No description available.

Las proteínas quinasas BRSK1 y BRSK2 (Brain-specific kinase 1 y 2) pertenecen a la familia de las AMPK-related kinases, y son activadas mediante fosforilación por la proteína kinasa supresora de tumores LKB1. Las BRSKs se expresan mayoritariamente en cerebro, donde juegan un papel clave en el establecimiento de la polaridad neuronal y sinaptogénesis. En la neurona madura, BRSK1 modularía la función sináptica controlando la liberación de neurotransmisores en la neurona madura. Así pues, LKB1-BRSK1/2 constituye una nueva vía de señalización crucial para la función neuronal. Sin embargo, se desconoce cómo las BRSKs ejercen su función, dado que no se han descrito los mecanismos que regulan su actividad (LKB1 es una quinasa constitutivamente activa) ni los sustratos a través de los cuales median su función. En este trabajo se han obtenido y puesto a punto las herramientas bioquímicas para el estudio de las BRSKs (anticuerpos, constructos de DNA, preparaciones puras y activas, etc.), que se han utilizado para mostrar que tanto el desarrollo ontogénico del cerebro como la diferenciación neuronal transcurren con un aumento de expresión de BRSK1 y BRSK2. Mediante ensayos de gene-reporter luciferase e inmunoblots demostramos que las neurotrofinas NGF y BNDF promueven el aumento de la transcripción y expresión de las BRSKs, en un proceso mediado por la vía de señalización de Ras-Raf-MEK1-ERK1/2 y el factor de transcripción Sp1. Por otra parte, se ha caracterizado el patrón de localización subcelular de las BRSKs en neuronas corticales maduras: BRSK1 presenta un marcaje puntuado (similar al que del marcador de vesículas sinápticas sinaptofisina), y BRSK2 presenta una distribución homogénea a lo largo del soma, axón y dendritas, donde co-localiza con el marcador MAP2. Por primera vez se describe que las BRSKs se expresan en células gliales. Por primera vez se muestra en sinaptosomas de cerebro de rata la existencia de un pool de BRSK1, pero de BRSK2 o LKB1, en microdominios de membrana ricos en colesterol lipid rafts, y que esta localización resulta en una BRSK1 que presenta una mayor actividad kinasa que la proteína citosólica. Mediante ensayos de purificación de los lípidos de lipid rafts y reconstitución de vesículas multilamelares lipídicas, así como utilizando lípidos sintéticos, se muestra que la actividad kinasa BRSK1 (pero BRSK2) es regulada por el entorno lipídico mediante un mecanismo nuevo, diferente al de fosforilación por LKB1. Por último, se ha identificado un nuevo sustrato para BRSK1: la proteína t-SNARE sintaxina-1A, que regula la liberación de neurotransmisores a nivel presináptico. Mediante ensayos de fosforilación, generación de mutantes de deleción y análisis por espectrometría de masas se ha identificado un residuo de Serina como el residuo de sintaxina-1A fosforilado por BRSK1. Esta fosforilación presenta una estequiometría de 1 mol de fosfato incorporado por mol de sintaxina, y ha sido confirmada mediante ensayos radiométricos de fosforilación utilizando diferentes formas mutantes de las proteínas BRSK1 y sintaxina. Se ha generado y caracterizado un anticuerpo que reconoce específicamente el residuo de Ser fosforilado, lo que ha permitido demostrar que BRSK1 fosforila a esta Ser en sistemas celulares. / Brain-specific kinase 1 and 2 (BRSK1 and BRSK2) belong to the AMPK-related family protein kinases, and they are activated by direct phosphorylation by the LKB1 tumor suppressor protein. BRSK proteins are expressed within the brain, where they play a crucial role for establishing neuronal polarity and synaptogenesis. BRSK1 also modulate synaptic function and neurotransmitter release in the mature neuron. Therefore, LKB1-BRSKs is a new signaling pathway that plays a pivotal role in neuronal function. However, how BRSKs exert their functions is unknown, since their substrates and the mechanisms modulating their activity remain to be described. In this study we have obtained new molecular and biochemical tools for the study of the BRSKs (antibodies, DNA constructs, etc), that allow us to show that neuronal differentiation and brain maturation processes take place with an increase of expression of BRSK1 and BRSK2 proteins. Using gene-reporter and immunoblot assays, we show that NGF and BDNF neurotrophins induce an increase on BRSKs transcription and translation, by a process which involves Ras-Raf-MEK1-ERK1/2 signaling pathway and the Sp1 transcription factor. We also study BRSKs subcellular localization from rat cultured cortical neurons by immunocytochemical assays. BRSK1 shows a punctuate pattern, similar to that observed for the synaptic vesicle protein marker synaptophysin, whereas BRSK2 mainly localize throughout the neuron in both axon and dendrites co-localizes with dendritic marker MAP2. We also report by the first time that glial cells express both, BRSK1 and BRSK2 proteins. We provide biochemical and pharmacological evidences demonstrating that a pool of BRSK1, but not BRSK2 or LKB1, localizes at membrane lipid rafts. We also show that raft-associated BRSK1 has higher activity than BRSK1 from non-raft environment. Further, recombinant BRSK1 activity increased 3-fold when assayed with small multilamellar vesicles (SMV) generated with both, lipids extracted from synaptosomal raft fractions or synthetic SMV made with authentic phosphatidylcholine, cholesterol and sphingomyelin. Interaction with lipid rafts represents a new mechanism of BRSK1 activity modulation, additional to T-loop phosphorylation. Finally, we have identified a new BRSK1 substrate, namely syntaxin-1A. Using phosphorylation and mass-spec analysis we have mapped and identified a residue of Ser that is mainly phosphorylated by BRSK1, showing a stechiometry of 1 mol P per mol of protein. We have generated an antibody that specifically recognizes this Ser residue and provided evidences showing that this phosphorylation occurs in vivo.

