Introduction: Les instillations nasales de solution saline isotonique (INSS) chez les enfants sont recommandées par les pédiatres et des oto-rhino-laryngologistes de notre institution dans le but de prévenir les otites moyennes aigues à répétition (OMAr). Cependant, aucune étude dans la littérature ne vient corroborer ou infirmer cette pratique. Objectifs: Déterminer l’efficacité des INSS dans la prévention des OMAr. Méthode: Projet pilote d’un essai clinique randomisé. Les enfants diagnostiqués avec des OMAr étaient éligibles. Les patients recrutés ont été randomisés en 2 groupes. Seul le groupe traitement procède aux INSS. L’issue primaire est l’incidence d’OMAr pendant une période de 3 mois. Résultats: Vingt-neuf patients satisfaisant les critères d’inclusion et d’exclusion ont consenti à participer. Le taux d’OMAr était inférieur dans le groupe traitement (p=0.03, chi-carré) Conclusion: Les INSS semblent efficaces dans la prévention des OMAr. Une étude multicentrique est indiquée pour vérifier la validité externe et confirmer la sécurité. / Introduction: Normal saline nasal cavity irrigations (NSNI) are commonly recommended by pediatricians and otolaryngologists to prevent recurrent acute otitis media (rAOM). However, no published scientific study corroborates or invalidates this practice. Goal: To determine the efficacy of NSNI to prevent rAOM. Method: Pilot randomized controlled clinical trial. All consecutive patients with a diagnosis of rAOM were eligible. Recruited patients were randomized in 2 groups. Only patients in the treatment group proceeded with NSNI. The primary outcome of the study was the incidence of rAOM observed during a 3 month period. Results: Twenty-nine patients met the inclusion and exclusion criteria and agreed to participate. There was a statistically significant lower incidence of rAOM in the treatment group (p=0.003, Fisher exact). Conclusion: Our results suggest that NSNI could effectively prevent rAOM. A larger scale randomized multicentre study is feasible, and it must be done in order to verify for external validity and to properly assess safety issues.

L’hépatite autoimmune (HAI) est une maladie grave affectant le foie et présentant un haut taux de mortalité lorsque non traitée. Les traitements disponibles sont efficaces, mais de graves effets secondaires leur sont associés. Ces effets secondaires sont généralement le résultat d'une forte immunosuppression et d’autres sont spécifiques à chaque médicament. Aucune immunothérapie spécifique n’est présentement disponible pour le traitement de l’HAI. Récemment, un modèle murin d’HAI a été développé dans notre laboratoire par xénoimmunisation des souris C57BL/6 avec les antigènes humains de l'HAI de type 2. Ce modèle présente la plupart des caractéristiques biochimiques et cliniques retrouvées chez les patients atteints d'HAI de type 2. Dans cette étude, nous avons évaluée l’efficacité de deux types de traitement pour l’HAI de type 2 à l’aide de notre modèle murin. Dans un premier temps, l’anticorps anti-CD3ε a été étudié en prophylaxie et en traitement. Nous avons montré qu’une posologie de 5µg d’anti-CD3 i.v. par jour pendant 5 jours consécutifs induit une rémission chez les souris avec HAI de type 2 établie (traitement). Cette rémission est caractérisée par une normalisation des niveaux d’alanine aminotransférase et une diminution significative de l’inflammation hépatique. Cette rémission semble être associée à une déplétion partielle et transitoire des lymphocytes T CD3+ dans la périphérie et une augmentation des lymphocytes T régulateurs CD4+, CD25+ et Foxp3+ dans le foie. La même posologie lorsqu’elle est appliquée en prophylaxie n’a pas réussi à prévenir l’apparition de l’HAI de type 2. La deuxième voie de traitement consiste en l’administration par voie intranasale d’un forte dose de formiminotransférase cyclodésaminase murin (mFTCD), un autoantigène reconnu dans l’HAI de type 2. Une administration en prophylaxie par voie intranasale de 100µg de mFTCD par jour durant 3 jours consécutifs arrive à prévenir l’HAI de type 2 en diminuant l’inflammation hépatique au bout de deux semaines post-traitement. / Autoimmune hepatitis (AIH) is a severe liver disease with high mortality rates if left untreated. Current treatments, while effective, are associated with deleterious side-effects. These side effects are specific to each drug and the result of broad immunosuppression. Recently, a murine model of type 2 AIH has been created in our laboratory in wild-type naïve mice. In this model, DNA immunization with type 2 AIH human autoantigens breaks immune tolerance and induces an autoimmune response against the liver. Lately, new therapeutic strategies based on depletion of specific immune cell populations have been proposed for the treatment of several diseases, including autoimmune diseases. Currently, no immunotherapies using biological agents are available for the treatment of autoimmune liver diseases. Therefore, the goal of this project is to study the efficacy of new immunotherapeutic agents for the treatment of type 2 AIH in an experimental model. We evaluated the effectiveness of two approaches for treating type 2 AIH. First, we tested the anti-CD3ε antibody in prophylaxis and in treatment of type 2 AIH. We showed that a dosage of 5µg i.v. of anti-CD3ε antibody per day for 5 consecutive days induced remission in mice with established type 2 AIH. This remission was defined as a normalization of serum alanine aminostransferase levels and a significant decrease of liver inflammation in treated mice. This remission seems to be associated with a transitory depletion of CD3+ T lymphocytes in peripheral blood mononuclear cells and increased CD4+CD25+Foxp3+ regulatory T lymphocytes in the liver. But when this dosage was applied in prophylaxis, it could not prevent the induction of type 2 AIH. The second approach was to induce tolerance by nasal administration of murin formiminotransferase cyclodeaminase (mFTCD), an autoantigen of type 2 AIH. We showed that nasal administration of 100µg of mFTCD for 3 consecutive days prevented development of type 2 AIH in prophylaxis by reducing liver inflammation.

