Neste trabalho foi transfectada uma linhagem de células embrionárias de rim humano (HEK293) com os genes das subunidades α e β da tireotrofina humana (hTSH), hormônio glicoproteico secretado pela hipófise. Após 5 dias de cultivo obteve-se uma concentração de hTSH no meio condicionado de 0,95μg/mL. O material foi concentrado e purificado utilizando uma estratégia envolvendo duas etapas, uma cromatografia de troca catiônica e uma cromatografia líquida de alta eficiência (HPLC) de fase reversa, que permitiu uma recuperação de 55% e uma pureza >90%. O produto purificado (hTSH-HEK) foi analisado e comparado a uma preparação comercial obtida em células CHO (hTSH-CHO) e a uma preparação hipofisária (hTSH-Pit). A identidade e a pureza do hTSH-HEK foram avaliadas por métodos físicoquímicos e imunológico (espectrometria de massa MALDI-TOF, HPLC de exclusão molecular e de fase reversa, SDS-PAGE e ensaio imunoradiométrico). A porção glicídica do hTSH-HEK foi avaliada pela análise do perfil dos N-glicanos e o comportamento biológico deste hormônio foi avaliado por bioensaio in vivo e estudo farmacocinético. As 3 preparações apresentaram pureza equivalente (97%) e a massa molecular relativa do hTSH-HEK foi 2,1% menor do que a do hTSH-CHO e 2,7% maior do que a do hTSH-Pit. A maior hidrofobicidade relativa, avaliada por RP-HPLC, foi a do hTSH-HEK. Os N-glicanos identificados no hTSH-HEK foram do tipo complexo, apresentando predominantemente estruturas tri-antenárias, enquanto no hTSH-CHO e no hTSH-Pit as estruturas bi-antenárias foram predominantes. Foram detectadas diferenças significativas relacionadas à composição dos carboidratos para estas preparações, um teor muito menor de ácido siálico e muito maior de fucose foram observados no hTSHHEK. Foi confirmada a atividade biológica das 3 preparações, sendo a bioatividade do hTSHHEK 39% e 16% inferior à do hTSH-CHO e hTSH-Pit, respectivamente. A meia-vida circulatória do hTSH-HEK foi menor (1,5 X) que a do hTSH-CHO e a do hTSH-Pit (1,2 X). De acordo com esses resultados o hTSH-HEK pode ser considerado uma alternativa viável para aplicações clínicas especialmente por sua origem humana e composição de carboidratos. / In this work a strain of embryonic human kidney cells (HEK293) was transfected with the genes of the α and β subunits of human thyrotropin (hTSH), a glycoproteic hormone secreted by the pituitary gland. After 5 days of culture, the concentration of hTSH in conditioned medium was 0.95μg/mL. The material was concentrated and purified utilizing a strategy involving two steps, a cation-exchange chromatography and a reversed phase high performance liquid chromatography (RP-HPLC), providing an overall yield of 55% and a purity level > 90%. The purified material (hTSH-HEK) was analyzed and compared to a recombinant commercial preparation obtained from CHO cells (hTSH-CHO) and to a human pituitary preparation (hTSH-Pit). Identity and purity of hTSH-HEK were evaluated through physicochemical and immunological methods (MALDI-TOF mass spectrometry, size exclusion HPLC, reversed-phase HPLC, SDS-PAGE, immunoradiometric assay). Glycidic portion of hTSHHEK was evaluated by N-glycoprofiling analysis and the biological behavior of this hormone was evaluated by an in vivo bioassay and via a pharmacokinetic study. The 3 preparations showed equivalent purity (97%) and hTSH-HEK molecular mass was 2.1% lower than hTSHCHO mass and 2.7% higher than hTSH-Pit mass. The highest relative hydrophobicity, evaluated by RP-HPLC, was shown by hTSH-HEK. Remarkable differences related to the carbohydrate moiety were found for these preparations, a much lower sialic acid content and a higher fucose content being observed in hTSH-HEK. Biological activity was confirmed for the three preparations, the hTSH-HEK bioactivity being 39% and 16% lower than hTSH-CHO and hTSH-Pit, respectively. The hTSH-HEK circulatory half-life (t1/2) was lower than that of hTSHCHO (1.5-fold) and hTSH-Pit (1.2-fold). According to these findings, HEK293-derived hTSH can be considered useful for clinical applications, also in view of its human origin and particular N-glycan composition.

