One major disadvantage of nucleic acid delivery systems is the low transfection or transduction efficiency of large-sized plasmids into cells. In this communication, we demonstrate the efficient transfection of a 15.5 kb green fluorescent protein (GFP)-fused HIV-1 molecular clone with a nucleic acid delivery system prepared from the highly potent peptide-mimicking cationic lipid OH4 in a mixture with the phospholipid DOPE (co-lipid). For the transfection, liposomes were loaded using a large-sized plasmid (15.5 kb), which encodes a replication-competent HIV type 1 molecular clone that carries a Gag-internal green fluorescent protein (HIV-1 JR-FL Gag-iGFP). The particle size and charge of the generated nanocarriers with 15.5 kb were compared to those of a standardized 4.7 kb plasmid formulation. Stable, small-sized lipoplexes could be generated independently of the length of the used DNA. The transfer of fluorescently labeled pDNA-HIV1-Gag-iGFP in HEK293T cells was monitored using confocal laser scanning microscopy (cLSM). After efficient plasmid delivery, virus particles were detectable as budding structures on the plasma membrane. Moreover, we observed a randomized distribution of fluorescently labeled lipids over the plasma membrane. Obviously, a significant exchange of lipids between the drug delivery system and the cellular membranes occurs, which hints toward a fusion process. The mechanism of membrane fusion for the internalization of lipid-based drug delivery systems into cells is still a frequently discussed topic.

Cancer is one of the leading causes of death in the world, often due to failed treatments because of drug resistance. Treatment is difficult as resistance is hard to detect before treatment and can develop during treatment. The fluorometric microculture cytotoxicity assay (FMCA) is a reliable, rapid method for testing drug cytotoxicity but requires large cell samples, which can be challenging to obtain. Patient-derived cancer cells (PDC) have proven challenging to culture in monolayer models, but recent studies have shown the possibility of using tumoroids. Tumoroids are three-dimensional models where cells are grown in basement membrane matrix hydrogel, allowing scaffold growth like in vivo tumors. This study aimed to culture colorectal PDC in the form of tumoroids, transfecting them, and examine cell cycle and tumor resistance for 5-Fluorouracil, Oxaliplatin and Irinotecan. Cells were deposited in gels with medium mimicking in vivo conditions, supporting growth and allowing extracellular signaling. The study succeeded in culturing both untransfected and transfected cells, resulting in cells expanding 48 and 42 times, respectively. Cell cycle remained unchanged. No changes were observed in 5-Fluorouracil, but a change was seen in transfected cells at passage 3 with oxaliplatin. The cells showed a 22% difference in survival indexes compared to naïve cells. Changes were seen in Irinotecan’s half maximal inhibitory concentration (IC50); all cell passage IC50 values differed >15.17 µM (p-value 0.0184). In conclusion, PDC can be cultured as tumoroids, but more studies are needed to determine if the model can generate reliable results representing PDC regarding tumor resistance.

“Lab on a Chip” technologies have revolutionized the field of bio-chemical analytics. The crucial role of optical techniques in this revolution resulted in the emergence of a field by itself, which is popularly termed as “optofluidics”. The miniaturization and integration of the optical parts in the majority of optofluidic devices however still remains a technical challenge. The works described in this thesis focuses on developing integration methods to combine various optical techniques with microfluidics in an alignment-free geometry, which could lead to the development of portable analytical devices, suitable for field applications. The integration approach was applied to implement an alignment-free optofluidic chip for optical chromatography; a passive optical fractionation technique fractionation for cells or colloids. This system was realized by embedding large mode area photonic crystal fiber into a microfluidic chip to achieve on-chip laser beam delivery. Another study on passive sorting envisages an optofluidic device for passive sorting of cells using an optical potential energy landscape, generated using an acousto-optic deflector based optical trapping system. On the analytical side, an optofluidic chip with fiber based microfluidic Raman spectroscopy was realized for bio-chemical analysis. A completely alignment-free optofluidic device was realized for rapid bio-chemical analysis in the first generation by embedding a novel split Raman probe into a microfluidic chip. The second generation development of this approach enabled further miniaturization into true microfluidic dimensions through a technique, termed Waveguide Confined Raman Spectroscopy (WCRS). The abilities of WCRS for online process monitoring in a microreactor and for probing microdroplets were explored. Further enhanced detection sensitivity of WCRS with the implementation of wavelength modulation based fluorescent suppression technique was demonstrated. WCRS based microfluidic devices can be an optofluidic analogue to fiber Raman probes when it comes to bio-chemical analysis. This allows faster chemical analysis with reduced required sample volume, without any special sample preparation stage which was demonstrated by analyzing and classifying various brands of Scotch whiskies using this device. The results from this study also show that, along with Raman spectroscopic information, WCRS picks up the fluorescence information as well, which might enhance the classification efficiency. A novel microfabrication method for fabricating polymer microlensed fibers is also discussed. The microlensed fiber, fabricated with this technique, was combined with a microfluidic gene delivery system to achieve an integrated system for optical transfection with localized gene delivery.