No description available.

Sarcocystis neurona is an apicomplexan parasite that is a major cause of equine protozoal myeloencephalitis (EPM). During intracellular development of S. neurona, many genes are temporally regulated. To better understand gene regulation, it is important to identify and characterize regulatory elements controlling gene expression in S. neurona. To perform this study, it was essential to establish transfection system for this parasite. Hence, the 5 flanking region of the SnSAG1 gene was isolated from a genomic library and used to construct expression plasmids. In transient assays, the reporter molecules -galactosidase (-gal) and yellow fluorescent protein (YFP) were expressed by electroporated S. neurona, thereby confirming the feasibility of performing molecular genetic experiments in this organism. Stable transformation of S. neurona was achieved using a mutant dihydrofolate reductase thymidylate synthase (DHFR-TS) gene of T. gondii that confers resistance to pyrimethamine. This selection system was used to create transgenic S. neurona that stably express -gal and YFP. These transgenic clones were shown to be useful for analyzing growth rate of parasites in-vitro and for assessing drug sensitivities. To uncover possible sequence elements involved in promoter activity, the 5 flanking regions of five S. neurona genes were subjected to comparative analysis. This revealed the presence of a 7-base conserved motif GCGTCTC. Using a dual luciferase assay system, the SnSAG1 promoter was subjected to functional analysis. The motif GAGACGC located between -136 and -129 upstream of the transcription start site was found to be essential for SnSAG1 expression. This motif functions in an orientation dependent manner and was shown to play a role in binding nuclear proteins of S. neurona.

Encephalitozoon cuniculi, Toxoplasma gondii and Sarcocystis neurona are intracellular parasites that infect a wide range of mammalian host species including domestic cats. The prevalence of antibodies to these parasites in cats was examined using an indirect immunofluorescence antibody assay. E. cuniculi targets the kidneys of rabbits but the prevalence of disease in cats is unknown. Chronic kidney disease (CKD) is a common cause of illness in cats. T. gondii is a widespread parasite of cats; however, it is not considered a major causative agent of CKD. The first hypothesis was that E. cuniculi and T. gondii are unrecognized causes of chronic kidney disease in domestic cats. Serum and plasma samples were examined for protozoal antibodies from 232 feline patients at the VMRCVM Teaching Hospital. Thirty-six of the 232 samples met the IRIS criteria for CKD. Antibodies to E. cuniculi were found in 15 samples, 4 of which came from cats with CKD. Antibodies to T. gondii were found in 63 samples; 10 cats of the 63 had CKD. These were not significantly different from cats with no CKD and the null hypothesis was rejected. Domestic cats, armadillos, raccoons and skunks are intermediate hosts (IH) for S. neurona while opossums are the definitive host (DH). The seroprevalence of S. neurona was examined in domestic cats from Virginia and Pennsylvania. The second hypothesis was that domestic cats are important IH for S. neurona transmission. A low seroprevalence was found in 32 of the 441 cats and the null hypothesis was rejected. / Master of Science in Life Sciences

