OBJETIVO: o Fator-C é normalmente usado para predizer a tensão de contração em interfaces compósito-dente. Foi delineado um estudo para contribuir a esclarecer os elementos que permitem prever a tensão em interfaces de restaurações adesivas. Teoricamente, a tensão varia em função da oposição à contração (rigidez do substrato), da complacência e das dimensões e propriedades mecânicas do compósito. MÉTODOS: foram obtidos pelo Método de Elementos Finitos (MEF) 8 modelos estilizados 3D não-lineares de cavidades Classe I mantendo constantes o volume (20 mm3) e a espessura das paredes (20 mm) e com variação do Fator-C (1,9 a 13,5). Foi simulado contato tipo glue entre compósito e dente. A contração de polimerização, por analogia térmica do compósito, foi 1%. Foram analisadas as tensões e deformações principais. Foi também idealizado um método para calcular por MEF a complacência regionalizada e foi avaliada sua validade pela comparação do resultado obtido por este método com o valor de complacência calculada analiticamente para um cubo de dentina de 1 mm de lado fixado numa de suas faces. Foi ainda idealizado um método para calcular, para cada formato de cavidade, o volume útil de compósito capaz de influir para aumentar o pico de tensão na interface. Por fim, com os elementos anteriores, foi calculado para cada cavidade um valor de Fator-CA, dependente da complacência na região de pico e do volume útil de compósito. Foi avaliada a capacidade de predizer o pico de tensão mediante o teste de correlação (Pearson) entre o pico de tensão e o Fator-CA, bem como entre o pico de tensão e o Fator-C. RESULTADOS: os picos de tensões provocadas pela contração em cavidades de classe I tendem a diminuir em função do aumento do Fator-C, embora o teste de Pearson evidenciou significância apenas para a tensão Máxima Principal na parede XZ e no eixo Z. O cálculo da complacência regional obtido por MEF foi de 0,0652 m/N, bem próximo ao valor analítico (0,0666 m/N). O Fator-CA correlacionou significantemente e positivamente com as três tensões obtidas na quina (Von Mises, Máxima Principal e Máxima Cisalhante). CONCLUSÕES: o aumento do Fator-C não conduz ao aumento dos picos de tensão em cavidades de classe I. O método de cálculo de complacência é válido para estruturas complexas. O Fator-CA permite predizer os picos de tensão em compósitos aderidos em cavidades de classe I. / OBJECTIVES: The C-Factor is normally used to predict the shrinkage stress at composite-tooth interfaces. A study was outlined for clarifying the elements which allows providing the stress at bonded restorations interface. Theoretically, the stress varies as a function of the opposition to the shrinkage (substrate stiffness), the compliance, dimensions and mechanical properties of the composite. METHODS: Eight 3D non-linear stylized models of Class I cavities were obtained by the Finite Element Method (FEM), maintaining the volume (20 mm3) and wall thickness (2 mm) constant and varying the C-Factor (1.9 through 13.5). A \'glue\' contact between the composite and teeth was simulated. A 1% polymerization shrinkage was simulated by using thermal analogy of the composite. Principal stresses and strains were analyzed. A method for calculating the local compliance by FEM was also idealized as well as its validity of comparing the obtained result with the analytical compliance value of a 1 mm dentin cube, with fixation at one of its faces. A method for calculating, for each cavity shape, the \'useful volume\' of the composite, which is able to influence on the stress peak increase at the interface, was further idealized. And finally, using elements mentioned before, a compliance dependent value, so-called CA-Factor, was calculated for each cavity, at peak and \'useful volume\' of the composite regions. The capability on predicting the stress peak by the correlation test (Pearson) between the stress peak and the CA-Factor as well as between the stress peak and the C-Factor was also evaluated. RESULTS: the stress peaks generated by the shrinkage on Class I cavities tend to decrease as a function of the C-Factor, though the Pearson only showed the significance for the maximum principal stress at the XZ wall and Z axis. The local compliance calculation obtained by FEM was 0.0652 m/N, which was very close to the analytical one (0.0666 m/N). The CA-Factor presented a positive and significant correlation with all three stresses obtained at the trihedral angle formed by two occlusal edges. (Von Mises, maximum principal and maximum shear stresses). CONCLUSION: the increase of the C-Factor does not lead to the increase of the stress peaks in Class I cavities. The method for calculating the compliance is valid for complex structures. The CA-Factor allows providing the stress peaks for bonded composites on Class I cavities.

