A produção de biofármacos segue um crescente interesse industrial e social. Atualmente existem vários tipos de tratamento contra o câncer, porém, a maioria desses tratamentos não atua unicamente no alvo terapêutico. Um estudo realizado por pesquisadores do Instituto Butantan identificou uma proteína com ação anticoagulante e antitumoral, codificada por um gene proveniente das glândulas salivares do carrapato Amblyoma cajennense . Esse gene foi inserido em um plasmídeo e expresso com sucesso na bactéria Escherichia coli BL21DE3. Estudos pré-clínicos mostraram que a injeção in vivo de Amblyomin-X reduziu a formação de massa tumoral induzida por células de melanoma B16F10 em camundongos. Este trabalho contempla o estudo das condições de cultivo para obtenção da proteína Amblyomin-X com a linhagem Escherichia coli BL21DE3 recombinante em biorreatores. Para se determinar a melhor condição de biossíntese da proteína recombinante Amblyomin-X estabeleceu-se uma estratégia de cultivo para a etapa de crescimento do microrganismo e estudaram-se três diferentes fatores na etapa posterior à do crescimento microbiano, ou seja, na etapa de biossíntese da proteína: i) a concentração do indutor IPTG (Isopropil β-D-1-tiogalactopiranosídeo) no momento de indução da proteína; ii) a temperatura de cultivo durante a etapa de biossíntese da proteína; e iii) a concentração celular no início da etapa de biossíntese da proteína. Para a realização dos ensaios foi proposto um planejamento experimental delineamento composto central rotacional (DCCR) escolhido para os três fatores em estudo e resultou na condução de 17 ensaios. Os ensaios foram realizados com a estratégia de condução proposta, mantendo o valor de &#956x entre 0,20 e 0,23 h-1 na etapa de batelada alimentada, o que possibilitou atingir a concentração celular desejada com a formação de ácido acético em concentrações não inibitórias. Como resultado do planejamento experimental, a obtenção dos valores ótimos dos fatores para a etapa de produção da proteína: temperatura de 38 °C, concentração celular de 35,27 g/L e concentração do indutor IPTG de 0,10 mM resultou em um protocolo de processo que obteve 8,07 g/L de proteína recombinante, valor máximo obtido e validado. / The production of biopharmaceuticals follows an increasing industrial and social interest. Currently there are several types of cancer treatment, however, most of these treatments do not only act on the therapeutic target. A study by researchers at the Butantan Institute identified a protein with anticoagulant and antitumor action, encoded by a gene from the salivary glands of the Amblyoma cajennense tick. This gene was inserted into a plasmid and successfully expressed on the bacterium Escherichia coli BL21DE3. Preclinical studies have shown that the in vivo injection of Amblyomin-X reduced the tumor mass formation induced by B16F10 melanoma cells in mice. This work includes the study of the culture conditions to obtain Amblyomin-X protein with the recombinant Escherichia coli strain BL21DE3 in bioreactors. In order to determine the best biosynthesis condition of the recombinant Amblyomin-X protein, a culture strategy was established for the growth stage of the microorganism and three different factors were studied in the post-microbial growth stage, that is, in the biosynthesis stage of the protein: i) the concentration of the IPTG (Isopropyl β-D-1-thiogalactopyranoside) inducer at the time of induction of the protein; ii) the culture temperature during the protein biosynthesis step; and iii) the cell concentration at the beginning of the protein biosynthesis step. For the accomplishment of the tests it was proposed an experimental design - central rotational compound design (DCCR) - chosen for the three factors under study and resulted in the conduction of 17 tests. The tests were performed with the proposed conduction strategy, maintaining the value of µ &x between 0.20 and 0.23 h-1 in the fed batch stage, which allowed to reach the desired cellular concentration with the formation of acetic acid in non-inhibitory concentrations. As a result of the experimental design, obtaining the optimum values of the factors for the protein production step: temperature of 38 °C, cell concentration of 35.27 g/L and concentration of the IPTG inductor of 0.10 mM resulted in a process protocol which obtained 8.07 g / L of recombinant protein, maximum value obtained and validated.

