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The long-term goal of the Laboratory of Biology of Cytoskeleton is to explain in molecular terms the role of microtubules in cellular processes and to characterize proteins associated with microtubules and its organizing centers (MTOCs). The main attention of our research is focused on -tubulin and its role in microtubule nucleation. We have shown previously that besides its localization on centrosomes, -tubulin is found on celullar membranes (Macůrek et al., 2008), plant kinetochores (Dryková et al., 2003) and marginal bend of embryonal chicken erytrocytes (Linhartová et al., 2002). g-Tubulin also interacts with protein tyrosine kinases, that can regulate microtubule nucleation (Sulimenko et al., 2006; Macůrek et al. 2008). There are growing evidence that centrosomal proteins can affect genetic stability of the cell. g-Tubulin is involved in the regulation of the cell cycle (Vardy et al., 2002) and can participate in activation of checkpoint mechanism controlling the integrity of DNA. Recently, it has been proved its interaction with nuclear protein ATR (Zhang et al., 2007) and Rad51 (Lesca et al., 2005). That's why g-tubulin has been connected with the process of cancerogenesis. Cancer cells also display the changes in expression of b-tubulin isotypes (Katsetos et al., 2003). Moreover, increased level of...

No description available.

Considerando os efeitos da radiação gama sobre proteínas e a capacidade do sistema imune de reconhecer macromoléculas modificadas, decidimos avaliar alguns aspectos imunológicos de camundongos B10.PL frente a ovalbumina e bothropstoxina–1 (BTHX-1), nas formas nativa e irradiada. Para avaliar prováveis modificações estruturais nas moléculas das proteínas após o processo de irradiação (radiação gama de 60Co), foi realizada a eletroforese em gel de poliacrilamida 15% para a ovalbumina e para a BTHX-1, nas formas nativa e irradiada. A ovalbumina também foi submetida à cromatografia de exclusão molecular, enquanto a BTHX-1 foi submetida à espectrometria de massa. Os resultados destes ensaios mostraram que a radiação gama foi capaz de promover alterações nas moléculas de ovalbumina e BTHX-1. A fim de avaliar a toxicidade da BTHX-1 irradiada em relação à nativa realizou-se um ensaio de citotoxicidade celular em células CHO. O resultado mostrou que a toxina na sua forma irradiada apresentou, aproximadamente, cinco vezes menos toxicidade do que a toxina na sua forma nativa. Com relação aos aspectos imunológicos, foram realizados ensaios de produção e identificação de anticorpos, nos quais, os animais foram imunizados com ovalbumina e BTHX-1, nas formas nativa ou irradiada. Observou-se que as proteínas nativas induziram, preferencialmente, uma resposta do tipo Th2, enquanto que as proteínas irradiadas induziram uma resposta do tipo Th1. Realizou-se um ensaio de proliferação celular para avaliar o comportamento de esplenócitos, retirados do baço de camundongos B10.PL, imunizados com ovalbumina e BTHX-1, nativas ou irradiadas, cultivados em presença de ambas as formas das proteínas. Em relação à ovalbumina, os resultados mostraram que tanto as células dos animais imunizados com a ovalbumina nativa como aquelas dos animais imunizados com a proteína na sua forma irradiada apresentaram crescimento semelhante. No caso da BTHX-1, os resultados mostraram que as células dos animais imunizados com a toxina irradiada foram capazes de reconhecer a forma nativa da toxina, pois apresentaram crescimento semelhante ao das células dos animais imunizados com a BTHX-1 nativa. / Considering the effects of gamma radiation on proteins and the capacity of the immune system to recognize modified macromolecules, we decided to evaluate some immunological aspects of B10.PL mice exposed to native or irradiated ovalbumin and bothropstoxin-1 (BthTx-1). In order to evaluate possible structural modifications of the molecules after being irradiated (60Co gamma rays), bothropstoxin-1 was analysed by electrophoresis, while ovalbumin was submitted to analytical size exclusion chromatography. The toxin was also analised by ESI-mass spectrometry. Our results indicate that radiation promoted modifications on both the molecules. Aiming to compare the toxicity of the native and irradiated forms of the toxin, an in vitro citotoxicity assay, using CHO cells, was performed. According to our results, the modified toxin was 5 folds less toxic than its native counterpart. Sera of animals immunized with the native and irradiated proteins were analyzed in order to evaluate levels of IgG, as well as to quantify specific isotypes. While the native proteins induced a predominant Th2 response, the irradiated molecules apparently promoted a switch towards a Th1 pattern. We also performed a cell proliferation assay with splenocites from mice immunized with either the native or the irradiated proteins, cultured in the presence of the antigens. Our results indicate that both the forms of the proteins induced a similar proliferative response. These data indicate a potential use of detoxified proteins as antigens for immunization.