Trichobilharzia regenti is a nasal avian schistosome which has during the initial phase of infection an affinity to the nervous system. Larvae migrate through the central nervous system to the nasal mucosa of waterfowl, where they mature and reproduce. Until now this infection phase has been described only marginally. Adults are located in the nasal mucosa approximately from 13th to 24th day post infection. During this life phase, they migrate through the vascularized connective tissue and lay eggs, the presence of which has been detected in the tissue only. Maturation and hatching of miracidia occur in the tissue (unlike human schistosomes). The parasite causes inflammation, and the tissue is infiltrated with immune cells. Lymphocytes, granulocytes, macrophages, plasma cells and giant multi-nuclear cells were described by histological methods. The thesis is focused on a more detailed description of cellular immune response and histopathological changes of the tissue by means of histological stains, and antibody/lectin probes. The flukes were observed more frequently in the blood vessel lumen, together with a higher number of immune cells compared to the healthy duck. Infiltration by a high number of lymphocytes occurred in the tissue, the macrophages were frequently observed in clusters around the...

Esta tese objetiva analisar processos de variação linguística em tempo real (LABOV, 1994) no português brasileiro de contato com variedades dialetais italianas, para verificar se existe coerência dialetal (GUY e HINSKENS, 2016) na comunidade de Flores da Cunha (RS). Para tanto, fizemos análise quantitativa conforme metodologia de análise de regra variável (LABOV, 1972), para cotejar o padrão de variação de -ão::-on em tempo real com o de outro processo na mesma comunidade. A pesquisa foi embasada em dados de fala provenientes de dois acervos distintos, o do projeto VARSUL (1990) e do Projeto BDSer (2008/2009). Na „troca‟ de -ão por –on, houve um decréscimo na frequência total de aplicação da regra variável: 30,7% nos dados do VARSUL e 21,6% nos do BDSer, indício de regressão de regra. Para verificar a existência de coerência dialetal, compararam-se os resultados da análise de ão::on com os do estudo de Azeredo (2012), sobre o uso variável de tepe em lugar de vibrante no português de contato com variedades dialetais italianas e observamos que as variantes atribuídas ao contato são, ambas, favorecidas pelos homens de mais idade e com nível mais baixo de escolaridade. Essa correlação entre as variáveis analisadas aponta para a existência de coerência dialetal na comunidade de fala em estudo. / This thesis aims at analyzing linguistic variation processes in real time (LABOV, 1994) in Brazilian Portuguese in contact with Italian dialectal varieties, to verify if there is lethal coherence (GUY and HINSKENS, 2016) in the community of Flores da Cunha (RS). We performed a quantitative analysis according to the methodology of variable rule analysis (LABOV, 1972), to compare the variation pattern of -ão::-on in real time with that of another process in the same community. Our research was based on speech data from two different collections, the VARSUL project (1990) and the BDSer Project (2008/2009). The analysis showed a decrease in the total frequency of use of -on in contexts where -ão is expected: 30.7% in the speech of VARSUL informants and 21.6% in the speech of BDSer informants, a sign of rule regression. We compared the results of the analysis of -ão::-on with the results of Azeredo (2012) about the use of tap where a trill is expected. We observed that older men with low level of schooling favor the variants under analysis. The correlation between the analyzed variables points to the existence of lethal coherence in the speech community under study.

