Host cell reactivation (HCR) of viruses has been used in the past to assess the DNA repair capacities of various mammalian cell types. In this study, a PCR-based HCR technique was developed for determining DNA repair capacity of mammalian cells. Many DNA lesions, including UV photoproducts, block DNA amplification by Taq polymerase, and the exponential nature of PCR imparts a tremendous potential for quantifying the remaining non-adducted DNA templates from small samples. Ad5HCMVspl ZacZ is a recombinant nonreplicating adenovirus (Ad) containing the lacZ reporter gene under the control of the human cytomegalovirus (HCMV) immediate early promoter inserted into the deleted El region of the viral genome. This virus is unable to replicate, but it can efficiently express the reporter gene in many types of mammalian cells, including human fibroblasts. Using quantitative PCR, the induction and repair of UV photoproducts was measured in a 2.6 kb region of the lacZ reporter gene inserted into the deleted El region of Ad5HCMVspllacZ and in a 2.8 kb region of the endogenous E4 region of the virus. Primers flanking the regions were added to equal amounts of DNA extracted from cells infected with unirradiated or UV-irradiated Ad5HCMVspl lacZ and each sample was amplified by PCR using radiolabelled nucleotides as substrates. PCR products were separated by agarose gel electrophoresis and quantified using a phosphorimaging system. Results show a simple exponential decrease in PCR product with increasing UV fluence to the virus. There was a significant removal of UV photoproducts by 24 hours after infection of normal human fibroblasts. A reduced capacity for lesion removal was detected after infection of nucleotide excision repair deficient fibroblasts derived from patients with xeroderma pigmentosum (XP) and Cockayne syndrome (CS). Previous work from our lab using a P-gal reporter gene assay has shown that both UV light and heat shock treatment of cells prior to infection with UV-damaged Ad5HCMVspl lacZ enhances HCR. Application of the quantitative PCR technique to the study of inducible repair shows there is an enhancement in the rate of lesion removal from both regions of the vector in UV-irradiated normal lung fibroblast cells, compared to unirradiated cells. This demonstrates that previous reports of enhanced host cell reactivation are indicative of a genuine enhancement of DNA repair. Also, the P-gal reporter gene assay was used to investigate inducibility of UV lesion repair by ionising radiation; no significant increase in HCR of p-gal activity was found in cells treated with y-rays compared to untreated cells. / Thesis / Master of Science (MS)

Thesis advisor: Abhishek Chatterjee / Thesis advisor: Jia Niu / Directed Evolution has emerged as an invaluable tool for advancing protein functions in both research and industry. Our lab has pioneered a directed evolution platform in mammalian cells, utilizing an AAV delivery vector to package a DNA library and linking the biomolecule of interest to AAV production. During my tenure in Prof. Chatterjee's lab, I focused on harnessing our lab’s directed evolution platform, known as Virus-Assisted Directed Evolution of tRNA (VADER), to develop highly efficient tRNAs for genetic code expansion. Additionally, I contributed to the development of the AAV-based selection platform, termed Virus-Assisted Mutational Profiling (VAMP), as a profiling tool. Through the utilization of VAMP, I conducted comprehensive profiling of tRNA and RNA polymerase III promoter sequences. This enabled me to gain insights into regions of flexibility and evolution, ultimately leading to the construction of improved constructs with enhanced activity relative to the starting sequence. / Thesis (PhD) — Boston College, 2024. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Microfluidic devices are ideal for analysis of biological systems. The small dimensions result to controlled handling of the flow profile and the cells in suspension. Implementation of additional forces in the system, such as an electric field, promote further manipulation of the cells. In this dissertation, I show novel, unique microfluidic approaches for manipulation and analysis of mammalian cells by the aid of electrical methods or the architecture of the device. Specifically, for the first time, it is shown, that adoption of electrical methods, using surface electrodes, promotes cell concentration in a microchamber due to isoelectric focusing (IEF). In contrast to conventional IEF techniques for protein separation, a matrix is not required in our system, the presence of which would even block the movement of the bulky cells. Electric field is, also, used to breach the cell membrane and gain access to the cell interior by electroporation (irreversible and reversible). Irreversible electroporation is used in a unique, integrated microfluidic device for cell lysis and reagentless extraction of DNA. The genomic material is subsequently analyzed by on-chip PCR, demonstrating the possible elimination of the purification step. On the other hand, reversible electroporation is used for the delivery of exogenous molecules to cells. For the first time, the effect of shear stress on the electroporation efficiency of both attached and suspended cells is examined. On the second part of my dissertation, I explore the capabilities of the architecture of microfluidic devices for cell analysis. A simple, unique method for compartmentalization of a microchamber in an array of picochambers is presented. The main idea of the device lies on the fabrication of solid supports on the main layer of the device. These features may even hold a dual nature (e.g. for cell trapping, and chamber support), in which case, single cell analysis is possible (such as single cell PCR). On the final chapter of my dissertation, a computational analysis of the flow and concentration profiles of a device with hydrodynamic focusing is conducted. I anticipate, that all these novel techniques will be used on integrated microfluidic systems for cell analysis, towards point-of-care diagnostics. / Ph. D.