Considerando os efeitos da radiação gama sobre proteínas e a capacidade do sistema imune de reconhecer macromoléculas modificadas, decidimos avaliar alguns aspectos imunológicos de camundongos B10.PL frente a ovalbumina e bothropstoxina–1 (BTHX-1), nas formas nativa e irradiada. Para avaliar prováveis modificações estruturais nas moléculas das proteínas após o processo de irradiação (radiação gama de 60Co), foi realizada a eletroforese em gel de poliacrilamida 15% para a ovalbumina e para a BTHX-1, nas formas nativa e irradiada. A ovalbumina também foi submetida à cromatografia de exclusão molecular, enquanto a BTHX-1 foi submetida à espectrometria de massa. Os resultados destes ensaios mostraram que a radiação gama foi capaz de promover alterações nas moléculas de ovalbumina e BTHX-1. A fim de avaliar a toxicidade da BTHX-1 irradiada em relação à nativa realizou-se um ensaio de citotoxicidade celular em células CHO. O resultado mostrou que a toxina na sua forma irradiada apresentou, aproximadamente, cinco vezes menos toxicidade do que a toxina na sua forma nativa. Com relação aos aspectos imunológicos, foram realizados ensaios de produção e identificação de anticorpos, nos quais, os animais foram imunizados com ovalbumina e BTHX-1, nas formas nativa ou irradiada. Observou-se que as proteínas nativas induziram, preferencialmente, uma resposta do tipo Th2, enquanto que as proteínas irradiadas induziram uma resposta do tipo Th1. Realizou-se um ensaio de proliferação celular para avaliar o comportamento de esplenócitos, retirados do baço de camundongos B10.PL, imunizados com ovalbumina e BTHX-1, nativas ou irradiadas, cultivados em presença de ambas as formas das proteínas. Em relação à ovalbumina, os resultados mostraram que tanto as células dos animais imunizados com a ovalbumina nativa como aquelas dos animais imunizados com a proteína na sua forma irradiada apresentaram crescimento semelhante. No caso da BTHX-1, os resultados mostraram que as células dos animais imunizados com a toxina irradiada foram capazes de reconhecer a forma nativa da toxina, pois apresentaram crescimento semelhante ao das células dos animais imunizados com a BTHX-1 nativa. / Considering the effects of gamma radiation on proteins and the capacity of the immune system to recognize modified macromolecules, we decided to evaluate some immunological aspects of B10.PL mice exposed to native or irradiated ovalbumin and bothropstoxin-1 (BthTx-1). In order to evaluate possible structural modifications of the molecules after being irradiated (60Co gamma rays), bothropstoxin-1 was analysed by electrophoresis, while ovalbumin was submitted to analytical size exclusion chromatography. The toxin was also analised by ESI-mass spectrometry. Our results indicate that radiation promoted modifications on both the molecules. Aiming to compare the toxicity of the native and irradiated forms of the toxin, an in vitro citotoxicity assay, using CHO cells, was performed. According to our results, the modified toxin was 5 folds less toxic than its native counterpart. Sera of animals immunized with the native and irradiated proteins were analyzed in order to evaluate levels of IgG, as well as to quantify specific isotypes. While the native proteins induced a predominant Th2 response, the irradiated molecules apparently promoted a switch towards a Th1 pattern. We also performed a cell proliferation assay with splenocites from mice immunized with either the native or the irradiated proteins, cultured in the presence of the antigens. Our results indicate that both the forms of the proteins induced a similar proliferative response. These data indicate a potential use of detoxified proteins as antigens for immunization.