Esta tese objetiva analisar processos de variação linguística em tempo real (LABOV, 1994) no português brasileiro de contato com variedades dialetais italianas, para verificar se existe coerência dialetal (GUY e HINSKENS, 2016) na comunidade de Flores da Cunha (RS). Para tanto, fizemos análise quantitativa conforme metodologia de análise de regra variável (LABOV, 1972), para cotejar o padrão de variação de -ão::-on em tempo real com o de outro processo na mesma comunidade. A pesquisa foi embasada em dados de fala provenientes de dois acervos distintos, o do projeto VARSUL (1990) e do Projeto BDSer (2008/2009). Na „troca‟ de -ão por –on, houve um decréscimo na frequência total de aplicação da regra variável: 30,7% nos dados do VARSUL e 21,6% nos do BDSer, indício de regressão de regra. Para verificar a existência de coerência dialetal, compararam-se os resultados da análise de ão::on com os do estudo de Azeredo (2012), sobre o uso variável de tepe em lugar de vibrante no português de contato com variedades dialetais italianas e observamos que as variantes atribuídas ao contato são, ambas, favorecidas pelos homens de mais idade e com nível mais baixo de escolaridade. Essa correlação entre as variáveis analisadas aponta para a existência de coerência dialetal na comunidade de fala em estudo. / This thesis aims at analyzing linguistic variation processes in real time (LABOV, 1994) in Brazilian Portuguese in contact with Italian dialectal varieties, to verify if there is lethal coherence (GUY and HINSKENS, 2016) in the community of Flores da Cunha (RS). We performed a quantitative analysis according to the methodology of variable rule analysis (LABOV, 1972), to compare the variation pattern of -ão::-on in real time with that of another process in the same community. Our research was based on speech data from two different collections, the VARSUL project (1990) and the BDSer Project (2008/2009). The analysis showed a decrease in the total frequency of use of -on in contexts where -ão is expected: 30.7% in the speech of VARSUL informants and 21.6% in the speech of BDSer informants, a sign of rule regression. We compared the results of the analysis of -ão::-on with the results of Azeredo (2012) about the use of tap where a trill is expected. We observed that older men with low level of schooling favor the variants under analysis. The correlation between the analyzed variables points to the existence of lethal coherence in the speech community under study.

Este estudo tem como objetivo verificar a eficácia do Modelo de Ontogenia (MAJOR, 1987), por meio da análise do processo de aquisição do português como segunda língua (L2) por falantes de espanhol no nível fonético-fonológico. Para tanto, reportamo-nos a pressupostos teóricos que antecedem o dito modelo: a Análise Contrastiva (LADO, 1957), o Modelo de Interlíngua (SELINKER, 1972) e a Hipótese da Marcação Diferencial (ECKMAN, 1987). O Modelo de Ontogenia descreve o processo da aquisição de L2 no nível fonético-fonológico dividindo-o em duas fases: interferência, quando o aprendiz passa a sofrer grande influência de sua L1, e desenvolvimento, quando o aprendiz pode passar por um processo semelhante ao da criança em fase de aquisição da L1. Nosso objeto de estudo foi o processo de aquisição das vogais médio-abertas e das vogais nasais do português, realizando uma análise comparativa entre o sistema fonético-fonológico do português e do espanhol, com enfoque no sistema vocálico. Nessa análise, incluímos o percurso histórico da origem das vogais e a questão da nasalidade em ambas as línguas. Embora este estudo seja centrado na observação da interferência da L1 na aquisição de L2, consideramos também, a interferência de fatores extralinguísticos (KRASHEN, 1982). Para elaboração do corpus foram realizadas duas gravações, com intervalo entre 8 a 18 meses, com quatro pós-graduandos da Universidade de São Paulo, denominados Informantes. A metodologia consistiu na aplicação de entrevista espontânea e leitura de palavras, frases e trecho de texto literário. Acreditamos que nosso trabalho possa contribuir para o desenvolvimento da área de Português para Falantes de Outras Línguas (PFOL) por agregar informações sobre as dificuldades dos aprendizes falantes de espanhol na aquisição do sistema vocálico. / This study aims to examine the efficiency of The Ontogeny Model (Major, 1987) through analysis of the phonetic and phonological aspects of the acquisition of Portuguese as a second language (L2) by native speakers of Spanish. To do this, we relied on the following theoretical assumptions which preceded the model: Contrastive Analysis (Lado, 1957), Interlanguage Model (Selinker, 1972) and Differential Markedness Hypothesis (Eckman, 1987). The Ontogeny Model describes the process of L2 acquisition in the phonetic and phonological levels by dividing it in two stages: interference, when the learner has great influence of his/her first language (L1), and development, when the learner may pass through a process similar to the child acquiring L1. Our object of study is the acquisition process of Portuguese open-mid vowels and nasal vowels, establishing a comparative analysis between the phonetic and