L'utilisation des ressources en fer et les variations du potentiel redox sont des processus impliqués dans la prolifération et la différenciation cellulaire. Ils participent à l'hématopoïèse normale et leur dérégulation peut être associée à des conditions pathologiques. Les hémopathies, telles que la leucémie aiguë myéloïde (LAM), témoignent du lien entre disponibilité en fer, signalisation redox et leucémogenèse. La déplétion en fer induit un arrêt de la prolifération suivi de la mort cellulaire, et pour des cellules primaires leucémiques (blastes) de patients LAM, elle peut conduire à un réengagement de la différenciation vers la lignée monocytaire. Cependant, les besoins en fer des clones leucémiques restent mal définis. Dans les cellules animales, le cœur du réseau de régulation du fer est organisé à travers le système régulateur IRE-IRP. Les Iron Regulatory Proteins (IRP), agissent sur la traduction de nombreuses protéines impliquées dans la gestion du fer par interaction avec les Iron Responsive Elements (IRE) localisés sur les régions non codantes des ARN messager (ARNm) régulés. A partir de lignées cellulaires leucémiques (KG1, K562), de blastes de patients LAM et de progéniteurs CD34+ contrôles issus de sang de cordon et de moelle osseuse de donneurs sains, le statut du système de gestion cellulaire du fer a été caractérisé pour les premières étapes de l'hématopoïèse normale et pathologique. A travers la manipulation des apports cellulaires en fer, notamment par l'utilisation de chélateurs à usage thérapeutique, la réponse du système homéostatique a été suivie. Nos données soulignent les faibles besoins en fer des progéniteurs hématopoïétiques, et d'autres cellules, pour proliférer. Dans les lignées cellulaires le régulateur IRP est en excès par rapport à ses cibles IRE, ce qui pourrait être une caractéristique générale du contrôle de la traduction pour des ARNm spécifiques par fixation de régulateurs translationnels. La régulation semble exclusivement le fait d' IRP1, puisqu' IRP2 n'a pas été détecté dans les progéniteurs hématopoïétiques, qu'ils soit pathologiques ou non. De subtiles différences ont été identifiées dans les quantités des composants du réseau gérant le fer dans les cellules leucémiques en comparaison des cellules saines témoins, ainsi que des capacités différentes à croître dans un milieu minimal comportant des concentrations en fer précisément définies. Les informations obtenues à travers ce travail pourraient bénéficier à l'élaboration de protocoles thérapeutiques, incluant notamment la manipulation du fer, dans les LAM ou d'autres pathologies. / Use of iron resources and variations of the redox balance are processes involved in cell proliferation and differentiation. They participate to normal hematopoiesis and their disturbance may be associated with pathological conditions. Hematological neoplasms, such as acute myeloid leukemia (AML), provide clinical evidence of the link between iron availability, redox signaling, and malignancy. Stringent iron depletion induces arrest of proliferation followed by cell death, and deprived primary leukemic cells of AML patients (blasts) have been previously shown to engage into the monocytic lineage. Yet, the iron needs of leukemic clones are unknown. The core network of cellular iron regulation in mammals is organized around the IRE-IRP system. The Iron Regulatory Proteins (IRP) act on the translation of many proteins involved in iron management by interacting with Iron Responsive Elements (IRE) located on the untranslated regions of messenger RNA (mRNA) coding these proteins. Using leukemic cell lines (KG1, K562), blasts of AML patients and CD34+ progenitors isolated from cord blood or the bone marrow of healthy donors, the status of the iron management system was established in the first stages of normal and pathological hematopoiesis. The response of the homeostatic system upon manipulation of iron provision, including with clinically implemented chelators, has been monitored. Our data emphasize the weak iron requirements of hematopoietic progenitors, and other cells, to proliferate. In cell lines the IRP regulator is in excess of its IRE targets, which may be a general feature of translational control for specific mRNA. The regulation seems exclusively mediated by IRP1, as the IRP2 regulator has not been detected in normal or malignant hematopoietic progenitors. Subtle differences have been found in the iron handling system of leukemic cells as compared to normal cells, together with different abilities to grow on a minimal medium containing precisely defined iron concentrations. The design of improved therapeutic regimens including iron manipulation, in AML and other pathologies, may benefit from considering the information obtained in this work.