Neste trabalho foi transfectada uma linhagem de células embrionárias de rim humano (HEK293) com os genes das subunidades α e β da tireotrofina humana (hTSH), hormônio glicoproteico secretado pela hipófise. Após 5 dias de cultivo obteve-se uma concentração de hTSH no meio condicionado de 0,95μg/mL. O material foi concentrado e purificado utilizando uma estratégia envolvendo duas etapas, uma cromatografia de troca catiônica e uma cromatografia líquida de alta eficiência (HPLC) de fase reversa, que permitiu uma recuperação de 55% e uma pureza >90%. O produto purificado (hTSH-HEK) foi analisado e comparado a uma preparação comercial obtida em células CHO (hTSH-CHO) e a uma preparação hipofisária (hTSH-Pit). A identidade e a pureza do hTSH-HEK foram avaliadas por métodos físicoquímicos e imunológico (espectrometria de massa MALDI-TOF, HPLC de exclusão molecular e de fase reversa, SDS-PAGE e ensaio imunoradiométrico). A porção glicídica do hTSH-HEK foi avaliada pela análise do perfil dos N-glicanos e o comportamento biológico deste hormônio foi avaliado por bioensaio in vivo e estudo farmacocinético. As 3 preparações apresentaram pureza equivalente (97%) e a massa molecular relativa do hTSH-HEK foi 2,1% menor do que a do hTSH-CHO e 2,7% maior do que a do hTSH-Pit. A maior hidrofobicidade relativa, avaliada por RP-HPLC, foi a do hTSH-HEK. Os N-glicanos identificados no hTSH-HEK foram do tipo complexo, apresentando predominantemente estruturas tri-antenárias, enquanto no hTSH-CHO e no hTSH-Pit as estruturas bi-antenárias foram predominantes. Foram detectadas diferenças significativas relacionadas à composição dos carboidratos para estas preparações, um teor muito menor de ácido siálico e muito maior de fucose foram observados no hTSHHEK. Foi confirmada a atividade biológica das 3 preparações, sendo a bioatividade do hTSHHEK 39% e 16% inferior à do hTSH-CHO e hTSH-Pit, respectivamente. A meia-vida circulatória do hTSH-HEK foi menor (1,5 X) que a do hTSH-CHO e a do hTSH-Pit (1,2 X). De acordo com esses resultados o hTSH-HEK pode ser considerado uma alternativa viável para aplicações clínicas especialmente por sua origem humana e composição de carboidratos. / In this work a strain of embryonic human kidney cells (HEK293) was transfected with the genes of the α and β subunits of human thyrotropin (hTSH), a glycoproteic hormone secreted by the pituitary gland. After 5 days of culture, the concentration of hTSH in conditioned medium was 0.95μg/mL. The material was concentrated and purified utilizing a strategy involving two steps, a cation-exchange chromatography and a reversed phase high performance liquid chromatography (RP-HPLC), providing an overall yield of 55% and a purity level > 90%. The purified material (hTSH-HEK) was analyzed and compared to a recombinant commercial preparation obtained from CHO cells (hTSH-CHO) and to a human pituitary preparation (hTSH-Pit). Identity and purity of hTSH-HEK were evaluated through physicochemical and immunological methods (MALDI-TOF mass spectrometry, size exclusion HPLC, reversed-phase HPLC, SDS-PAGE, immunoradiometric assay). Glycidic portion of hTSHHEK was evaluated by N-glycoprofiling analysis and the biological behavior of this hormone was evaluated by an in vivo bioassay and via a pharmacokinetic study. The 3 preparations showed equivalent purity (97%) and hTSH-HEK molecular mass was 2.1% lower than hTSHCHO mass and 2.7% higher than hTSH-Pit mass. The highest relative hydrophobicity, evaluated by RP-HPLC, was shown by hTSH-HEK. Remarkable differences related to the carbohydrate moiety were found for these preparations, a much lower sialic acid content and a higher fucose content being observed in hTSH-HEK. Biological activity was confirmed for the three preparations, the hTSH-HEK bioactivity being 39% and 16% lower than hTSH-CHO and hTSH-Pit, respectively. The hTSH-HEK circulatory half-life (t1/2) was lower than that of hTSHCHO (1.5-fold) and hTSH-Pit (1.2-fold). According to these findings, HEK293-derived hTSH can be considered useful for clinical applications, also in view of its human origin and particular N-glycan composition.

