Orientador: Eliane Maria Ingrid Amstalden / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-08T21:39:33Z (GMT). No. of bitstreams: 1 Cavalcanti_JosemaraNeves_D.pdf: 3609104 bytes, checksum: 434c28eb652f05aaf48e1669c6773623 (MD5) Previous issue date: 2007 / Resumo: O osteossarcoma destaca-se por ser o sarcoma mais comum dentre os tumores ósseos malignos, na espécie canina, e pelo seu alto potencial de agressividade. Do ponto de vista histológico é um tumor de padrão celular heterogêneo, que se caracteriza pela produção de matriz osteóide e/ou osso imaturo por osteoblastos. Apenas dois estudos sistemáticos, relativos ao osteossarcoma canino, foram identificados na literatura nacional. Um deles refere-se a dados epidemiológicos e o outro aborda achados clínico-morfológicos. Estudos objetivando a avaliação dos aspectos histogenéticos e do índice de proliferação celular não foram ainda descritos na literatura nacional. A proposta deste estudo foi determinar a diferenciação dos diversos elementos celulares constituintes do osteossarcoma canino e avaliar o índice de proliferação celular destes tumores, correlacionando-os com seus diferentes subtipos morfológicos, utilizando-se de imunomarcadores, tais como: vimentina, osteocalcina, proteína S -100, 1A4, HHF35, AE1/AE3, CD31, CD34 e Ki-67. Foi realizado estudo retrospectivo de 65 casos de cães portadores de osteossarcoma, os quais foram avaliados clinicamente quanto ao sexo, raça, idade e localização topográfica das lesões. Os tumores foram classificados quanto ao subtipo histológico em osteoblásticos pouco produtivos ou osteoblásticos produtivos (de acordo com a quantidade de osteóide produzido), condroblásticos, fibroblásticos, do tipo células gigantes, telangiectásicos e indiferenciados. Cães machos, da raça Rottweiler, com idade média de 7,4 anos, foram mais freqüentemente afetados por osteossarcoma. Em 77,4 % dos casos as lesões ocorreram no esqueleto apendicular (fêmur). Os subtipos histológicos identificados foram: osteoblásticos (47,7%), fibroblásticos (23,1%), condroblásticos (21,5 %), do tipo células gigantes (4,7%), telangiectásicos (1,5%) e indiferenciados (1,5%). Em 98,2 % dos osteossarcomas houve reatividade para vimentina. Imunorreatividade à osteocalcina ocorreu em células osteoblásticas, condroblásticas e fibroblásticas de 91% dos osteossarcomas. Proteína S-100 foi expressa em 79,7% dos tumores, tendo ocorrido em todos os tumores condroblásticos. Proporção significativa de osteossarcomas reagiu positivamente aos anticorpos 1A4 e HHF35. Todos os osteossarcomas foram negativos para AE1/AE3e para CD31 e CD34. A imunomarcação com Ki-67 revelou alto índice de proliferação celular em osteossarcomas osteoblásticos pouco produtivos. Os osteossarcomas caninos são constituídos por diversificada população de células, representadas por elementos osteoblásticos, condroblásticos, miofibroblásticos e mioblásticos, os quais possivelmente, originam-se de uma única célula mesenquimal pluripotente ou de osteoblastos imaturos, que sofrem diferenciação diversificada. Osteossarcomas osteoblásticos pouco produtivos apresentaram índice expressivo de proliferação celular, o qual pode estar correlacionado a um caráter de agressividade mais acentuado / Abstract: Osteosarcoma is the most common of all malignant canine bone tumors and has a high aggressiveness potential. From the histological point of view it is a tumor with a heterogeneous cell pattern that is characterized by the production of bone matrix and/or immature bone by osteoblasts. Systematic studies with a clear objective to evaluate histogenetic aspects and the cellular proliferation index in canine osteosarcomas have not yet been published. The objective of this study was to determine the differentiation of the constituting cellular elements and evaluate the cellular proliferation index of these tumors correlating them with their different morphological subtypes, using markers such as: vimentin, osteocalcin, S-100 protein, 