Verificando a importância da morfologia dos arcos dentários no tratamento ortodôntico, a proposta deste estudo foi avaliar e comparar as formas e dimensões de arcos de 30 indivíduos com oclusão normal e 30 com má-oclusão de Classe IIdivisão 1ª, geradas a partir de três categorias de pontos de referência. Foram demarcados nos modelos de estudo: os pontos médios das faces vestibulares dos dentes (1ª categoria de pontos); pontos no rebordo alveolar, perpendiculares aos anteriores e sobre a junção muco-gengival (2ª categoria de pontos); e pontos mesiais e distais das superfícies oclusais dos dentes. Todos os pontos foram digitalizados e convertidos automaticamente para o sistema de coordenadas x, y e z pela máquina de medidas tridimensional da Mitutoyo (modelo Crysta-Apex/C). Um software, especialmente desenvolvido para este trabalho, criou um conjunto de pontos de referência virtuais (3ª categoria de pontos) a partir dos pontos mesiais e distais das superfícies oclusais previamente demarcados nos modelos, que representou o fundo dos canais de encaixe de braquetes. Além disso, gerou curvaturas por meio de equações matemáticas (parábola, elipse, catenária e função beta) que se ajustaram, pelo método dos mínimos quadrados, às 3 diferentes categorias de pontos de referência, e mediu as dimensões transversais dentárias e dos rebordos alveolares. A seleção da equação matemática que melhor descreveu cada categoria de pontos de referência foi realizada por meio de avaliação do menor valor do erro médio. A curvatura do rebordo selecionada foi deslocada sobre o eixo y até tangenciar o ponto médio virtual do incisivo central mais anterior, simulando a inserção de um arco ortodôntico, e foram medidas as distâncias entre esta curvatura e os pontos médios virtuais dos outros dentes. Todos os dados foram organizados em tabelas de acordo com a categoria dos pontos de referência, os arcos (superior ou inferior) e o tipo de oclusão. Verificou-se que todas as dimensões transversais dentárias e dos rebordos superiores, e posteriores inferiores são estatisticamente maiores nos indivíduos com oclusão normal do que nos indivíduos com má-oclusão de Classe II; e que as distâncias transversais dos rebordos alveolares são estatisticamente maiores do que as dentárias. Quanto às formas dos arcos, a catenária, seguida da elipse, foram as que melhor descreveram as suas curvaturas, independentemente da categoria de pontos e do tipo de oclusão. As outras formas, como a parábola e a gerada pela função beta, foram observadas com baixo percentual de ocorrência. As formas dos arcos não caracterizaram diferencialmente os tipos de oclusão e as 3 categorias de pontos de referência estudadas. Praticamente todos os pontos médios virtuais apresentaram-se internamente posicionados em relação à curvatura do rebordo alveolar quando esta foi deslocada até o ponto médio virtual do incisivo central mais vestibularizado, sendo encontrados valores estatisticamente maiores no arco superior dos indivíduos com má-oclusão de Classe II-divisão 1ª. Este dado revela maior tendência a vestibularização dos dentes superiores nestes indivíduos, quando o rebordo alveolar é utilizado como guia para a construção do arco ortodôntico, embora as diferenças entre os grupos não nos pareçam clinicamente importantes. / Given the importance of dental arch morphology in orthodontic treatment, we have evaluated and compared, based on three categories of reference points, the shapes and dimensions of the arches of 30 subjects with normal occlusion and 30 subjects with Class II, division 1 malocclusions. .The points marked on the study casts were: the middle points of the buccal surfaces of teeth (first category of points); points on the alveolar ridge, perpendicular to those of the first category and on the mucogingival junction (second category of points); and mesial and distal points of the occlusal surfaces of the teeth. All points were digitized and automatically converted to the x, y and z system of coordinates by a three-dimensional measurement machine (Mitutoyo - Crysta-Apex/C model). A computer software program especially developed for our study created a set of virtual reference points (third category of points) based on the distal and mesial points on the occlusal surfaces previously marked on the casts, which represented the most inner point of the bracket slot. In addition, the software generated curvatures using mathematical equations that were adjusted to the three categories of reference points by the method of minimum squares, and measured the transversal dimensions of teeth and alveolar ridges. Selection of the mathematical equation that best described each category of reference points was performed by evaluating the smallest value for the mean error. The selected ridge curvature was shifted on the y-axis until it became tangential to the virtual middle point of the most anterior central incisor, simulating the insertion of an orthodontic arch, and the distances between this curvature and the virtual middle point on the other teeth were measured. All data were tabulated according to reference point category, arch (upper or lower), and type of occlusion. We observed that all transversal dimensions of teeth, upper ridges and lower posterior ridges were statistically greater in subjects with normal occlusion than in subjects with Class II malocclusions; and that the transversal distances of the alveolar ridges were statistically greater than those of the teeth. As to arch shape, the catenary, followed by the ellipse, were the shapes that best described the arch curvatures, regardless of reference point category and occlusion type. The other shapes, such as the parabola and the shape generated by the Beta function, were observed at lower frequencies. The arch shapes did not differentially characterize the several types of occlusion or the 3 reference point categories studied. Practically all virtual middle points were located internally to the curvature of the alveolar ridge when this curvature was shifted up to the virtual middle point of the most buccaly projected central incisor, and the subjects with Class II division 1 malocclusion were found to have statistically greater values for the upper arch. This observation reveals a greater trend for buccally projected upper teeth in these subjects when the alveolar ridge is used as a guide to construct the orthodontic arch, although the differences observed between the groups do not seem to be clinically important.