ATP-sensitive K+ channels (KATP) channels were first described in the cardiac muscles. KATP channels are a complex of regulatory sulphonylurea receptor subunits and pore-forming inward rectifier subunits such as Kir6.1. Nicotine, an exogenous substance, adversely affects cardiovascular function in humans. Acetylcholine (ACh) is well known as a key neurotransmitter of the parasympathetic nervous system. ACh effects are usually related to binding to muscarinic receptors and stimulating second messengers that relay and direct the extracellular signals to different intracellular destinations, resulting in modulated cellular activity. We hypothesize that nicotine and ACh may modulate Kir6.1 channels via different mechanisms. Using the whole cell patch-clamp technique, the interactions of nicotine and ACh with Kir6.1 subunit permanently expressed in Human Embryonic Kidney (HEK-293) cells as well as the underlying mechanisms were studied.<p> Non-transfected HEK-293 cells possess an endogenous K+ current with current density of 3.2 ± 1.4 pA/pF at 150 mV (n = 9). Stable expression of Kir6.1 subunits cloned from rat mesenteric artery in HEK-293 cells yielded a detectable inward rectifier KATP current (-23.9 ± 1.6 pA/pF at 150 mV, n = 6). In the presence of 0.3 mM ATP in the pipette solution, nicotine at 30 and 100 µM increased the expressed Kir6.1 currents by 42 ± 11.8 and 26.2 ± 14.6%, respectively (n = 4-6, p<0.05). In contrast, nicotine at 1-3 mM inhibited Kir6.1 currents (p<0.05). Nicotine at 100 µM increased the production of superoxide anion (O2.-) by 20.3 ± 5.7% whereas at 1 mM it significantly decreased the production of O2.- by 37.7 ± 4.3%. The hypoxanthine/xanthine oxidase (HX/XO) reaction was used as a source of O2.-. Co-application of HX and XO to the transfected HEK-293 cells resulted in a significant and reproducible increase in Kir6.1 currents. Tempol, a scavenger of O2.-, abolished the stimulatory effect of HX/XO on Kir6.1 currents. Tempol also abolished the stimulatory effect of 30 mM nicotine on Kir6.1 currents (-28.3 ± 6.1 pA/pF vs. -31.2 ± 7.3 pA/pF at -150 mV, n = 6-9 for each group, p>0.05). <p> In the presence of 0.3 mM ATP in the pipette solution, ACh concentration-dependently increased the expressed Kir6.1 currents. At 1 µM, ACh increased Kir6.1 currents from -19 ± 2.5 to 31.7 ± 2.1 pA/pF (n = 8, p < 0.05). Pretreatment of the transfected HEK-293 cells with either 2 or 20 µM atropine, 100 nM a-bungarotoxin, 100 µM mecamylamine, 2 µM prazosin, 1 µM propranolol, or 10 µM dihydro-b-erythroidine hydrobromide did not alter the stimulatory effect of ACh on Kir6.1 currents (n = 4 - 5 for each group, p<0.05). When intracellular ATP was increased to 5 mM, ACh at 10 µM still exhibited its stimulatory effect (-16.4 ± 2.3 to 25.5 ± 3.8 pA/pF, n = 8, p<0.05). For the first time, the present study provides an insight for the interactions of nicotine and ACh with Kir6.1 subunits. Our data demonstrate that micromolar concentration of nicotine and ACh stimulated Kir6.1 channels. Nicotine at millimolar concentrations inhibited Kir6.1 channels. The dual effect of nicotine, not mediated by nAChR, are mediated partially by O2.- levels in the cells. The ACh excitatory effect is mediated neither by an AChR-dependent mechanism, nor by alteration in ATP metabolism. This study challenges the traditional explanations for the receptor-mediated effects of nicotine and ACh on ion channels and opens a new door to understand the effects of nicotine and ACh on KATP channels in many cellular systems.

Serine proteases are a family of enzymes with a wide array of functions across both eukaryotes and prokaryotes. Here we have attempted to produce the serine proteases rat mast cell protease 2 and mouse mast cell protease 5 in a culture of HEK 293 cells; and mouse mast cell protease 4, platypus granzyme B-like protease and platypus hypothetical protease in a baculovirus expression system. Following production we wanted to analyse these serine proteases using a phage display assay and a battery of chromogenic substrates.

