo: Avaliar o fenótipo e a função das mDC (IDEC-like) em células mononucleares do sangue periférico (PBMC) na DA do adulto. Métodos: Foram selecionados 21 pacientes com DA (idades entre18 e 65 anos, sendo 13 homens e oito mulheres) e 21 controles (idades entre 21 e 41 anos, sendo oito homens e 13 mulheres), nos quais foram realizadas as avaliações fenotípica e funcional das mDC (IDEC-like) em PBMC. Para tal, foram analisadas as expressões de: Fc?RI, TNF, IFN-y, IL-10, CD36 e CD83 nas mDC, estimuladas com enterotoxina estafilocócica B (SEB), agonistas de TLR2 (Pam3CSK4), TLR4 (LPS) e de TLR7/8 (CL097) através da citometria de fluxo. Resultados: Os principais achados nos pacientes com DA foram: aumento da frequência de células IDEC-like frente ao estímulo com agonista de TLR2 (Pam3CSK4); aumento da frequência de IFN-y em condição não estimulada, e de IL-10 frente a estímulo com agonista de TLR7/8 (CL097) nesta população de células dendríticas. Conclusão: A caracterização das mDC circulantes na DA evidencia perfil pró-inflamatório em condição não estimulada, impactando na resposta imune adaptativa. O aumento significativo na frequência de células IDEC-like nos pacientes com DA sugere sua participação na perpetuação do processo inflamatório da DA / Introduction: Atopic dermatitis (AD) is an inflammatory skin disease with a chronic course, with constant pruritus and marked xerosis. It usually starts during childhood, and a personal or familial history of skin and/or respiratory allergy may be present. Around 80-100% of the patients show a cutaneous colonization of Staphylococcus aureus (S. aureus), which produces enterotoxins that may exacerbate the inflammatory response in AD. In this disease, there are distinct subtypes of antigen-presenting cells or dendrytic cells (DC), either circulating or present in the affected tissue. DC exert a relevant role in AD inflammation, especially a subgroup of myeloid cells (mDC), known as epidermal inflammmatory dendritic cells (IDEC). Objective: To evaluate the phenotype and function of mDC (IDEC-like) in mononuclear cells of the peripheral blood (PBMC) of adults with AD. Methods: Twenty-one adults with AD (age 18/65; male/female: 13/8) and 21 healthy controls (age 21/41; male/female: 8/13) were selected for the current study. Phenotypical and functional analysis of mDC (IDEC-like) of PBMC were performed, through the expression of Fc?RI ,TNF, IFN-y, IL-10, CD36 and CD83 in mDC, stimulated with enterotoxin B (SEB) and with agonists of TLR2 (Pam3CSK4), TLR4 (LPS) and TLR7/8 (CL097) by flow cytometry. Results: Main findings of AD patients included: elevation of IDEC-like cell frequency with TLR2 (Pam3CSK4) agonist, augmented unstimulated frequency of IFN-y, and of IL-10 with TLR7/8(CL097) agonist of this population of dendritic cells. Conclusion: Characterization of circulating mDC on AD shows proinflammatory profile in unstimulated conditions, therefore causing impact on the adaptive immune response. The significant increase in the frequency of IDEC-like cells in AD patients suggest a role in the maintenance of inflammation in AD

La dermatite atopique, mieux connue sous le nom d’eczéma, est une condition chronique fréquente dont la prise en charge est souvent complexe et difficile à comprendre. Cette étude prospective vise la conception et la validation d'un guide pratique écrit ainsi qu'un questionnaire de connaissances en eczéma dans le but de combler un besoin concernant la disponibilité d'un programme d'éducation thérapeutique accessible à la population souffrant de cette maladie. Les objectifs secondaires sont d'évaluer l'impact de ces outils sur les connaissances en eczéma à court terme ainsi que l'impact du niveau de littératie en santé et des variables sociodémographiques sur ces mêmes connaissances. Un projet pilote a d’abord été conduit auprès d’onze futurs parents d'un cours prénatal de l'Université de Sherbrooke, suivi d'une phase expérimentale auprès de 107 patients âgés entre 18 et 45 ans de la clinique de dermatologie du Centre Hospitalier Universitaire de Sherbrooke (CIUSSS de l’Estrie-CHUS). Les analyses de validité et de fiabilité performées lors du projet pilote ont permis de faire les modifications nécessaires sur le guide pratique écrit ainsi que sur le questionnaire de connaissances afin de les rendre applicables à plus grande échelle. Ces outils modifiés ont par la suite été utilisés dans les études comparatives menées dans la phase expérimentale. Ces dernières ont démontré une amélioration statistiquement significative de la connaissance en eczéma à court terme par une comparaison des scores moyens au questionnaire de connaissances en pré-test et en post-test. Dans l’ensemble, aucune différence de progression de connaissance (delta entre le pré-test et le post-test) entre les différents sous-groupes de chaque variable sociodémographique n’a été démontrée