Este estudo caso-controle teve por objetivo comparar as angulações e inclinações dentárias em casos de má oclusão de Classe I pré e pós-tratamento ortodôntico e em casos de oclusão normal. A amostra consistiu nos modelos dentários em gesso de 18 indivíduos com má oclusão de Classe I (3 homens e 15 mulheres; idade 15,12 ± 1,42 anos), tratados com exodontias de primeiros pré-molares e mecânica de deslize por meio da técnica MBT® com ancoragem por mini-implantes. Estes indivíduos foram pareados por idade com outros 18 indivíduos com oclusão normal não tratada (7 homens e 11 mulheres; idade 14,9 ± 1,0 anos). Os modelos dentários em gesso foram escaneados tridimensionalmente e, em seguida, os modelos digitais foram encaminhados para o programa Power Shape-e® (Delcam®, Birminghan, Reino Unido) para mensuração das angulações e inclinações dentárias. O teste t de Student foi empregado para análise dos dados. Os resultados não apontaram diferenças significantes entre os lados direito e esquerdo (p > 0,05). O tratamento da má oclusão de Classe I impôs uma significativa diminuição nas angulações de caninos e segundos molares superiores e um aumento das inclinações vestibulares das coroas de incisivos laterais superiores, segundos pré-molares, primeiros e segundos molares inferiores. Verificou-se que uma maior angulação dos caninos e pré-molares inferiores contribuiu para a caracterização da má oclusão de Classe I, assim como uma redução na angulação dos incisivos laterais superiores. Os casos de má oclusão de Classe I pós-tratamento, em comparação com os indivíduos com oclusão normal, apresentaram significativas diminuições nas angulações de segundos molares, caninos e incisivos laterais superiores e de segundos molares inferiores e aumentos significativos nas angulações de segundos pré-molares, caninos e incisivos inferiores. Houve também redução das inclinações dentárias de primeiros e segundos molares inferiores no grupo tratado. Portanto, as alterações nas angulações e inclinações contribuíram para a correção da má oclusão de Classe I e para a aproximação com os valores do grupo com oclusão normal. / This case-control study aimed at comparing dental angulation and inclination in cases with Class I malocclusion pretreatment and posttreatment and in cases with normal occlusion. The sample consisted of dental plaster models of 18 subjects with Class I malocclusion (3 males and 15 females, aged 15.12 ± 1.42 years), treated by first premolars extractions and sliding mechanics by MBT® technique with miniimplants for anchorage. These individuals were age-matched with 18 patients with untreated normal occlusion (7 men and 11 women, aged 14.9 ± 1.0 years). The dental plaster models were scanned three-dimensionally, and then the digital models were referred to the software Power Shape-e® (Delcam®, Birmingham, UK) for measurement of angulations and inclinations. The Student t test was used for statistical analysis. The results did not show significant differences between the right and left sides (p> 0.05). The treatment of Class I malocclusion imposed a significant decrease in the angulation of upper canines (U3) and second molars (U7) and an increase in the buccal inclination of the crowns of upper lateral incisors (U2), lower second premolars (L5), first (L6) and second molars (L7). It was found that an increase in the angulation of lower canines (L3) and premolars (L4 and L5) contributed to the characterization of the Class I malocclusion, as well as a decrease in the angulation of U2. Posttreatment cases of Class I malocclusion, compared with normal occlusion, showed significant decreases in the angulation of U7, U3, U2 and L7 and significant increases in the angulation of L5, L3 and lower incisors (L1 and L2). There was also a decrease in inclinations of L6 and L7 in the treated group. Therefore, changes in angulations and inclinations contributed to correction of Class I malocclusion.

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.

Orientador: Alexandre Leite Rodrigues Oliveira / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-18T14:50:45Z (GMT). No. of bitstreams: 1 Inacio_RodrigoFabrizzio_M.pdf: 6359187 bytes, checksum: 67cbbe4ec81cf9bbd0b48c1dd3b13843 (MD5) Previous issue date: 2011 / Resumo: Foi demonstrado que a regulação positiva do MHC I por tratamento exógeno com interferon beta (IFN beta) influencia no processo de eliminação das sinapses. Também, o aminoácido taurina mostrou ter influencia positiva na sobrevivência e plasticidade neuronal. No entanto, o estabelecimento de um modelo in vitro para estudo do processo de formação/eliminação sináptica e sua relação com a expressão de MHC I ainda não foi proposto. Portanto, o presente estudo tem como objetivo investigar os efeitos do tratamento com IFN e taurina, sozinhos ou diluídos em meio glial (derivado do glioma NG97), na expressão de MHC classe I e na formação de sinapses em células PC12. Células PC12 foram tratadas com NGF para indução do fenótipo semelhante a neurônio e as culturas estabelecidas foram submetidas ao tratamento com IFN beta (500 e 1000 IU) e taurina (0.025 and 0.050mg/mL) por 15 dias em meio normal e por 10 dias em meio condicionado. Finalizado o período de cultivo, as células foram fixadas e processadas para imunocitoquímica com anticorpos anti-MHC I (OX18) e anti-sinaptofisina. A imunomarcação foi mensurada com o software Image J. Nesse contexto, quatro campos representativos foram usados, a partir de cada poço de cultivo. Os resultados mostraram que o IFNbeta (500UI) e a taurina (0.025 mg) modulam a expressão de MHC em células PC12, especialmente após 10 