INTRODUÇÃO: O receptor tipo Toll 4 (TLR4) pertencente a uma família de receptores do sistema imune inato, reconhece o padrão molecular de lipopolissacarídeos (LPS), expressos por bactérias Gram negativas. Sua cascata de sinalização, nas células apresentadoras de antígeno, ocorre por duas vias principais: MyD88-dependente, que resulta na ativação de NF-B e na expressão de genes de resposta inflamatória e MyD88-independente, responsável pela ativação dos fatores IRF3 e IRF7, culminando na síntese de interferons e , envolvidos na resposta anti-viral e anti-bacteriana. Células não-imunes, de diversos tecidos, também expressam TLR4, incluindo células pancreáticas murinas e humanas. Devido ao seu papel nos processos inflamatórios, os TLR estão implicados em doenças crônicas como obesidade e diabetes. Estudo anterior do grupo identificou TLR4 como uma molécula que ativa sinais inflamatórios e provoca alterações na homeostase das células . Neste trabalho, investigamos qual via é ativada por LPS e quais os efeitos da expressão do TLR4 na viabilidade celular e na produção de insulina em células murinas. MÉTODOS: Células MIN6 (linhagem celular de insulinoma de camundongo) foram cultivadas em condições de hipo (2,8mM glicose), normo (5,6mM glicose) e hiperglicemia (11,2mM glicose), por 4 dias. Após esse período, foi adicionado LPS (50 ng/mL) por 48h e foram feitas análises por PCR em tempo real, Western Blot, ELISA e citometria de fluxo. RESULTADOS: Os resultados confirmam o aumento de TLR4 em células em condições de hiperglicemia e a via de sinalização ativada por LPS é a via MyD88-dependente, envolvida na produção de citocinas pró-inflamatórias. A via de indução de intérferons tipo 1 está ausente nestas células. Além disso, TLR4 ativado por LPS aumentou secreção de insulina em resposta a glicose, mas não induziu a morte celular. CONCLUSÃO: A expressão de TLR4 em células pancreáticas murinas é induzida em resposta ao aumento da glicemia, constituindo um novo elo entre a agressão à célula causada por altos níveis de glicose e a alteração da função celular induzida por LPS / INTRODUCTION: Toll-like receptor 4 (TLR4) belongs to a family of innate immunity receptors and recognizes the molecular pattern present in lipopolysaccharides (LPS), typical of Gram-negative bacteria. There are two TLR4 signaling pathways, typically in antigen-presenting cells: one is MyD88-dependent, activating NF-kB transcription factor and triggering inflammatory cytokine production and the other is MyD88-independent, leading to activation of IRF3 and IRF-7 and production of interferons e , involved in antiviral and antibacterial immune responses. Non-immune cells in several tissues also express TLR4, including human and murine pancreatic cells. Due to their role in inflammatory processes, TLRs have been implicated in chronic diseases like obesity and diabetes. Our previous study identified TLR4 as a molecule which activates inflammatory signals and induces changes in cell homeostasis. In this study, we investigated which of the TLR4 pathways is activated by LPS and the effects of glucose levels on cell viability and insulin production in a mouse insulinoma cell line. METHODS: MIN6 cells were maintained in low (2,8mM), normal (5,6mM) and high (11,2mM) glucose levels for 4 days, and then incubated with LPS (50 ng/mL) for 48 hours. Analyses were done by real-time PCR, Western Blot, ELISA and flow cytometry. RESULTS: Analysis confirmed increase in TLR4 gene expression in hyperglycemic conditions and showed that the signaling pathway activated by LPS is MyD88-dependent. The interferon induction pathway is absent in these cells. Furthermore, upon activation by LPS, TLR4 impacts on insulin secretion in response to glucose, but without triggering cell death. CONCLUSION: We conclude that TLR4 expression in mouse pancreatic cells is induced in response to increased glucose levels, constituting a new link in the chain of events leading to cell stress caused by high glucose levels with concomitant changes in cell function induced by LPS