phonological systems of Portuguese and English, with focus on the vowel system. In this analysis, we include the historical progression of the vowel origins and the issue of nasality in both languages. Despite this study being centered in the observation of L1 interference in L2 acquisition, we also considered the influence of extra-linguistic factors (Krashen, 1982). To compose the corpus, two recordings were done, with an interval between 8 to 18 months, with four informers, postgraduate students at Universidade de São Paulo. The methodology consisted of spontaneous speech interview and the reading of words, sentences and a literary excerpt. We believe that our work can contribute to the development of the area of Portuguese to Speakers of Other Languages (PSOL) as it assembles information about the difficulties that speakers of Spanish have in the acquisition of the vowel system

Orientador: Lucy Seki / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Estudos da Linguagem / Made available in DSpace on 2018-08-19T17:23:42Z (GMT). No. of bitstreams: 1 Pessoa_KatiaNepomuceno_D.pdf: 32464697 bytes, checksum: d889efd1819683032b3b3b0071e3161c (MD5) Previous issue date: 2012 / Resumo: Botocudo é um nome genérico aplicado aos índios que tinham como padrão de cultura o uso de botoques de madeira, auriculares e labiais, prezando por adornos corporais (pulseiras, perneiras, colares e pintura no corpo) (MARCATO, 1979). Esses índios ocupavam as regiões do interior do leste e sudeste brasileiro, vivendo em faixas florestais da Mata Atlântica e Zona da Mata e foi sobre eles que se deu a maior repressão oficial e extraoficial do homem branco contra uma população indígena já registrada no Brasil, o que levou ao desaparecimento de quase todos os membros pertencentes a este grupo. Atualmente, os únicos representantes sobreviventes são os índios Krenak, que habitam a Aldeia Krenak, às margens do rio Doce, entre as cidades de Conselheiro Pena e Resplendor, no Estado de Minas Gerais. A língua Krenak é considerada como uma língua em situação de extremo risco de extinção. Mesmo assim, assumimos que a língua continua viva e que deve, portanto ser descrita, a fim de que possamos contar com estudos cada vez mais recentes e que ofereçam mais informações linguísticas sobre as línguas da família Botocudo/ Krenak Neste trabalho, temos como objetivo principal descrever aspectos fonéticos e fonológicos da língua Krenak. De modo complementar, apresentamos uma descrição da morfossintaxe da língua, segundo Seki (2000, 2001, 2004) e, a partir destes estudos, propomos uma análise preliminar de contos Botocudo, coletados pelo pesquisador russo Heri Herikovich Manizer, em 1915. Os dados de áudio foram coletados por Seki, entre os anos de 1979 e 1981. O estudo fonético revelou características articulatórias das nasais surdas e dos segmentos de contorno (as nasais pré e pós oralizadas) da língua. A descrição fonológica permitiu distinguir vogais orais e nasais, diferentemente do que fora proposto anteriormente no estudo de Silva (1986). As reflexões fonológicas tiveram por base a Geometria dos Traços (Clements e Hume, 1995) e a tipologia proposta por Wetzels (2008), a qual nos permitiu argumentar em favor da existência de uma classe /B/ como subjacente no sistema da língua, mesmo que sua manifestação superficial seja predominantemente realizada por meio da forma bifásica [mb]. Na coda, foi possível identificar a presença de segmentos de contorno parcialmente orais/nasais, os quais ocorrem como uma estratégia da língua para preservar a distinção entre vogais orais x nasais. Estas considerações foram determinantes para a definição do padrão silábico e dos processos fonológicos observáveis na língua. Ainda, o estudo fonológico permitiu verificar características referentes ao acento do Krenak. Ainda, a fim de apresentar uma visão mais ampla da língua, incluímos em nossa discussão um resumo do estudo de Seki acerca das características morfossintáticas da língua Krenak. A partir dos estudos apresentados, passamos para uma apreciação dos contos Botocudo coletados por Manizer em 1915. Estes contos apresentam-se como um material inédito e podem vir a contribuir para a recuperação de aspectos lingüísticos e culturais do grupo Krenak. Veremos que, apesar de ser possível ter uma compreensão geral sobre o funcionamento da língua, existem ainda vários aspectos que precisam ser esclarecidos, como a função de alguns morfemas e partículas. No entanto, estes avanços talvez sejam viáveis apenas por meio de uma nova coleta que conte com a colaboração direta dos falantes atuais / Abstract: Botocudo is a common name applied to the natives who has a cultural pattern used to use wooden, lip and auricular botoques, valuing body adornments (bracelets, necklaces, body painting and others) (MARCATO, 1979). These natives used to occupy the Brazilian east and southeast hinterland, living in forest belts of the Atlantic Forest and Forest Zone and it was over them that there was the greater official and "off the record" white man repression against an indigenous population ever registered in Brazilian records, which led to the disappearance of almost all the members belonging to this