ATP-sensitive K+ channels (KATP) channels were first described in the cardiac muscles. KATP channels are a complex of regulatory sulphonylurea receptor subunits and pore-forming inward rectifier subunits such as Kir6.1. Nicotine, an exogenous substance, adversely affects cardiovascular function in humans. Acetylcholine (ACh) is well known as a key neurotransmitter of the parasympathetic nervous system. ACh effects are usually related to binding to muscarinic receptors and stimulating second messengers that relay and direct the extracellular signals to different intracellular destinations, resulting in modulated cellular activity. We hypothesize that nicotine and ACh may modulate Kir6.1 channels via different mechanisms. Using the whole cell patch-clamp technique, the interactions of nicotine and ACh with Kir6.1 subunit permanently expressed in Human Embryonic Kidney (HEK-293) cells as well as the underlying mechanisms were studied.<p> Non-transfected HEK-293 cells possess an endogenous K+ current with current density of 3.2 ± 1.4 pA/pF at 150 mV (n = 9). Stable expression of Kir6.1 subunits cloned from rat mesenteric artery in HEK-293 cells yielded a detectable inward rectifier KATP current (-23.9 ± 1.6 pA/pF at 150 mV, n = 6). In the presence of 0.3 mM ATP in the pipette solution, nicotine at 30 and 100 µM increased the expressed Kir6.1 currents by 42 ± 11.8 and 26.2 ± 14.6%, respectively (n = 4-6, p<0.05). In contrast, nicotine at 1-3 mM inhibited Kir6.1 currents (p<0.05). Nicotine at 100 µM increased the production of superoxide anion (O2.-) by 20.3 ± 5.7% whereas at 1 mM it significantly decreased the production of O2.- by 37.7 ± 4.3%. The hypoxanthine/xanthine oxidase (HX/XO) reaction was used as a source of O2.-. Co-application of HX and XO to the transfected HEK-293 cells resulted in a significant and reproducible increase in Kir6.1 currents. Tempol, a scavenger of O2.-, abolished the stimulatory effect of HX/XO on Kir6.1 currents. Tempol also abolished the stimulatory effect of 30 mM nicotine on Kir6.1 currents (-28.3 ± 6.1 pA/pF vs. -31.2 ± 7.3 pA/pF at -150 mV, n = 6-9 for each group, p>0.05). <p> In the presence of 0.3 mM ATP in the pipette solution, ACh concentration-dependently increased the expressed Kir6.1 currents. At 1 µM, ACh increased Kir6.1 currents from -19 ± 2.5 to 31.7 ± 2.1 pA/pF (n = 8, p < 0.05). Pretreatment of the transfected HEK-293 cells with either 2 or 20 µM atropine, 100 nM a-bungarotoxin, 100 µM mecamylamine, 2 µM prazosin, 1 µM propranolol, or 10 µM dihydro-b-erythroidine hydrobromide did not alter the stimulatory effect of ACh on Kir6.1 currents (n = 4 - 5 for each group, p<0.05). When intracellular ATP was increased to 5 mM, ACh at 10 µM still exhibited its stimulatory effect (-16.4 ± 2.3 to 25.5 ± 3.8 pA/pF, n = 8, p<0.05). For the first time, the present study provides an insight for the interactions of nicotine and ACh with Kir6.1 subunits. Our data demonstrate that micromolar concentration of nicotine and ACh stimulated Kir6.1 channels. Nicotine at millimolar concentrations inhibited Kir6.1 channels. The dual effect of nicotine, not mediated by nAChR, are mediated partially by O2.- levels in the cells. The ACh excitatory effect is mediated neither by an AChR-dependent mechanism, nor by alteration in ATP metabolism. This study challenges the traditional explanations for the receptor-mediated effects of nicotine and ACh on ion channels and opens a new door to understand the effects of nicotine and ACh on KATP channels in many cellular systems.