La sialylation des N-glycanes du fragment Fc des immunogobulines G (IgG) est une modification peu fréquente des IgG humaines. Pourtant, elle est l’objet de beaucoup d’attention depuis que deux articles fondateurs ont été publiés, qui montrent l’un que la sialylation des IgG diminue leur capacité à déclencher la cytotoxicité cellulaire dépendant de l’anticorps (ADCC), et l’autre que les IgG sialylées en α2,6 seraient la fraction efficace des IgG intraveineuses (IgIV) anti-inflammatoires. Les anticorps monoclonaux thérapeutiques, qui sont le plus souvent des IgG recombinantes produites en culture de cellules de mammifère, connaissent depuis la fin des années 90 un succès et une croissance phénoménaux sur le marché pharmaceutique. La maîtrise de la N-glycosylation du Fc des IgG est une clé de l’efficacité des anticorps monoclonaux. Si les IgG sialylées sont des molécules peu fréquentes in vivo, elles sont très rares en culture cellulaire. Dans cette étude, nous avons développé une méthode de production d’IgG avec une sialylation de type humain en cellules CHO. Nous avons travaillé principalement sur la mise au point d’une stratégie de production d’IgG sialylées par co-expression transitoire d’une IgG1 avec la β1,4-galactosyltransférase I (β4GTI) et la β-galactoside-α2,6-sialyltransférase I (ST6GalI). Nous avons montré que cette méthode permettait d’enrichir l’IgG1 en glycane fucosylé di-galactosylé mono-α2,6-sialylé G2FS(6)1, qui est le glycane sialylé présent sur les IgG humaines. Nous avons ensuite adapté cette méthode à la production d’IgG présentant des profils de glycosylation riches en acides sialiques, riches en galactose terminal, et/ou appauvris en fucosylation. L’analyse des profils de glycosylation obtenus par la co-expression de diverses combinaisons enzymatiques avec l’IgG1 native ou une version mutante de l’IgG1 (F243A), a permis de discuter des influences respectives de la sous-galactosylation des IgG1 en CHO et des contraintes structurales du Fc dans la limitation de la sialylation des IgG en CHO. Nous avons ensuite utilisé les IgG1 produites avec différents profils de glycosylation afin d’évaluer l’impact de la sialylation α2,6 sur l’interaction de l’IgG avec le récepteur FcγRIIIa, principal récepteur impliqué dans la réponse ADCC. Nous avons montré que la sialylation α2,6 augmentait la stabilité du complexe formé par l’IgG avec le FcγRIIIa, mais que ce bénéfice n’était pas directement traduit par une augmentation de l’efficacité ADCC de l’anticorps. Enfin, nous avons débuté le développement d’une plateforme d’expression stable d’IgG sialylées compatible avec une production à l’échelle industrielle. Nous avons obtenu une lignée capable de produire des IgG enrichies en G2FS(6)1 à hauteur de 400 mg/L. Cette étude a contribué à une meilleure compréhension de l’impact de la sialylation sur les fonctions effectrices des IgG, et a permis d’augmenter la maîtrise des techniques de modulation du profil de glycosylation des IgG en culture cellulaire. / Only a fraction of the N-glycans present on the Fc fragment of the human IgGs is sialylated. However, a new interest for sialylation has risen since two major articles were published, one showing that sialylation reduces the capacity of the antibody to trigger antibody-dependent cell cytotoxicity (ADCC), whereas the other showed that the IgGs carrying α2,6-sialic acids on their Fc N-glycans were responsible for the anti-inflammatory activity of intravenous immunoglobulins (IVIGs) injected at high doses. Therapeutic monoclonal antibodies (mAbs) are in majority recombinant IgGs produced in mammalian cell culture. Since the end of the nineties, mAbs have become a major class of pharmaceutical products, and their success is still growing. The control of Fc N-glycosylation is a key parameter for the improvement of the therapeutic efficacy of mAbs. Sialylated IgGs are found only as traces in the classic CHO cell culture processes. In this study, we developed a method for the production of IgGs with a human-like sialylation in CHO cells. We focused on a production strategy relying on the transient