1A4, HHF53, factor VIII, AE1/AE3, CD31, CD34 and Ki-67. A retrospective study was performed using 65 cases of canine osteosarcoma, clinically evaluated according to sex, breed, age and topography of the lesions. Tumors were classified according to their histological subtype: moderately productive osteoblastic tumor, productive tumor (both according to the production of osteoid matrix), chondroblastic tumor, fibroblastic tumor, giant cell tumor, telangiectatic tumor and undifferentiated tumors. Males from the Rottweiler breed with an average age of 7,4 years were more frequently affected by osteosarcoma. In 77,4% of cases, the lesions were on the appendicular skeleton (femur). The Identified histological subtypes were: osteoblastic (47,7%), fibroblastic (23,1%), chondroblastic (21,5 %), giant cell (4,7%), telangiectatic (1,5%) and undifferentiated (1,5%). In 98,2% of all tumors there was vimentin reactivity. Immunoreactivity to osteocalcin occurred in osteoblastic, chondroblastic and fibroblastic cells of 91% of all osteosarcomas. The S-100 protein was expressed in 79,7% of all tumors, and in 100% of the chondroblastic tumors. A significant proportion of osteosarcomas reacted positively to the 1A4 and HHF35 antibodies. All osteosarcomas were negative for AE1/AE3 and CD31 and CD34 testing. Marking essays with Ki-67 revealed a high cell proliferation index in moderately productive osteoblastic osteosarcomas. Canine osteosarcomas are constituted by a diverse cell population, composed of osteoblastic, chondroblastic, myofibroblastic and mioblastic elements, which are probably originated from a single pluripotent mesenchymal cell or from immature osteoblasts that go thru a differentiation process. Moderately productive osteoblastic osteosarcoma presented a considerably high cell proliferation index that may relate to a more aggressive behavior / Doutorado / Ciencias Biomedicas / Doutor em Ciências Médicas

Larvas de cisticercos de Taenia crassiceps foram deixadas em repouso em TRIS 9mM pH 7,2 com 1mM EDTA até 180 minutos, os sobrenadantes coletados e processados nos 30, 60, 120 e 180 minutos, dando origem aos antígenos de excreção e secreção (ES 30; ES 60; ES 120 e ES 180). A caracterização do antígeno de excreção e secreção de larvas de Taenia crassiceps foi feito por SDS-PAGE e imunoblot utilizando anticorpos monoclonais (AcMos) anti-T. crassiceps e anti-T. solium e anticorpos humanos. Os resultados mostraram que o rendimento do antígeno ES foi menor nos primeiros 30 minutos (0,4 µg) por larva quando comparado os demais ES (ES 60: 2,4 µg; ES 120: 2,9 µg; ES 180: 2,5 µg). O SDS-PAGE confirmou que no ES 30 há menos proteínas. No imunoblot, o ES 180 mostrou que 6 AcMos (anti-LV-Tcra; anti-ES-Tcra; anti-LV-Tso: A3; anti-T-Tso: B4, B11 e A6) reconheceram apenas as frações 18- e 14-kDa do antígeno ES 180. Os AcMos anti-E-Tso (B8) e anti-LV-Tso (B6) não reconheceram frações do antígeno ES 180. Anticorpos presentes em amostras humanas de pacientes com NC reconheceram as frações protéicas entre 94- a 30-kDa e as de 18- e 14-kDa. Utilizando antígeno ES 180 e amostras de soros de pacientes supostamente saudáveis (GC), foram identificadas proteínas acima de 30-kDa e somente uma amostra reconheceu a de 16- kDa, anômala em relação ao perfil 18- e 14-kDa. As amostras de soro de pacientes com outras parasitoses mostraram reatividade com frações ≥ de 30-kDa do ES 180 e o maior índice de reatividade foi com a proteína 71-KDa. Um total de 77%; 70%; 60% e 70% das amostras de pacientes com toxocaríase, esquistossomose mansônica, hidatidose e Chagas, respectivamente, reconheceram a fração 71-kDa do ES 180. O antígeno ES pode contribuir com futuros estudos abordando a complexa relação parasito hospedeiro na cisticercose e na produção de vacinas para o uso em suínos. / Cysticercus Larvae of Taenia crassiceps were maintained in TRIS 9mM pH 7,2 with 1mM EDTA for 180 minutes; the supernatant was collected and processed at 30; 60; 120 and 180 minutes, originating