de façon statistiquement significative. De plus, la courbe de progression de connaissances en pré-test et post-test est comparable entre les différents niveaux de littératie en santé. Ces derniers résultats démontrent l’absence d’impact des variables sociodémographiques étudiées ainsi que du niveau de littératie sur les outils à l’étude, ce qui supporte leur applicabilité à grande échelle. Mots clés: dermatite atopique, eczéma, éducation thérapeutique, guide pratique écrit, éducation en santé. / Atopic dermatitis, also known as eczema, is a common chronic disease for which treatments are often complex and difficult to understand. This prospective study aims to develop and validate a written action plan and a knowledge questionnaire about eczema in order to fill an existing gap in literature concerning the availability of a self-management training program that would be accessible to patients suffering from this condition. The secondary end-points aim to evaluate the impact of the written action plan as well as the impact of health literacy and other demographic variables on eczema knowledge over a short period of time. A pilot over 11 subjects was firstly conducted in a prenatal class at the University of Sherbrooke. Validity and reliability analyses were performed and allowed for alterations on the written action plan as well as the knowledge questionnaire so that they could be used on a larger scale. These modified assessment tools were then used on the 107 patients recruited at the dermatology clinic of the Centre Hospitalier Universitaire de Sherbrooke (CIUSSS de l’Estrie-CHUS) for comparative analyses. Results demonstrate a significant increase on the short-term eczema knowledge when comparing mean knowledge scores on the pre-test and post-test. On a general basis, there is no significant difference on the knowledge progression for the many demographic variables analysed (delta between pre-test and post-test mean scores). Also, knowledge progression between the different levels of health literacy is comparable. These results demonstrate a lack of impact of health literacy and other demographic variables on the studied assessment tools, which support their applicability on a larger scale. Keywords : Atopic Dermatitis, Eczema, Self-Management, Written Action Plan, Health Education.

Depuis plusieurs années, la vectorisation de molécules est considérée comme la stratégie la plus prometteuse pour améliorer la pénétration cutanée des principes actifs et pour cibler et contrôler leur libération, augmentant ainsi l'efficacité thérapeutique des traitements tout en limitant leurs effets secondaires. Les nanoparticules polymériques ont fait l'objet de nombreuses études car elles possèdent une très grande stabilité et une capacité supérieure aux autres vecteurs à libérer les principes actifs de façon prolongée. Récemment, le laboratoire de Pharmacie Galénique de Besançon a montré, in vivo, que les nanoparticules polymériques de diamètre inférieure ou égale à 100 nm pénétraient spécifiquement dans la peau inflammée de la souris, alors qu'aucune pénétration n'était observée dans la peau saine. Le premier objectif de cette thèse était de confirmer ces hypothèses dans la peau inflammée d'un autre animal, le porc. Les résultats de notre étude in vivo confirment l'absence de pénétration des nanoparticules polymériques dans la peau saine du porc et montrent une pénétration taille dépendante dans la peau inflammée. Le second objectif poursuivi était de vérifier ces résultats chez l'Homme. Pour cela, une preuve de concept a été mise en place dans le service de Dermatologie du CHRU de Besançon. Les premiers résultats de cette étude clinique semblent confirmer l'absence de pénétration des nanoparticules polymérique de I 00 nm dans la peau des volontaires sains et dans la peau non lésée des patients souffrant de dermatite atopique. A l'inverse, une forte pénétration des nanoparticules est observée au niveau des plaques d'eczéma des patients. Si les résultats de l'étude clinique se confirment, nous prévoyons d'encapsuler un anti­inflammatoire ou un immunosuppresseur pour vérifier l'intérêt thérapeutique de ces vecteurs en médecine humaine dans le traitement de la dermatite atopique. Parallèlement, une évaluation pourrait être réalisée en médecine vétérinaire pour le traitement de cette pathologie fréquente chez le chien, et dont le traitement actuel repose sur l'administration per os de corticoïdes à l'origine de nombreux effets secondaires. / For several years now, nanocarriers have been considered as the most promising strategy to improve skin penetration of active ingredients. Moreover, these carriers are more efficient at targeting and controlling drug release into the skin, which leads to increased treatment efficiency and reduced side effects. Polymeric nanoparticles have been the object of an increasing number of