co-expression of an IgG1 with the β1,4-galactosyltransferase I (β4GTI) and the β-galactoside-α2,6-sialyltransferase I (ST6GalI). We showed that this method allowed the enrichment of the IgG1 glycoprofile in the fucosylated di-galactosylated mono-α2,6-sialylated glycane G2FS(6)1, which is the main sialylated glycan found in human IgGs. We then adapted this method to the production of highly galactosylated or highly sialylated IgGs with and without core-fucosylation. The analysis of the glycosylation profiles obtained using the various enzyme combinations co-expressed with the native IgG1 or the mutant IgG1 F243A allowed us to discuss the influence of the under-galactosylation found in IgGs produced in CHO cells versus the Fc structural constraints on the limitation of IgG sialylation in CHO cells. We used the IgG1 glycovariants produced with our method to assess the impact of Fc α2,6-sialylation on the interaction of the IgG with the receptor FcγRIIIa, which is the main receptor mediating the ADCC response. We showed that the presence of α2,6-sialylation in the Fc increased the stability of the IgG-FcγRIIIa complex. This benefit however did not translate into an improved ADCC capacity. Finally, we initiated the development of a stable expression platform for the production of sialylated IgGs at yields relevant for the industry. We obtained a cell line capable of producing IgGs enriched in G2FS(6)1 at 400 mg/L. This may eventually represent a novel approach to manufacture a recombinant IVIG surrogate. With this work, we contributed to a better understanding of the impact of sialylation on the effector functions of IgGs. We also improved our understanding of the techniques allowing for the modification and control of the glycosylation profile of IgGs in cell culture.

This diploma thesis deals with the analysis of structural and dynamic organization of thyrotropin releasing hormone receptor (TRH-R) and δ-opioid receptor (DOR) within plasma membrane (PM) in relation to the specific sub-compartments of PM denominated as domains or membrane rafts. Modern fluorescence microscopy techniques FLIM, FRAP and RICS were used for this purpose. The experiments were performed on the live cells derived from HEK293 cell line. To reach the main goal of this work, the integrity of PM structure was altered by depletion of cholesterol which was performed by incubation of cells with β cyclodextrin. Results clearly support our previously suggested idea that the vast majority of TRH-R is localized in non-raft regions of plasma membrane. This work also compared different modes of performance of FRAP and results obtained by FRAP and RICS because these methods are to some extent analogous. This is one of the first works that used the RICS approach to characterize the G protein-coupled receptors. In the second part of this work, the setup of transient transfection of the HEK293 cells with DOR-ECFP and DOR EYFP constructs was established. Simultaneously, the functionality of these constructs, i.e. the ability of DOR to activate the cognate G protein was determined. Powered by TCPDF (www.tcpdf.org)

Therapeutic proteins, a.k.a. biopharmaceuticals, are most commonly produced in expression systems derived from Chinese Hamstery Ovary (CHO) cells, thanks to great capacity of post-translational modifications like secretation, folding and glycosylation. The engineering of cells for regulation of protein expression has many options including knock-in and knock-out of genes, epigenetic studies or improvement of the expression casette of the protein of interest by e.g. promotor variants or modifications of the 5’ and 3’ untranslated region (UTR). The 3’-UTR is therefore a good optimization candidate for attempting to achieve increased expression of therapeutic proteins. The final aim of this study was to identify and design 3’-UTRs for improved expression of therapeutic proteins in HyClone™ CHO cells from GE Healthcare Bio-Sciences AB (GEHC). The impact goal is to increase the efficiency and lower the costs for pharmaceutical companies when producing biopharmaceuticals in the