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by joint inflammation eventually leading to the destruction of cartilage and bone. The aetiopathogenesis is not completely understood, but previous studies have shown that the disease is multifactorial with genetic, environmental and hormonal factors involved. Immune cells, e.g., T- and B-cells, and macrophages, migrate into the joints, with increased expression of numerous soluble factors such as cytokines, chemokines and adhesion molecules functionally active both locally and systemically. Analyses of blood samples from the Medical Biobank in Umeå from individuals before the onset of symptoms of joint disease showed that anti-citrullinated protein/peptide antibodies (ACPA) preceded the development of disease by years and this finding has been confirmed by other studies.                                         The aim of this thesis was to identify signs of activation of the immune system analysed as up-regulation of pro- and anti-inflammatory cytokines, sero-positivity for autoantibodies, and genetic factors identified as relevant for the development and disease progression of RA. The concentrations of 30 cytokines and chemokines were measured in blood samples from individuals before the onset of symptoms, and when diagnosed with RA, together with population-based matched controls using a multiplex system. The predictive value of different isotypes (IgG, IgA, and IgM) of ACPA and rheumatoid factor (RF) before onset of symptoms and different types of ACPA (e.g., mutated citrullinated vimentin, MCV) were analysed for disease development and progression in patients with early RA and controls from Northern Sweden. These factors were related to the genetic markers, HLA- shared epitope (SE) alleles and the 1858C/T polymorphism of the protein tyrosine phosphatase non-receptor type 22 (PTPN22) gene.                            In paper I, it was shown that in individuals who later developed RA (i.e., pre-patients) the levels of several cytokines and related factors that represent the adaptive immune system (Th1, Th2, and T regulatory cell related factors) were significantly elevated compared with controls, whereas, after the onset of disease the involvement of the immune system was more general and widespread. In paper II, the presence of different isotypes (IgM, IgA and IgG) of ACPA in pre-patients, patients and controls was evaluated showing that both the IgG and IgA isotype predicted the onset of RA by years with the IgG isotype having the highest predictive value. In paper III, the association of the 1858T variant of PTPN22 with RA was confirmed. Furthermore, the association was restricted to autoantibody positive disease and this variant was correlated with an earlier age for disease onset. In paper IV, anti-MCV antibodies were identified as being associated with a more severe disease course of RA, measured by disease activity score, erythrocyte sedimentation rate, and swollen joint count over time compared with anti-CCP2, anti-CCP3, and anti-CCP3.1 antibodies.                                                                                                In conclusion, individuals who later developed RA had increased concentrations of inflammatory markers reflecting an activation of the immune system years before the clinical symptoms of the disease developed. Also, the presence of ACPA of IgG and IgA isotype prior to disease onset predicted the development of RA. The PTPN22 1858T variant was associated with sero-positive RA and anti-MCV antibodies were associated with a higher inflammatory activity compared with anti-CCP2, -CCP3 and -CCP3.1 antibodies. These findings together present a possibility to better predict the development and progression of RA.

Abstract The characteristics of humoral immunity have been proposed to reflect the bias between two T helper (Th) lymphocyte subsets: Th1 cells, which activate cell-mediated immunity, and Th2 cells, which mediate humoral immunity. The present study aimed to characterize the humoral immunity to beta-cell autoantigens insulin and glutamic acid decarboxylase (GAD65) in preclinical and clinical type 1 diabetes. Insulin antibodies were analyzed in pregnant women with or without type 1 diabetes and their newborn infants and in prediabetic children. Epitope or/and isotype-specific GAD65 antibodies (GAD65Abs) were analyzed in prediabetic children, in children and adolescents diagnosed with type 1 diabetes, and in patients with the autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) syndrome. Antibodies were determined by radioligand immunoassays. The humoral immune response to insulin and GAD65 was observed to be a highly dynamic process, comprising mainly the IgG1 subclass and, less frequently, other IgG subclasses. GAD65Abs were directed primarily to the middle region and secondarily to the C-terminal region of GAD65 as a consequence of epitope spreading. Young children who progressed to overt type 1 diabetes were characterized by a broad initial isotype response to insulin and GAD65 and by a strong IgG1 and IgG3 response to insulin. Children who did not progress to clinical type 1 diabetes were characterized by an emerging IgG4 response to GAD65. Rising levels of GAD65Abs targeted to the middle region of GAD65 were associated with high titers of islet cell antibodies and a decreased requirement for exogenous insulin, probably reflecting a persistent residual beta-cell mass, in patients with manifest type 1 diabetes. Non-immunoglobulin insulin-binding activity was observed to be induced by pregnancy. APECED-associated humoral autoimmunity to GAD65 did not differ markedly from that observed in subjects with type 1 diabetes alone. In conclusion, isotype-specific GAD65 and especially insulin antibodies are valuable markers of the risk of progression to type 1 diabetes in young children. The appearance of an initial IgG3 subclass response and a strong IgG3 response to insulin in children who progressed to overt type 1 diabetes may reflect the role of cytotoxic Th1-biased immunity in the disease process leading to clinical presentation of type 1 diabetes.