Sabe-se há décadas que mutações nos genes ras estão presentes em cerca de 20% dos cânceres humanos, mas o desenvolvimento de terapias eficazes para o tratamento de câncer dependente dos oncogenes ras permanece um desafio científico importante. Nesse contexto, o nosso grupo publicou recentemente resultados interessantes mostrando que FGF2 exógeno ou PMA, contrariamente à expectativa geral, inibem a proliferação de células de camundongo malignas dependentes dos oncogenes H- ou K-Ras. Para dar continuidade a estes estudos o projeto desta tese foi planejado para investigar os mecanismos subjacentes a possíveis efeitos citotóxicos de FGF2 e PMA em células humanas transformadas por ras. Para esse fim, a linhagem humana imortalizada HEK 293 foi condicionalmente transformada pela expressão ectópica da construção quimérica de DNA ER:H-rasV12, que codifica a oncoproteína de fusão ER:H-RasV12, cuja atividade é induzível por 4-hidroxi-tamoxifen (4OHT). Essa abordagem nos permitiu verificar os efeitos de FGF2 e PMA em sublinhagens HEK/ER:HrasV12 fenotipicamente \"normais\" ou transformadas por níveis crescentes da oncoproteína H-RasV12. Os principais resultados mostraram que tanto FGF2 como PMA tem efeito dual promovendo ou inibindo a proliferação das células transformadas em função da concentração intracelular crescente de H-RasV12. Ensaios de crescimento de colônias em suspensão de agarose mostraram que: a) as células parentais HEK293 não desenvolveram colônias mesmo quando tratadas com FGF2 ou PMA, resultados que estão de acordo com seu fenótipo não tumoral; b) mas, as sublinhagens HEK/ER:HrasV12 deram origem a colônias mesmo quando tratadas com concentrações pequenas de 4OHT, que condicionaram níveis intracelulares baixos de ER:HRasV12; nestas condições experimentais, FGF2 foi um forte promotor do crescimento de colônias, condizente com sua reconhecida atividade promotora do crescimento de células tumorais em suspensão; ainda nestas condições, PMA não teve efeito significante sobre o crescimento de colônias; c) coerentemente, concentrações elevadas de 4-OHT levaram aos níveis intracelulares mais altos de ER:HRasV12 e, por conseguinte, a desenvolvimento máximo de colônias de células HEK/ER:HrasV12, no entanto, nestas condições, ambos FGF2 e PMA inibiram completamente o crescimento de colônias. Por outro lado, transformação de HEK293 com um vetor de expressão constitutiva de HrasV12 levou à seleção e isolamento das sublinhagens tumorais HEK/HrasV12, cujo fenótipo se caracterizou por: a) nenhum efeito de FGF2 sobre a sua proliferação e b) forte inibição de sua proliferação por PMA. A ação citotóxica de PMA exclusivamente observada em células HEK 293 transformadas por H-rasV12 se caracterizou por: a) total dependência de PKC, provavelmente mediada pela ativação proteolítica específica de PKC &#948;; b) envolvimento de níveis elevados e sustentados de ROS com disparo tardio de apoptose. / It is known for nearly 20 years that mutated ras oncogenes are found in 20% of human malignancies, however efficacious therapies are not yet available for Ras-driven cancer. Along of these lines, our group recently published provocative results showing, against common belief, that FGF2 and PMA inhibited proliferation of Ras-dependent malignant mouse cells. Aiming to gain insight into this intriguing phenomenon, the present thesis project was planned to investigate the possible cytotoxicity of FGF2 and PMA in human Ras-driven malignant cells. To this end an immortalized non-tumorigenic human cell line (HEK293) was stably transformed with the DNA construction ER:H-rasV12, which encodes the fusion protein ER:H-RasV12, whose activity requires activation by 4-hidroxitamoxifen (4-OHT). This approach allowed us to evaluate FGF2 and PMA effects on HEK/ER:HrasV12 sublines under switching from \"normal\" to transformed phenotypes upon 4-OHT induction. Our main results have shown that both FGF2 and PMA displayed dual effects promoting or inhibiting proliferation of HEK/ER:HrasV12 cells in function of ER:HRasV12 intracellular levels. Clonogenic assays in agarose suspension have shown: a) parental HEK293 line did not develop colonies under FGF2 and PMA treatment or not, in agreement with its non-tumorigenic nature; b) however, HEK/ER:HrasV12 sublines developed colonies even under low 4-OHT concentrations, which led to low ER:HRasV12 intracellular levels; under these conditions FGF2 strongly promoted colony growth and PMA had no effect; c) furthermore, in HEK/ER:HrasV12 sublines, elevated 4-OHT concentrations led to high ER:HRasV12 intracellular levels and maximal colony growth; but, under these experimental conditions both FGF2 and PMA abolished colony growth. On the other hand, HEK293 transformation with a vector that constitutively express HrasV12 yielded HEK/ER:HrasV12 sublines displaying the following phenotypic traits: a) non FGF2 effects on proliferation and b) severe proliferation inhibition by PMA. PMA toxicity, exclusively observed in HrasV12 -transformed HEK293 cells, was characterized by: a) total dependency on PKC, likely mediated by specific proteolytic activation of PKC&#948;; b) involvement of high and sustained ROS levels correlated with late apoptosis triggering.