A hemofilia A é uma doença ligada ao cromossomo X causada pela deficiência do fator VIII da coagulação sanguínea (FVIII). O tratamento disponível consiste na terapia de reposição da proteína do fator VIII derivada do plasma (FVIIIdp) ou recombinante (FVIIIr). Atualmente, dos 7 produtos recombinantes disponíveis no mercado, 6 são produzidos em linhagens celulares de roedores. A expressão dessa proteína em sistemas celulares não-humanos pode gerar uma molécula com perfil de modificações diferente do endógeno, podendo levar a reações imunogênicas e geração de inibidores anti-FVIIIr. Em função disso, novas estratégias de produção têm sido avaliadas, como a utilização de células hospedeiras mais eficientes no que diz respeito ao potencial de expressão da proteína de interesse. Dentre as linhagens de interesse, a linhagem hepática SK-HEP-1 tem se destacado por apresentar altos níveis de expressão do FVIIIr e potencial para o cultivo em suspensão em meios livres de soro fetal bovino (SFB). Dessa forma, o objetivo deste trabalho foi avaliar a produção de FVIIIr na linhagem celular humana SK-Hep-1 comparando duas estratégias para o estabelecimento de processos de produção em condições livres de soro e em suspensão: Estratégia 1 - adaptação para tais condições da linhagem já modificada geneticamente e Estratégia 2 - modificação gênica para a expressão da proteína já em células previamente adaptadas à tais condições. Para a estratégia 1, foram geradas duas linhagens recombinantes produtoras de FVIIIr, SK-HEP-F8/Neo-E1 e SK-HEP-F8/GFP-E1 aderentes e em cultivos suplementados com SFB. Na caracterização da cinética e produção do FVIIIr as linhagens apresentaram taxas específicas máxima de crescimento (?max) de 0,064 e 0,0031h-1 produzindo 1,0 e 0,78UI/mL de FVIIIr, respectivamente. Diversos protocolos de adaptação foram empregados, entretanto, não foi possível obter sucesso na adaptação das linhagens recombinantes para condições livres de soro e em suspensão. Para a estratégia 2, as células SK-HEP-1 selvagens adaptadas ao meio de cultura livre de SFB SFMII apresentaram um valor de ?max de 0,0186h-1 e Xmax de 1,9x106cels/mL. Para as etapas de modificação gênica da linhagem selvagem foram utilizados os mesmos vetores lentivirais empregados para a geração das células recombinantes aderentes, pLVmpsvFVIII?B-Neo e pLVCMVFVIII?B-GFP. Para o primeiro, não foi possível gerar uma linhagem produtora do FVIIIr. Para o segundo, foi possível obter duas linhagens produtoras do FVIIIr com 0.14 e 0.12IU/mL de atividade pelo ensaio cromogênico. O presente trabalho mostrou que a linhagem humana Sk-Hep-1 é apropriada para a produção de altos níveis de FVIIIr. No entanto, maiores esforços devem ser voltados ao desenvolvimento de meios de cultura livres de soro específicos para a linhagem para possibilitar a produção eficiente do FVIIIr em suspensão em meios livre de soro. / Hemophilia A is a genetic X-linked disorder caused by the coagulation factor VIII (FVIII) deficiency. The current treatment is the replacement therapy with plasma derived FVIII (pdFVIII) or recombinant FVIII (rFVIII) products. Nowadays, of the seven products available in the market, six are produced in rodent expression systems, which can result in a rFVIII molecule with different post-translational modifications and may lead to immune responses to non-human epitopes. Therefore, new production strategies have been evaluated, as the use of more efficient hosts in terms of protein expression potential. Among potential cell lines, the hepatic SK-HEP-1 cell line features high levels of rFVIII production and potential for serum-free suspension culture. In face of the exposed above, the goal of this study was to evaluate rFVIII production in the SK-HEP-1 human cell line comparing two strategies for the establishment of production process in a suspension serum-free condition: strategy 1 - adaptation to these conditions of a genetic modified cell line; strategy 2 - genetic modification of an already adapted cell line to rFVIII protein expression. For strategy 1, two adherent rFVIII producer cell lines were established in serum containing medium, SK-HEP-F8/Neo-E1 e SK-HEP-F8/GFP-E1. Characterization of cell growth and rFVIII production showed a maximum specific growth rate (?max) of 0.064 and 0.00311h-1 with rFVIII production of 1.0 and 0.78UI/mL, respectively. Different adaptation protocols were used; however, it was not possible to adapt the recombinant cell lines to growth in suspension serum-free conditions. For strategy 2, the wildtype SK-HEP-1 cell line adapted growth in SFMII BSF medium, showed a ?max of 0.0186h-1 and a maximum cell concentration (Xmax) of 1.9x106cells/mL. For the genetic modification, it were employed the same lentiviral vectors used for the recombinant adherent cells generation, pLVmpsvFVIII?B-Neo and pLVCMVFVIII?B-GFP. For the first, no attempts were successful. For the second, it was possible to generate two rFVIII producer populations with 0.14 and 0.12IU/mL activity, measured by chromogenic assay. These results demonstrate that the SK-HEP-1 cell line is appropriate for the production of high levels of rFVIII. Nevertheless, efforts should be made in developing specific medium to support efficient rFVIII production in suspension and suspension serum-free conditions.