O câncer cervical é um dos tipos de câncer mais comuns entre as mulheres, e a infecção persistente pelos HPV-16 e HPV-18 é responsável por 70% dos casos. As vacinas profiláticas disponíveis possuem alta eficácia na prevenção da infecção pelos tipos mais prevalentes de HPV. No entanto, este tipo de abordagem não beneficia mulheres que já apresentam lesões precursoras ou tumores cervicais avançados, e a busca por abordagens terapêuticas para esse tipo de câncer é considerada uma necessidade. A qualidade do antígeno representa um aspecto fundamental para o sucesso de vacinas terapêuticas baseadas em proteínas recombinantes. Neste sentido, os sistemas de expressão em células eucarióticas, como leveduras e células de mamíferos são considerados adequados para a produção de proteínas com aplicação biotecnológica. O objetivo principal deste trabalho contemplou a expressão das proteínas de fusão gDE7E6 do HPV-16 e do HPV-18 e a oncoproteína E7 do HPV-16 em células da levedura Pichia pastoris e expressão da gDE7E6 do HPV-16 e do HPV-18 em células de mamífero HEK293T e CHODG-44 para obtenção de antígenos purificados com futura aplicação em vacinas terapêuticas contra tumores associados ao HPV-16 e HPV-18. Os genes que codificam as proteínas gDE7E6 dos HPV-16 e HPV-18 e da E7 do HPV-16 foram clonados no vetor pPIC9K, os quais foram linearizados por digestão enzimática e utilizados na transformação da P. pastoris. A expressão das proteínas foi analisada nos tempos de 24, 48, 72 e 96 horas, no entanto, não foi observada a produção das proteínas no sobrenadante e nem no lisado celular. Diante desta constatação, iniciamos a expressão das proteínas gDE7E6 do HPV-16 e gDE7E6 do HPV-18 em células de mamíferos HEK293T e CHODG-44. As sequências genéticas das proteínas gDE7E6 do HPV-16 e do HPV-18 foram clonadas no vetor de expressão pNU1 e analisadas por digestão enzimática. Análises de SDS-PAGE e western blot demonstraram a expressão das proteínas gDE7E6 do HPV-16 e do HPV-18 em até 96 horas em células HEK293T. Em paralelo, realizamos a transfecção estável dos plasmídeos contendo as sequencias da gDE7E6 do HPV-16 e gDE7E6 do HPV-18 em células CHO-DG44. Com o intuito de aumentar a expressão das proteínas de interesse na população mista de CHODG-44, realizamos amplificação genômica com metotrexato (MTX), sendo possível observar aumento da expressão das proteínas, conforme aumento gradativo nas concentrações de MTX. Posteriormente, foram feitas tentativas para isolar um clone produtor das proteínas gDE7E6 HPV-16 e HPV-18, através de clonagem por diluição limitante e sistema automatizado, sendo possível isolar um clone para cada construção através de matriz semisólida, confirmado por western blot e citometria de fluxo. Apesar de demonstrar a expressão das proteínas de interesse em sistema de expressão baseado em células de mamífero, o rendimento obtido após a purificação por afinidade ao níquel foi extremamente baixo, o que dificulta a obtenção dos antígenos para fins vacinais. / Cervical cancer is one of the most common cancers among women, and persistent infection with HPV-16 and HPV-18 accounts for 70% of the cases. Available prophylactic vaccines are highly effective in preventing infection by the most prevalent types of HPV. However, this type of approach does not benefit women who already have precursor lesions or advanced cervical tumors, and the search for therapeutic approaches to this type of cancer is considered a necessity. Antigen quality represents a key aspect for the success of therapeutic vaccines based on recombinant proteins. In this sense, expression systems based in eukaryotic cells such as yeast and mammalian cells are considered suitable for the production of proteins with biotechnological applications. The main objective of this work was to express the gDE7E6 fusion proteins HPV-16 and HPV-18 and the E7 oncoprotein HPV-16 in Pichia pastoris and expression of gDE7E6 HPV-16 and HPV-18 in mammalian cells HEK293T and CHODG-44 to obtain purified antigens with future applications in therapeutic vaccines against HPV-16 and HPV-18 associated tumors. The genes encoding the gDE7E6 proteins HPV-16 and HPV-18 and E7 