studies due to their good physicochemical stability and prolonged release of active ingredients which is superior to any other carriers. Recently, the Laboratory of Pharmaceutical Engineering at Besançon proved in vivo, that polymeric nanoparticles with a diameter smaller or equivalent to I 00 nm specifically penetrated in inflamed skin of mice whilst no penetration was observed in healthy skin. The first aim ofthis thesis was to confirm this hypothesis on another animal's inflamed skin, in instance the pig. Our results confirm the poor penetration of polymeric nanoparticles in healthy skin ofpigs and show various degree ofpenetration depending on the size of the nanoparticles into the inflamed skin area. The second objective ofthis work was to evaluate the skin penetration of our polymeric nanoparticles in humans. A proof of concept has been developed in the Department of Dermatology at Besancon University Hospital. The first results ofthis clinical trial tend to confim1 the greater penetration ofour carrier, specifically in inflamed skin. In fact, no penetration of polymeric nanoparticles with a size close to 100 nm was observed in healthy skin ofvolunteers or in the non-inflamed skin of patients suffering from atopic dennatitis. Conversely, a high penetration ofthese carriers was observed in the skin lesions of patients with atopic dermatitis. If the results ofthis clinical trial are confirmed, we plan to load an anti-inflammatory or an immunosuppressive drug into the nanoparticles to evaluate the therapeutic value ofthese nanocarriers in human medicine in the treatment ofatopic dermatitis. Meanwhile, a similar study may be undertaken in veterinary medicine for the treatment of atopic dermatitis in dogs which is a common disease whose current treatment is based on the oral administration of corticosteroids and cause many undesirable side effects.

La dermatite atopique (DA) est une dermatose inflammatoire dont la prévalence élevée est en constante augmentation dans les pays industrialisés. La physiopathologie de la DA est complexe et associe des facteurs immunologiques, environnementaux et génétiques. L'ensemble de ces facteurs touchent principalement l'épiderme et sont responsables d'une altération de la fonction barrière, ce qui facilite la pénétration transcutanée des molécules en contact avec la peau et la sensibilisation des individus. Ce travail de thèse vise à mieux caractériser le rôle de l'épiderme dans la physiopathologie de la DA. Il repose sur l'utilisation d'un modèle in vitro d'épiderme reconstruit humain généré à partir de cellules progénitrices de follicules pileux prélevés chez des patients atteints de DA, ainsi que chez des individus sains. Ce travail a tout d'abord montré que les épidermes DA générés à partir de patients non porteurs de mutations pour le gène de la filaggrine (FLG+/+) se comportaient comme des épidermes normaux, (i) tant à l'homéostasie, (ii) que suite à une stimulation par de l'interleukine-1beta (IL-1 β) ou par un allergène majeur de la DA, tel qu'un extrait d'acarien (Dermatophagoïdes farinae). Cependant, de façon très intéressante, les épidermes- DA se sont avérés davantage sensibles à l'apoptose induite par une exposition aux ultraviolets (UV), que les épidermes normaux. A cet égard, nous avons observé une modulation différente de certains gènes impliqués dans l'induction/régulation de l'apoptose par les épidermes provenant de patients DA après exposition aux UV. En outre, ce travail a démontré que des épidermes normaux sont profondément affectés par l'IL-1 β qui induit un phénotype atopique caractéristique associant (i) la production de quantité notable de TSLP (thymic stromal lymphopoietin) et (ii) une altération de la fonction barrière. L'impact de l'IL-1 β sur la biologie de l'épiderme et la sensibilité accrue exprimée par les épidermes-DA à l'apoptose induite par les UV sont donc de nouveaux éléments qui confirment l'importance de l'épiderme dans la physiopathologie de la DA ce qui permettra certainement de nouvelles stratégies thérapeutiques / Atopic dermatitis (AD) is an inflammatory skin disease with a high prevalence constantly increased in developed countries. The AD physiopathology is complex and associated with immunological, environmental and genetic factors. These factors impact mainly the epidermis and are responsible of the impaired barrier function which increases antigen penetration and people sensitization. The aim of this work is to improve the understanding of the epidermis role in AD physiopathology. For this, we used an in vitro reconstructed human epidermis model generated from outer root sheath cells taken off AD patients or normal individuals.First of all, this work has shown that epidermis generated from AD patients (AD- epidermis) with no mutation on filaggrine gene (FLG+/+) behave like those from normal individuals (Normal- epidermis), (i) at