Sabe-se há décadas que mutações nos genes ras estão presentes em cerca de 20% dos cânceres humanos, mas o desenvolvimento de terapias eficazes para o tratamento de câncer dependente dos oncogenes ras permanece um desafio científico importante. Nesse contexto, o nosso grupo publicou recentemente resultados interessantes mostrando que FGF2 exógeno ou PMA, contrariamente à expectativa geral, inibem a proliferação de células de camundongo malignas dependentes dos oncogenes H- ou K-Ras. Para dar continuidade a estes estudos o projeto desta tese foi planejado para investigar os mecanismos subjacentes a possíveis efeitos citotóxicos de FGF2 e PMA em células humanas transformadas por ras. Para esse fim, a linhagem humana imortalizada HEK 293 foi condicionalmente transformada pela expressão ectópica da construção quimérica de DNA ER:H-rasV12, que codifica a oncoproteína de fusão ER:H-RasV12, cuja atividade é induzível por 4-hidroxi-tamoxifen (4OHT). Essa abordagem nos permitiu verificar os efeitos de FGF2 e PMA em sublinhagens HEK/ER:HrasV12 fenotipicamente \"normais\" ou transformadas por níveis crescentes da oncoproteína H-RasV12. Os principais resultados mostraram que tanto FGF2 como PMA tem efeito dual promovendo ou inibindo a proliferação das células transformadas em função da concentração intracelular crescente de H-RasV12. Ensaios de crescimento de colônias em suspensão de agarose mostraram que: a) as células parentais HEK293 não desenvolveram colônias mesmo quando tratadas com FGF2 ou PMA, resultados que estão de acordo com seu fenótipo não tumoral; b) mas, as sublinhagens HEK/ER:HrasV12 deram origem a colônias mesmo quando tratadas com concentrações pequenas de 4OHT, que condicionaram níveis intracelulares baixos de ER:HRasV12; nestas condições experimentais, FGF2 foi um forte promotor do crescimento de colônias, condizente com sua reconhecida atividade promotora do crescimento de células tumorais em suspensão; ainda nestas condições, PMA não teve efeito significante sobre o crescimento de colônias; c) coerentemente, concentrações elevadas de 4-OHT levaram aos níveis intracelulares mais altos de ER:HRasV12 e, por conseguinte, a desenvolvimento máximo de colônias de células HEK/ER:HrasV12, no entanto, nestas condições, ambos FGF2 e PMA inibiram completamente o crescimento de colônias. Por outro lado, transformação de HEK293 com um vetor de expressão constitutiva de HrasV12 levou à seleção e isolamento das sublinhagens tumorais HEK/HrasV12, cujo fenótipo se caracterizou por: a) nenhum efeito de FGF2 sobre a sua proliferação e b) forte inibição de sua proliferação por PMA. A ação citotóxica de PMA exclusivamente observada em células HEK 293 transformadas por H-rasV12 se caracterizou por: a) total dependência de PKC, provavelmente mediada pela ativação proteolítica específica de PKC &#948;; b) envolvimento de níveis elevados e sustentados de ROS com disparo tardio de apoptose. / It is known for nearly 20 years that mutated ras oncogenes are found in 20% of human malignancies, however efficacious therapies are not yet available for Ras-driven cancer. Along of these lines, our group recently published provocative results showing, against common belief, that FGF2 and PMA inhibited proliferation of Ras-dependent malignant mouse cells. Aiming to gain insight into this intriguing phenomenon, the present thesis project was planned to investigate the possible cytotoxicity of FGF2 and PMA in human Ras-driven malignant cells. To this end an immortalized non-tumorigenic human cell line (HEK293) was stably transformed with the DNA construction ER:H-rasV12, which encodes the fusion protein ER:H-RasV12, whose activity requires activation by 4-hidroxitamoxifen (4-OHT). This approach allowed us to evaluate FGF2 and PMA effects on HEK/ER:HrasV12 sublines under switching from \"normal\" to transformed phenotypes upon 4-OHT induction. Our main results have shown that both FGF2 and PMA displayed dual effects promoting or inhibiting proliferation of HEK/ER:HrasV12 cells in function of ER:HRasV12 intracellular levels. Clonogenic assays in agarose suspension have shown: a) parental HEK293 line did not develop colonies under FGF2 and PMA treatment or not, in agreement with its non-tumorigenic nature; b) however, HEK/ER:HrasV12 sublines developed colonies even under low 4-OHT concentrations, which led to low ER:HRasV12 intracellular levels; under these conditions FGF2 strongly promoted colony growth and PMA had no effect; c) furthermore, in HEK/ER:HrasV12 sublines, elevated 4-OHT concentrations led to high ER:HRasV12 intracellular levels and maximal colony growth; but, under these experimental conditions both FGF2 and PMA abolished colony growth. On the other hand, HEK293 transformation with a vector that constitutively express HrasV12 yielded HEK/ER:HrasV12 sublines displaying the following phenotypic traits: a) non FGF2 effects on proliferation and b) severe proliferation inhibition by PMA. PMA toxicity, exclusively observed in HrasV12 -transformed HEK293 cells, was characterized by: a) total dependency on PKC, likely mediated by specific proteolytic activation of PKC&#948;; b) involvement of high and sustained ROS levels correlated with late apoptosis triggering.