HPV-16 were cloned into the pPIC9K vector, which were linearized by enzymatic digestion and used in the transformation of P. pastoris. Expression of the proteins was analyzed at 24, 48, 72 and 96 hours, however, the production of the proteins in the supernatant and in the cell lysate was not observed. In light of this finding, we initiated the expression of gDE7E6 proteins HPV-16 and HPV-18 in mammalian cells HEK293T and CHODG-44. The genetic sequences of gDE7E6 proteins HPV-16 and HPV-18 were cloned into the pNU1 expression vector and analyzed by enzymatic digestion. SDSPAGE and western blot analyzes demonstrated expression of gDE7E6 proteins HPV-16 and HPV-18 within 96 hours in HEK293T cells. In parallel, we performed stable transfection of plasmids containing gDE7E6 HPV-16 and HPV-18 sequences into CHODG44 cells. In order to increase the expression of the proteins in the mixed population of CHODG-44, we performed genomic amplification with methotrexate (MTX), and it was possible to observe an increase in protein expression, as a gradual increase in MTX concentrations. Therefore, attempts were made to isolate a clone producing gDE7E6 proteins HPV-16 and HPV-18 by limiting dilution and automated system, being possible to isolate one clone for each construct through a semisolid matrix, confirmed by western blot and flow cytometry. Despite observing protein expression in mammalian cell-based expression system, the yield obtained after nickel affinity purification was extremely low, which makes it difficult to obtain the antigens for vaccine purposes.

Les conditions du corps humain ne sont pas reproduites fidèlement par la culture cellulaire traditionnelle en 2D. Dans cette thèse, des cultures cellulaires 3D sont réalisées dans une plateforme microfluidique hautement intégrée. Des cellules mammifères adhérentes sont encapsulées dans des gouttes immobilisées dans un tableau de pièges capillaires à haute densité. Dans chaque goutte, les cellules se réorganisent pour former un unique microtissu 3D et fonctionnel appelé sphéroïde. L'utilisation d'un hydrogel permet d'alonger le temps de culture et de perfuser le tableau avec des solutions aqueuses, par exemple pour de l'immuno-cyto-chimie. Un unique sphéroïde, viable, peut aussi être extrait de cette puce microfluidique. Des données quantitatives sont extraites à haut débit au niveau de la population, du sphéroïde (dizaines de miliers de sphéroïdes) et au niveau cellulaire emph{in situ} (centaines de miliers de cellules) grâce à de l'imagerie de fluorescence et au dévelopement d'un code d'analyse d'image. Une première preuve de concept a été obtenue en démontrant la viabilité, la prolifération et la fonctionalité de sphéroïdes d'hépatocytes et en les corrélant à des paramètres morphologiques. Ensuite, des aggrégats de cellules souches mésenchymales ont été produits et les hétérogénéités spatiales dans l'expression de protéines impliquées dans leurs propriétés thérapeutiques ont été étudiées. Enfin, cette technologie a été encore dévelopée pour permettre d'appliquer des conditions biochimiques différentes dans chaque goutte. La production et la culture de sphéroïdes dans cette plateforme microfluidique peut mener à des dévelopements importants dans beaucoup de domaines tels que l'analyse de la toxicité des médicaments, le criblage de médicaments à haut débit, le traitement personnalisé du cancer, l'ingénierie tissulaire ou la modélisation de maladies. / Conventional 2D cell culture fails to reproduce emph{in vivo} conditions. In this PhD thesis, 3D cell culture is implemented into a highly integrated microfluidic platform. Adherent mammalian cells are encapsulated in droplets immobilized on a high density array of capillary traps called anchors. In each droplet, the cells reorganize into a single functional 3D microtissue called spheroid. The use of an hydrogel allows to extend the culturing time in microdroplets and to perfuse the array with aqueous solutions, for instance for immuno-cyto-chemistry. A single and viable spheroid can also be selectively retrieved from the microfluidic chip. High throughput and quantitative data is extracted at the population, spheroid (tens of thousands of spheroids) and cellular level emph{in situ} (hundreds of thousands of cells) thanks to fluorescent imaging and a custom image analysis software. As a first proof of concept, the viability, proliferation and functionality of hp sh s were demonstrated and correlated with morphological parameters. Drug toxicity experiments were also performed on this liver model. Then, human mesenchymal stem cell aggregates were produced and the spatial heterogeneities of the expression of proteins involved in their therapeutic properties were investigated. Finally, this technology was further developed to enable applying different biochemical conditions in each droplet. The production and culture of spheroids in this microfluidic platform could lead to major advances in many fields such as drug toxicity, high throughput drug screening, personalized cancer treatment, tissue engineering or disease modeling.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19048号 / 農博第2126号 / 新制||農||1032(附属図書館) / 学位論文||H27||N4930(農学部図書室) / 31999 / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 植田 充美, 教授 植田 和光, 教授 小川 順 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

O sistema de expressão gênica derivado do Semliki Forest Vírus (SFV) vem sendo muito utilizado nos últimos tempos para expressão em grandes quantidades de inúmeras proteínas, quando comparado com outros sistemas. O objetivo desse estudo foi otimizar a capacidade desse vetor viral de expressar proteínas em diferentes linhagens celulares de mamíferos, utilizando como alvo, a glicoproteína do vírus rábico (RVGP). Foram avaliadas formas de obtenção do vetor SFV recombinante, através de diferentes métodos de transfecção, como eletroporação e lipofecção, utilizando um lipossomo comercial chamado Transmessenger (Qiagen, Valencia, CA., U.S.A.). Foi estabelecido, um método rápido e preciso de quantificação das partículas virais, através da técnica de qPCR, para padronizar a relação entre a quantidade de vírus recombinante a ser utilizada em um processo de infecção, visando aumentar os níveis de produção da proteína heteróloga. Diferentes proporções entre vírus e células foram utilizadas em cinco linhagens distintas: BHK-21, Huh-7, VERO, L929 e HEK-293T; sendo avaliados dois tempos de coleta da RVGP após a infecção (24 e 48 h). A proteína gerada foi avaliada através de diferentes métodos como Western Blot, Dot blot e imunofluorescência indireta (IFI), sendo a quantificação da proteína realizada através de ensaio imunoenzimático (ELISA). Esse trabalho contribui para o desenvolvimento de abordagens que utilizam o SFV como vetor de expressão, indicando as melhores metodologias e linhagens celulares, que podem ser utilizadas para aplicação na produção das mais variadas proteínas. / The expression system based on Semliki Forest virus (SFV) is a system which has been widely used in recent times for expression of many proteins in large quantities as compared with other systems. The aim of this study was to optimize the capacity of this vector to express viral proteins in different mammalian cell lines, using as target, the rabies virus glycoprotein (RVGP). We assessed two different methods of transfection to obtain recombinant SFV vector, such as electroporation and liposome commercial Transmessenger (Qiagen, Valencia, CA., U.S.A.). It was established also a fast and accurate quantification of viral particles by qPCR technique, to improve the relation between the amount of recombinant virus to be used in a process of infection, to increase production levels of the heterologous protein. Different proportions between viruses and cells were used in five distinct lineages: BHK-21, Huh-7, Vero, L929 and HEK-293T; being evaluated two sampling times after infection of RVGP (24 e 48 h). The protein was assessed by various methods such as Western blot, Dot blot and indirect immunofluorescence (IIF), and the protein quantification performed by enzyme immunoassay (ELISA). This work contributes to the development of approaches to using the SFV expression vector indicating the best methods and cell lines that can be used for application in the production of various proteins.