Le circovirus porcin de type 2 (PCV2) est un pathogène majeur pour l’industrie porcine et est associé à une longue liste de maladies associées au circovirus porcin (MACVP). Les premières tentatives pour reproduire ces maladies ont montré que le virus doit être combiné à d’autres agents pathogènes du porc ou à différents stimulants du système immunitaire. De ces agents, le virus du syndrome reproducteur et respiratoire porcin (VSRRP) est celui qui est le plus souvent co-isolé avec le PCV dans les fermes. Une grande partie des efforts faits pour étudier les interactions entre ces deux virus ont été menés in vivo. Les interactions in vitro ont jusqu’à maintenant été peu étudiées du fait qu’il n’existe pas de modèle cellulaire permettant la réplication efficace des deux virus. L’objectif de ce projet était donc de développer un modèle cellulaire propice à la réplication des deux virus et d’étudier leur interaction en co-infection. Une lignée cellulaire provenant de la trachée d’un porcelet nouveau-né (NPTr), permissive au PCV, a été génétiquement modifiée pour exprimer la protéine CD163, un récepteur majeur du VSRRP. Ce projet a montré que cette nouvelle lignée cellulaire (NPTr-CD163) est permissive au VSRRP ainsi qu’à plusieurs génotypes de PCV (PCV1, PCV2a, PCV2b et PCV1/2a). De plus, les résultats obtenus lors d’infections mixtes suggèrent que la réplication du VSRRP et du PCV conditionne de façon génotype-dépendante celle du PCV puisque la réplication du PCV1 est inhibée en présence de VSRRP, alors que celle du PCV2b est significativement augmentée dans les mêmes conditions. Ni la mortalité cellulaire, ni la réponse cellulaire en cytokines n’a permis d’expliquer ces résultats. La modulation de la réplication du PCV par le VSRRP serait donc liée à un mécanisme spécifique qui demeure inconnu. De plus, cet effet varierait en fonction du génotype de PCV. / Porcine circovirus (PCV) type 2 (PCV2) is a major pathogen in the swine industry and has been described as the causative agent of a long list of conditions under the designation of porcine circovirus-associated diseases (PCVAD). Attempts to replicate PCVAD initially failed, as it was discovered that an immune trigger could facilitate the reproduction of clinical signs, either by co-infecting with other swine pathogens or using immune stimulants. Of these, porcine reproductive and respiratory syndrome virus (PRRSV) is the most frequently co-isolated agent in the field. Most effort has been made to understand this interaction in vivo since most in vitro cellular models lack the ability to efficiently replicate both viruses. To answer the lack of an in vitro model, we developed a cell line that allows the replication of both PRRSV and PCV. A neonate porcine tracheal cell line (NPTr) was genetically modified to stably express CD163 (NPTr-CD163), a major PRRSV receptor. NPTr-CD163 cells were able to replicate all PCV genotypes (PCV1, PCV1/2a, PCV2a and PCV2b) and PRRSV. A significant effect of PRRSV on PCV replication was found to be genotype dependent, as PCV1 replication was down regulated in the presence of PRRSV and PCV2b replication was up regulated in the same conditions. Neither cell mortality assays nor cytokine expression analysis were able to provide an explanation for these results. The effect of PRRSV on PCV1 and PCV2b replication is suggestive of a more specific, yet still unknown, mechanism. Furthermore, this effect is PCV-genotype dependant.