A estabilidade pós-tratamento sempre foi um assunto delicado da ortodontia. A maioria dos pacientes e até mesmo os ortodontistas, julgam o sucesso do tratamento ortodôntico pela estabilidade dos resultados em longo prazo. Por este motivo a literatura é repleta de estudos acerca deste assunto, principalmente da recidiva do apinhamento ântero -inferior. Já o interesse pelo apinhamento ânterosuperior e sua recidiva é crescente por conta de uma maior preocupação com a estética do sorriso. Desta forma, o propósito deste estudo é comparar a recidiva do apinhamento ântero-superior em pacientes com má oclusão de Classe I e Classe II de Angle. A amostra consiste em 70 pacientes divididos em 3 grupos. O Grupo 1 apresenta 30 pacientes (12 do gênero masculino e 18 do gênero feminino) com má oclusão de Classe I, idade média inicial de 13,16 anos e tratados com extrações dos 4 primeiros pré -molares. O Grupo 2 apresenta 20 pacientes (11 do gênero masculino e 9 do gênero feminino) com má oclusão de Classe II divisão 1, idade média inicial de 12,95 anos e também tratados com extrações dos 4 primeiros pré-molares. O Grupo 3 apresenta 20 pacientes (11 do gênero masculino e 9 do gênero feminino) com má oclusão de Classe II divisão 1, idade média inicial de 13,09 anos e tratados com extrações dos 2 primeiros pré -molares superiores. Foram avaliados os modelos de estudo nas fases inicial (T1), final (T2) e no mínimo 5 anos pós -tratamento (T3) de cada pac iente. As variáveis do arco superior avaliadas e comparadas estatisticamente pela Análise de Variância (ANOVA) foram: Índice de Irregularidade de Little superior (IRLS), comprimento do arco (CAS), distância intercaninos (DICS), interpré-molares (DI2PMS) e intermolares (DIMS). Como os resultados entre os grupos não mostraram diferenças estatisticamente significantes nas fases avaliadas, a amostra foi unificada. O Teste de Correlação de Pearson e o Teste de Regressão Linear Múltipla foram utilizados para veri ficar se alguma variável estudada teria influência sobre o apinhamento nas três fases (IRLS1, IRLS2, IRLS3). Os resultados mostraram que a recidiva do apinhamento superior (IRLS3-2) é influenciada pelo apinhamento inicial (IRLS1) e que os dentes tendem a voltar à posição original. O gênero feminino apresentou mais recidiva do apinhamento ântero -superior que o gênero masculino. / The posttreatment stability was always a delicate issue in Orthodontics. Most of the orthodontic patients and even the orthodontists judge their treatment as successful based on the treatment outcomes stability in the long-term. Because of this reason the literature has a lot of studies about this issue, mostly on relapse of mandibular anterior crowding. However the interest on maxillary anterior crowding and its relapse has been growing because of the greater conscious on smile esthetic among patients. Therefore, the purpose of this study was to compare the relapse of maxillary anterior crowding in cases presenting Angles Class I and Class II malocclusions. The experimental sample consisted of 70 patients divided into 3 groups. Group 1 comprised 30 patients (12 male; 18 female) at a mean initial age of 13.16 years, with Class I malocclusion, treated with all first premolars extraction. Group 2 comprised 20 patients (11 male; 9 female) at a mean initial age of 12.95 years, with Class II division 1 malocclusion, also treated with all first premolars extraction. Group 3 comprised 20 patients (10 male; 10 female) at a mean initial age of 13.09 years, with Class II division 1 malocclusion, treated with extraction of two maxillary first premolars. Dental casts measurements were obtained at three stages (pretreatment, posttreatment and postretention) and the variables assessed were Little Irregularity Index, maxillary arch length, intercanine, interpremolar and intermolar widths. The statistical analysis was performed by one-way ANOVA and Tukey tests if necessary (intragroup comparison) and by independent t-tests (intergroup comparison). As the results among the groups did not show statistically significant difference, the experimental sample was unified in order to inve stigate, using Pearson correlation coefficient and Multiple linear regression, if some studied variable would have influence the crowding in the three stages (IRLS1, IRLS2, IRLS3). The results showed that the maxillary crowding relapse (IRLS3-2) is influenced by the initial (IRLS1), and the teeth tend to return to their pretreatment position. The females presented more maxillary anterior crowding relapse than males.