Breast cancer is a world health problem and is a leading cause of cancer-related death among women in the United States. However, breast cancer risks were reported to be reduced through exposure to Vitamin D through its Receptors identified as the p53 target gene. The purpose of this study was to assess the associations between VDR gene polymorphisms knowledge/awareness and decisions to reduce breast cancer risks and likelihood of mammogram screening among women in Texas. Data from survey were used. Roy adaptation model was the theoretical framework that guided this quasi- experimental, quantitative research. The dependent variables were decisions to reduce breast cancer risks and likelihood of mammogram screening. The independent variables were knowledge about VDR gene polymorphisms and exposure to vitamin D. The covariates were level of education, awareness, lifestyle, breast self-exams, mammograms, age, early menarche, late menopause, and family history of breast cancer. The chi-square test and regression analysis were used to test the stated research hypotheses and to answer the research questions. Knowledge of VDR gene polymorphisms and exposure to vitamin D were not significantly associated with breast cancer risk, ï?£2 (3, N= 250) =3.84, p > 0.05. Also, awareness of the risk factors for breast cancer was not significantly associated with decisions to go for mammogram screenings or to enroll in breast cancer risk-reduction programs, ï?£2 (3, N= 250) =1.58, p > 0.05. To advocate for the promotion of awareness of the importance of pharmacogenetic testing for VDR gene polymorphisms for early detection of breast cancer, which would help to undertake appropriate therapeutic measures in a timely manner to prevent cancer metastasis, further research is warranted.

Les anticorps monoclonaux (AcM) sont des protéines ayant une reconnaissance antigénique spécifique utilisée pour le développement de réactifs thérapeutiques et diagnostiques. La production commerciale est réalisée en cultivant des cellules hôtes dans des bioréacteurs spécifiques. La densité cellulaire et le métabolisme cellulaire sont des paramètres clés pour le rendement élevé des AcM. Bioréacteur à fibres creuses (HFB), une cartouche contenant des fibres poreuses emballées, est l'un des systèmes de production disponibles dans le commerce. Si la densité de cellules obtenue peut conduire à un rendement élevé, le coût de l'ensemble du dispositif, y compris les pompes et les cartouches très coûteuses, empêche son utilisation de petites unités. Comme une alternative économique, nous avons proposé ici d'étudier le potentiel des modules de dialyse de polysulfone du commerce, classiquement employés dans le traitement de l'insuffisance rénale en phase terminale. Cependant, la membrane de polysulfone native a démontré une adsorption de protéines non spécifique significative préjudiciable à la production d'AcMs. De plus des enjeux normatifs viennent se greffer à ces problématiques scientifiques et technico-économiques, avec le cas des normes (ISO 13485/ AC S99-104/ GMP FDA /BPF…) qui imposent des méthodes de travail normalisées. Ce travail de thèse consiste en la conception d’un bioréacteur jetable, sur la base d’une cartouche de dialyse médicale à fibres creuses. Ce système doit offrir toutes les garanties en termes de production, de facilité d’utilisation, de stérilité, et permettre de concentrer les produits de cytoculture. La méthodologie scientifique a été couplée à une démarche qualité. La gestion de ce projet a été couplé à l’analyse de risques. En effet ce projet a été divisé en ses 5 composantes élémentaires décrite par Ishikawa par la méthode des 5 M. L’analyse de risque a consisté au calcul d’indice de criticité par la méthode AMDEC de chacune de ses familles de risques. Cette approche nous a permis de formaliser deux axes de recherche : i) la mise en œuvre d’une oxygénation efficace du milieu de culture (chapitre 4) et ii) les moyens de limiter le colmatage dans le module à fibres creuses pour obtenir une culture cellulaire conforme aux objectifs (chapitre 5). Le taux d’oxygénation est un facteur à prendre en compte dans un processus de culture cellulaire. L’oxygène