excretion-secretion antigens (ES 30; ES 60; ES 120 and ES 180). The characterization of the ES antigen was conducted through SDS-PAGE and immunoblot using anti-T. crassiceps and anti-T. solium monoclonal antibodies (AcMos) and human antibodies. The results showed that the production of ES antigen was lower in the first 30 min. (0,4 µg) compared with the others (ES 60: 2,4 µg; ES 120: 2,9 µg; ES 180: 2,5 µg). The SDS-PAGE confirmed that ES 30 presented less protein. By immunoblot,6 AcMos (anti-LV-Tcra; anti-ES-Tcra;anti-LV-Tso: A3; anti-T-Tso: B4, B11 and A6) have recognized only the 18- and 14-kDa fractions of the ES 180. The anti-E-Tso (B8) and the anti-LV-Tso (B6) AcMos did not recognize any fractions. Antibodies from human samples NC recognized the proteins from 94- to 30-kDa and from 18- and 14-kDa. Using serum samples of apparently healthy individuals (GC), the ES 180 antigen showed proteins > 30-kDa and one sample recognized the 16-kDa fraction, anomalous when compared to the 18- and 14-kDa fractions. The serum samples of subjects with other parasitoses showed reactivity ≥ 30-kDa, more frequently with 71-KDa protein. A total of 77%; 70%; 60% and 70% of the samples from subjects presenting toxocariasis, esquistossomose mansonic, hydatidosis and Chagas disease, respectively, recognized the 71-kDa fraction of ES 180. The ES antigen may contribute to further studies on the complex cysticercosis parasite/host relation as well as for the production of vaccines for swine.

Made available in DSpace on 2014-08-20T13:32:51Z (GMT). No. of bitstreams: 1 tese_claudia_fernandes.pdf: 1692742 bytes, checksum: 5395743de930fc1ac7ec3c75eaab4e86 (MD5) Previous issue date: 2008-02-15 / Leptospirosis is a zoonotic disease that occurs all over the world and is caused by pathogenic bacteria of the genus Leptospira. Clinical manifestations of leptospirosis are similar to other febrile illnesses and this fact frequently retards beginning of antibiotic therapy. Thus, early and accurate diagnosis is a prerequisite for proper treatment of leptospirosis. Pathogenic serovars of Leptospira have a wide antigenic diversity attributed mainly to the lipopolysacharide present in the outer membrane. In contrast, antigens conserved among pathogenic serovars are mainly represented by outer membrane proteins. Surface exposure of a major and highly conserved outer membrane lipoprotein (LipL32) was recently demonstrated on pathogenic Leptospira. LipL32 on its recombinant form (rLipL32) was used to immunize BALB/c mice to develop murine monoclonal antibodies (mAbs). Three mAbs against rLipL32 were produced, isotyped and evaluated for further use in diagnostic tests of leptospirosis using different approaches. The mAbs were conjugated to peroxidase and evaluated in a native protein ELISA with intact and heat-treated leptospiral cells, conjugated to fluorescein isothiocyanate (FITC) for direct immunofluorescence with intact and methanol fixed cells and were used for LipL32 immunoprecipitation from leptospiral cells. rLipL32 mAbs conjugated to peroxidase or used as primary antibody bounded to intact and heat-treated cells in ELISA, proving that they could be used in enzyme immunoassays for detection of the native protein. On immunofluorescence assay, mAbs labeled bacterial cells either intact or methanol fixed. Two mAbs were able to immunoprecipitate the native protein from live and motile leptospiral cells and, adsorbed onto magnetic beads, captured intact bacteria from artificially contaminated human sera for detection by PCR amplification. One mAb was utilized for the development of an immunoseparation assay coupled to PCR test (IMS/PCR) for diagnosis of leptospirosis. The antibody adsorved onto magnetic beads captured leptospires from urine and human sera artificially contaminated for further amplification of the lipL32 gene by PCR. To ensure PCR accuracy, an internal amplification control (IAC) was constructed using as amplification targets sequences