O objetivo deste trabalho foi comparar a frequência e a quantidade da reabertura dos espaços das extrações de primeiros pré-molares em longo prazo nas más oclusões de Classe II tratadas com o protocolo de extração de dois pré-molares superiores, com aquelas tratadas com extrações de quatro pré-molares e com casos de Classe I tratados com extrações de quatro pré-molares. Para isso foi selecionada do arquivo da Disciplina de Ortodontia da Faculdade de Odontologia de Bauru USP, uma amostra composta pelas documentações ortodônticas de 105 pacientes com más oclusões de Classe I e Classe II completa. Em seguida a amostra foi dividida em 3 grupos proporcionais entre os gêneros e as idades ao início do tratamento: grupo 1- constituído por 33 pacientes com má oclusão de Classe II completa tratados com extrações de dois primeiros pré-molares superiores; grupo 2- constituído por 34 pacientes com má oclusão de Classe II completa tratados com extrações de quatro primeiros pré-molares e grupo 3- constituído por 38 pacientes com má oclusão de Classe I tratados com extrações de quatro primeiros pré-molares. As fichas cadastrais e clínicas dos pacientes foram utilizadas para obtenção do gênero e das idades ao início e fim do tratamento, assim como para obtenção dos tempos de tratamento e pós-tratamento. O índice oclusal PAR foi utilizado para avaliar a severidade inicial da má oclusão e a qualidade oclusal ao final do tratamento mensurado nos modelos de gesso das fases correspondentes. A frequência e a quantidade de reabertura dos espaços das extrações dos primeiros pré-molares foram mensuradas com paquímetro digital nos modelos finais e de pós-tratamento, com média de 9,79 anos após o término do tratamento. A comparação entre os grupos das variáveis estudadas foi feita por meio dos testes qui-quadrado e ANOVA. Os grupos foram proporcionais entre os gêneros e à quantidade de locais das extrações que não possuíam pontos de contato ao final do tratamento. Foram compatíveis quanto às idades ao início e fim do tratamento, PAR inicial e PAR final, assim como quanto aos tempos de tratamento e de pós-tratamento. A frequência e a quantidade de reabertura dos espaços das extrações de primeiros pré-molares não apresentaram diferença estatisticamente significante entre os três grupos estudados, concluindo que os protocolos de tratamento estudados apresentam semelhança em relação à recidiva dos espaços das extrações. / The aim of this retrospective study was to compare the frequency and quantity of reopening extraction spaces in long term of Class II malocclusion treated with two premolar extraction protocol, with those treated with four premolar extractions and Class I malocclusion cases treated with extractions of four premolars. The sample, from files of the Department of Orthodontics, School of Dentistry of Bauru - USP, comprised 105 subjects presenting complete Class I and Class II malocclusion. This sample was divided into three groups in proportion between genders and ages: group 1 - comprising 33 patients presenting complete Class II malocclusion treated with two premolar extraction protocol, group 2 - consisting 34 patients presenting complete Class II malocclusion treated with extraction of four first premolars and group 3 - comprising 38 patients presenting Class I malocclusion treated with extraction of four first premolars. The registration and clinical forms were used to obtain the gender and age at beginning and end of treatment, so as to obtain the time of treatment and post-treatment. The PAR occlusal index was used to assess the initial malocclusion severity and occlusal quality at the end of treatment measured on dental casts. The frequency and amount of extraction spaces were measured with a digital caliper rule in the final and post-treatment dental casts, with an average of 9.79 years after treatment. Comparison of variables between groups was performed by chi-square and ANOVA tests. There were no significant differences between groups variables considered in this study and it was concluded that these treatment protocols show similarity in recurrence of extraction spaces in long term.