dias de tratamento. IFN e taurina apresentaram efeitos opostos, sendo que o IFN induz o aumento do MHC I, enquanto a taurina causa sua diminuição. Em ambos os casos, o aumento das doses causa degeneração da cultura. Interessantemente, a regulação diferencial do MHC I ocorreu paralelamente a um aumento ou diminuição da plasticidade sináptica, respectivamente. O uso do meio condicionado de NG97, juntamente com IFNbeta ou taurina, leva a uma diminuição da estabilidade sináptica. De uma maneira geral, os presentes dados indicam que as células PC12 podem ser usadas como modelo in vitro para estudos de modulação de MHC I e plasticidade sináptica. Também, reforçam o papel do IFNbeta na eliminação sináptica e indicam que a taurina é capaz de aumentar a formação da rede sináptica / Abstract: It has been demonstrated that MHC I up regulation by exogenous treatment with interferon beta (IFN beta) influences the glial reaction and the synaptic elimination process. Also, the amino acid taurine has been shown to positively influence neuronal survival and plasticity. Nevertheless, the establishment of an in vitro model for studying the synaptic formation/elimination process and its relationship with MHC I expression has not yet been proposed. Therefore, the present study aimed to investigate the effects of the IFN beta and taurine treatments, alone or diluted in glial medium (derived from the NG97 gliome), on the expression of MHC I and synaptic formation in PC12 cells. Established cultures were subjected to the IFN beta (500 and 1000 IU) and taurine treatments (0.025 and 0.050mg/ml) for 5 and 10 days. Finally the cells were fixed and processed for immuno-cytochemistry with antisera against MHC I (OX18) and synaptophysin. The results were compared with control cultures only treated with basal or conditioned medium. The results showed that IFNbeta (500 IU) and taurine (0.025 mg) modulated the MHC I expression in PC12 cells, especially after 10 days of treatment. IFN and taurine displayed opposite effects, such that IFN induced MHC I up regulation, while taurine induced down regulation. In both cases, the highest doses caused culture degeneration. Interestingly, the differential regulation of MHC I was paralleled by enhancement or a decrease in synaptic plasticity, respectively. The use of the NG97 conditioned medium together with IFNbeta or taurine led to a decrease in synaptic stability. Altogether, the present data indicate that PC12 cells may be used as an in vitro model for studying MHC I modulation and synaptic plasticity. It also reinforced the role of IFNbeta on synaptic elimination and indicated that taurine was able to increase the synaptic network formation / Mestrado / Anatomia / Mestre em Biologia Celular e Estrutural

Orientador: Luiz Gonzaga Gandini Júnior / Resumo: Objetivo: Avaliar as alterações dentárias em indivíduos biprotrusos submetidos a extração dos quatro primeiros pré-molares. Materiais e métodos: A amostra constituise de 50 pacientes adultos Classe I de Angle, biprotrusos, com apinhamento até 4mm por arco dentário e com necessidade de extração de quatro primeiros pré-molares, que foram randomizados em 2 técnicas de tratamento: a retração em massa (n = 25) e a retração em duas etapas (n = 25, cada paciente teve colado um braquete convencional ou autoligado em cada canino de forma aleatória). Foram usados modelos digitais obtidos antes, um, dois e três meses após o início da retração dos caninos, e após o fechamento dos espaços das extrações. Foram avaliados a inclinação, angulação e rotação dos caninos (durante a retração dos caninos), e a rotação dos primeiros molares superiores (na retração em massa ou duas etapas). Resultados: Nenhuma diferença foi encontrada entre a técnica de retração ou tipo de braquete. Conclusão: Conclui-se que não houve diferença na angulação, inclinação e rotação entre os braquetes convencionais ou autoligados, além disso a rotação dos molares foi similar na retração em massa e duas etapas. / Abstract: Objetive: To evaluate the dental changes in patients bimaxillary protrusion with firstpremolar extraction. Methods: Fifty adults patients Class I malocclusion, dentalprotrusion, arch length discrepancy bellow for 4mm, and need for first premolarextractions were randomized to 2 treatment techniques: en-mass retraction (n = 25)and two-steps retraction (n = 25, patients had either conventional or self-ligatingbrackets bonded to canines randomly). Digital models before, and one, two, threemonths after starting the canine retraction, and after the closing of space. Wereevaluated the canine inclination, angulation and rotation (canine retraction), androtation of the upper first molars (en-mass or two-steps retraction). Results: Nodifferences were found between retraction techniques or the type of bracket.Conclusions: Angulation, inclination and rotation showed no diferences betweenconventional or self-ligating brackets, besides that the rotation molar was similar in enmassor two-steps retraction. / Mestre