group. Nowadays, the only remaing survivors are the Krenak indians who live in the Krenak village, on the banks of Rio Doce, between the cities of Conselheiro Pena and Resplendor, in the state of Minas Gerais. The Krenak language is considered as in extreme extinction risk. Even though, it is assumed that it is still alive and, therefore, it must be described hence we can count on new and recent studies which provide more linguistic data on the Botocudo/Krenak family. In this dissertation, the main goal is to describe phonetic and phonologic aspects of the Krenak language. As a complement, we present a description of the language's morphosyntax, according to Seki (2000, 2001, 2004) and, from these studies, we propose a preliminary analysis of Botocudo tales, collected by the Russian researcher Heri Herikovich Manizer, in 1915. The audio data was collected by Seki, between the years of 1979 and 1981. The phonetic study revealed articulatory characteristics of voiceless nasals and the contour segments (the pre and post oralized nasals) of the language. The phonological description allowed distinguishing oral and nasal vowels, differently from what was posed by Silva (1986). The phonological analysis were based on the Feature Geometry (Clements e Hume, 1995) and the typology on Wetzels (2008), which granted us talking about the existence of a class /B/ as an underlying segment of the language system, even if its superficial manifestation is mostly done through its biphasic form [mb]. In the coda it was possible to identify the presence of contour segments partially oral/nasal, which occur as a language strategy to preserve the distinction between oral and nasal vowels. These considerations were determinant to define the syllabic pattern and the language observable phonological processes. Yet, the phonological study allowed verifying characteristics related to the Krenak accent. In order to present a wider perspective of the language, we included in this work a summarized Seki's study concerning morphossyntatic characteristics of Krenak language. From the present studies, we go for an analysis of the Botocudo tales collected by Manizer in 1915. These tales are presented as an unpublished material and might contribute to the recovery of cultural and linguistic aspects of Krenak. We will see that, besides the possibility of having a general understanding of the language functionality, there are, still, many aspects which must be clarified such as, the function of certain morphemes and particles. However, these improvements may become impossible to happen until its done a new collection relied on the directly contribution of the current speakers / Doutorado / Linguistica / Doutor em Linguística

Esta tese objetiva analisar processos de variação linguística em tempo real (LABOV, 1994) no português brasileiro de contato com variedades dialetais italianas, para verificar se existe coerência dialetal (GUY e HINSKENS, 2016) na comunidade de Flores da Cunha (RS). Para tanto, fizemos análise quantitativa conforme metodologia de análise de regra variável (LABOV, 1972), para cotejar o padrão de variação de -ão::-on em tempo real com o de outro processo na mesma comunidade. A pesquisa foi embasada em dados de fala provenientes de dois acervos distintos, o do projeto VARSUL (1990) e do Projeto BDSer (2008/2009). Na „troca‟ de -ão por –on, houve um decréscimo na frequência total de aplicação da regra variável: 30,7% nos dados do VARSUL e 21,6% nos do BDSer, indício de regressão de regra. Para verificar a existência de coerência dialetal, compararam-se os resultados da análise de ão::on com os do estudo de Azeredo (2012), sobre o uso variável de tepe em lugar de vibrante no português de contato com variedades dialetais italianas e observamos que as variantes atribuídas ao contato são, ambas, favorecidas pelos homens de mais idade e com nível mais baixo de escolaridade. Essa correlação entre as variáveis analisadas aponta para a existência de coerência dialetal na comunidade de fala em estudo. / This thesis aims at analyzing linguistic variation processes in real time (LABOV, 1994) in Brazilian Portuguese in contact with Italian dialectal varieties, to verify if there is lethal coherence (GUY and HINSKENS, 2016) in the community of Flores da Cunha (RS). We performed a quantitative analysis according to the methodology of variable rule analysis (LABOV, 1972), to compare the variation pattern of -ão::-on in real time with that of another process in the same community. Our research was based on speech data from two different collections, the VARSUL project (1990) and the BDSer Project (2008/2009). The analysis showed a decrease in the total frequency of use of -on in contexts where -ão is expected: 30.7% in the speech of VARSUL informants and 21.6% in the speech of BDSer informants, a sign of rule regression. We compared the results of the analysis of -ão::-on with the results of Azeredo (2012) about the use of tap where a trill is expected. We observed that older men with low level of schooling favor the variants under analysis. The correlation between the analyzed variables points to the existence of lethal coherence in the speech community under study.