of standardized primers specific for pathogenic Leptospira and for a not-related DNA sequence. The IMS/PCR IAC method developed was able to detect 102 cells per mL of sera or urine, corresponding to approximately 25 genomic copies per reaction. These results suggest that the association of LipL32-based immunochemical and molecular techniques could yield a novel method for the diagnosis of leptospirosis. Moreover, immunomagnetic separation with mAbs against LipL32 can be used previous to amplification of other targets in the Leptospira genome by PCR. / A Leptospirose é uma zoonose de ocorrência mundial causada por bactérias do gênero Leptospira. As manifestações clínicas da leptospirose são similares a outras doenças febris e este fato frequentemente atrasa o diagnóstico e o início do tratamento. Portanto, o diagnóstico precoce e acurado da doença é um prerequisito para o tratamento adequado. Sorovares patogêncios de Leptospira possuem uma grande variação antigência e esta diversidade é atribuída principalmente ao lipopolissacarídeo presente na membrana externa. Contrastando com esta característica, antígenos conservados de sorovares patogênicos são principalmente representados por proteínas de membrana externa. Recentemente foi comprovada a exposição da proteína LipL32 na superfície da membrana externa de leptospiras patogênicas. Neste estudo, LipL32 em sua forma recombinante (rLipL32) foi utilizada para imunizar camundongos BALB/c e produzir anticorpos monoclonais (mAbs). Três mAbs contra rLipL32 foram produzidos e caracterizados quanto ao seu potencial para uso em testes diagnósticos usando diferentes metodologias. Os mAbs foram conjugados à peroxidase e avaliados quanto a reação com proteina nativa em células de leptospiras íntegras e rompidas, conjugados com isotiocianato de fluoresceína (FITC) para uso em imunofluorescência para marcar células de leptospira intactas e tratadas com metanol, e usados para imunoprecipitar células de leptospira. Os anticorpos monoclonais anti-LipL32, utilizados em ELISA tanto conjugados com peroxidase ou como anticorpo primário, ligaram-se às células de leptospiras intactas ou rompidas pelo calor, provando que podem ser usados em testes imunoenzimáticos para detecção da proteína nativa. Na imunofluorescência, os mAbs foram capazes de marcar células da bactéria tanto intactas como fixadas com metanol. Dois mAbs foram capazes de imunoprecipitar proteina nativa de leptospiras vivas e móveis, e quando adsorvidos em partículas magnéticas foram capazes de capturar bactérias para amplificação por PCR. Na seqüência deste estudo, o mAb 1D9 foi utilizado em estudos de padronização da metodologia de imunoseparação magnética associada a PCR (IMS/PCR) para diagnóstico de leptospirose. O anticorpo 1D9 foi adsorvido em partículas magnéticas e utilizado para capturar leptospiras em soro e urina humanas artificialmente contaminadas com leptospiras para posterior amplificação. Para assegurar a acurácia da PCR foi construído um controle interno de amplificação (IAC) específico para a metodologia desenvolvida utilizando como alvo sequências de primers já padronizados para exclusiva amplificação de leptospiras patogências e uma sequencia de DNA não relacionada. A metodologia de IMS/PCR IAC permitiu usar somente um par de primers na reação de PCR e mostrou ser promissora para diagnóstico de leptospirose, pois foi capaz de detectar 102 células por mL em amostras de soro e urina artificialmente contaminadas, correspondendo a amplificação a partir de aproximadamente 25 cópias do genoma. Os resultados obtidos evidenciam uma nova perspectiva no diagnóstico da leptospirose através da utilização da proteína LipL32 em métodos imunoquímicos e moleculares ou pela associação destas metodologias. A metodologia de imunoseparação com o mAb anti-LipL32 pode ser utilizada previamente a amplificação de outros alvos do genoma bacteriano por PCR, já que ela possibilita a separação e concentração exclusiva de Leptospira patogênica.