Presque toutes les cellules nucléées expriment des peptides associés au CMH I (HLA I chez l’humain)(MAP) qui sont échantillonnés à partir du protéome cellulaire et transportés vers la surface cellulaire pour inspection par les lymphocytes T CD8. En tant que tel, la collection de MAP à la surface des cellules, ou immunopeptidome, informe les lymphocytes T CD8 de l’état cellulaire interne. L’immunosurveillance du cancer repose sur la capacité des lymphocytes T CD8 à reconnaître les MAP anormaux sur les cellules tumorales et à les éliminer tout en épargnant les cellules saines. Par conséquent, l’existence du cancer indique que bien souvent, les lymphocytes T CD8 spécifiques à la tumeur sont impuissants, dysfonctionnels ou incapables d’exercer leur fonction. Les vaccins anticancéreux peuvent actionner la destruction des tumeurs en stimulant la reconnaissance des MAP anormaux. Toutefois, le développement de vaccins anticancéreux efficaces est entravé par le manque de MAP exploitables, ou antigènes tumoraux (TA), exprimés exclusivement sur les cellules tumorales. La recherche et l’identification de TA ont été largement limitées aux MAP dérivés de mutations non synonymes situées dans des exons canoniques codant pour des protéines. Ces régions génomiques ne représentent que 2% du génome humain. Le fait que les MAP puissent potentiellement dériver de la traduction non canonique de toutes les régions génomiques n’a été pleinement compris que récemment. Ici, nous avons utilisé la protéogénomique pour découvrir des TA exploitables dérivés de produits de traduction canoniques et non canoniques partagés au sein ou entre divers types de cancers humains. Premièrement, nous avons utilisé des cellules souches pluripotentes induites (iPSC) pour identifier les MAP associés à la pluripotence (paMAP) étant partagés par les cellules cancéreuses. Les antigènes pluripotents sont exprimés dans les tissus embryonnaires et absents des tissus adultes sains, mais anormalement réexprimés par les cellules cancéreuses. Ainsi, bien qu'ils ne soient pas mutés, les paMAP constituent des cibles idéales et spécifiques au cancer. Nous avons identifié un ensemble de 48 paMAP dérivés de transcrits codants et non codants (48 %) impliqués dans le maintien de la pluripotence et exprimés de manière aberrante dans plusieurs types de cancer. Ainsi, bien qu’elles proviennent de différents types de cellules et de tissus, des tumeurs 4 distinctes convergent vers un programme transcriptionnel associé à la pluripotence. En effet, l’expression des paMAP dans les cancers est corrélée à l’hypométhylation récurrente de leurs gènes sources, la présence d’aberrations génomiques courantes et l’adoption par les tumeurs de stratégies d’évasion immunitaire communes. Enfin, comme plusieurs paMAP sont immunogènes, leur utilisation comme cibles dans des vaccins anticancéreux pourrait entrer en synergie avec les inhibiteurs disponibles des voies d'évasion immunitaire et améliorer le traitement de plusieurs cancers agressifs. Ensuite, nous avons évalué l’ensemble des TA ayant un potentiel thérapeutique dans deux types de tumeurs présentant une charge mutationnelle particulièrement élevée, le mélanome et le cancer du poumon non à petites cellules (NSCLC). Nous avons constaté que les TA mutés (mTSAs) représentent une minorité (1 %) des TA exploitables dans ces deux types de cancer. Cela peut s'expliquer par une faible expression d'ARN de la plupart des mutations non synonymes ainsi que par leur localisation en dehors des régions génomiques les plus efficaces pour la génération de MAP. En revanche, 99 % des TA dérivent de séquences génomiques non mutées spécifiques au cancer (aeTSA), surexprimées dans le cancer (TAA) ou spécifiques à la lignée cellulaire d'origine (LSA, exprimés par les mélanocytes ou par les cellules épithéliales pulmonaires, pour le mélanome et le NSCLC, respectivement). Tout comme les paMAP, environ 50 % des aeTSA identifiés dans le mélanome et le NSCLC proviennent de séquences non canoniques et sont régulés de manière épigénétique. Alors que les mTSA sont exclusivement spécifiques à chaque patient patient, les aeTSA sont partagés entre les échantillons tumoraux. De plus, leur absence dans les tissus normaux, leur abondance et leur capacité à activer les lymphocytes T CD8 en font des cibles idéales pour traiter les mélanomes et les NSCLC. En conclusion, cette thèse fournit un aperçu de la biogenèse de différents types de TA dans diverses cohortes de patients et ouvre la voie au développement d’immunothérapies ciblées et efficaces contre une grande variété de cancers. / Nearly all nucleated cells express MHC I (HLA I in humans)-associated peptides (MAPs) which are sampled from the cellular proteome and transported to the cell surface for inspection by CD8 T cells. As such, the collection of cell-surface MAPs, or the immunopeptidome, informs CD8 T cells on the inner cell state. Cancer immunosurveillance relies on the capacity of CD8 T cells to recognize abnormal MAPs on tumor cells and eliminate them while sparing healthy cells. Hence, the existence of cancer indicates that tumor-specific CD8 T cells are underpowered, dysfunctional or inhibited from exerting their function. Anti-cancer vaccines can boost tumor killing by stimulating the recognition of abnormal MAPs. The development of effective anti-cancer vaccines is limited by the identification of actionable MAPs, or tumor antigens (TAs), expressed exclusively on tumor cells. The TA search space has been largely limited to MAPs derived from non-synonymous mutations in canonical protein-coding exons which represent a mere 2% of the human genome. That MAPs can derive from the non-canonical translation of potentially all genomic regions has only recently been fully appreciated. Herein, we used proteogenomics to discover actionable TAs derived from canonical and non-canonical translation products shared within or across different types of human cancer. First, we used induced pluripotent stem cells (iPSCs) to identify pluripotency-associated MAPs (paMAPs) shared by cancer cells. Pluripotency antigens are restricted to embryonic tissues and absent from healthy adult tissues but abnormally re-expressed by cancer cells, which makes them ideal tumor-specific targets despite being unmutated. We identified a set of 46 paMAPs derived from coding and allegedly non-coding (48%) transcripts involved in pluripotency maintenance and aberrantly expressed in multiple cancer types. Thus, despite originating from different cell types and tissues, distinct tumor types converged towards a pluripotency-associated transcriptional program. Indeed, the expression of paMAPs across cancers correlated with recurrent source gene hypomethylation, genomic aberrations, and immune evasion properties. Several paMAPs were immunogenic, thus their targeting could synergize with available inhibitors of immune evasion pathways to improve the outcome of multiple aggressive cancers. 