O objetivo do presente estudo foi determinar os parâmetros morfológicos, faciais e dentários, que devem ser utilizados como referência pelo profissional, ortodontista e cirurgião, para indicação do tratamento ortodôntico-cirúrgico em pacientes com deformidades dentofaciais do Padrão II. Foram selecionados 40 pacientes brasileiros, com crescimento finalizado, 26 do gênero feminino e 14 do masculino, com ângulo de convexidade facial maior que 12º e exposição gengival no sorriso igual ou menor a 3 mm. Foram excluídos os indivíduos xantodermas e melanodermas ou aqueles que já se submeteram a qualquer cirurgia facial. Foram obtidas fotografias extrabucais de frente, de perfil e do sorriso, fotografias intrabucais frontal e laterais direita e esquerda, modelos de gesso e telerradiografias do perfil facial de todos os pacientes. Toda essa documentação foi digitalizada e os arquivos enviados para 30 professores de cursos de Pós-graduação em Ortodontia. Os avaliadores foram solicitados a determinar, em uma lista de fatores relacionados ao diagnóstico ortodôntico, os que eles consideravam de extrema, moderada ou pequena/nenhuma importância na indicação da cirurgia ortognática. Posteriormente, deveriam avaliar os arquivos com as documentações ortodônticas para classificar a estética facial (agradável; aceitável; desagradável) e a melhor opção de tratamento (tratamento ortodôntico compensatório; tratamento ortodôntico-cirúrgico; nenhum tratamento) para cada paciente da amostra. Foram obtidas as medidas das variáveis da análise facial numérica do perfil nas fotografias do perfil facial, da sobressaliência e da sobremordida nos modelos de gesso, das inclinações dos incisivos superiores e inferiores, além do tipo facial, nas telerradiografias do perfil. Testou-se, então, a associação entre cada uma das variáveis acima, além da idade e do gênero, e a indicação do tratamento ortodôntico-cirúrgico e, a partir das variáveis que apresentaram associação, estabeleceu-se uma equação matemática que permite prever a probabilidade de indicação da cirurgia para pacientes Padrão II. Os fatores considerados de extrema importância na indicação da cirurgia ortognática, por pelo menos 50% dos avaliadores, em ordem decrescente de importância, foram os seguintes: estética facial, assimetria facial, convexidade do perfil facial, proporção entre os terços faciais médio e inferior, comprimento da linha queixo-pescoço, idade, exposição gengival no sorriso, projeção anterior do mento, exposição dos incisivos em repouso e sobressaliência. As variáveis que apresentaram associação com a indicação do tratamento ortodôntico-cirúrgico foram a estética facial, o ângulo de convexidade facial total, a sobressaliência e a inclinação do incisivo superior (1.PP). O modelo matemático proposto para prever a porcentagem de indicação de cirurgia ortognática em pacientes com deformidades dentofaciais do Padrão II inclui a estética facial, a sobressaliência e o ângulo de convexidade facial total. A equação visa prover o clínico de uma ferramenta auxiliar na decisão do tratamento dos pacientes Padrão II, principalmente nos casos limítrofes. / The aim of the present study was to determine the morphological parameters, facial and dental, that may be used as references by the professional, either orthodontist or surgeon, to indicate compensatory orthodontic treatment or orthodontic-surgical treatment to patients with Class II dentofacial deformities. Forty brazilian leucoderma patients have been selected, 26 female and 14 male, all of them with growth finished, convexity facial angle higher than 12° and gingival exposure at smile equal or less than 3mm. It has been excluded the ones who have gone through facial surgery. Extrabucal photographs at the frontal, profile and smile views and intrabucal at the frontal, right lateral and left lateral views, plaster casts and lateral cephalometric radiograph have been taken of all patients. This documentation was digitalized and the archives sent to 30 post-graduation course orthodontic teachers of different parts of the country. The appraisers were asked to determine, in a list of factors related to orthodontic diagnosis, the ones they considered of extreme, moderate, little or no importance at the indication of orthognathic surgery. Subsequently, they evaluated the archives with the orthodontic documentation to classify the facial esthetic (pleasant, acceptable, and unpleasant) and the best treatment option (orthodontic compensatory treatment; orthodontic-surgical treatment; no treatment) to each patient of the sample. The measurements of the variables of the numerical facial analysis of the profile have been obtained at the photographs of the facial profile, the overjet and overbite at the plaster casts, and the inclination of the upper and lower incisors and the facial type at the lateral cephalometric radiograph. Then, the association between each of the variables mentioned above, age and gender, and the indication of the orthodontic-surgical treatment were tested. From the variables that showed association, a mathematic equation that allowed to foresee the indication of surgery to patients with Class II dentofacial deformities was established. The factors considered of extreme importance to indicate orthognathic surgery for at least fifty percent of the appraisers, in decreasing order of importance were: facial esthetics, facial assimetry, convexity of the facial profile, proportion between medium and lower facial thirds, length of the chin-neck line, age, gingival exposure at smile, chin anterior projection, incisor exposure at rest and overjet. The variables that showed association with the indication of orthodontic-surgical treatment were: facial esthetics, total facial convexity angle, overjet and upper incisor inclination (1.PP). The mathematic model proposed to foresee the percentage of indication of orthognathic surgery to patients with Class II dentofacial deformities includes facial esthetics, total facial convexity angle and overjet. The equation aims to provide the clinicians with an auxiliary tool at the treatment decision of the patients with Class II dentofacial deformities, especially at borderline cases.