Orientador: Maria Palmira Daflon Gremião / Resumo: Glioblastomas (GBM) representam 77% dos tumores malignos do sistema nervoso central (SNC) e ainda hoje, apesar de todos os avanços na terapia, continua com prognóstico limitado. A existência de barreiras fisiológicas como a barreira hematoencefálica (BHE) representa o principal obstáculo que impede que concentrações adequadas do fármaco atinjam o local de ação. Por suas vantagens anatômicas, uma estratégia proposta para a administração de fármacos destinados ao SNC consiste no uso da via nasal. Além disso, o uso de terapias combinadas utilizando fármacos capazes de agir em diferentes alvos moleculares deve ser considerada para o tratamento de doenças complexas como GBM. O candidato a fármaco ácido alfa-ciano-4-hidroxicinâmico (CHC) e o anticorpo monoclonal cetuximab (CTX) já são explorados devido à capacidade de agir em diferentes alvos moleculares nas células tumorais e aplicados em conjunto, como uma nova abordagem combinada, podem melhorar os resultados terapêuticos. De forma complementar, a utilização de sistemas de liberação baseados em nanotecnologia trará inevitavelmente ganhos terapêuticos à combinação proposta, permitindo que atributos específicos sejam agregados ao sistema e possibilite não somente a administração nasal, como também a associação de diferentes fármacos em um único carreador. Assim, o presente estudo propõe o desenvolvimento de diferentes plataformas poliméricas baseadas em poli(ácido láctico-co-glicólico) (PLGA) e quitosana trimetilada (TMC) ou quito... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Glioblastomas (GBM) account for 77% of malignant tumors in the central nervous system (SNC), and today, despite all advances in therapy, remains with a limited prognosis. The existence of physiological barriers as the blood brain barrier (BBB) represents the main obstacle that limits appropriate concentrations of drugs designed to therapy. Due to their anatomical advantages, a strategy proposed for direct delivery to SNC involves the use of the nose-to-brain route. Besides, combination therapy that uses multiple drugs against different molecular targets should be considered for complex diseases such as GBM. Drugs like alpha-cyano-4-hydroxycinnamic acid (CHC) and the monoclonal antibody cetuximab (CTX) are already explored for their capacity to act against different hallmarks of cancer and applied together, as a novel combining approach, might improve therapeutic outcomes. Therefore, advances in nanotechnology-based delivery systems will inevitably bring therapeutic gains to the proposed combination since they enable acquisition of important characteristics desired and also the association of different drugs into a single carrier. Thus, the current study proposes the development of different polymeric platforms based on poly(lactic-co-glycolic acid) (PLGA) and trimethyl chitosan (TMC) /chitosan oligosaccharide (OCS) for CHC encapsulation. Both CHC-loaded developed systems (PLGA/TMC and PLGA/OCS) exhibited nanostructure organization of about 300 to 400 nm, containing chitosan o... (Complete abstract click electronic access below) / Doutor

Einleitung: Die Osteoarthritis ist für einen Großteil der Lahmheiten beim Pferd verantwortlich. Durch die starken Belastungen der equinen Gelenke schreitet die Osteoarthritis unweigerlich fort und setzt diese Tiere einem hohen Leidensdruck aus. Bisherige Therapien reichen nicht aus, um in osteoarthritischen Gelenken die physiologische Integrität des Knorpels wiederherzustellen. Humane und caprine nasale Knorpelzellen zeigten in präklinischen und klinischen Studien ein hohes Regenerationspotential und eine hohe Integrität nach autologer Implantation in Defekte des Gelenkknorpels. Dies wurde auf ihr „Hox-Gen-negatives“ Expressionsprofil zurückgeführt, das sich nach der Implantation dem des artikulären Knorpels anpasste. Ziel der Studie: Das Hox-Genexpressionsprofil nasaler Chondrozyten sollte mit denen artikulärer Chondrozyten in der Zellkultur verglichen werden, um den nasalen Knorpel auch beim Pferd als mögliche autologe Knorpelquelle zu identifizieren. Tiere, Material und Methoden: Knorpelgewebe wurde von 7 verstorbenen Pferden aus dem Nasenseptum und einem vorderen sowie hinterem Fesselgelenk entnommen, Chondrozyten isoliert und bis zur vierten Passage zweidimensional kultiviert. Während der Kultivierung wurden die Chondrozyten alle 3 bis 4 Tage mittelseines eigens erstellten Beurteilungsbogens evaluiert. Zellen aus der ersten (T1) und der dritten (T2) Subkultivierung wurden lysiert, die RNA extrahiert und in cDNA umgeschrieben. Es folgte eine qPCR, um die Expressionslevel von drei Hox-Genen (A3, D1, D8) und zwei knorpeltypischen Genen (SOX9, Kollagen II) an den drei verschiedenen Lokalisationen während T1 und T2 zu bestimmen. Die Quantifizierung der relativen Genexpression erfolgte anschließend mit der ΔΔCT-Methode unter Verwendung von RPL32 und GAPDH als Housekeeping-Gene. Zur statistischen Auswertung wurden die Multiple Lineare Regression, eine einfaktorielle Varianzanalyse (ANOVA) und ein zweiseitiger t-Test herangezogen. Die Signifikanzniveaus aller statistischen Tests wurden auf α= 0,05 festgesetzt. Ergebnisse: Die Hox-Genexpressionen unterschieden sich in Bezug auf die drei Lokalisation und die Messzeitpunkte nicht signifikant voneinander. Die nasalen Chondrozyten wiesen während der ersten Subkultivierung gegenüber den artikulären Chondrozyten signifikant höhere Kollagen-II-Expressionen auf. Eine „Hox-Gen-negative“ Expression konnte für die Tierart Pferd nicht bestätigt werden. Die vorliegende Arbeit zeigt, dass Pferde wahrscheinlich ein speziesspezifisches Hox- Gen-Expressionsmuster aufweisen und dass die equinen Hox-Genexpressionsprofile statistisch signifikanten individuellen Einflüssen unterliegen. Schlussfolgerung: Das equine Hox-Genexpressionsprofil unterliegt statistisch signifikanten individuellen Einflüssen, die bei einer potenziellen Zelltherapie zu beachten sind. Nasale Chondrozyten eignen sich beim Pferd aufgrund ihrer genetischen Ähnlichkeit, bezogen auf die Expressionen der untersuchten Hox-Gene, zu artikulären Chondrozyten und ihrer hohen Bereitschaft zur Kollagen-II-Bildung wahrscheinlich als potenzielle Quelle für die autologe chondrozytäre Implantation (ACI).:1. Einleitung ...................................................................................................... 