7 Next, we evaluated the actionable TA landscape of two tumor types with particularly high mutational load, melanoma and non-small cell lung cancer (NSCLC). We found that mutated TAs (mTSAs) represent a minority (1%) of actionable TAs in both cancer types, which can be explained by a low RNA expression of most non-synonymous mutations and their localization outside genomic regions proficient for MAP generation. By contrast, 99% of TAs derived from unmutated genomic sequences specific to cancer (aeTSAs), overexpressed in cancer (TAAs), or specific to the cell lineage of origin (LSAs, expressed by melanocytes or by lung epithelial cells, for melanoma and NSCLC LSAs, respectively). As for paMAPs, around 50% of aeTSAs in melanoma and NSCLC were non-canonical and were epigenetically regulated. Whereas mTSAs were exclusively patient-specific, aeTSAs were shared among tumor samples and exhibited all characteristics of targetable TAs, including tumor-specificity, high abundance, and immunogenicity. Altogether, this thesis provides insights into the biogenesis of different TA types in various patient cohorts and paves the way for the development of effective TA-based immunotherapies against a large variety of cancers.

L’immunité adaptive et la discrimination entre le soi et le non-soi chez les vertébrés à mâchoire reposent sur la présentation de peptides par les récepteurs d’histocompatibilité majeur de classe I. Les peptides antigéniques, présentés par les molécules du complexe d’histocompatibilité (CMH), sont scrutés par les lymphocytes T CD8 pour une réponse immunitaire appropriée. Le répertoire des peptides du CMH de classe I, aussi appelé immunopeptidome, est généré par la dégradation protéosomale des protéines endogènes, et a un rôle essentiel dans la régulation de l’immunité cellulaire. La composition de l’immunopeptidome dépend du type de cellule et peut présenter des caractéristiques liées à des maladies comme le cancer. Les peptides antigéniques peuvent être utilisés à des fins immunothérapeutiques notamment dans le traitement voire la prévention de certains cancers. La spectrométrie de masse est un outil de choix pour l’identification, le séquençage et la caractérisation de ces peptides. Cependant, la composition en acides aminés, la faible abondance et la diversité de ces peptides compliquent leur détection et leur séquençage. Nous avons développé un programme appelé StatPeaks qui permet de calculer un certains nombres de statistiques relatives à la fragmentation des peptides. À l’aide de ce programme, nous montrons sans équivoque que les peptides du CMH classe I, en mode de fragmentation par dissociation induite par collision (CID), fragmentent très différemment des peptides trypsiques communément utilisés en protéomique. Néanmoins, la fragmentation par décomposition induite par collision à plus haute énergie (HCD) proposée par le spectromètre LTQ-Orbitrap Velos améliore la fragmentation et fournit une haute résolution qui permet d’obtenir une meilleure confiance dans l’identification des peptides du CMH de classe I. Cet avantage permet d’effectuer le séquençage de novo pour identifier les variants polymorphes qui ne sont normalement pas identifiés par les recherches utilisant des bases de données. La comparaison des programmes de séquençage Lutefisk, pepNovo, pNovo, Vonode et Peaks met en évidence que le dernier permet d’identifier un plus grand nombre de peptides du CMH de classe I. Ce programme est intégré dans une chaîne de traitement de recherche d’antigènes mineurs d’histocompatibilité. Enfin, une base de données contenant les informations spectrales de plusieurs centaines de peptides du CMH de classe I accessible par Internet a été développée. / Adaptive immunity and discrimination between self and nonself in jawed vertebrates relies on the presentation of peptides by the major histocompatibility (MHC) class I receptors. Foreign or self peptide antigens presented by the MHC molecules are probed by CD8 T-cell lymphocyte for proper immune response. The repertoire of MHC I peptides collectively referred to as the immunopeptidome is generated through the proteasomal degradation of endogenous proteins and plays an important role in the regulation of cellular immunity. The composition of the immunopeptidome is cell specific and can harbor important hallmark of human diseases including cancer. Antigenic peptides can also be used in immunotherapy to mount an appropriate immune response against cancer cells displaying these peptides. Mass spectrometry is a tool of choice for the identification, sequencing and characterization of these peptides. However, the amino acid composition, the low abundance and diversity of these peptides make their detection and sequencing more challenging. We developed a software, called StatPeaks, that calculates statistics relative to the fragmentation of peptides. Using this software, we demonstrate