Tumour immunity is a balance between immune mediators that promote tumor progression versus mediators that promote tumor rejection. Infiltrating lymphocytes in human colorectal cancer tissues are independent prognostic factors for a better survival and a high number of cytotoxic CD8+ T-cells have been associated with a better prognosis in terms of a longer and disease free survival for the patient. In our syngeneic rat model we induce colon carcinoma subperitoneally by injecting a colon cancer cell line BN7005, a cell line expressing the epitope (Lewis Y) for the BR96 antibody. Tumours are dissected out and treated with different fixatives and then either frozen, snap-frozen or embedded in paraffin followed by sectioning. Immunohistochemistry using monoclonal antibodies against the tumour infiltrating leukocytes was performed on the tissue. The results were seen as an infiltration of different leukocytes in the tumours.

La quantification des anticorps monoclonaux (mAbs) dans le plasma est un pré-requis essentiel pour les études PK/PD. Les méthodes de références pour quantifier actuellement les mAbs sont de type ELISA mais les difficultés rencontrées notamment lorsque l’analyse porte sur des mAbs dont la cible pharmacologique est circulante, suggèrent que la spectrométrie de masse serait une alternative intéressante. Appliquée au bevacizumab, la stratégie développée fait appel à la spectrométrie de masse en tandem utilisée en mode MRM (HPLC-ESI-QqQ) et porte sur l’analyse des peptides spécifiques du bevacizumab obtenus à l’issu d’une protéolyse trypsique. La quantification absolue est réalisée à l’aide d’une droite de calibration obtenue à partir du ratio des aires des peptides du bevacizumab et de l’étalon interne. Afin de proposer une méthodologie de quantification de référence, nous avons définie les points clés du développement pour la transposition à d’autre mAbs et comparé les deux stratégies d’étalonnage interne les plus employées : l’une utilisant une protéine analogue et l’autre un peptide marqué par des isotopes stables (SIL-peptide). A travers ce développement la stratégie proposée présente un caractère universel vis-à-vis des anticorps monoclonaux de type IgG dont le traitement des échantillons repose sur une purification par protéine A suivit d’une concentration par ultrafiltration et dont la quantification fait appel à l’approche d’étalonnage interne SIL-peptide. Validée selon les recommandations de la FDA, notre méthode présente les performances analytiques attendues en termes de sensibilité, répétabilité et spécificité pour être appliquée à des études cliniques. / The quantification in plasma of monoclonal antibodies (mAbs) is an essential prerequisite to any PK/PD preclinical and clinical study. To date, reference techniques used to quantify mAbs, rely on enzyme-linked immunosorbent assay (ELISA) but the difficulties encountered in particular when the analysis focuses on the mAbs whose pharmacological target is circulating, suggest that mass spectrometry would be an interesting alternative. Applied to bevacizumab, the quantification developed strategy involves tandem mass spectrometry (HPLC-ESI-QqQ) used in MRM mode and focuses on the analysis of specific peptides bevacizumab obtained after tryptic proteolysis. Absolute quantification is achieved through calibration curve obtained from peak area ratios of bevacizumab surrogate peptide and internal standard. To propose a reference quantification methodology, we have identified the key points of development for transposition to other mAbs and compared the two most commonly used internal calibration approaches: one using protein analogue and the other a stable isotope labeled surrogate peptide (SIL-peptide). Through this development, the proposed strategy has a universal character with respect to IgG monoclonal antibodies subclasses which is based on sample processing purification using protein A followed by concentration by ultra filtration and whose quantification involves the internal calibration approach SIL-peptide. Validated according to FDA guidelines, our method shows the expected analytical performance in terms of sensitivity, specificity and repeatability for application in clinical studies