Der neuronale Zelluntergang bei einer Vielzahl von Krankheiten des ZNS, wie z.B. Morbus Alzheimer (AD) und Temporallappenepilepsie (TLE), wird mit oxidativem Stress sowie Fehlfunktionen von Kaliumkanälen in Verbindung gebracht. In dieser Studie soll die selektive neuronale Sensitivität auf oxidativen Stress durch die Messung der oxidativen Modulation von Kaliumströmen untersucht werden. Dabei werden sternförmige Neuronen der zweiten Schicht des entorhinalen Kortex (EC) (bei AD bereits früh geschädigt) mit pyramidalen Neuronen der dritten Schicht des EC (früh geschädigt bei TLE) sowie hippocampalen pyramidalen Neuronen der CA1 Region (bei AD und TLE erst spät geschädigt) miteinander verglichen. Mittels patch-clamp Ganzzellmessung zeigt diese Studie die differentielle Hemmung spannungsabhängiger transienter (IA) und „delayed-rectifier“ K+-Ströme (IK(V)) durch Arachidonsäure (AA) und Wasserstoffperoxid (H2O2). Die intrazelluläre Applikation von AA (1 pM) reduzierte IA in Neuronen des entorhinalen Kortex signifikant stärker verglichen mit Neuronen des CA1. ETYA imitiert diesen Effekt, dies schliesst die Metabolite der AA als Mediatoren des Effekts auf Kaliumkanäle aus. Weder AA noch ETYA reduzierten IK(V). Im Gegensatz dazu reduzierte H2O2 IA in Neuronen des CA1 effektiver als in Neuronen der Schichten II und III des entorhinalen Kortex. Die Reduktion des IA, vermittelt durch AA, wurde durch Radikalfänger (Glutathion, Ascorbinsäure, Vitamin E Analogon Trolox) blockiert. Dabei verstärkten manche dieser Antioxidantien den Effekt der AA, dies legt eine komplexere Modulation dieser Ströme in Schnitten verglichen mit Kulturen nahe. Dies sollte bei der Entwicklung antioxidativer Therapien von AD und TLE berücksichtigt werden. Bei der heterologer Expression von Kv1.4 und Kv4.2 in HEK-293 Zellen wurden funktionelle Kanäle gebildet und A-Typ Ströme ausgelöst. Diese Ströme wurden nach der Applikation von 1 pM AA stark reduziert. ROS scheinen neben ihrer zellschädigenden Wirkung physiologische Prozesse zu regulieren, indem sie eine Reihe von Signalwegen beeinflussen. Da spannungsabhängige Kaliumkanäle vielen wichtigen zellulären Funktionen zugrundeliegen, könnte die Modulation dieser Kanäle durch ROS einen Mechanismus für die Feinabstimmung zellulärer Prozesse darstellen. / Oxidative stress and dysfunction of potassium channels are believed to play a role in neuronal death in a number of CNS diseases (e.g. Alzheimer’s disease, epilepsy). The present study addresses selective neuronal vulnerability to oxidative stress by studying oxidative modulation of potassium channels in entorhinal cortex (EC) layer II stellate neurons (cell loss early in AD) and layer III pyramidal neurons (early damage in TLE), in comparison to hippocampal CA1 pyramidal neurons (late damage in TLE and AD). Using whole-cell patch-clamp, differential inhibition of transient IA and delayed rectifier K+-currents IK(V) by arachidonic acid (AA) and H2O2 was demonstrated. Intracellular AA (1 pM) reduced IA in EC neurons significantly stronger than in CA1 neurons. AA affected the voltage dependence of steady-state inactivation as well. ETYA mimicked the effect of AA, excluding its metabolites as mediators of IA modulation. Neither AA nor ETYA reduced IK(V). In contrast, a non-lipid oxidizing agent, H2O2 reduced IA more effectively and robustly attenuated IK(V) in CA1, compared to EC neurons. AA-mediated reduction of IA was blocked by free radical scavengers (glutathione, ascorbic acid, Trolox). Antioxidants did not simply inhibit AA and H2O2 effects. In particular, they even enhanced AA effects, suggesting more complex modulation of these currents in slices, compared to culture. Moreover, intracellular antioxidants, themselves, influenced maximal conductance and voltage-conductance characteristics of IA and IK(V). This should be considered in design of anti-oxidative therapies in AD and TLE. Heterologous expression of Kv1.4 and of Kv4.2 cDNA in HEK-293 cells formed functional channels and elicited A-type currents, which shared similar biophysical characteristics with native IA from the hippocampus. These currents were strongly decreased upon administration of 1pM AA, demonstrating that at least one of multiple sites for AA action is situated on the pore-forming alfa-subunit of the A-channel. In conclusion, beside contribution to cell damage, ROS may regulate physiological processes by acting on different signalling pathways. Since voltage-gated K+-channels underlie many important cellular functions modulation of these channels by ROS would represent a mechanism for fine tuning of cellular processes.