that under collision induced dissociation (CID) MHC class I peptides fragment in a very different fashion than tryptic peptides, commonly used in proteomics. However, the higher-energy collisional dissociation (HCD) mode available on the LTQ-Orbitrap Velos enhances peptide fragmentation and provides high resolution fragment information that significantly improves the confidence in MHC class I peptide identification. This inherent advantage confers the ability to perform de novo sequencing to identify polymorphic variants that would normally elude conventional database searches. The comparison of de novo peptide sequencing software Lutefisk, pepNovo, pNovo, Vonode and Peaks indicated that the later software enabled higher rates of correct identification for MHC class I peptides. This software was integrated into a data analysis pipeline for the identification minor histocompatibility antigens (MiHAs). A web-based library that stores spectral information of hundreds of synthetic MHC class I peptides was developed in support to the needs of the immunopeptidome discovery program.

Le système ubiquitine-protéasome est le principal mécanisme par lequel les protéines intracellulaires sont dégradées. Le protéasome dit constitutif (PC) est donc essentiel à l’homéostasie mais aussi à la régulation de la majorité des processus cellulaires importants. La découverte d’un deuxième type de protéasome, appelé immunoprotéasome (IP), soulève toutefois de nouvelles questions. Pourquoi existe-t-il plus d’un type de protéasome ? L’IP a-t-il des rôles redondants ou complémentaires avec le PC ? L’IP étant présent principalement dans les cellules immunitaires ou stimulées par des cytokines, plusieurs groupes ont tenté de définir son rôle dans la réponse immunitaire. Or, l’implication de son homologue constitutif dans un éventail de processus non spécifiquement immunitaires nous laisse croire que l’IP pourrait lui aussi avoir un impact beaucoup plus large. L’objectif de cette thèse était donc de caractériser certains rôles cellulaires de l’IP dans les cellules dendritiques. Nous avons d’abord étudié l’impact global de l’IP sur la présentation antigénique de classe I. Ce faisant, nous avons pu déterminer ses deux contributions principales, soit l’augmentation drastique du nombre et de la diversité des peptides présentés sur les complexes majeurs d’histocompatibilité de classe I. Les différences de clivage entre le PC et l’IP pourraient expliquer en partie cette diversité du répertoire peptidique, notamment par l’affinité apparente de l’IP pour les régions protéiques non structurées. Dans un deuxième temps, nous avons dévoilé un nouveau rôle de l’IP sur un processus dépassant le cadre immunitaire : la transcription. Nous avons découvert que l’IP modifie l’abondance des ARNm en agissant principalement au niveau de leur synthèse. L’impact de l’IP sur le transcriptome est majeur et serait dû en partie à une dégradation différente de facteurs de transcription des familles IRF, STAT et NF-kB. Les cellules dendritiques IP-déficientes activent moins efficacement les lymphocytes T CD8+ et nous croyons que cette défaillance est causée (du moins en partie) par la perturbation transcriptomique provoquée par l’absence d’IP. Il importe donc de comprendre les différents rôles moléculaires de l’IP afin de mieux définir sa contribution globale au fonctionnement de la cellule et comprendre l’avantage évolutif, au niveau de l’organisme, procuré par une telle plasticité du système ubiquitine-protéasome. / The ubiquitin-proteasome system is the major mechanism by which intracellular proteins get degraded. Constitutive proteasomes (CPs) are thus essential for cellular homeostasis but also to regulate the majority of important cellular processes. However, the discovery of a second type of proteasome, named immunoproteasome (IP), raises new questions. Why are there more than one type of proteasome? Does the IP perform redundant or complementary roles with the CP? The IP is predominantly expressed in immune or cytokine-stimulated cells and several groups worked at defining its role during the immune response. Yet, the implication of its constitutive homolog in a variety of processes suggests that the IP may also have a much broader impact. The objective was to characterize cellular roles of the IP in dendritic cells. We first studied the global impact of the IP on class I antigen presentation. We discovered that the IP drastically increases the number and the diversity of peptide presented by class I major histocompatibility complexes. Cleavage differences between the CP and the IP are likely part of the explanation for this peptide repertoire diversity, notably due to IP’s apparent affinity for unstructured protein regions. Second, we discovered a new role for the IP in a process unrestricted to the immune system: transcription. We found that the IP affects transcript abundance mostly at the level of mRNA synthesis. The impact of IPs on the transcriptome is major and would be partly based on a different degradation of IRF, STAT and NF-kB transcription factor family members by the two types of proteasomes. IP-deficient dendritic cells are less potent activators of CD8+ T cells and we believe that this defect is at least partly caused by the transcriptome alterations induced by the absence of IPs. It is therefore important to understand the different molecular roles of the IP in order to better define its global contribution to cellular functions and to understand the evolutionary advantage, at the level of the organism, brought by such plasticity of the ubiquitin- proteasome system.