Au cours de ce travail, nous avons mis au point une nouvelle interface CE/MALDI-MS automatisée, équipée d’une cellule UV/visible déportée, et d’une distribution automatique de matrice intégrée. Ce nouveau système a été évalué sur des mélanges différents de protéines entières, de digestats de protéines et d’anticorps monoclonaux (mAbs). Les résultats obtenus lors de cette évaluation ont montré la complémentarité de la nouvelle interface avec les systèmes analytiques classiques. De plus, nous avons montré la première séparation et analyse de mAbs entier par CE/MALDI-MS. Dans un second travail, la nouvelle interface a été utilisée pour effectuer la première analyse Top Down de proteine entière et de mAbs par collecte et enrichissement de fraction. Cette stratégie a montré la répétabilité du système permettant de séparer des analytes d’intérêt et d’enrichir les dépôts MALDI jusqu'à de très hautes quantités d’analytes permettant l’obtention de spectre Top Down. Au cours du troisième travail, le nouveau système CE/MALDI-MS a été utilisé dans une stratégie originale à 2 dimensions de séparation et de collecte d’isoformes de mAbs entiers ou partiellement digérés suivi d’infusions et analyses à l’aide du nanosprayer CESI. Pour cela, nous avons mis au point des conditions électrophorétiques dit « asymétriques » séparant les mAbs dans des conditions très salées mais collectés dans un milieu compatible avec l’ESI-MS. Cette stratégie inédite a permis d’effectuer la première séparation et caractérisation de mAbs par CE-MS. Parallèlement, nous avons mis au point le premier dosage plasmatique d’ITPP par MALDI-TOF MS et surtout la création de CEToolbox, une application Androïd gratuite pour smartphone et tablette permettant le calcul des principales grandeurs mathématiques pour la caractérisation et l’optimisation des séparations par électrophorèse capillaire. / In this work, we developed a new interface CE/MALDI-MS automated, equipped with a UV/visible cell remote, and integrated automatic distribution of matrix. This new system has been evaluated on different mixtures of intact protein, digested protein and monoclonal antibodies (mAbs). The results obtained during this evaluation showed the complementarity of the new interface with conventional analytical systems. Furthermore, we have shown the first separation and analysis of mAbs by CE/MALDI-MS. In a second work, the new interface was used to perform the first Top Down analysis for intact protein and mAbs by fraction collection and enrichment. This strategy has shown the repeatability of the system for separating analytes and the enrichissment the MALDI deposits up to very high amounts compatible for Top Down approach. In the third work, the new system CE/MALDI-MS has been used in an original 2-dimensional strategy of separating and collecting intact mAbs isoforms or partially digested followed by infusions and analyzes with CESI nanosprayer. For this, we have developed electrophoretic condition so-called "asymmetric" allowing the separation of mAbs under very salty conditions but collected in a totally compatible solution with ESI-MS. This novel strategy allowed for the first separation and characterization of mAbs by CE-MS. Meanwhile, we have developed the first plasma level of ITPP by MALDI-TOF-MS and particularly the creation of CEToolbox as a Free Android application for smartphone and tablet enabling the calculation of the main mathematical quantities for characterization and optimization of CE separations.

Ce travail de thèse porte sur le développement de méthodes en spectrométrie de masse (MS) et mobilité ionique (IM-MS) supramoléculaires pour la caractérisation fine de complexes protéine-ligand et d’assemblages protéiques hétérogènes de hauts poids moléculaires. L’optimisation instrumentale apportée à l’étude de ces systèmes, permet d’étendre le potentiel de ces deux approches en biologie structurale. Le criblage de complexes protéine-ligand permet ici une détermination de leurs propriétés d’interaction et la mise en évidence de subtils changements de conformation induits, pouvant être suivis au cours du temps. L’application de ce couplage à l’analyse de complexes multi-protéiques, réfractaires aux techniques conventionnelles, donne accès à la topologie de ces assemblages, facilitant la proposition de modèles structuraux. Enfin, l’apport récent de la haute résolution en MS native est ici illustré à travers l’étude de protéines complexes et hétérogènes : les anticorps thérapeutiques et leurs conjugués. Ces développements permettent de repousser certaines limites en MS native et IM-MS native, élargissant leurs perspectives d’application dans la recherche et l’industrie pharmaceutique. / This PhD thesis aims at developing methods in native mass spectrometry (MS) combined with ion mobility (IM-MS) to characterize protein-ligand complexes and large protein assemblies. Fine-tuning of instrumental settings allowed expanding the scope of these approaches in structural biology. Real-time monitoring of protein-ligand complexes by native MS and IM-MS enabled to screen their binding properties while depicting subtle conformational changes induced upon binding. Applying these methods to refractory multi-protein complexes provided insights about their topology, making structural modeling easier. Finally, benefits from high-resolution native MS were highlighted through the characterization of heterogeneous systems, including monoclonal antibodies and their drug conjugates. Here, these developments enable to push some technical limits one step forward, increasing the potential of native MS and IM-MS both in academic research and pharmaceutical industry.