O objetivo do presente estudo foi avaliar a estabilidade das alterações cefalométricas dentoesqueléticas e do perfil tegumentar obtidas durante a correção da má oclusão de Classe II divisão 1, com o aparelho Herbst com cantiléver seguido do aparelho fixo, 11 anos após o término de tratamento. O grupo tratado foi composto por 13 pacientes (3 feminino; 10 masculino) com idade média inicial de 12,87 anos os quais foram avaliados em três estágios: inicial (T1), final (T2), e pós-tratamento em longo prazo (T3). O grupo controle foi composto por 15 indivíduos (5 feminino; 10 masculino) com oclusão normal e idade média compatível com a do grupo tratado. A análise de variância para medidas repetidas (ANOVA), seguida por testes de Tukey (p<0,05) foram utilizadas para comparação intragrupo entre os três estágios. A comparação intergrupos das alterações no período pós-tratamento em longo prazo (T3-T2) e alterações do crescimento normal em um período comparável foram realizadas por meio do teste t (p<0,05). A maior parte das alterações dentoesqueléticas e dos tecidos tegumentares obtidas durante o tratamento se mantiveram estáveis em longo prazo. No entanto, a sobremordida demonstrou recidiva significativa, enquanto o lábio inferior tornou-se mais retrusivo em relação ao grupo controle. Apesar de alterações sutis terem ocorrido na sobremordida e na posição do lábio inferior em longo prazo, essas podem ser consideradas de menor relevância clínica. O tratamento da Classe II com Herbst com cantiléver seguido de aparelho fixo demonstrou estabilidade em longo prazo. / The aim of this study was to assess the stability of the dentoskeletal and soft-tissue cephalometric changes obtained during Class II division 1 malocclusion correction with the Cantilever Bite Jumper (CBJ), followed by fixed appliances, 11 years after treatment. The treatment group comprised 13 patients (3 female; 10 male) with an initial mean age of 12.87 years who were evaluated in three stages: initial (T1), final (T2), and long-term posttreatment (T3). The control group comprised 15 subjects (5 female; 10 male) with normal occlusion and compatible mean age with the treated group. Repeated measures analysis of variance (ANOVA), followed by Tukey tests (p<0.05) were used for intratreatment comparisons among the three stages. Intergroup comparisons of long-term posttreatment changes (T3-T2) and normal growth changes in a comparable period were performed with t tests (p<0.05). Most of dentoskeletal and soft-tissue changes obtained during treatment remained stable in the long-term. However, overbite demonstrated significant relapse while lower lip become more retrusive in relation to control group. Although mild changes have occured in overbite and lower lip position in the long-term, these can be considered of less clinical relevance. Treatment of Class II with Cantilever Bite Jumper followed by fixed appliances showed long-term stability.

March1 joue un rôle essentiel dans la régulation de la réponse immunitaire. Cette ubiquitine ligase régule à la baisse l’expression de certaines protéines intervenant dans les fonctions des cellules présentatrices des antigènes, telles que le CMH de classe II et le CD86. March1 ubiquitine aussi quelques protéines impliquées dans le métabolisme cellulaire, comme le transporteur des acides aminées CD98 et le récepteur de l’insuline. L'ubiquitination du CMH de classe II et du CD98 par March1 affecte les fonctions de présentation des antigènes par les cellules dendritiques et la capacité de prolifération des TCD8+, respectivement. Cependant, le rôle de l’ubiquitination de CMH de classe II dans le développement et la migration des cellules dendritiques, n’est pas connu, et les implications physiologiques liées au rôle de March1 dans d’autres cellules, telles que les lymphocytes T CD8+, ne sont pas encore claires. Nos travaux démontrent que l'ubiquitination de CMH de classe II par March1 est spécifiquement requise pour la migration des cellules dendritiques dérivées de monocytes (moDCs) de la peau. L’effet de March1 sur la migration est intrinsèque à ces cellules et corrèle avec les niveaux d’expression des protéines impliquées dans la migration, notamment l’IRF4 et le CCR7. Dans un modèle de mélanome chez la souris, la déficience en ubiquitination du CMH de classe II est associée à une exacerbation de la croissance des tumeurs et un défaut de migration des moDCs vers les ganglions drainant les tumeurs. L’utilisation de cellules tumorales exprimant le GM-CSF augmente l’expression de l’IRF4 et du CCR7 dans les moDCs et la migration celles-ci vers les ganglions. D’autre part, nous avons démontré que la déficience en March1 exacerbe la résistance à l’insuline induite par l’obésité. Cet effet est associé à un enrichissement en lymphocytes T CD8+ ayant un phénotype effecteur/mémoire dans le tissu adipeux des souris obèses. Les expériences de transfert adoptif de lymphocytes T CD8+ montrent que March1 exacerbe la résistance à l’insuline en affectant intrinsèquement le phénotype de ces cellules. Nos résultats indiquent une augmentation de l’activité métabolique des lymphocytes T CD8+ est en absence de March1, ce qui est en accord avec le rôle de ce dernier dans l’ubiquitination de CD98 et du récepteur de l’insuline. Dans l’ensemble, nos travaux montrent que March1 régule la capacité migratoire des moDCs et