INTRODUÇÃO: O penfigoide bolhoso é uma doença autoimune, vesicobolhosa com incidência de 0,2 a 1,4 por 100.000 hab. Sua fisiopatologia caracteriza-se pela ação de autoanticorpos na junção dermoepidérmica dos hemidesmossomos, promovendo a formação de bolhas subepidérmicas na pele e mucosas. Estudos vêm sendo publicados demonstrando a associação de penfigoide com alelos do sistema HLA classe II em diferentes populações do mundo, entretanto não há dados sobre a população brasileira, uma das mais heterogêneas do mundo. PACIENTES E MÉTODOS: O grupo de estudo incluiu 17 pacientes brasileiros com diagnóstico confirmado de PB de um hospital na cidade de São Paulo, sudeste do Brasil. O DNA foi extraído a partir de sangue periférico utilizando kits Qiagen (QIAamp DNA Mini Kit®) e a tipagem HLA loci A, B, C, DR e DQ foi realizada por meio de PCR e a amplificação utilizando o oligonucleótido de sequência específica (SSO) contido nos kits LABType®. O grupo controle foi composto por um banco de dados de 297 doadores falecidos da cidade de São Paulo. Este banco de dados é parte do Sistema de Transplantes da Secretaria de Saúde do Governo do Estado de São Paulo. RESULTADOS: Os resultados mostram que os alelos HLA C*17, DQB1*03:01, DQA1*01:03 e DQA1*05:05 estão associados com o aparecimento da doença na população brasileira, com risco relativo de 8,31 (2,46 a 28,16), 3,76 (1,81 a 7,79), 3,57 (1,53 a 8,33) e 4,02 (1,87 a 8,64), respectivamente (p < 0,005). O nível de significância estatística foi ajustado utilizando a correção de Bonferroni, dependendo das frequências fenotípicas avaliadas para HLA de classe I (A, B e C) e classe II (DRB1, DQB1 e DQA1). DISCUSSÃO: Os dados indicam que pacientes brasileiros com PB apresentam a mesma predisposição genética ligada ao HLA-DQB1*03:01 relatado anteriormente em caucasianos e indivíduos iranianos e o estudo apresenta três novos alelos (C *17, DQA1*01:03 e DQA1* 05:05) envolvidos na fisiopatologia da PB. CONCLUSÕES: Os resultados mostram que os alelos HLA C*17, DQB1*03:01, DQA1*01:03 e DQA1*05:05 estão associados com o aparecimento da doença na população brasileira / BACKGROUND: Bullous pemphigoid (BP) is an autoimmune disease with bullous vesicles and an incidence of 0.2 to 1.4 per 100,000 inhabitants. Its pathophysiology is characterized by the action of autoantibodies on hemidesmosomes at the dermalepidermal junction, promoting subepidermal blister formation in the skin and mucous membranes. Many studies have been published demonstrating the association of pemphigoid with HLA class II system alleles in different populations, however there are no data on the Brazilian population, one of the most heterogeneous in the world. PATIENTS AND METHODS: The study group included 17 Brazilian patients with a confirmed diagnosis of BP from a hospital in Sao Paulo city, southeast Brazil. DNA was extracted from peripheral blood using Qiagen kits (QIAamp DNA Mini Kit®) and HLA A, B, C, DR and DQ typing was performed using PCR and amplification using Sequence-Specific Oligonucleotide (SSO) contained in LABType® kits. The control group was composed of a database of 297 deceased donors from the city of São Paulo. This database is part of the Transplants State System of the Government\'s Health Secretary from the State of Sao Paulo. RESULTS: Our findings show that alleles HLA C*17, DQB1*03:01, DQA1*01:03 and DQA1*05:05 are associated with the onset of the disease in the Brazilian population, with relative risks of 8.31 (2.46 to 28.16), 3.76 (1.81 to 7.79), 3.57 (1.53 to 8.33), and 4.02 (1.87 to 8.64), respectively (p < 0.005). The statistical significance level was adjusted using the Bonferroni correction depending on the phenotypic frequencies evaluated for HLA class I (A, B and C) and class II (DRB1, DQB1 and DQA1). DISCUSSION: Our data indicate that Brazilian patients with BP present the same genetic predisposition linked to HLA-DQB1*03:01 previously reported in Caucasian and Iranian individuals and our study introduces three new alleles (C*17, DQA1*01:03 and DQA1*05:05) involved in the pathophysiology of BP. CONCLUSIONS: Our findings show that alleles HLA C*17, DQB1*03:01, DQA1*01:03 and DQA1*05:05 are associated with the onset of the disease in the Brazilian population