The calcium-sensing receptor (CaR) is a G-protein coupled receptor that controls mammalian extracellular calcium (Ca2+o) homeostasis. CaR downstream signalling involves intracellular calcium (Ca2+i) mobilisation which can be negatively modulated by protein kinase C (PKC)-mediated phosphorylation of CaR residue Thr-888 (CaRT888). The nature of this regulation was investigated here using siRNA-based knockdown of individual PKC isotypes. Knocking down PKCα expression increased CaR-induced Ca2+i mobilisation in CaR-HEK cells, significantly lowering the EC50 for Ca2+o relative to control siRNA-transfected cells. In accordance, PKCα knockdown also decreased CaRT888 phosphorylation which also permitted the triggering of Ca2+i mobilisation in CaR-HEK cells at sub-threshold Ca2+o concentrations. Interestingly, PKCε knockdown attenuated CaR-induced Ca2+i mobilisation in CaR-HEK cells, significantly increasing the EC50 for Ca2+o. However, this knockdown was also also found to inhibit CaRT888 phosphorylation and this is the first time that CaRT888 phosphorylation has been shown to be dissociate from Ca2+i mobilisation. The results show the complexity of the interactions that potentially underlie the CaR’s pleiotropic signalling and provides novel targets for examining signal bias. Classically an increase in cAMP is known to trigger PTH seceretion. The observation in this study shows that raising intracellular cAMP levels with forskolin also decreased CaRT888 phosphorylation permitting increased Ca2+i mobilisation. This suggests that cAMP may stimulate the phosphatase (most likely protein phosphatase 2A (PP2A)). Nevertheless, knocking down Gα12, which has been shown to activate PP2A, resulted in increased CaRT888 phosphorylation and lower Ca2+i mobilisation (increased EC50 for Ca2+o). This suggests the possibility of CaR as a cAMP sensor that can detect an increase in intracellular cAMP in order to stop PTH serection. Three novel CaR effectors, P70 ribosamal protein S6 kinase, insulin-like growth factor receptor-1 and nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, were identified in CaR-HEK cells. It was shown that a) high Ca2+o stimulated the activation of these effectors and b) each effector was inhibited by knockdown of PKCα and Gα12, which further confirmed the association of these signals with CaR. These data show that CaR also plays an important role outside Ca2+o homeostasis, such as growth and inflammation. Finally, five CaR mutations associated with autosomal dominant hypocalcaemia (ADH) were found to increase Ca2+o-induced Ca2+i mobilisation, as well as ERK and p38MAPK activation, when transfected stably in HEK-293 cells. Cotreatment with the calcilytic NPSP795 inhibited ERK and p38MAPK phosphorylation in all 5 gain-of-function mutants and in the wild type CaR cells, with IC50s for the compound in the nanomolar range. These data highlight the potential utility of CaR negative allosteric modulators in the treatment of gain-of-function CaR mutations. Together these data enhance our understanding of CaRT888 phosphorylation and CaR signalling.