HyClone™ CHO cell line, leading to increased accessibility of monoclonal antibodies (mAbs) on the pharmaceutical market. The study was initiated with bioinformatic analysis of the CHO cell transcriptome from a set of RNA-seq data of HyClone™ CHO to find high expressing, context independent genes. The 3’-UTRs from the best candidate genes were used for construction of plasmids for expression of a Fc-eGFP fusion protein. Nine selected 3’-UTRs were designed, synthesized and cloned into a parent plasmid (pGE0520) creating nine plasmid variants (pGE0523-531). The constructed plasmids were used for evaluation with site directed integration (SDI) into the HyClone™ CHO cell line and expression analysis were performed by flow cytometry and antibody titer measurements from cells with successfully integrated plasmid sorted by fluorescence-activated cell sorting (FACS).   Result show a significant effect on protein expression when using different variants of 3’-UTRs. Two variants, pGE0524 and pGE0526, competing with the parent plasmid in expression levels and integration efficiency from SDI, making them candidates for further investigations against the parent plasmid. Results also show good correlation between flow cytometry data from pre- and post-sorting, which can make research for further 3’-UTRs more efficient by evaluations and prediction of expression levels before cell sorting.

Modelos humanizados de porco são muito importantes para pesquisa biomédica e desenvolvimento de novas drogas e tratamentos. Além de ser um melhor modelo para doenças humanas do que animais de menor porte devido sua maior semelhança fisiológica, anatômica, de metabolismo e tempo de vida, o modelo suíno ainda permite suprimento ilimitado de órgãos para transplante. Apesar dessas vantagens, a expressão gênica inconsistente de animais transgênicos tornam a criação e avaliação desses animais muito dispendiosas, imprevisível e não permite a comparação de resultados de animais diferentes de maneira apropriada. Nesse estudo descrevemos uma nova técnica utilizando o promoter endógeno para a geração de um protocolo de substituição de genes com padrão clonal (transplante clonal de genes) sem clonagem de células, preservando a expressão genética e sua regulação intactas. Esse protocolo é reprodutível e pode ser aplicado para mais de um alvo genético, permitindo geração rápida de linhas transgênicas de animais (14-20 dias) com potencial de se tornar o novo padrão para geração de animais transgênicos de grande porte Suínos / Humanized pig models are very important for biomedical research, and drugs and treatment development. Not only it is a better model for diseases than smaller animals because of its closer physiology, anatomy, metabolism and life span, it also may provide unlimited organs for transplantation. In spite of all this advantages, inconsistent gene expression in transgenic animals make its generation and evaluation expensive, unpredictable and do not allow proper outcome comparison between different animals. In this report we describe a reproducible technique utilizing the endogenous promoter for generation of a clonal pattern gene replacement protocol (clonal gene transplant) without cell cloning, maintaining the normal gene expression and its regulation. This protocol is reproducible and applicable to more than one gene target, allowing fast generation of transgenic animals cell lines (as low as 14-20 days) and could become the new standard for transgenic large animal generation

