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La lutte contre le cancer est l'un des défis majeurs du XXème siècle. Pour que les tumeurs puissent se développer dans l'organisme, elles ont besoin d'un apport en nutriment par le biais de vaisseaux sanguins pour se faire, elles vont avoir recours au processus angiogénique. Lors de ce processus, les cellules endothéliales qui tapissent la paroi des vaisseaux sanguins vont se multiplier et créer de nouveaux vaisseaux sanguins qui vont permettre la vascularisation des tumeurs. L'angiogenèse constitue donc aujourd'hui un axe de recherche pour la lutte contre la progression tumorale et donc contre le cancer. Lors de ce développement tumoral, une enzyme, l'aminopeptidase neutre APN est surexprimée sur les parois des cellules endothéliales. Différentes études ont été menées et montrent que l'inhibition de cette enzyme bloque la progression tumorale. Mon travail au sein de l'équipe du Pr Céline Tarnus consistait en la conception et la synthèse d'inhibiteurs de l'APN. Une relation structure activité de nos composés vis-à-vis de l'APN a tout d'abord été effectuée. Le développement de synthèse du composé le plus actif ont été faite, puis la synthèse d'inhibiteurs d'APN ayant pour objectif l'utilisation de la BNCT a été abordée. / The fight against the cancer is one of the most important struggles of this century. For the development of the tumors inside the body, they need to receive nutriments by the blood vessels and they use the angiogenic process. During this process, the endothelial cells being shown on the wall of the blood vessel will multiply and design new blood vessel, which will allow the tumor's vascularisation. Today, the angiogenesis is an axis of research for the fight against the cancer. During the tumoral development, the aminopeptidase N (APN) is overexpressed on the wall of endothelial cells. Various studies have shown that the inhibition of this enzyme stops the tumoral progression. My work in the Pr. Céline Tarnus Team consists in the conception and the synthesis of APN's inhibitors. In a first time, a structure activity relationship has been realized. Syntheses of a subnamolar compound have been developed, and then the synthesis of APN's inhibitors with the use of BNCT has been got onto.

Les aminopeptidases sont des cibles thérapeutiques importantes pour plusieurs maladies, car elles sont impliquées dans divers processus physiologiques et pathologiques comme la progression tumorale, l'angiogenèse, et certaines infections (virales, bactériennes, et parasitaires). Il en existe deux classes : les aminopeptidases avec un ion métallique (Aminopeptidase N [APN ou CD13] et leukotrien A4 hydrolase [LTA4H]) et les aminopeptidases avec deux ions métalliques (Aminopeptidase de l'Aeromonas proteolytica [APaero], Leucine Aminopeptidase cytosolique [LAPc] et Méthionine aminopeptidase 1 ou 2 [MetAP]). Deux types de composés dérivés des amino-benzosubérones ont été envisagés pour inhiber sélectivement chacune de ces classes d'aminopeptidases. L'étude des relations structures-activités (RSA) nous a permis de découvrir une molécule très puissante et sélective de l'APN (Ki 60 pM). L'APN est une enzyme monométallique considérée aujourd'hui comme une nouvelle cible pour la lutte contre le cancer car son inhibition bloque le processus de l'angiogenèse et donc la progression tumorale. L'étude d'une nouvelle classe de molécules trisubstituées dérivées des amino­benzosubérones a abouti à la découverte d'une seconde molécule active et sélective des enzymes bimétalliques notamment l'APaero (Ki 10 nM). / The aminopeptidases are important therapeutic targets for several diseases, because they are implied in various physiological and pathological processes like the tumoral progression, the angiogenesis, and certain infections (viral, bacterial, and parasitic). There are two classes: aminopeptidases with one metal ion (Aminopeptidase N [APN or CD13] and leukotriene A4 hydrolase [LTA4H]) and aminopeptidases with two metal ions (Aminopeptidase of Aeromonas proteolytica [APaero], cytosolic leucine aminopeptidase [LAPc] and Methionine aminopeptidase 1 or 2 [MetAP]). Two types of compounds of amino-benzosuberone derivatives were envisaged to inhibit selectively each one of these classes of aminopeptidases. The study of the structure-activity relationship (SAR) enabled us to discover a very powerful and selective molecule of the APN (Ki 60 pM). The APN is a monometallic enzyme considered today as a new target for the fight against cancer because its inhibition blocks the angiogenesis process and thus the tumoral progression. The study of a new class of trisubstituted molecules derived from the amino-benzosuberone led us to discover another molecule which is active and selective of the bimetallic enzymes in particular APaero (Ki 10 nM).