peut être supplémenté selon deux modes, le mode passif ou le mode actif [Ozturk et Palsson 1990; Zhang S. et al 1992]. Il existe 2 types de système d’oxygénation : les système dit passif ou les échanges se font a travers une paroi de silicone et un système actif par aération directe dans le milieu de culture. Ce système est de loin l'opération la plus simple pour fournir de l'oxygène. Cependant, lorsque celui-ci est utilisé pour apporter de l’oxygène à des cultures de cellules mammifères, cela peut engendrer des altérations cellulaires. Des agents protecteurs chimiques peuvent être utilisés pour réduire les dommages cellulaires et la formation de mousse [Kamase et Moo-yung 1990; van Der pol L.A et al 1993]. Nos études ont démontré que l'addition d'agents anti-mousse peut entraîner une diminution du coefficient de transfert de masse d’O2 en phase liquide (Kl) [Kamase et Moo-yung 1990]. Nous avons établi l’efficacité de l’utilisation d’un polymère silice/silicone pour éliminer la mousse sur des cultures bactériennes et de cellules mammifères. Afin de limiter ces phénomènes de colmatage, les fibres de polysulfone ont été traitées avec plusieurs tensioactifs (acide pluronique F127, D-limonen et différentes huiles de silicone) qui ont conduit à une diminution significative de l'adsorption protéique. L'effet de ces surfactants sur les performances de filtration et sur la cytotoxicité a été étudié. Certains d'entre eux n'ont pas influencé ces paramètres alors que d'autres ont présenté des effets négatifs. / Monoclonal Antibodies (mAbs) are proteins with specific antigen recognition used for development of both therapeutic and diagnostic reagents. Commercial production is achieved by growing host cells in specific bioreactors. Cell density and cell metabolism are key parameters for high yield of mAbs. Hollow fiber bioreactor (I-IFB), a cartridge containing packed porous fibres, is one of the system for production commercially available. If the cell density achieved can lead to high yield, the cost of the whole device, including pumps and very expensive cartridges prevents its use of small units. As an economical alternative, we proposed here to investigate the potential of commercial polysulfone dialysis modules, classically employed in the treatment of end stage renal failure. However, the native polysulfone membrane demonstrated a significant non-specific protein adsorption detrimental to mAbs production. Moreover normative issues are added to these scientific and techno-economic issues, with the case of standards (ISO 13485 / AC S99-104 / GMP FDA / BPF ...) which impose standard working methods. This thesis consists of the design of a disposable bioreactor, based on a hollow-fiber medical dialysis cartridge. This system must offer all the guarantees in terms of production, ease of use, sterility, and allow to concentrate the cytoculture products. Scientific methodology has been coupled with a quality approach. The management of this project was coupled with the risk analysis. Indeed this project was divided into its 5 elementary components describe by Ishikawa by the 5M method. The risk analysis consisted in the calculation of the criticality index by the AMDEC method of each of its families of risks. This approach allowed us to formalize two research axes: i) the implementation of an effective oxygenation of the culture medium (chapter 4) and ii) the means of limiting the clogging in the hollow fiber module to obtain a culture consistent with the objectives (Chapter 5). The rate of oxygenation is a factor to be taken into account in a cell culture process. Oxygen can be supplemented in two modes, passive mode or active mode [Ozturk and Palsson 1990; Zhang S. et al 19921. There are two types of oxygenation system: the so-called passive system or the exchanges are made through a silicone wall and an active system by direct aeration in the culture medium. This system is by far the simplest operation for providing oxygen. However, when it is used to supply oxygen to mammalian cell cultures, this can cause cellular damage. Chemical protective agents can be used to reduce cell damage and DKamase and Moo-yung 1990 foam formation; van