A importância em conhecer o padrão alimentar em mosquitos da Família Culicidae permite esclarecer alguns aspectos relacionados à transmissão de zoonoses e estimar o grau de contato humano-vetor que é fator relevante em estudos epidemiológicos. Com o objetivo de explorar o comportamento alimentar dessa Família, em área epizoótica de febre amarela silvestre, foram coletados exemplares nos municípios de Santo Antônio das Missões e Garruchos, Estado do Rio Grande do Sul. Fêmeas ingurgitadas foram obtidas por aspiração em ambiente de mata, no período de setembro de 2005 a abril de 2007 e identificadas segundo fonte de sangue ingerido através da técnica imunoenzimática ELISA de captura no sistema avidinabiotina. Foram testadas seis fontes de alimento: ave, bovino, eqüino, humano, macaco e rato. Os resultados obtidos mediante a padronização de anticorpos monoclonais possibilitaram demonstrar pela primeira vez o reconhecimento de sangue humano ingerido nesses mosquitos pelo emprego da subclasse IgG1 e comprovar a sensibilidade e especificidade da técnica ELISA de captura. No município de Santo Antônio das Missões, de um total de 190 amostras, 60,9% reagiram para sangue de boi, 23,6% para humano, 9,9% para ave, 1,9% para macaco e 3,7% para combinações de dois hospedeiros. Quanto às amostras referentes ao município de Garruchos, das 158 fêmeas capturadas na área Cachoeirinha pode-se observar reatividade para ave (16%), boi (29,6%), humano (36,8%), cavalo (4%), macaco (0,8%) e combinações de hospedeiros (12,8%), enquanto que para as 149 fêmeas pertencentes à área de São José, detectou-se sangue ingerido de boi em (51,5%), ave e humano (11,5%), macaco (6,2%), cavalo (0,8%) e mistos (18,5%). Aedes scapularis, Aedes crinifer, Culex (Culex) spp., Haemagogus leucocelaenus apresentaram maior número de fêmeas ingurgitadas nos dois municípios. Os resultados obtidos com Aedes scapularis sugerem ecletismo, conforme combinações detectadas em amostras de sangue de diferentes fontes. Haemagogus leucocelaenus apresentou a maior proporção de amostras contendo sangue humano em relação às demais fontes e essa característica traz implicações, por ser espécie incriminada na transmissão e por se tratar de área de ocorrência de epizootias de febre amarela. / The knowledge of mosquitoes Culicidae host feeding patterns permits to clarify some aspects related to zoonosis transmission and to estimate the degree of human-vector contact which is relevant in epidemiological studies. Aiming to explore the feeding behavior of these mosquitoes, specimens were collected in the municipalities of Santo Antônio das Missões and Garruchos, Rio Grande do Sul, an epizootic area of sylvatic yellow fever. Engorged females were collected by aspiration from forested areas from September 2005-April 2007 and their blood meals were identified using the avidin-biotin system of immunoenzymatic ELISA capture. Six blood meal sources were tested: bird, cattle, horse, human, monkey and rat. The result achieved with the species-specific IgG1 mAb was unprecedented for mosquito blood meal identification and reinforced the sensibility and specificity of the immunoenzymatic ELISA capture. Of the 190 samples from Santo Antônio das Missões, 60.9% reacted to cattle, 23.6% to human, 9.9% to bird, 1.9% to monkey and 3.7% to mixed blood meals. In Garruchos, of the 158 females collected in Cachoeirinha, 16.0% reacted to bird, 29.6% to cattle, 36.8% to human, 4.0% to horse, 0.8% to monkey and 12.8% to mixed blood, while of the 149 engorged females from São José, blood from cattle accounted for 51.5%, of blood identified, bird and human 11.5%, monkey 6.2%, horse 0.8% and mixed blood 18.5%. Blood engorged females of Aedes scapularis, Aedes crinifer, Culex (Culex) spp., Haemagogus leucocelaenus predominated in the two municipalities. The results obtained with Aedes scapularis suggests its eclecticism, according to the combinations of blood which were detected from different sources. Haemagogus leucocelaenus was found to have the highest proportion of samples containing human blood in comparison with other sources, which has implications, on account of being incriminated in the transmission and also for taking into consideration the outbreaks reported that underline the risk of yellow fever.