O desenvolvimento da imunoterapia do câncer baseada em células dendríticas (DCs) é alvo de vários estudos. Para tumores sólidos, a abordagem baseada no uso de DCs alogenêicas fundidas com células tumorais tem se mostrado relativamente eficaz. Por outro lado, esta estratégia necessita uma massa tumoral considerável de cada paciente para a geração das células híbridas. Para contornar este problema o uso de DCs transfectadas com mRNA tumoral, o qual pode ser amplificado in vitro a partir de uma pequena amostra inicial do tumor, tem sido investigado. Para isto, uma transfecção eficiente e tradução correta do mRNA tumoral nas DCs são etapas críticas. Sabendo-se que as DCs de pacientes com câncer possuem atividade aloestimuladora defeituosa, DCs derivadas de doadores saudáveis poderiam ser uma alternativa para induzir uma resposta imune mais eficiente. Assim, este trabalho pretendeu aprimorar a metodologia de transfecção de DCs alogenêicas, derivadas de monócitos de doadores saudáveis, com mRNA de antígenos tumorais (survivina e RPSA) super-expressos na leucemia linfóide crônica (LLC) e avaliar sua capacidade em estimular a resposta linfocitária. Ao mesmo tempo, foram estabelecidas as metodologias para amplificação e síntese do RNA destes antígenos tumorais específicos, assim como do RNA mensageiro total, contidos nas células tumorais de pacientes com LLC. Os resultados mostraram ser possível a amplificação do mRNA total extraído das células leucêmicas com manutenção da expressão dos antígenos tumorais. Ainda, várias condições de transfecção com mRNA da survivina, transcrito in vitro, foram testadas, encontrando-se na lipofecção, a melhor maneira de transfectar as DCs. A lipofecção mostrou-se com baixa toxicidade quando comparada à técnica de eletroporação. Observou-se uma eficiência em torno de 40% de células transfectadas num intervalo de tempo entre 12 e 48 horas. Estas células foram usadas como estimuladoras em ensaios de proliferação usando-se linfócitos T alogenêicos como células respondedoras. As células transfectadas com mRNA da survivina foram capazes de estimular resposta linfoproliferativa com maior produção de IFN-gama, avaliado por ELISA. Além disso, a transfecção não alterou o padrão de expressão dos marcadores de superfície característicos das DCs. Estes dados mostram que a transfecção das DCs com mRNA pode afetar a resposta imune induzida por estas APCs. Nossos resultados suportam o uso de DCs transfectadas com mRNA para produção de vacinas anti-tumorais e mostram a survivina como um potente antígeno indutor da resposta linfocitária. / The development of dendritic cell (DC)-based cancer immunotherapy has been the target of many studies. For solid tumors, a promising approach based in allogeneic DCs fused to tumor cells has been relatively effective. On the other hand, this approach needs large tumor samples to generate enough DC-tumor cell hybrids. To overcome this problem, tumor mRNA-transfected DCs can be used, since mRNA can be amplified in vitro and allow unlimited vaccine production. For this, efficient transfection and optimal translation of tumor mRNA in DCs are critical. Moreover, DC derived from cancer patients has defective alostimulatory activity. In this case, DC derived from healthy donors may be an alternative to induce immune response more efficiently. Here, we established the methodology of allogeneic DC transfection with mRNA for tumor antigens (survivin and RPSA) overexpressed in chronic lymphocytic leukemia (CLL), and evaluated their ability for T cell stimulation. At the same time, we established mRNA amplification and mRNA in vitro transcription methodology for specific tumor antigens and total messenger RNA, present in CLL tumor cells. Our results showed it to be possible to amplify total mRNA derived from leukemic cells maintaining tumor antigen expression. Moreover, several transfection conditions using survivin mRNA obtained from in vitro transcript reactions were evaluated, defining lipofection as the better way to transfect DC. We obtained nearly 40% of transfected DCs between 12 and 48 hours. Transfected DCs were used as stimulator cells in proliferation assays using allogeneic T cells as responder cells. Survivin mRNA transfected DCs were able to stimulate T cell proliferation with increased IFN-gama production, measured by ELISA. Furthermore, the transfection did not change the pattern of surface molecules expression characteristic of DC. These data show that mRNA DC transfection can affect immune responses induced by these APCs. These findings support the use of tumor mRNA transfected DCs for anti-cancer vaccine production and show survivin as a potent antigen to induce T cell responses.