Em um trabalho anterior, foi avaliada a variabilidade de Sarcocystis spp. isolados de gambás oriundos do estado do Rio Grande do Sul pesquisando sequências gênicas codificadoras de antígenos de superfície (SAGs). Os resultados indicaram haver linhagens de isolados de Sarcocystis (relacionadas geneticamente a S. falcatula) que trocam genes em prováveis processos de recombinação sexual. A proposta deste estudo foi de conhecer as variações gênicas e relações filogenéticas em Sarcocystis spp. isolados de gambás através da análise de loci gênicos de genoma mitocondrial (CytB), de genoma de apicoplasto (ClpC) e das regiões espaçadoras transcritas internas (ITS-1), comparando a diversidade encontrada nestes grupos com a observada em integrantes da sub-familia Toxoplasmatinae. E também de conhecer a diversidade de genes codificadores de SAG-2, SAG-3 e SAG-4. Neste estudo foram obtidos esporocistos de Sarcocystis spp. através de raspado intestinal e fezes provenientes de gambás do gênero Didelphis, oriundos do Estado de São Paulo, Rio Grande do Sul e Rio Grande do Norte. Os marcadores moleculares CytB, ClpC e ITS-1 revelaram uma ampla variabilidade genotípica e alelos inéditos entre os isolados do Brasil. Os resultados levam a crer que é possível a existência de espécies “híbridas” de Sarcocystis no Brasil, com mistura de genes de ambas as espécies. Em relação aos genes codificadores de SAGs, foi encontrada uma diversidade ainda maior do que trabalho similar realizado. Os resultados sugerem uma possível ocorrência de reassortment e recombinação de alelos em sequências SAGs, contribuindo ainda mais para a geração de variabilidade e consequentemente na configuração antigênica do parasito. Estes resultados demonstram que isolados brasileiros possuem uma composição genética diferente do reportado em demais localidade, o que pode ter reflexos importantes no conhecimento da diversidade das espécies de Sarcocystis que infectam gambás em nosso meio e em toda a epidemiologia das infecções produzidas pelos protozoários deste grupo / A great variability in surface antigens genes (SAGs) of Sarcocystis spp. sporocysts was found in a previous study, through the intestinal scraping technique of opossums from the south region of Brazil. The results indicated probable sexual recombination between lineages of Sarcocystis isolates (genetically related to Sarcocystis falcatula). The aim of this research is the study of genetic variability and phylogenetic relationships among Sarcocystis spp. isolates from captured opossums in Brazil, by the analysis of molecular markers less subjected to selective pressure, such as DNA barcode, for this purpose, genetic loci from mitochondrial (CytB) and apicoplast genome (ClpC), and internal transcribed spacer regions (ITS-1) were applied and compare the results with the diversity found within members of the Toxoplasmatinae sub-family. The diversity among the SAG-2, SAG-3 and SAG-4 genes was also studied. The Sarcocystis sporocysts were obtained from opposum’s intestinal scraping and faeces from São Paulo State, Rio Grande do Sul State and Rio Grande do Norte State, Brazil. The molecular markers CytB, ClpC and ITS-1 revealed a vast genotypic variability and novel alleles among the Brazilian isolates. The results indicate possible existence of hybrid species of Sarcocystis in Brazil, with a gene mixture from both species. Regarding the SAG genes, an even greater variability was found than previously reported. The results also suggest the occurrence of reassortment and allele recombination of SAG sequences, contributing even more to the variability and thus in the parasites antigenic configuration. In this study, the results demonstrate that isolates from Brazil have a different genetic composition than reported in other locations, which may have important impacts in the knowledge of the diversity of Sarcocystis species shed by opossums in our environment and in all of the epidemiology of infections of protozoa of this particular group