<p>Amyotrophic lateral sclerosis is a fatal, progressive neurodegenerative disease with unknown ethiology. The aim of this study was to increase understanding of the pathophysiological mechanisms of dying motor neurons and wasting muscle tissue in this particular disorder.</p><p>Quantitative receptor autoradiographic methodology was applied on cervical spinal cord sections from patients with ALS to evaluate the specific binding of the acetylcholine transporter ³H-vesamicol in motor neurons. Despite a significant reduction of the number of ventral motor neurons in ALS, the ³H-vesamicol binding was not reduced in ALS compared to control cases, which suggests an increased metabolic activity in remaining motor neurons.</p><p>Motor neurons dying in ALS might go through apoptosis (programmed cell death), so immunohistochemical and TUNEL techniques were applied on thoracic spinal cord from ALS patients to evaluate the possibility of an apoptotic process. The increased Bax expression indicates an apoptotic process and further, motor neurons were TUNEL-positive, indicating DNA degradation caused by programmed cell death.</p><p>Muscle biopsies were obtained from ALS patients, and mRNA levels for the neurotrophic factors GDNF and BDNF were measured and compared to control subjects. GDNF levels were increased in muscle tissue in ALS whereas BDNF levels were unaltered.</p><p>Levels of GDNF and BDNF were also measured in cerebrospinal fluid from ALS patients and controls using ELISA methodology. Levels of BDNF were unaltered in ALS cornpared to controls. GDNF however was not detectable in controls whereas 12 out of 15 ALS patients had measurab1e levels of GDNW. A marked upregulation of endogenous GDNF and GDNF mRNA in ALS CSF and muscle respectively is of special interest in relation to clinical trials where GDNF is administered to this group of patients.</p>

Amyotrophic lateral sclerosis is a fatal, progressive neurodegenerative disease with unknown ethiology. The aim of this study was to increase understanding of the pathophysiological mechanisms of dying motor neurons and wasting muscle tissue in this particular disorder. Quantitative receptor autoradiographic methodology was applied on cervical spinal cord sections from patients with ALS to evaluate the specific binding of the acetylcholine transporter 3H-vesamicol in motor neurons. Despite a significant reduction of the number of ventral motor neurons in ALS, the 3H-vesamicol binding was not reduced in ALS compared to control cases, which suggests an increased metabolic activity in remaining motor neurons. Motor neurons dying in ALS might go through apoptosis (programmed cell death), so immunohistochemical and TUNEL techniques were applied on thoracic spinal cord from ALS patients to evaluate the possibility of an apoptotic process. The increased Bax expression indicates an apoptotic process and further, motor neurons were TUNEL-positive, indicating DNA degradation caused by programmed cell death. Muscle biopsies were obtained from ALS patients, and mRNA levels for the neurotrophic factors GDNF and BDNF were measured and compared to control subjects. GDNF levels were increased in muscle tissue in ALS whereas BDNF levels were unaltered. Levels of GDNF and BDNF were also measured in cerebrospinal fluid from ALS patients and controls using ELISA methodology. Levels of BDNF were unaltered in ALS cornpared to controls. GDNF however was not detectable in controls whereas 12 out of 15 ALS patients had measurab1e levels of GDNW. A marked upregulation of endogenous GDNF and GDNF mRNA in ALS CSF and muscle respectively is of special interest in relation to clinical trials where GDNF is administered to this group of patients.