As proteínas que contem domínios ricos em leucina (LRR) mediam interações macromoleculares que estão envolvidas em muitos processos biológicos como infecção bacteriana em células hospedeiras e respostas imunológicas de plantas. Estudos anteriores em nosso laboratório identificaram um gene que codifica uma proteína contendo 6 LRRs (LaLRR17) em L. (L.) amazonensis. O LaLRR17 é um gene com expressão estágio regulada sendo abundantemente expresso na fase amastigota. Seqüências homólogas ao gene LaLRR17 foram encontradas em todas as espécies de Leishmania analisadas. Esse trabalho tem como objetivo a caracterização da proteína homóloga em L. (L.) major (LmLRR17). Anticorpos obtidos contra seqüências conservadas das proteínas LaLRR17 e LmLRR17 permitiram o estudo da abundância protéica em diferentes estágios do parasita. Curiosamente, a proteína LmLRR17 foi encontrada em maior abundância em promastigotas procíclicos em vez de amastigotas. Linhagens hiperexpressoras da proteína LmLRR17 ou expressoras da proteína LaLRR17 em fusão com o epitopo viral myc foram obtidas. As proteínas quiméricas foram expressas seguindo o mesmo padrão observado na cepa selvagem. O fenótipo desses mutantes foi avaliado mediante infecções de macrófagos in vitro. A hiperexpressão da proteína LmLRR17 em L. (L.) major não alterou o fenótipo da infecção in vitro. Por outro lado, a expressão da proteína heteróloga, LaLRR17, em promastigotas de L. (L.) major levou a incremento na virulência com maior número de células infectadas e de parasitas por célula. Esses resultados indicam que a expressão da proteína LmLRR17 em L. (L.) major é fortemente regulada. Esse trabalho também mostra que a expressão da proteína LaLRR17 em L. (L.) major leva a um aumento na infectividade. / Proteins containing leucine rich repeats (LRR) are known to be involved in macromolecular interactions in many processes such as signal transduction, cell-adhesion, RNA processing, apoptosis, disease resistance and immune response. A previous study in our laboratory identified a L. (L.) amazonensis gene encoding a protein containing 6 LRRs (LaLRR17). LaLRR17 is a stage-regulated gene expressed with increased abundance in the amastigote stage. Highly conserved homologues of LaLRR17 were found in all Leishmania species analyzed. Therefore, the aim of this study was to characterize the homologous protein of L. major (LmLRR17). Antibodies raised against peptide sequences common to LaLRR17 and LmLRR17 allowed the study of the steady-state protein abundance. Interestingly, LmLRR17 protein was found to be up-regulated in procyclic promastigotes, instead of amastigotes. Mutants of L. (L.) major overexpressing a myc-tagged version of LmLRR17 or of LaLRR17 protein were obtained. In these parasites, the chimeric proteins were expressed following the same pattern of expression observed in the wild type parasites. The phenotype of these mutants was assessed in vitro through macrophage infections. Overexpression of LmLRR17 protein in L. (L.) major resulted in an unaltered phenotype. On the other hand, overexpression of LaLRR17 in L. (L.) major induced an increase in virulence with a higher number of infected cells and intracellular parasites. These results indicate that the expression of LmLRR17 protein in L. major is tightly regulated and the expression of the heterologous LaLRR17 protein increased infectivity in vitro.

This thesis work describes the role of Rab proteins in amyloid processing and clearance in different cell pathways. It also describes an experimental approach used to analyze the expression effects of Rab10 in amyloid beta production. Since the main theory behind neurodegeneration in Alzheimer's disease claims that high levels of amyloid beta 42 (Aβ42) molecules trigger widespread neuronal death, control of Aβ42 has been a main target in Alzheimer's disease research. In addition, several studies show increased levels of particular Rab proteins in Alzheimer's pathogenesis. However, no review consolidates current findings in neurodegeneration of Alzheimer's with Rab protein dysfunction. The first chapter of this thesis aims to address this need by providing a current review of Rab proteins associated with APP and neurodegeneration. The second chapter constitutes an experimental approach used to characterize the effects of Rab10 and Sar1A GTPases in APP and amyloid processing. We found that Rab10 expression does not affect APP production but significantly changes Aβ generation, particularly the toxic Aβ42 and Aβ42:40 ratio. On the other hand, we found no significant effect of Sar1A expression on either APP or amyloid beta generation. These findings partially confirm the work done by Kauwe et al (2015) and provide preliminary evidence for two potential targets for protective effects in neurodegeneration.