the steady state, and (ii) after stimulation with interleukin-1beta (IL-1 β) or a major allergen found in AD, Dermatophagoïdes farinae. However, interestingly, AD-epidermis are more sensitized to ultra-violets radiation-induced (UVR) apoptosis than Normal-epidermis. In this regard, we observed a higher expression of genes involved in the induction / regulation of apoptosis by the epidermis from AD patients after UV exposure. Moreover, this work has demonstrated that normal epidermis are profoundly affected by IL-1 β which induces an AD like phenotype associating (i) production of large amount of TLSP (thymic stromal lymphopoietin) and (ii) alterated barrier function. The impact of IL-1 β on epidermis biology and the increased sensitivity that seems to express AD-epidermis to UVR-induced apoptosis are new evidences that confirm the importance of the epidermis in pathophysiology of AD which certainly will lead to new therapeutic strategies

A prevalência de doenças alérgicas incluindo asma e dermatite atópica aumentou nos últimos anos. A asma é uma doença crônica caracterizada por hiper-responsividade das vias aéreas e limitação variável ao fluxo aéreo, reversível espontaneamente ou com tratamento. A elevada prevalência, mortalidade e custos associados tornam a doença um importante problema de saúde pública que requer atenção. A dermatite atópica (DA) é uma doença inflamatória crônica da pele, caracterizada por prurido intenso, eritema, escoriações, liquenificação na pele entre outras características. A DA causa profundo impacto na vida do indivíduo e da família e geralmente mostra os primeiros sintomas durante a infância. Ambas as doenças podem ser associadas a sensibilização a alérgenos. Nosso grupo mostrou que entre pacientes com rinite e/ou asma alérgicos a barata a tropomiosina da barata Periplaneta americana é um alérgeno principal. Em invertebrados, a tropomiosina induz resposta IgE e reatividade cruzada entre invertebrados incluindo ácaros, barata, camarão e parasitas. No presente estudo, produzimos tropomiosina recombinante de barata (alérgeno Per a 7) com elevado grau de pureza e com quantidades mínimas de endotoxina. Em modelo experimental de asma em camundongos, com sensibilização e desencadeamento com Per a 7 recombinante, houve aumento do número de células totais, e de eosinófilos, neutrófilos e linfócitos no lavado broncoalveolar. Alérgeno Per a 7 recombinante foi também utilizado em ELISA quimérico para investigar a resposta IgE a este alérgeno em 112 pacientes com dermatite atópica. A idade dos pacientes variou de 3 a 67 anos com média de 24,9 (± 15,4) anos, com 75 indivíduos do sexo feminino (67%). Nos 112 pacientes, o SCORAD apresentou média de 43,1 (± 18,1) e foram relatadas asma e rinite em 42 e 85 indivíduos, respectivamente. Níveis de IgE total apresentaram ampla variação, de 14,2 a 63.000 UI/mL, com média geométrica de 2.193 UI/mL. A idade de início dos sintomas e o tempo de doença apresentaram médias, respectivamente, de 9,5 (± 12,9) e 15,4 (± 12,3) anos. Trinta pacientes (26,8%) apresentaram níveis detectáveis de IgE para Per a 7 recombinante, com variação de 2,3 a 3.191 UI/mL. A razão de IgE específica para Per a7/IgE total nestes pacientes variou de 0,03% a 33,8%. Dividindo os pacientes em sensibilizados e não sensibilizados ao alérgeno de barata, observamos que não houve diferença significante entre os grupos com relação à idade de início dos sintomas, tempo de doença, SCORAD, presença de rinite ou asma e níveis de IgE total. Nossos resultados mostraram que o alérgeno Per a 7 recombinante induziu resposta inflamatória com caraterísticas semelhante à observada em humanos, em modelo experimental de asma em camundongos. Frequência menor de reatividade IgE ao alérgeno Per a 7 foi observada entre pacientes com dermatite atópica em nosso meio, quando comparada à observada previamente em pacientes com asma e/ou rinite. Não houve associação entre a presença de sensibilização IgE ao alérgeno Per a 7 e a gravidade da dermatite atópica, presença de asma ou rinite, idade de início e tempo de doença, e níveis de IgE total. Entretanto, a investigação do perfil de sensibilização IgE tem importância ao se considerar o uso de imunoterapia alérgenoespecífica em pacientes com dermatite atópica. / The prevalence of allergic diseases has increased in recent years, including asthma and atopic dermatitis. Asthma is a chronic disease resulting from airway hyperresponsiveness and variable airflow limitation, reversible spontaneously or with treatment. The high prevalence, mortality, and associated costs make the disease an important public health problem which requires attention. Atopic dermatitis (AD) is a chronic inflammatory skin disease, as asthma has high prevalence, and it is characterized by intense itching, erythema, excoriations, lichenification in the skin among other characteristics. The