The detection and analysis of biomolecules, such as proteins, are of great interest since these molecules are fundamental for life and our health. Due to the complexity of biological processes, there is a great advantage of studying proteins in their natural context, for example by using bioimaging. The objective of this doctoral thesis has been to develop, implement and evaluate techniques for the use of proteinspecific affinity reagents in diverse bioimaging platforms for analysis of protein expression in situ in cells and tissues. To be able to visualize a desired protein in situ using affinity reagents, reporter labels are needed. A novel technique for labeling of antibodies on solid phase was developed. This method offers simultaneous purification, concentration and labeling of an antibody sample, giving highly predictable and reproducible results, in a miniaturized format. Another study demonstrates the use of an alternative affinity reagent, the Affibody molecule, in bioimaging as well as other immunoassays. As a relevant proof-of-principle, an Affibody molecule binding the HER2 receptor was site-specificly labeled and employed for analysis of HER2 protein expression in cells and tissue using immunofluorescence (IF), immunohistochemistry (IHC), immunoprecipitation and flow cytometry. Furthermore, it is shown how antibody-based bioimaging approaches can be applied for systematic analysis of protein expression in terms of subcellular localization and expression levels in cell lines. The systematic subcellular localization of nearly 500 proteins was performed using IF and confocal microscopy. Global analysis of expression levels of nearly 2000 proteins in a panel of cell lines using IHC and automated image analysis, revealed that most proteins are expressed in a cell size dependent manner. Two normalization approaches were evaluated and found to allow for protein profiling across the panel of morphologically diverse cells, revealing patterns of protein over- and underexpression, and proteins with stable as well as with lineage specific expression were identified. Finally, the value of antibody-based, bioimaging proteomics as a platform for biomarker discovery is demonstrated. The identification and in depth study of a candidate biomarker for colorectal cancer, SATB2, is described using both IHC and IF bioimaging. Results from extended analyses of tumor biopsies showed that detection of SATB2 protein using IHC provides a clinically relevant diagnostic tool with high specificity and sensitivity to aid in diagnosis of colorectal cancer. Furthermore, the study demonstrated a potential prognostic role of SATB2, as decreased expression was associated with a significantly shorter overall survival in patients with advanced colorectal cancer. / QC 20100824

Chronic hyperlipidemia (lipotoxicity) and hyperglycemia (glucotoxicity) have recently been shown to induce Endoplasmic Reticulum (ER) stress, which may contribute to pancreatic beta-cell dysfunction in type 2 diabetes. This thesis examined the involvement of ER stress in beta-cell lipotoxicity and glucotoxicity. Although chronic treatment with saturated free fatty acids (FFA) in vitro induced ER stress, altering ER stress by increasing or knocking-down GRP78 chaperone expression had no effect on apoptosis induction. Conversely, overexpression of ER chaperones rescued the reduction in proinsulin protein levels caused by chronic exposure to high glucose, although it had no effect on the decreased insulin mRNA levels and proinsulin translation rate. Thus, ER stress is likely not the main mechanism involved in saturated FFA-induced beta-cell apoptosis in vitro, but it may contribute to glucotoxic effects on proinsulin levels. These findings have increased our understanding of the link between ER stress and beta-cell dysfunction in type 2 diabetes.

Chronic hyperlipidemia (lipotoxicity) and hyperglycemia (glucotoxicity) have recently been shown to induce Endoplasmic Reticulum (ER) stress, which may contribute to pancreatic beta-cell dysfunction in type 2 diabetes. This thesis examined the involvement of ER stress in beta-cell lipotoxicity and glucotoxicity. Although chronic treatment with saturated free fatty acids (FFA) in vitro induced ER stress, altering ER stress by increasing or knocking-down GRP78 chaperone expression had no effect on apoptosis induction. Conversely, overexpression of ER chaperones rescued the reduction in proinsulin protein levels caused by chronic exposure to high glucose, although it had no effect on the decreased insulin mRNA levels and proinsulin translation rate. Thus, ER stress is likely not the main mechanism involved in saturated FFA-induced beta-cell apoptosis in vitro, but it may contribute to glucotoxic effects on proinsulin levels. These findings have increased our understanding of the link between ER stress and beta-cell dysfunction in type 2 diabetes.

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