Les récents progrès instrumentaux en spectrométrie de masse, notamment en terme de- rapidité de balayage et de résolution, ont permis l'émergence de l'approche « data independent acquisition» (DIA). Cette approche promet de combiner les points forts des approches « shotgun » et ciblées,mais aujourd'hui l'analyse des données DIA reste compliquée. L'objectif de cette thèse a été de développer des méthodes innovantes de spectrométrie de masse, et en particulier d'améliorer l'analyse des données DIA. De plus, nous avons développé une approche originale Top 3-ID-DIA, permettant à la fois un profilage complet des protéines de la cellule hôte (HCP) ainsi qu'une quantification absolue d'HCP clés dans les échantillons d'anticorps monoclonaux (mAb), au sein d'une même analyse.Cette méthode est prête à être implémentée en industrie, et pourrait fournir un support en temps réel aux développements du procédé de production de mAb, ainsi que pour évaluer la pureté des biomédicaments. / Recent instrumental developments in mass spectrometry, notably in terms of scan speed and resolution, allowed the emergence of “data independent acquisition” (DIA) approach. This approach promises to combine the strengths of both shotgun and targeted proteomics, but today DIA data analysis remains challenging. The objective of my PhD was to develop innovative mass spectrometry approaches, and in particular to improve DIA data analysis. Moreover, we developed an original Top 3-ID-DIA approach, allowing both a global profiling of host cell proteins (HCP) and an absolute quantification of key HCP in monoclonal antibodies samples, within a single analysis. This method is ready to be transferred to industry, and could provide a real time support for mAb manufacturing process development, as well as for product purity assessment.

Les fonctions catalytiques de l'intégrase (IN) du Virus de l'Immunodéficience humaine (VIH-1) sont strictement nécessaires pour l'intégration du génome viral dans les cellules hôtes. Aujourd'hui, trois inhibiteurs anti-IN appartenant à la famille des dikétoacides sont utilisés en thérapie : le raltegravir, l'elvitegravir et le dolutegravir. Cependant, les patients traités par ces médicaments développent des mutations de résistance. Dans ce travail, nous cherchons à mieux comprendre le mécanisme d'interaction de ces drogues avec l'ADN viral. Ce travail a également contribué à la conception de molécules qui devraient être dotées d'une affinité augmentée pour l'ADN, permettant de surmonter le problème de la résistance virale. La compréhension du mécanisme d'inhibition de IN s'est poursuivie par l'étude de deux anticorps monoclonaux anti-K159 (peptide 147-175 du coeur catalytique de IN), 4C6 et 4F4. Les résultats montrent que les anticorps reconnaissent leurs épitopes dans l'IN. D'autre part, du fait de son implication dans de nombreuses étapes du cycle du VIH-1, nous ciblons la protéine 7 de la nucléocapside (NCp7). Pour ce faire, nous avons étudié la structure de nos systèmes (NCp7 et NCp7-ADN) et nous avons pu déterminer les interactions clés responsables de la structuration, ainsi que des fonctions, de ces systèmes. Dans un second temps, nous avons évalué les interactions de NCp7 avec un inhibiteur éjecteur de zinc (C247) de la famille des thioesters. Enfin, sur le plan méthodologique, nous avons raffiné dans le potentiel SIBFA (Sum of Interaction Between Fragments Ab initio computed) la représentation des doublets libres de type sp et sp2 dans les molécules conjuguées. / The catalytic functions of integrase (IN) of Human Immunodeficiency Virus (HIV-1) are strictly necessary for the integration of the viral genome into the host cells. To this day, three anti-IN inhibitors belonging to the diketoacids are used in therapy: raltegravir, elvitegravir and dolutegravir. However, under treatments with these drugs, patients develop resistance mutations. In this work, we seek to better