Em um trabalho anterior, foi avaliada a variabilidade de Sarcocystis spp. isolados de gambás oriundos do estado do Rio Grande do Sul pesquisando sequências gênicas codificadoras de antígenos de superfície (SAGs). Os resultados indicaram haver linhagens de isolados de Sarcocystis (relacionadas geneticamente a S. falcatula) que trocam genes em prováveis processos de recombinação sexual. A proposta deste estudo foi de conhecer as variações gênicas e relações filogenéticas em Sarcocystis spp. isolados de gambás através da análise de loci gênicos de genoma mitocondrial (CytB), de genoma de apicoplasto (ClpC) e das regiões espaçadoras transcritas internas (ITS-1), comparando a diversidade encontrada nestes grupos com a observada em integrantes da sub-familia Toxoplasmatinae. E também de conhecer a diversidade de genes codificadores de SAG-2, SAG-3 e SAG-4. Neste estudo foram obtidos esporocistos de Sarcocystis spp. através de raspado intestinal e fezes provenientes de gambás do gênero Didelphis, oriundos do Estado de São Paulo, Rio Grande do Sul e Rio Grande do Norte. Os marcadores moleculares CytB, ClpC e ITS-1 revelaram uma ampla variabilidade genotípica e alelos inéditos entre os isolados do Brasil. Os resultados levam a crer que é possível a existência de espécies “híbridas” de Sarcocystis no Brasil, com mistura de genes de ambas as espécies. Em relação aos genes codificadores de SAGs, foi encontrada uma diversidade ainda maior do que trabalho similar realizado. Os resultados sugerem uma possível ocorrência de reassortment e recombinação de alelos em sequências SAGs, contribuindo ainda mais para a geração de variabilidade e consequentemente na configuração antigênica do parasito. Estes resultados demonstram que isolados brasileiros possuem uma composição genética diferente do reportado em demais localidade, o que pode ter reflexos importantes no conhecimento da diversidade das espécies de Sarcocystis que infectam gambás em nosso meio e em toda a epidemiologia das infecções produzidas pelos protozoários deste grupo / A great variability in surface antigens genes (SAGs) of Sarcocystis spp. sporocysts was found in a previous study, through the intestinal scraping technique of opossums from the south region of Brazil. The results indicated probable sexual recombination between lineages of Sarcocystis isolates (genetically related to Sarcocystis falcatula). The aim of this research is the study of genetic variability and phylogenetic relationships among Sarcocystis spp. isolates from captured opossums in Brazil, by the analysis of molecular markers less subjected to selective pressure, such as DNA barcode, for this purpose, genetic loci from mitochondrial (CytB) and apicoplast genome (ClpC), and internal transcribed spacer regions (ITS-1) were applied and compare the results with the diversity found within members of the Toxoplasmatinae sub-family. The diversity among the SAG-2, SAG-3 and SAG-4 genes was also studied. The Sarcocystis sporocysts were obtained from opposum’s intestinal scraping and faeces from São Paulo State, Rio Grande do Sul State and Rio Grande do Norte State, Brazil. The molecular markers CytB, ClpC and ITS-1 revealed a vast genotypic variability and novel alleles among the Brazilian isolates. The results indicate possible existence of hybrid species of Sarcocystis in Brazil, with a gene mixture from both species. Regarding the SAG genes, an even greater variability was found than previously reported. The results also suggest the occurrence of reassortment and allele recombination of SAG sequences, contributing even more to the variability and thus in the parasites antigenic configuration. In this study, the results demonstrate that isolates from Brazil have a different genetic composition than reported in other locations, which may have important impacts in the knowledge of the diversity of Sarcocystis species shed by opossums in our environment and in all of the epidemiology of infections of protozoa of this particular group

Equine protozoal myeloencephalitis (EPM) is a progressive neurological disease of horses caused by Sarcocystis neurona. Two projects were conducted to identify factors involved in the development of EPM. The first study explored a possible genetic susceptibility to EPM by attempting a genome-wide association study (GWAS) on formalin-fixed, paraffin-embedded (FFPE) tissue from 24 definitively-positive EPM horses. DNA extracted from tissues older than 14 months was inadequate for SNP analysis on the Illumina Equine SNP50 BeadChip probably due to degradation and formalin cross-linking. Results were inconclusive as analysis was not possible with the small sample set. The second study evaluated an artificial infection method in creating a reliable equine EPM model. Five horses were injected intravenously at 4 time points with autologous blood incubated with 1,000,000S. neurona merozoites. Challenged horses progressively developed mild to moderate clinical signs and had detectable S. neurona serum antibodies on day 42 post challenge. Horses appeared to have produced a Th1 immune response and cleared the infection by the conclusion of the study on day 89. No histopathological evidence of S. neurona infection was found within central nervous system tissue. This artificial infection method was not effective in replicating the severe clinical EPM seen in natural infections.