Une analyse génomique (Comparative Genomic Hybridization) a été réalisée sur une cohorte d’adénomes et de tumeurs coliques et a mis en évidence, parmi d’autres altérations, la délétion de la région 1p36.12 dans 23% des adénomes et 47% des carcinomes. Parmi les 15 gènes ayant une fonction connue retrouvés dans cette zone, le gène codant pour le facteur de transcription E2F2 a été retenu en raison de son implication dans des processus cellulaires clés. Une analyse de Kaplan- Meier a montré que la délétion de E2F2 est un facteur de bon pronostic de survie sans progression. Afin de mieux cerner l’implication des gènes ciblés par la micro-délétion en 1p36.12, une étude fonctionnelle in vitro et in vivo de la perte de fonction de E2F2 a été réalisée, et étendue à celle de ID3 dont le gène est le voisin direct de E2F2. Nos observations indiquent qu’in vivo, la perte d’E2F2 favorise la croissance tumorale et bloque le développement de métastases. Dans le cadre d’une collaboration avec l’équipe de biochimiste du Pr Céline TARNUS (U.H.A.), une étude pilote a été réalisée pour prouver l’efficacité anti-tumorale de nouveaux inhibiteurs hautement sélectifs pour l’aminopeptidase N. / A genomic analysis (Comparative Genomic Hybridization) evidenced, among other chromosomic alterations, a microdeletion at 1p36.12 locus in 23% and 47% of colon adenomas and carcinomas, respectively. Among the 15 genes located in the deleted region, we focused on E2F2 gene involvedin various cellular processes. The Kaplan-Meier curve analysis showed that E2F2 deletion is associated with a better progression-free survival. To better understand the involvement of genes targeted by the microdeletion in colon tumors, we assessed the functional impact of the underexpression of E2F2 and of its direct neighbor gene ID3, coding for a dominant-negative repressor involved in cell differentiation. Our results indicated that E2F2 loss favors tumor growth and prevent metastatic spread. In collaboration with the biochemical team directed by the Pr Céline TARNUS, we started a pilotstudy to prove the anti-tumor potential of new chemical inhibitors highly selective of the aminopeptidase N.

The primary contributor to Vibrio fischeri aminopeptidase activity is aminopeptidase N, PepN. Colonization assays revealed the pepN mutant strain to be deficient at forming dense aggregates and populating the host’s light organ compared to wildtype within the first 12 hours of colonization; however the mutant competed normally at 24 hours. To address the role of PepN in colonization initiation and establish additional phenotypes for the pepN mutant strain, stress response and other physiological assays were employed. Marked differences were found between pepN mutant and wildtype strain in response to salinity, acidity, and antibiotic tolerance. This study has provided a foundation for future work on identifying a putative role for V. fischeri PepN in regulating stress response.

Les aminopeptidases sont des cibles thérapeutiques importantes pour plusieurs maladies, car elles sont impliquées dans divers processus physiologiques et pathologiques comme la progression tumorale, l'angiogenèse, et certaines infections (virales, bactériennes, et parasitaires). Il en existe deux classes : les aminopeptidases avec un ion métallique (Aminopeptidase N [APN ou CD13] et leukotrien A4 hydrolase [LTA4H]) et les aminopeptidases avec deux ions métalliques (Aminopeptidase de l'Aeromonas proteolytica [APaero], Leucine Aminopeptidase cytosolique [LAPc] et Méthionine aminopeptidase 1 ou 2 [MetAP]). Deux types de composés dérivés des amino-benzosubérones ont été envisagés pour inhiber sélectivement chacune de ces classes d'aminopeptidases. L'étude des relations structures-activités (RSA) nous a permis de découvrir une molécule très puissante et sélective de l'APN (Ki 60 pM). L'APN est une enzyme monométallique considérée aujourd'hui comme une nouvelle cible pour la lutte contre le cancer car son inhibition bloque le processus de l'angiogenèse et donc la progression tumorale. L'étude d'une nouvelle classe de molécules trisubstituées dérivées des amino­benzosubérones a abouti à la découverte d'une seconde molécule active et sélective des enzymes bimétalliques notamment l'APaero (Ki 10 nM).

