Ocorrência e caracterização molecular de Cryptosporidium spp. em caprinos jovens do município de Quixadá-Ceará

Pós-graduação em Medicina Veterinária - FCAV

Avaliação mitocondrial em células mononucleares periféricas caninas infectadas com o vírus da cinomose

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Chemopreventive actions of blond and red-fleshed sweet orange juice on the loucy leukemia cell line

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Cytotoxic effects of neem oil in the midgut of the predator Ceraeochrysa claveri

Studies of morphological and ultrastructural alterations in target organs have been useful for evaluating the sublethal effects of biopesticides regarded as safe for non-target organisms in ecotoxicological analyses. One of the most widely used biopesticides is neem oil, and its safety and compatibility with natural enemies have been further clarified through bioassays performed to analyze the effects of indirect exposure by the intake of poisoned prey. Thus, this study examined the cellular response of midgut epithelial cells of the adult lacewing, Ceraeochrysa claveri, to neem oil exposure via intake of neem oil-contaminated prey during the larval stage. C. claveri larvae were fed Diatraea saccharalis eggs treated with neem oil at concentrations of 0.5%, 1% and 2% throughout the larval stage. The adult females obtained from these treatments were used at two ages (newly emerged and at the start of oviposition) in morphological and ultrastructural analyses. Neem oil was found to cause pronounced cytotoxic effects in the adult midgut, such as cell dilation, emission of cytoplasmic protrusions, cell lysis, loss of integrity of the cell cortex, dilation of cisternae of the rough endoplasmic reticulum, swollen mitochondria, vesiculated appearance of the Golgi complex and dilated invaginations of the basal labyrinth. Epithelial cells responded to those injuries with various cytoprotective and detoxification mechanisms, including increases in cell proliferation, the number of calcium-containing cytoplasmic granules, and HSP 70 expression, autophagic processes and the development of smooth endoplasmic reticulum, but these mechanisms were insufficient for recovery from all of the cellular damage to the midgut. This study demonstrates that neem oil exposure impairs the midgut by causing sublethal effects that may affect the physiological functions of this organ, indicating the importance of studies of different life stages of this species and similar species to evaluate the safe and compatible integrated use of biopesticides.

Diagnóstico laboratorial de Mycobacterium spp. em Botucatu e região, utilizando a técnica Reação em Cadeia da Polimerase (PCR) em material biológico e avaliação de condições associadas

Pós-graduação em Saúde Coletiva - FMB

Avaliação da capacidade de renovação do epitélio do mesêntero e seus efeitos no potencial reprodutivo de fêmeas adultas de Ceraeochrysa claveri (Neuroptera: Chrysopidae) alimentadas durante a fase larval com óleo de nim (azadirachta indica A. JUSS)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Caracterização do Pythium insidiosum por abordagem proteômica

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Cytoplasmic and extracellular expression of pharmaceutical-grade mycobacterial 65-kDa heat shock protein in Lactococcus lactis

Lactic acid bacteria (LAB) are an attractive and safe alternative for the expression of heterologous proteins, as they are nonpathogenic and endotoxin-free organisms. Lactococcus lactis, the LAB model organism, has been extensively employed in the biotechnology field for large-scale production of heterologous proteins, and its use as a "cell factory" has been widely studied. We have been particularly interested in the use of L. lactis for production of heat shock proteins (HSPs), which reportedly play important roles in the initiation of innate and adaptive immune responses. However, this activity has been questioned, as LPS contamination appears to be responsible for most, if not all, immunostimulatory activity of HSPs. In order to study the effect of pure HSPs on the immune system, we constructed recombinant L. lactis strains able to produce and properly address the Mycobacterium leprae 65-kDa HSP (Hsp65) to the cytoplasm or to the extracellular medium, using a xylose-induced expression system. Approximately 7 mg/L recombinant Hsp65 was secreted. Degradation products related to lactococcal HtrA activity were not observed, and the Limulus amebocyte lysate assay demonstrated that the amount of LPS in the recombinant Hsp65 preparations was 10-100 times lower than the permitted levels established by the U. S. Food and Drug Administration. These new L. lactis strains will allow investigation of the effects of M. leprae Hsp65 without the interference of LPS; consequently, they have potential for a variety of biotechnological, medical and therapeutic applications.

Protection conferred by heterologous vaccination against tuberculosis is dependent on the ratio of CD4(+)/CD4(+) Foxp3(+) cells