INTRODUÇÃO: O penfigoide bolhoso é uma doença autoimune, vesicobolhosa com incidência de 0,2 a 1,4 por 100.000 hab. Sua fisiopatologia caracteriza-se pela ação de autoanticorpos na junção dermoepidérmica dos hemidesmossomos, promovendo a formação de bolhas subepidérmicas na pele e mucosas. Estudos vêm sendo publicados demonstrando a associação de penfigoide com alelos do sistema HLA classe II em diferentes populações do mundo, entretanto não há dados sobre a população brasileira, uma das mais heterogêneas do mundo. PACIENTES E MÉTODOS: O grupo de estudo incluiu 17 pacientes brasileiros com diagnóstico confirmado de PB de um hospital na cidade de São Paulo, sudeste do Brasil. O DNA foi extraído a partir de sangue periférico utilizando kits Qiagen (QIAamp DNA Mini Kit®) e a tipagem HLA loci A, B, C, DR e DQ foi realizada por meio de PCR e a amplificação utilizando o oligonucleótido de sequência específica (SSO) contido nos kits LABType®. O grupo controle foi composto por um banco de dados de 297 doadores falecidos da cidade de São Paulo. Este banco de dados é parte do Sistema de Transplantes da Secretaria de Saúde do Governo do Estado de São Paulo. RESULTADOS: Os resultados mostram que os alelos HLA C*17, DQB1*03:01, DQA1*01:03 e DQA1*05:05 estão associados com o aparecimento da doença na população brasileira, com risco relativo de 8,31 (2,46 a 28,16), 3,76 (1,81 a 7,79), 3,57 (1,53 a 8,33) e 4,02 (1,87 a 8,64), respectivamente (p < 0,005). O nível de significância estatística foi ajustado utilizando a correção de Bonferroni, dependendo das frequências fenotípicas avaliadas para HLA de classe I (A, B e C) e classe II (DRB1, DQB1 e DQA1). DISCUSSÃO: Os dados indicam que pacientes brasileiros com PB apresentam a mesma predisposição genética ligada ao HLA-DQB1*03:01 relatado anteriormente em caucasianos e indivíduos iranianos e o estudo apresenta três novos alelos (C *17, DQA1*01:03 e DQA1* 05:05) envolvidos na fisiopatologia da PB. CONCLUSÕES: Os resultados mostram que os alelos HLA C*17, DQB1*03:01, DQA1*01:03 e DQA1*05:05 estão associados com o aparecimento da doença na população brasileira / BACKGROUND: Bullous pemphigoid (BP) is an autoimmune disease with bullous vesicles and an incidence of 0.2 to 1.4 per 100,000 inhabitants. Its pathophysiology is characterized by the action of autoantibodies on hemidesmosomes at the dermalepidermal junction, promoting subepidermal blister formation in the skin and mucous membranes. Many studies have been published demonstrating the association of pemphigoid with HLA class II system alleles in different populations, however there are no data on the Brazilian population, one of the most heterogeneous in the world. PATIENTS AND METHODS: The study group included 17 Brazilian patients with a confirmed diagnosis of BP from a hospital in Sao Paulo city, southeast Brazil. DNA was extracted from peripheral blood using Qiagen kits (QIAamp DNA Mini Kit®) and HLA A, B, C, DR and DQ typing was performed using PCR and amplification using Sequence-Specific Oligonucleotide (SSO) contained in LABType® kits. The control group was composed of a database of 297 deceased donors from the city of São Paulo. This database is part of the Transplants State System of the Government\'s Health Secretary from the State of Sao Paulo. RESULTS: Our findings show that alleles HLA C*17, DQB1*03:01, DQA1*01:03 and DQA1*05:05 are associated with the onset of the disease in the Brazilian population, with relative risks of 8.31 (2.46 to 28.16), 3.76 (1.81 to 7.79), 3.57 (1.53 to 8.33), and 4.02 (1.87 to 8.64), respectively (p < 0.005). The statistical significance level was adjusted using the Bonferroni correction depending on the phenotypic frequencies evaluated for HLA class I (A, B and C) and class II (DRB1, DQB1 and DQA1). DISCUSSION: Our data indicate that Brazilian patients with BP present the same genetic predisposition linked to HLA-DQB1*03:01 previously reported in Caucasian and Iranian individuals and our study introduces three new alleles (C*17, DQA1*01:03 and DQA1*05:05) involved in the pathophysiology of BP. CONCLUSIONS: Our findings show that alleles HLA C*17, DQB1*03:01, DQA1*01:03 and DQA1*05:05 are associated with the onset of the disease in the Brazilian population