Equine Protozoal Myeloencephalitis (EPM) is a serious neurologic disease of horses in the United States. The primary etiologic agent is Sarcocystis neurona (S. neurona). Currently, there is limited knowledge regarding the protective or pathologic immune response to infection to the intracellular protozoa S. neurona. The objective of these studies was to determine the effects of S. neurona infection on the immune response of horses that had EPM due to natural infection (experiment 1) and experimental infection (experiment 2). In experiment 1, twenty-two horses with naturally occurring cases of EPM, which were confirmed positive based on detection of antibodies in the serum and/or CSF and clinical signs, and 20 clinically normal horses were included to determine whether S. neurona altered the immune responses, as measured by immune cell subsets (CD4, CD8, B-cell, monocytes, and neutrophils) and leukocyte proliferation (antigen specific and non-specific mitogens). Our results demonstrated that naturally infected horses had significantly higher percentages of CD4 and neutrophils (PMN) in peripheral blood mononuclear cells (PBMCs) than clinically normal horses. Leukocytes from naturally infected EPM horses had a significantly lower proliferation response, as measured by thymidine incorporation, to a non-antigen specific mitogen phorbol 12-myristate 13-acetate (PMA) / ionomycin (I) than did clinically normal horses (p=0.04). The implications of these findings will be discussed. In experiment 2, 13 horses were randomly divided into two groups. Baseline neurologic examinations were performed and all horses were confirmed negative for S. neurona antibodies in the CSF and serum. Then, one group with 8 clinically normal seronegative horses was inoculated intravenously with approximately 6000 S. neurona infected autologous leukocytes daily for 14 days. All the challenged horses showed neurologic signs consistent with EPM. PBMCs were isolated from the control and infected horses to determine how S. neurona alters the immune responses based on changes in immune cell subsets and immune function. There were no significant differences in the percentage of CD4 cells in peripheral blood lymphocytes or IFN-γ production by CD4 and/or CD8 cells. PMA/I stimulated proliferation responses in PBMCs appeared suppressed compared to that of uninfected controls. Additional studies are necessary to determine the role of CD4 and CD8 cells in disease and protection to S. neurona in horses, as well as to determine the mechanism associated with suppressed in vitro proliferation responses. This project was funded by Patricia Stuart Equine grants and paramutual racing funds from Virginia Tech. / Master of Science

Equine Protozoal Myeloencephalitis is the most frequently diagnosed neurologic disorder of horses in the united states, which is caused by the protozoan organism Sarcocystis neurona. The disease has a profound impact on the American Horse Industry. This impact includes prolonged and expensive treatment without a guaranteed return to a previous level of use for the individual horse. Poor respponse to and prolonged duration of treatment may suggest an immune mediated impariement of host response. There is limited information about the direct interaction between the pathogen and the host. In two in vitro experiments we investigated a) whether the presence of the protozoan organism can influence mitogen-stimulated peripheral blood mononuclear cells (PBMCs), suggesting a direct influence of the protozoan organism on cells of the immune system, and b) if cerebrospinal fluid (CSF) from horses with EPM has an effect on mitogen-stimulated PBMCs, suggesting that the microenvironment of the site of infection influences the course of disease. Experiment 1: Mitogen simulated PBMCs from EPM affected and control horses were co-cultured with fragments of freeze thawed bovine turbinate cells that were infected with S. neurona merozoites. Compared to controls PBMCs co-cultured with S. neurona fragments were the only cells that showed a decreased proliferation (p<0.05). A difference between EPM affected and control horses could not be detected (p>0.05). These results may imply that the persistence of S. neurona infection in the horses CNS is, in part, due to a pathogen-derived mechanism that attentuates the hosts immune response. Experiment 2: Mitogen stimulated PBMCs from a horse affected with EPM and a control were co-cultured n the presence of CSF from EPM affected and uninfected controls. Prior to co-culture the CSF was fractionated by a filtration process over two microfilter units. An identical volume of NaCl (0.9%) served as a control for the volume of CSF that was added. The proliferation assay revealed a deviation of the response depending on cell donor and CSF fraction used. The effect was independant of the protein concentration of the CSF fraction, and a decrease in lymphocyte proliferation was not caused by increased cellular death. This suggests the presence of subsets within the CSF which have a stimulatory of suppressive influence on the cells in culture. The effect was cell donor dependant which implies a difference in lymphocyte subsets between the two horses that were used. / Master of Science