le métabolisme des lymphocytes T CD8+. L’implication de cette régulation dans le développement du cancer de mélanome et du diabète de type de II suggère que March1 pourrait être ciblé dans le cadre de stratégies thérapeutiques contre ces pathologies. / March1 plays a critical role in the immune response regulation. This ubiquitin ligase downregulates the expression of antigen presentation and costimulatory proteins, such as MHC class II and CD86, as well as other proteins involved in the cellular metabolism, such as the amino acid transporter CD98 and the insulin receptor. The ubiquitination of these proteins by March1 alters the antigen presentation and proliferative capacities of dendritic cells and CD8+ T cells, respectively. However, the effect of MHC class II ubiquitination by March1 on the development and the migration of dendritic cells are not known, and the physiological implications of March1 in other cells, such as CD8+ T cells, are not completely understood. In this thesis, we show that MHC class II ubiquitination by March1 is specifically required for the migration of monocyte-derived dendritic cells (moDCs) from skin to skin draining lymph nodes (sdLN). This effect is cell intrinsic and correlates with the expression level of proteins involved in immune cell migration, IRF4 and CCR7. In a melanoma mouse model, the deficiency of MHC class II ubiquitination is associated with exacerbated tumor growth and impaired moDCs migration from tumor to tumors-draining LNs. Using GM-CSF producing tumors, we found that this cytokine increases the expression of IRF4 and CCR7 in moDCs and improves their migration. On the other hand, we show that March1 deficiency exacerbates obesity-induced insulin resistance. Adipose tissue from these mice was enriched in CD8+ T cells with an effector/memory phenotype. Adoptive transfer of splenic CD8+ T cells showed that March1 intrinsically affects the phenotype of these cells in obese adipose tissue and exacerbates insulin resistance. Consistent with the role of March1 in the ubiquitination of CD98 and insulin receptor, the metabolic activity of CD8+ T cells was increased in absence of March1. Overall, we showed that March1 regulates the migratory capacity of moDCs and the metabolic activity of CD8+ T cells. The involvement of these effects in the development of melanoma cancer and type II diabetes suggests that March1 can be a target for therapeutic strategies against these pathologies.

MHC II proteins are heterodimeric glycoproteins that form complexes with antigenic peptides in order to elicit a CD4+ adaptive immune response. Even though there have been numerous MHC II-peptide crystal structures solved, there is little insight into the dynamic process of peptide loading. Through biochemical and biophysical studies, it has been shown that MHC II adopt multiple conformations throughout the peptide loading process. At least one of these conformations is stabilized by the MHC II-like homologue, HLA-DM. The main focus of this thesis is to elucidate alternate conformers of MHC II in an effort to better understand the structural features that enable HLA-DM catalyzed peptide loading. In this thesis, two altered conformations of HLA-DR were investigated, one modeled in the absence of peptide using molecular dynamics, and one stabilized by the mutation αF54C. The model for the peptide-free form of HLA-DR1 was derived from a molecular dynamics simulation. In this model, part of the alpha-subunit extended-strand region proximal to the peptide binding groove is folded into the peptide-binding groove such that the architecture of the critical peptide binding pocket, P1, as well as the invariant hydrogen bonding network were maintained. Biochemical studies aimed at validating the predicted structural changes were consistent with the model generated from the simulations. Next, structural studies were carried out on an MHC II mutant, αF54C, which was shown to have unique peptide binding characteristics as well as enhanced susceptibility to HLA-DM. Although this mutation did not affect the affinity for peptide, there was a striking increase in the rate of intrinsic peptide release. Both αF54C and αF54A were over 100-fold more susceptible to HLADM catalyzed peptide release than wild type as well as other mutants introduced along the peptide binding groove. In addition, mutation of the αF54 position results in a higher affinity for HLA-DM, which, unlike wild type, is detectable by surface plasmon resonance. Crystallographic studies resulted in a 2.3 Å resolution structure for the αF54C-Clip complex. There were two molecules in the asymmetric unit, one of which had no obvious deviations from other MHC II-pep complexes and one which had a conformational change as a result of a crystal contact on the αF51 residue, a residue which has been shown to be involved in the HLA-DM/HLA-DR binding interface. The crystal structure of wild type HLA-DR1- Clip was also solved, but did not have the altered conformation even though there was a similar crystal contact at the αF51. These data suggest the altered conformation seen in the mutant structure, results from increased lability in the extended stand region due to the αF54C mutation. As a result of this work, we have developed a new mechanistic model for how structural features of MHC II influence DM mediated peptide release.