1 2. Literaturübersicht .......................................................................................... 2 2.1 Knorpel ..................................................................................................... 2 2.1.1 Allgemeiner Überblick ....................................................................... 2 2.1.2 Chondrogenese und Regeneration .................................................. 3 2.1.3 Intraartikulärer Hyaliner Knorpel ....................................................... 3 2.1.4 Extraartikulärer Hyaliner Knorpel....................................................... 6 2.2 Osteoarthritis bei Mensch und Pferd ........................................................ 8 2.2.1 Bedeutung und Ätiologie ................................................................... 8 2.2.2 Pathogenese ..................................................................................... 9 2.2.3 Diagnostik ........................................................................................ 10 2.2.4 Bisherige Therapieansätze .............................................................. 12 2.3 Knorpelgewebe in der Forschung .............................................................13 2.3.1 Kultivierung von Chondrozyten ......................................................... 13 2.3.2 Tiermodelle in der Osteoarthritisforschung ....................................... 15 2.3.3 Neue Therapieansätze ..................................................................... 17 2.3.3.1 Stammzellbasierte Verfahren ...................................................... 17 2.3.3.2 Artikuläre autologe Chondrozyten .............................................. 19 2.3.3.3 Nasale Chondrozyten ................................................................. 21 2.4 Hox-Gene ................................................................................................ 22 2.4.1 Allgemeiner Überblick ..................................................................... ..22 2.4.2 Regulation der Hox-Gene ................................................................. 23 2.4.3 Erkrankungen im Zusammenhang mit Hox-Genen ........................... 25 3. Ziel der Studie ........................................................................................... 27 4. Publikation ................................................................................................ 28 5. Diskussion................................................................................................. 45 5.1 Primer ....................................................................................................46 5.2 Auswahl der Messzeitpunkte und Proben ............................................. 47 5.3 Hox-Genprofile kultivierter equiner Chondrozyten ................................ 49 5.4 Individuelle Hox-Genprofile ................................................................... 50 5.5 SOX9 und Kollagen II .............................................................................51 5.6 Hox-Genprofile im Vergleich verschiedener Spezies ............................. 53 5.7 Ausblick ................................................................................................ 53 6. Schlussfolgerung ...................................................................................... 55 7. Zusammenfassung ................................................................................... 56 8. Summary .................................................................................................. 58 9. Referenzen .............................................................................................. 60 9.1 Literaturverzeichnis .............................................................................. 60 9.2 Abbildungsverzeichnis.......................................................................... 71 10. Anhang .................................................................................................. 72 10.1 Evaluierungsbogen Chondrozytenkulturen ........................................ 72 10.2 Auszug aus der Korrelationsmatrix ..................................................... 73 10.3 Publikationsverzeichnis Christiane Storch .......................................... 74 11. Danksagung .......................................................................................... 75 / Introduction: Osteoarthritis is responsible for most of the lameness in horses. Due to severe stress on equine joints, osteoarthritis inevitably progresses and results in a high degree of suffering. Current therapeutic options are not sufficient to restore the physiological integrity of the cartilage in osteoarthritic joints. Human and caprine nasal chondrocytes demonstrated high regenerative potential and integrity after autologous implantation into articular cartilage defects in preclinical and clinical studies. This was attributed to their “Hox gene negative” expression profile, matching the profile of articular cartilage after implantation. Aim of the study: The Hox gene expression profile of nasal chondrocytes was compared with those of articular chondrocytes in a cell culture to address nasal cartilage as a possible autologous source also in horses. Animals, Material and Methods: Cartilage was harvested from the nasal septum, one anterior and one posterior fetlock joint of deceased 7 horses, chondrocytes were isolated and cultured two-dimensionally until the fourth passage. During cultivation, chondrocytes were evaluated every 3 to 4 days using a specially designed assessment sheet. Cells were harvested during the first (T1) and third (T2) subcultivation. RNA was extracted and transcribed into cDNA. Subsequently, qPCR was performed to determine the expression levels of three Hox genes (A3, D1, D8) and two tissue-identifying genes (SOX9, collagen II) of the three locations after T1 and T2. Quantification of relative gene expression was performed with the ΔΔCT method using RPL32 and GAPDH as housekeeping genes. Multiple linear regression, one-way analysis of variance (ANOVA), and two-tailed t-test were used for statistical analysis. The significance levels of all statistical tests were set at α= 0.05. Results: Hox gene expressions were not significantly different in terms of localization and measurement time points. Nasal chondrocytes exhibited significantly higher collagen II expression than articular chondrocytes during the first subcultivation. 