Submitted by IGOR HENRIQUE SENA DA SILVA null (igor.sena@outlook.com.br) on 2017-09-05T13:59:07Z No. of bitstreams: 1 DISSERTAÇÃO_Igor_Henrique_Sena_Silva.pdf: 1536012 bytes, checksum: da24e2e008037696caee4c8842fc8f05 (MD5) / Approved for entry into archive by Luiz Galeffi (luizgaleffi@gmail.com) on 2017-09-06T13:13:51Z (GMT) No. of bitstreams: 1 silva_ihs_me_jabo.pdf: 1536012 bytes, checksum: da24e2e008037696caee4c8842fc8f05 (MD5) / Made available in DSpace on 2017-09-06T13:13:51Z (GMT). No. of bitstreams: 1 silva_ihs_me_jabo.pdf: 1536012 bytes, checksum: da24e2e008037696caee4c8842fc8f05 (MD5) Previous issue date: 2017-07-26 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Helicoverpa armigera é uma praga altamente polífaga e ataca culturas de grande importância agrícola em diversos países do mundo. O controle desta praga é realizado primariamente por inseticidas químicos. Porém, o uso indiscriminado do controle químico levou a resistência de populações desta praga a maioria dos inseticidas químicos usados para seu controle, dificultando o seu manejo no campo. Além do controle químico, o controle de H. armigera tem sido realizado com uso de plantas transgênicas que expressam a proteína Cry1Ac de Bacillus thuringiensis (Bt) ou por bioinseticidas que contem esta e outras proteínas. No entanto, estudos têm demonstrado uma diminuição significativa na susceptibilidade de H. armigera às proteínas Cry com o aumento de seu desenvolvimento larval. O mecanismo de resistência mais comum dos insetos às proteínas Cry é a redução de ligação da proteína aos receptores presentes na membrana, levando a uma menor afinidade de ligação da proteína aos receptores intestinais. Desta forma, o objetivo deste trabalho foi avaliar a susceptibilidade de lagartas de diferentes ínstares de H. armigera à Cry1Ac e correlacionar com a capacidade de ligação da proteína Cry1Ac às microvilosidades apicais das células colunares do intestino médio (BBMVs) isoladas de todos ínstares larvais. Além disso, por meio de ensaios de imunoprecipitação e análise por cromatografia líquida acoplada a espectrofotometria de massa, identificar as proteínas envolvidas na interação com a proteína Cry1Ac no segundo e quinto ínstares de H. armigera. Foi observada uma redução significativa na susceptibilidade dos últimos ínstares larvais de H. armigera à proteína Cry1Ac comparado aos ínstares iniciais. Os valores estimados de CL50 variaram de 31,1 a 2525,7 ng de proteína/cm² de dieta, em lagartas de primeiro e sexto ínstar, respectivamente. Estes resultados evidenciam uma diferença de 80 vezes na susceptibilidade à proteína Cry1Ac do último para o primeiro ínstar. Nos testes de ligação de ELISA da proteína Cry1Ac às BBMVs dos diferentes ínstares, foi constatada uma diminuição total na capacidade de ligação da proteína Cry1Ac as BBMVs dos estádios mais tardios comparados aos iniciais, com afinidade de ligação aparente de 3,88 vezes menor no último ínstar comparado ao primeiro. Assim, uma clara correlação direta entre toxicidade de Cry1Ac e a afinidade de ligação da proteína às BBMVs de H. armigera foi demonstrada. Os resultados dos ensaios de imunoprecipitação demonstraram um padrão diferenciado de interação com a proteína Cry1Ac no segundo e quinto ínstar. A proteína fosfatase alcalina (ALP) foi identificada apenas no segundo ínstar, bem como, outras proteínas de membrana, como proibitina e uma proteína de canal iônico, que podem estar envolvidas para a alta toxicidade de Cry1Ac em ínstares iniciais de H. armigera. A identificação e o papel funcional das proteínas de ligação nos diferentes estádios de desenvolvimento de H. armigera, facilitará a elucidação do mecanismo de ação da proteína Cry1Ac e poderá ajudar a propor estratégias que retardem a evolução da resistência dos insetos às cultivares transgênicas que expressam esta proteína. / Helicoverpa armigera is a highly polyphagous pest and attacks important crops worldwide. The control of this pest is carried out primarily by chemical insecticides. However, the indiscriminate use of chemical control, led to pest populations to develop resistance to most of the chemical insecticides used for their control, making it difficult to management in the field. In addition to chemical control, H. armigera has been done by transgenic crops expressing Cry1Ac toxin from Bacillus thuringiensis (Bt) or by biopesticides that contains Cry1Ac or other toxins. However, studies have demonstrated a susceptibility decrease of H. armigera to Cry toxins with their larval development increase. The most common mechanism of resistance used by insects against Cry toxins is the reduced toxin binding to receptors present on the membrane, leading to a lower binding affinity of the toxin to intestinal receptors. Thus, the objective of this work was to evaluate the susceptibility of different instar larvae of H. armigera to Cry1Ac toxin and to correlate with the Cry1Ac toxin binding capacity to BBMV isolated from all larval instar. Furthermore, by pull-down techniques and liquid chromatography coupled to mass spectrometry analysis, to identify the proteins involved in the Cry1Ac toxin interaction in the second and fifth instars of H. armigera. A significant reduction in the susceptibility of the late instars of H. armigera to Cry1Ac toxin was observed compared to early instars. LC50 estimated values ranged from 31.1 to 2525.7 ng of toxin/cm2 of diet in first and sixth instar larvae, respectively. These results point a difference of 80-fold in the susceptibility to Cry1Ac toxin from late to first larval instar. In the ELISA binding assays results to BBMV of the different instars was found a total decrease in the binding capacity of Cry1Ac toxin to BBMVs from late instars compared to BBMV from early instars, presenting an apparent binding affinity of 3.88 times lower in the last instars than the first. Thus, a clearly correlation between Cry1Ac toxicity and binding toxin affinity to H. armigera BBMV has been demonstrated. The pull-down assays demonstrated a different pattern of the interaction between Cry1Ac toxin with the second and fifth instars. The protein phosphatase alkaline (ALP) was identified only in the second instar, as well as, other membrane proteins, as prohibitin and an ion channel protein, which may be involved for higher toxicity of Cry1Ac in early instars of H. armigera. The identification and functional role of binding proteins in the different stages of development of H. armigera will facilitate the elucidation of the Cry1Ac toxin mechanism of action and will may help to propose strategies that delay the insect resistance evolution to transgenic crops that express this protein. / FAPESP: 2015/24330-5