understand the interaction of the three drugs with viral DNA. This work also contributed to the design of novel molecules. These should be endowed with increased DNA binding affinities as a step towards overcoming viral resistance. The understanding of the inhibition mechanism of IN was pursued by the study of two monoclonal antibodies, 4F4 and 4C6, which are directed against sequence 147-175 of the catalytic core of HIV-1 IN, a peptide denoted K159. The results show that the antibodies recognize their epitopes in IN. We also aim to target an HIV-1 nucleocaspid protein NCp7 involved in many stages of the viral cycle. We have thus studied the structure of NCp7 and its viral DNA complex. We were able to determine the interactions responsible for the structuring and thus the functions of these complexes. Then, we evaluated the interactions of NCp7 with an inhibitor of the thioester family, C247, which acts as a zinc ejector. Finally, from the methodological standpoint, we have refined in SIBFA (Sum of Interaction Between Fragments Ab initio computed) the representation of the sp2 and sp lone pairs in conjugated molecules.

Les Neuropilines (NRPs) sont des récepteurs transmembranaires non tyrosine-kinase identifiés à l'origine comme des récepteurs pour les sémaphorines de la classe 3. Ces glycoprotéines sont particulièrements impliquées dans la migration de la crête neurale et dans la croissance axonale au cours du développement embryonnaire du système nerveux. En outre, les NRPs sont exprimées par une large variété de tumeurs et de nombreuses molécules solubles semblent interagir avec ces protéines pour moduler la progression tumorale. Parmi elles, les facteurs angiogéniques de la famille du facteur de croissance de l'endothélium vasculaire (VEGF) semblent être à l'origine d'une angiogénèse médiée par les NRPs.Tandis que la NRP1 a été largement étudiée et reconnue comme une cible intéressante dans le cadre du développement de thérapies anti-angiogéniques, très peu d'études s'étaient intéressées au rôle de la NRP2 dans la progression tumorale jusqu'à présent. La NRP2 semble réguler la progression tumorale par de nombreux mécanismes, non seulement l'angiogénèse mais aussi la lymphangiogénèse, la Transition-Epithélio-Mésenchymateuse (TEM) et la formation de métastases. A la vue de ces multiples rôles dans la progression tumorale, la NRP2 apparaît donc comme une cible intéressante dans le cadre du développement de thérapies ciblées innovantes en cancérologie. Au cours de notre thèse, nous nous sommes attachés à caractériser le rôle de la NRP2 dans la progression tumorale dans le cadre du cancer colorectal puis nous avons développé un anticorps monoclonal thérapeutique ciblant spécifiquement cette protéine. / Neuropilins (NRPs) are transmembrane non tyrosine-kinase glycoproteins first identified as receptors for class-3 semaphorins. They are particularly involved in neural crest migration and axonal growth during development of thé nervous System. Since many types of tumor and endothelial cells express NRP receptors. various soluble molécules were also found to interact with thèse receptors to modulate cancer progression. Among them, angiogenic factors belonging to thé Vascular Endothelial Growth Factor (VEGF) family seem to be responsible for NRPs-related angiogenesis.While NRP1 was intensively studied from many years and identified as an attractive angiogenesis target for cancer therapy. NRP2 signaling pathway has just recently been studied. NRP2 may regulate tumor progression by several concurrent mechanisms, not only angiogenesis but lymphangiogenesis, epithelial-mesenchymal transition and metastasis. In view of their multiples functions in cancer promotion, NRP2 fulfills ail thé criteria of a therapeutic target for innovative anti-tumor thérapies. Our thesis focuses on NRP2-specific rôles in tumor progression and subséquent development of a NRP2-neutralizing monoclonal antibody.