'Hox gene negative' expression could not be confirmed in horses. This study demonstrates that equine Hox gene expression pattern is likely species-specific and that equine Hox gene expression profiles are subject to statistically significant individual influences. Conclusion: The equine Hox gene expression profile is subject to statistically significant individual influences that should be considered in potential cell therapy. Nasal chondrocytes are probably suitable as a potential source for autologous chondrocyte implantation (ACI) in horses due to their genetic similarity, in terms of the expressions of the examined Hox genes, to articular chondrocytes and their high propensity for collagen II formation.:1. Einleitung ...................................................................................................... 1 2. Literaturübersicht .......................................................................................... 2 2.1 Knorpel ..................................................................................................... 2 2.1.1 Allgemeiner Überblick ....................................................................... 2 2.1.2 Chondrogenese und Regeneration .................................................. 3 2.1.3 Intraartikulärer Hyaliner Knorpel ....................................................... 3 2.1.4 Extraartikulärer Hyaliner Knorpel....................................................... 6 2.2 Osteoarthritis bei Mensch und Pferd ........................................................ 8 2.2.1 Bedeutung und Ätiologie ................................................................... 8 2.2.2 Pathogenese ..................................................................................... 9 2.2.3 Diagnostik ........................................................................................ 10 2.2.4 Bisherige Therapieansätze .............................................................. 12 2.3 Knorpelgewebe in der Forschung .............................................................13 2.3.1 Kultivierung von Chondrozyten ......................................................... 13 2.3.2 Tiermodelle in der Osteoarthritisforschung ....................................... 15 2.3.3 Neue Therapieansätze ..................................................................... 17 2.3.3.1 Stammzellbasierte Verfahren ...................................................... 17 2.3.3.2 Artikuläre autologe Chondrozyten .............................................. 19 2.3.3.3 Nasale Chondrozyten ................................................................. 21 2.4 Hox-Gene ................................................................................................ 22 2.4.1 Allgemeiner Überblick ..................................................................... ..22 2.4.2 Regulation der Hox-Gene ................................................................. 23 2.4.3 Erkrankungen im Zusammenhang mit Hox-Genen ........................... 25 3. Ziel der Studie ........................................................................................... 27 4. Publikation ................................................................................................ 28 5. Diskussion................................................................................................. 45 5.1 Primer ....................................................................................................46 5.2 Auswahl der Messzeitpunkte und Proben ............................................. 47 5.3 Hox-Genprofile kultivierter equiner Chondrozyten ................................ 49 5.4 Individuelle Hox-Genprofile ................................................................... 50 5.5 SOX9 und Kollagen II .............................................................................51 5.6 Hox-Genprofile im Vergleich verschiedener Spezies ............................. 53 5.7 Ausblick ................................................................................................ 53 6. Schlussfolgerung ...................................................................................... 55 7. Zusammenfassung ................................................................................... 56 8. Summary .................................................................................................. 58 9. Referenzen .............................................................................................. 60 9.1 Literaturverzeichnis .............................................................................. 60 9.2 Abbildungsverzeichnis.......................................................................... 71 10. Anhang .................................................................................................. 72 10.1 Evaluierungsbogen Chondrozytenkulturen ........................................ 72 10.2 Auszug aus der Korrelationsmatrix ..................................................... 73 10.3 Publikationsverzeichnis Christiane Storch .......................................... 74 11. Danksagung .......................................................................................... 75