Der pol L.A. et al 1993 Cl. Our studies have shown that the addition of antifoaming agents can lead to a decrease in the liquid phase (K2) mass transfer coefficient of D Kamase and Moo-yung 1990C]. We have established the effectiveness of using a silica / silicone polymer to remove foam on bacterial and mammalian cell cultures. In order to limit these adsorption phenomena, polysulfone fibers were treated with several surfactants (pluronic acid F 127, D-limonen, and different silicone oils) which led to a significant decrease in protein adsorption. The effect of such surfactants on the filtration performances and on cytotoxicity were investigated. Some of the them did not influence these parameters while some presented negative effects. Finally, different cell culture parameters (cells densities, production yield, flow properties, fouling) were studied, as well as the performance of the bioreactor in perfusion continuous mode. The bioreactor was maintained in continuous mode for fifteen days and the production yield per batch was 250 mg of AcMs. The results obtained in this work allowed us to define the next steps to be taken, and are the subject of the Perspectives section.

O sistema de expressão gênica derivado do Semliki Forest Vírus (SFV) vem sendo muito utilizado nos últimos tempos para expressão em grandes quantidades de inúmeras proteínas, quando comparado com outros sistemas. O objetivo desse estudo foi otimizar a capacidade desse vetor viral de expressar proteínas em diferentes linhagens celulares de mamíferos, utilizando como alvo, a glicoproteína do vírus rábico (RVGP). Foram avaliadas formas de obtenção do vetor SFV recombinante, através de diferentes métodos de transfecção, como eletroporação e lipofecção, utilizando um lipossomo comercial chamado Transmessenger (Qiagen, Valencia, CA., U.S.A.). Foi estabelecido, um método rápido e preciso de quantificação das partículas virais, através da técnica de qPCR, para padronizar a relação entre a quantidade de vírus recombinante a ser utilizada em um processo de infecção, visando aumentar os níveis de produção da proteína heteróloga. Diferentes proporções entre vírus e células foram utilizadas em cinco linhagens distintas: BHK-21, Huh-7, VERO, L929 e HEK-293T; sendo avaliados dois tempos de coleta da RVGP após a infecção (24 e 48 h). A proteína gerada foi avaliada através de diferentes métodos como Western Blot, Dot blot e imunofluorescência indireta (IFI), sendo a quantificação da proteína realizada através de ensaio imunoenzimático (ELISA). Esse trabalho contribui para o desenvolvimento de abordagens que utilizam o SFV como vetor de expressão, indicando as melhores metodologias e linhagens celulares, que podem ser utilizadas para aplicação na produção das mais variadas proteínas. / The expression system based on Semliki Forest virus (SFV) is a system which has been widely used in recent times for expression of many proteins in large quantities as compared with other systems. The aim of this study was to optimize the capacity of this vector to express viral proteins in different mammalian cell lines, using as target, the rabies virus glycoprotein (RVGP). We assessed two different methods of transfection to obtain recombinant SFV vector, such as electroporation and liposome commercial Transmessenger (Qiagen, Valencia, CA., U.S.A.). It was established also a fast and accurate quantification of viral particles by qPCR technique, to improve the relation between the amount of recombinant virus to be used in a process of infection, to increase production levels of the heterologous protein. Different proportions between viruses and cells were used in five distinct lineages: BHK-21, Huh-7, Vero, L929 and HEK-293T; being evaluated two sampling times after infection of RVGP (24 e 48 h). The protein was assessed by various methods such as Western blot, Dot blot and indirect immunofluorescence (IIF), and the protein quantification performed by enzyme immunoassay (ELISA). This work contributes to the development of approaches to using the SFV expression vector indicating the best methods and cell lines that can be used for application in the production of various proteins.