Antigen presented to CD4+ T cells by major histocompatibility complex class II molecules (MHCII) plays a key role in adaptive immunity. Antigen presentation is initiated by the proteolytic cleavage of pathogenic or self proteins and loading of resultant peptides to MHCII. The loading and exchange of peptides to MHCII is catalyzed by a nonclassical MHCII molecule, HLA-DM (DM). It is well established that DM promotes peptide exchange in vitro and in vivo. However, the mechanism of DM-catalyzed peptide association and dissociation, and how this would affect epitope selection in human responses to infectious disease remain unclear. The work presented in this thesis was directed towards the understanding of mechanism of DM-mediated peptide exchange and its role in epitope selection. In Chapter II, I measured the binding affinity, intrinsic dissociation half-life and DM-mediated dissociation half-life for a large set of peptides derived from vaccinia virus and compared these properties to the peptide-specific CD4+ T cell responses. These data indicated that DM shapes the peptide repertoire during epitope selection by favoring the presentation of peptides with greater DM-mediated kinetic stability, and DM-susceptibility is a strong and independent factor governing peptide immunogenicity. In Chapter III, I computationally simulated peptide binding competition reactions and found that DM influences the IC50 (50% inhibition concentration) of peptides based on their susceptibility to DM, which was confirmed by experimental data. Therefore, I developed a novel fluorescence polarization-based method to measure DM-susceptibility, reported as a IC50 (change in IC50 in the absence and presence of DM). Traditional assays to measure DM-susceptibility based on differential peptide dissociation rates are cumbersome because each test peptide has to be individually labeled and multiple time point samples have to be collected. However, in this method developed here only single probe peptide has to be labeled and only single reading have to be done, which allows for fast and high throughput measure of DM-susceptibility for a large set of peptides. In Chapter IV, we generated a series of peptide and MHCII mutants, and investigated their interactions with DM. We found that peptides with non-optimal P1 pocket residues exhibit low MHCII affinity, low kinetic stability and high DM-susceptibility. These changes were accompanied with conformational alterations detected by surface plasmon resonance, gel filtration, dynamic light scattering, small-angle X-ray light scattering, antibody-binding, and nuclear magnetic resonance assays. Surprisingly, all these kinetic and conformational changes could be reversed by reconstitution with a more optimal P9 pocket residue. Taken together, our data demonstrated that conformation of MHCII-peptide complex constrained by interactions throughout the peptide binding groove is a key determinant of DM-susceptibility. B cells recognizing cognate antigen on the virion can internalize and process the whole virion for antigen presentation to CD4+ T cells specific for an epitope from any of the virion proteins. In turn, the epitope-specific CD4+ T cells provide intermolecular (also known as noncognate or heterotypic) help to B cells to generate antibody responses against any protein from the whole virion. This viral intermolecular help model in which CD4+ T cells provide help to B cells with different protein specificities was established in small size influenza virus, hepatitis B virus and viral particle systems. For large and complex pathogens such as vaccinia virus and bacteria, the CD4+ T cell-B cell interaction model may be complicated because B cells might not be able to internalize the large whole pathogen. Recently, a study in mice observed that CD4+ T cell help is preferentially provided to B cells with the same protein specificity to generate antibody responses against vaccinia virus. However, for larger pathogens such as vaccinia virus and bacteria the CD4+ T cell-B cell interaction model has yet to be tested in humans. In Chapter V, I measured in 90 recently vaccinated and 7 long-term vaccinia-immunized human donors the CD4+ T cell responses and antibody responses against four vaccinia viral proteins (A27L, A33R, B5R and L1R) known to be strongly targeted by cellular and humoral responses. We found that there is no direct linkage between antibody and CD4+ T cell responses against each protein. However, the presence of immune responses against these four proteins is linked together within donors. Taken together, our data indicated that individual viral proteins are not the primary recognition unit and CD4+ T cells provide intermolecular help to B cells to generate robust antibody responses against large and complicated vaccinia virus in humans.