A importância em conhecer o padrão alimentar em mosquitos da Família Culicidae permite esclarecer alguns aspectos relacionados à transmissão de zoonoses e estimar o grau de contato humano-vetor que é fator relevante em estudos epidemiológicos. Com o objetivo de explorar o comportamento alimentar dessa Família, em área epizoótica de febre amarela silvestre, foram coletados exemplares nos municípios de Santo Antônio das Missões e Garruchos, Estado do Rio Grande do Sul. Fêmeas ingurgitadas foram obtidas por aspiração em ambiente de mata, no período de setembro de 2005 a abril de 2007 e identificadas segundo fonte de sangue ingerido através da técnica imunoenzimática ELISA de captura no sistema avidinabiotina. Foram testadas seis fontes de alimento: ave, bovino, eqüino, humano, macaco e rato. Os resultados obtidos mediante a padronização de anticorpos monoclonais possibilitaram demonstrar pela primeira vez o reconhecimento de sangue humano ingerido nesses mosquitos pelo emprego da subclasse IgG1 e comprovar a sensibilidade e especificidade da técnica ELISA de captura. No município de Santo Antônio das Missões, de um total de 190 amostras, 60,9% reagiram para sangue de boi, 23,6% para humano, 9,9% para ave, 1,9% para macaco e 3,7% para combinações de dois hospedeiros. Quanto às amostras referentes ao município de Garruchos, das 158 fêmeas capturadas na área Cachoeirinha pode-se observar reatividade para ave (16%), boi (29,6%), humano (36,8%), cavalo (4%), macaco (0,8%) e combinações de hospedeiros (12,8%), enquanto que para as 149 fêmeas pertencentes à área de São José, detectou-se sangue ingerido de boi em (51,5%), ave e humano (11,5%), macaco (6,2%), cavalo (0,8%) e mistos (18,5%). Aedes scapularis, Aedes crinifer, Culex (Culex) spp., Haemagogus leucocelaenus apresentaram maior número de fêmeas ingurgitadas nos dois municípios. Os resultados obtidos com Aedes scapularis sugerem ecletismo, conforme combinações detectadas em amostras de sangue de diferentes fontes. Haemagogus leucocelaenus apresentou a maior proporção de amostras contendo sangue humano em relação às demais fontes e essa característica traz implicações, por ser espécie incriminada na transmissão e por se tratar de área de ocorrência de epizootias de febre amarela. / The knowledge of mosquitoes Culicidae host feeding patterns permits to clarify some aspects related to zoonosis transmission and to estimate the degree of human-vector contact which is relevant in epidemiological studies. Aiming to explore the feeding behavior of these mosquitoes, specimens were collected in the municipalities of Santo Antônio das Missões and Garruchos, Rio Grande do Sul, an epizootic area of sylvatic yellow fever. Engorged females were collected by aspiration from forested areas from September 2005-April 2007 and their blood meals were identified using the avidin-biotin system of immunoenzymatic ELISA capture. Six blood meal sources were tested: bird, cattle, horse, human, monkey and rat. The result achieved with the species-specific IgG1 mAb was unprecedented for mosquito blood meal identification and reinforced the sensibility and specificity of the immunoenzymatic ELISA capture. Of the 190 samples from Santo Antônio das Missões, 60.9% reacted to cattle, 23.6% to human, 9.9% to bird, 1.9% to monkey and 3.7% to mixed blood meals. In Garruchos, of the 158 females collected in Cachoeirinha, 16.0% reacted to bird, 29.6% to cattle, 36.8% to human, 4.0% to horse, 0.8% to monkey and 12.8% to mixed blood, while of the 149 engorged females from São José, blood from cattle accounted for 51.5%, of blood identified, bird and human 11.5%, monkey 6.2%, horse 0.8% and mixed blood 18.5%. Blood engorged females of Aedes scapularis, Aedes crinifer, Culex (Culex) spp., Haemagogus leucocelaenus predominated in the two municipalities. The results obtained with Aedes scapularis suggests its eclecticism, according to the combinations of blood which were detected from different sources. Haemagogus leucocelaenus was found to have the highest proportion of samples containing human blood in comparison with other sources, which has implications, on account of being incriminated in the transmission and also for taking into consideration the outbreaks reported that underline the risk of yellow fever.