AD causes profound impact on individual and family´s life and usually shows the first symptoms during childhood. Both diseases could be related and associated with sensitization to allergens. In the case of asthma, allergy to cockroach is well known to be related with the disease. In 1999, a Brazilian study in our group has shown that among patients with allergic rhinitis and /or asthma the tropomyosin of the American cockroach Periplaneta americana (Per a 7) is a major allergen. This protein is present in vertebrates and invertebrates, and was related to induction of IgE response and cross-reactivity against among invertebrates, including mites, cockroaches, shrimp and parasites. In the present study, we produced recombinant tropomyosin from cockroach (Per a 7 allergen) with high purity and minimal amounts of endotoxin. In an experimental model of asthma in mice, with sensitization and triggering with recombinant Per a 7, there was an increase in the number of total cells, and eosinophils, neutrophils and lymphocytes in the bronchoalveolar lavage. Per a 7 allergen was also used in chimeric ELISA to investigate IgE response to this allergen in 112 patients with atopic dermatitis. Patients\' ages ranged from 3 to 67 years, mean of 24.9 (± 15.4) years, 75 female subjects (67%). In the 112 patients, a mean of SCORAD presented 43.1 (± 18.1) and asthma and rhinitis were reported in 42 and 85 individuals, respectively. Total IgE levels varied from 14.2 to 63.000 IU / mL, with a geometric mean of 2,193 IU / mL. The age at onset of symptoms and disease time presented averages, respectively, of 9.5 (± 12.9) and 15.4 (± 12.3) years. Thirty patients (26.8%) had detectable IgE recombinant Per a 7 levels, ranging from 2.3 to 3191 IU / mL. The ratio of IgA specific for Per a7 / total IgE in these patients ranged from 0.03% to 33.8%. Dividing the patients into sensitized and not sensitized to the cockroach allergen, we observed that there was no significant difference between the groups regarding the age of onset of symptoms, disease time, SCORAD, presence of rhinitis or asthma, and total IgE levels. Our results showed that recombinant Per a 7 allergen induced an inflammatory response with characteristics similar to that observed in humans in an experimental model of asthma in mice. Minor frequency reactivity of IgE Per a 7 allergen was observed among patients with atopic dermatitis in our group, when compared to that previously observed in patients with asthma and / or rhinitis. There was no association between the presence of IgE sensitization to the Per 7 allergen and the severity of atopic dermatitis, presence of asthma or rhinitis, age at onset and disease time, and total IgE levels. However, the investigation of the IgE sensitization profile is important when considering the use of allergen-specific immunotherapy in patients with atopic dermatitis

Termovizní zobrazování i 3D skenování jsou v současné době rychle se rozvíjející technologie. Obě technologie mají mnoho výhod, které by mohly být užitečné v medicíně. Jejich datová fúze přináší ještě více nových diagnostických informací, než kdyby byly použity samostatně. Cílem této práce je vývoj multispektrálního 3D skenovacího systému založeného na novém způsobu snímání pomocí robotického manipulátoru vybaveného laserovým snímačem, teplotní kamerou a barevnou kamerou. Navržené řešení přináší jak flexibilitu, tak přesnost. Tento systém skenování je dále využit v klinických aplikacích, aby byly ověřeny jeho schopnosti a ukázány přínosy nad rámec současného stavu techniky.

Oncogenic microRNAs (miRNAs) are small RNA molecules that inhibit post-translational regulatory mechanisms at the epigenetic level. miRNAs are often deregulated in malignancies and due to their stability are detectable in non-cellular fractions of peripheral blood. In our laboratory, we have performed several studies that have investigated and utilized miRNAs as biomarkers for various hematological tumors (e.g., chronic lymphocytic leukemia, Hodgkin`s lymphoma) and solid tumors (e.g., breast cancer). The aim of these studies was to find the association of miRNAs with pathophysiological and clinical aspects of each disease. Here, we confirmed the importance of particular miRNA or its complex during disease monitoring. Combining clinical, molecular biological and statistical analyses, we were able to find miRNA sets that fulfilled not only a diagnostic role but also a prognostic role beyond expectations. The main focus of this thesis is on the investigation of microRNAs in the diagnosis of a hematological malignancy - primary cutaneous T-cell lymphoma (CTCL). Tumor specificity of some miRNAs has been demonstrated. Their aberrant expression in tissue samples of CTCL patients obtained from skin biopsies, correctly distinguished malignant disease from control samples of benign skin lesions. Here, we...