Ce projet a accompagné le développement pré-clinique de l'hormone humaine Relaxine-2 (Rln2) ayant induit des anticorps durant des essais cliniques, il est axée autour de 2 problématiques : (1) comprendre son immunogénicité, (2) étudier l’impact de modifications chimiques sur l’immunogénicité afin d'augmenter sa stabilité.Compte tenu du rôle des lymphocytes T CD4 dans les réponses immunitaires, la fréquence de cellules T spécifiques de la Rln2 dans un large panel de donneurs sains a été estimée et a permis d’expliquer le développement d’anticorps anti-Rln2. La cartographie des épitopes T a ensuite identifié les zones portant son immunogénicité. Puis, 6 modifications chimiques (acide aminé D, acide aminé isobutyrique, peptoïde, N-méthylation, C-méthylation et réduction de la liaison peptidique) utilisées pour augmenter la demi-vie ont été introduites à la plupart des positions d’un peptide hautement immunogène. La reconnaissance par des cellules T, la liaison aux molécules de présentation et la capacité à induire des lymphocytes T CD4 ont été étudiées pour les peptides analogues modifiés. La plupart des modifications se sont révélées être très efficaces pour minimiser les propriétés immunogéniques.Ce projet de thèse se situe donc à la croisée des chemins entre l’acquisition de connaissances nouvelles en immunologie et leur application dans des processus de conception et de gestion des risques de peptides thérapeutiques. / This project has accompanied the pre-clinical development of the human hormone Relaxin-2 (Rln2) that induced antibodies during clinical trials, it focuses on two issues: (1) to understand its immunogenicity, (2) to study the impact of unnatural modifications on immunogenicity to increase its stability.Given the role of CD4 T-cells in immune responses, the frequency of Rln2-specific T-cells in a large panel of healthy donors was estimated, and explained the development of anti-Rln2 antibodies. The T epitope mapping then identified the areas responsible for its immunogenicity. Then, 6 unnatural modifications (D amino acid, amino isobutyric acid, peptoid, N-methylation, C-methylation & reduced peptide bond) used to increase the half-life were introduced at most positions of a highly immunogenic peptide. T-cell recognition, binding to HLA molecules and the ability to induce CD4 T-cells were studied for modified analog peptides. Most of the modifications were very effective in minimizing immunogenic properties.This thesis project is at the crossroads between the acquisition of new knowledge in immunology and its application in the process of design & risk management of therapeutic peptides.

Indiana University-Purdue University Indianapolis (IUPUI) / Globally, protein malnutrition remains problematic, adversely affecting several systems including the immune system. Although poorly understood, protein restriction severely disrupts host immunity and responses to infection. Induction of high-affinity, long-lasting immunity depends upon interactions between B and T lymphocytes. B lymphocytes exploit several pathways including endocytosis, macroautophagy, and chaperone-mediated autophagy to capture and deliver antigens to the endosomal network. Within the endosomal network antigens are processed and loaded onto major histocompatibility complex (MHC) class II molecules for display and recognition by T lymphocytes. To examine the effect of macronutrient malnutrition on MHC class II antigen presentation, we grew B lymphocytes in media containing amino acids, sugars and vitamins but lacking serum, which contains several types of macronutrients. Our studies show macronutrient stress amplified macroautophagy, favoring MHC class II presentation of cytoplasmic antigens targeted to autophagosomes. By contrast, macronutrient stress diminished MHC class II presentation of membrane antigens including the B cell receptor (BCR) and cytoplasmic proteins that utilize the chaperone-mediated autophagy pathway. The BCR plays a critical role in MHC class II antigen presentation, as it captures exogenous antigens leading to internalization and degradation within the endosomal network. While intracellular protease activity increased with macronutrient stress, endocytic trafficking and proteolytic turnover of the BCR was impaired. Addition of high molecular mass macronutrients restored endocytosis and antigen presentation, evidence of tightly regulated membrane trafficking dependent on macronutrient status. Cytosolic chaperone HSC70 has been shown to play a role in endocytosis, macroautophagy, chaperone-mediated autophagy and proteolysis by the proteasome, potentially connecting distinct routes of antigen presentation. Here, altering the abundance of HSC70 was sufficient to overcome the inhibitory effects of nutritional stress on BCR trafficking and antigen presentation suggesting macronutrient deprivation alters the availability of HSC70. Together, these results reveal a key role for macronutrient sensing in regulating immune recognition and the importance of HSC70 in modulating distinct membrane trafficking pathways during cellular stress. These results offer a new explanation for impaired immune responses in protein malnourished individuals.