Recombinant proteins belong to considerable biofarmaceutics products used in biomedical research and in the treatment of human disease. Recombinant protines can be produced by stable transfection in big amount or by faster transient transfection with smaller amounts. To provide regular biological activity, it is necessary for the protein to be properly folded and post-translationally modified. As these modifications can be accurately performed only in mammalian cells, they have become the major host for complex r-protein expression. In this thesis is described transient transfection HEK 293 EBNA1 cells with linear polyethylenimines. These cells has been adapted to suspension cultivation in serum free medium. The cells were transfected with pcDNA3.1, pCI, pEBSV1, pCEP4, pEAK8 a pcDNA5/FRT/TO plasmids, everyone contained repoter gene SEAP. Concentration of SEAP in cell culture supernatants were determined in order to compare efficiencies of individual transfections. DNA:PEI ratio was another factor which was optimised and two different PEIs were compared. Highest achieved expresion was 50 mg per litre with transfection in 24 well plate when DNA:PEI ratio was 1:5. Comparison of six different plasmids give the bigest expresion pCEP4/SEAP, in well plate as well as in scaled up system.

Protein solubility plays an important role whether for biophysical and structural studies, or for production and delivery of therapeutic proteins. Poor solubility could lead to protein aggregation, which is an undesired physicochemical mechanism at any stage of recombinant proteins production. To date, more than half of all recombinant therapeutic proteins are produced in mammalian cells, mainly due to the high similarity of the final product to human protein structures. However, poor secretion can occur, due to misfolded proteins or aggregates leading to cellular stress and proteolysis. Another widely-used expression system is E. coli, which can offer a cost-efficient alternative. This system has an important limitation, since proteins tends to form insoluble protein aggregates in the cytoplasm upon heterologous overexpression. Several strategies are being implemented to improved soluble expression, ranging from culture conditions to solubility enhancing tags. However, there is no universal approach or technology that solves protein aggregation. In this thesis two recently published hypotheses from our group have been applied. One stated that soluble expression of proteins was inversely correlated with the size of the largest positively-charged patch on the protein surface. The second hypothesis (of protein solubility), arose from the finding that the relative content of lysine and arginine residues separated E. coli proteins by solubility. Both hypotheses arose from a study of an extensive dataset of experimental solubilities determined for cell-free expression of E. coli proteins. In combination with other widely used strategies, such as lowering expression temperature and inducer concentration, decreasing non-charged (hydrophobic) patches and addition of helical capping for increasing stability, a rational understanding for directed alteration of solubility in a variety of recombinant proteins has been explored. This includes three protein models to test: (i) recombinant human erythropoietin (rHuEPO) (one of the top selling therapeutics) (ii) recombinant 6-Phosphofructo-2-Kinase/fructose-2,6-bisphosphatase (rPFKFB3) (a product for which over-expression has been sought for characterisation and insight into possible cancer therapy) and (iii) a set of three selected E. coli proteins containing high ratios of lysines to arginines: thioredoxin-1 (TRX), cold shock-like protein cspB (cspB), and the histidine-containing phosphocarrier protein (HPr). It was found that single or multiple point mutations (changing amino acids from positive to negative charge or vice versa; or lysines to arginines) verified the predicted effect on rHuEPO, rPFKFB3, TRX, cspB, and HPr solubility (experimentally defined as the distribution between soluble and total fractions) for expression in E. coli. In addition, the redesigned set of rHuEPO transiently expressed in HEK 293-EBNA cells, suggesting that positively-charged patch size may also influence protein secretion. Further application of these computational and experimental approaches could provide a valuable tool in the design and engineering of proteins, with enhanced solubility, stability and secretion.