CD4(+) Foxp3(+) regulatory T cells inhibit the production of interferon-?, which is the major mediator of protection against Mycobacterium tuberculosis infection. In this study, we evaluated whether the protection conferred by three different vaccines against tuberculosis was associated with the number of spleen and lung regulatory T cells. We observed that after homologous immunization with the 65 000 molecular weight heat-shock protein (hsp 65) DNA vaccine, there was a significantly higher number of spleen CD4(+) Foxp3(+) cells compared with non-immunized mice. Heterologous immunization using bacillus Calmette Guerin (BCG) to prime and DNA-hsp 65 to boost (BCG/DNA-hsp 65) or BCG to prime and culture filtrate proteins (CFP)-CpG to boost (BCG/CFP-CpG) induced a significantly higher ratio of spleen CD4(+)/CD4(+) Foxp3(+) cells compared with non-immunized mice. In addition, the protection conferred by either the BCG/DNA-hsp 65 or the BCG/CFP-CpG vaccines was significant compared with the DNA-hsp 65 vaccine. Despite the higher ratio of spleen CD4(+)/CD4(+) Foxp3(+) cells found in BCG/DNA-hsp 65-immunized or BCG/CFP-CpG-immunized mice, the lungs of both groups of mice were better preserved than those of DNA-hsp 65-immunized mice. These results confirm the protective efficacy of BCG/DNA-hsp 65 and BCG/CFP-CpG heterologous prime-boost vaccines and the DNA-hsp 65 homologous vaccine. Additionally, the prime-boost regimens assayed here represent a promising strategy for the development of new vaccines to protect against tuberculosis because they probably induce a proper ratio of CD4(+) and regulatory (CD4(+) Foxp3(+)) cells during the immunization regimen. In this study, this ratio was associated with a reduced number of regulatory cells and no injury to the lungs.

Recombinant DNA immunotherapy ameliorate established airway allergy in a IL-10 dependent pathway

Background Previous studies have established that mycobacterial infections ameliorate allergic inflammation. However, a non-infectious approach that controls allergic responses might represent a safer and more promising strategy. The 60-65 kDa heat shock protein (Hsp) family is endowed with anti-inflammatory properties, but it is still unclear whether and how single mycobacterial Hsp control allergic disorders. Objective Therefore, in this study we determined whether the administration of Mycobacterial leprae Hsp65 expressed by recombinant a DNA plasmid could attenuate a previously established allergic response. Methods We used an experimental model of airway allergic inflammation to test the effects of immunotherapy with DNA encoding Hsp65. Allergic mice, previously sensitized and challenged with ovalbumin, were treated with tree intramuscular doses of recombinant DNA encoding Hsp65. After treatment, mice received a second allergen challenge and the allergic response was measured. Results We found that immunotherapy attenuated eosinophilia, pulmonary inflammation, Th2 cytokine and mucus production. Moreover, we showed that the inhibition of allergic response is dependent on IL-10 production. Both Hsp65 and allergen-specific IL-10-producing cells contributed to this effect. Cells transferred from DNA-immunized mice to allergic mice migrated to allergic sites and down-modulated the Th2 response. Conclusions and Clinical Relevance Our findings clearly show that immunotherapy with DNA encoding Hsp65 can attenuate an established Th2 allergic inflammation through an IL-10-dependent mechanism; moreover, the migration of allergen-and Hsp65-specific cells to the allergic sites exerts a fundamental role. This work represents a novel contribution to the understanding of immune regulation by Hsp65 in allergic diseases.

Molecular diversity of Primula apennina and phylogeny of Primula Subsection Euauricula

Primula apennina Widmer is endemic to the North Apennines (Italy). ISSR were used to detect the genetic diversity within and among six populations representative of the species distribution range. High levels of genetic diversity were revealed both at population (PPB = 75.92%, HS = 0.204, Hpop = 0.319) and at species level (PPB = 96.95%, HT = 0.242, Hsp = 0.381). Nei gene diversity statistics (15.7%), Shannon diversity index (16.3%) and AMOVA (14%) detected a moderate level of interpopulation diversity. Principal coordinate and bayesian analyses clustered the populations in three major groups along a geographic gradient. The correlation between genetic and geographic distances was positive (Mantel test, r = 0.232). All together, these analyses revealed a weak but significant spatial genetic structure in P. apennina, with gene flow acting as a homogenizing force that prevents a stronger differentiation of populations. Conservation measures are suggested based on the observed pattern of genetic variability. P. apennina belongs to Primula subsect. Euauricula which includes 15 species distributed on the whole Alps and Apennines. A phylogenetic analysis was carried out using AFLP markers in order both to clarify the relationships among the species of subsection Euauricula that remained unresolved in previous works and to make some hypoteses on their evolutive dynamics. NJ, PCO and BAPS analyses strongly confirmed the monophyly of P. subsect. Euauricula and all the species form strongly supported clades. NJ tree topology suggested a simultaneous fragmentations of ancestral species in a large number of isolated populations that survived in refugia along the unglaciated margins of the Alps in response to the Pleistocene climatic oscillations.

Untersuchung der Modulation adaptiver Immunantworten in Abhängigkeit der Reifung dendritischer Zellen

Im ersten Teil dieser Arbeit wurde die Bildung von DALIS während der Reifung von dendritischen Zellen untersucht. Dabei konnte festgestellt werden, dass die DALIS-Bildung mit dem Aktivierungszustand der Zelle eng verknüpft ist. Es konnten verschiedene Hitzeschockproteine in Zusammenhang mit der DaLIS-Bildung gebracht werden und ein Molekül der Siganlgebung identifiziert werden. Im zweiten Teil der Arbeit wurde die Veränderung von dendritischen Zellen in Anwesenheit von regulatorischen T-Zellen untersucht. Hierbei zeigte sich, dass zwischen den Zellen Gap Junctions entstehen, durch die cAMP von den regulatorischen T-Zellen auf die dendritischen Zellen übertragen werden kann. Weiter konnte gezeigt werden, dass regulatorische T-Zellen IL-10 sezernieren und so die dendritischen Zellen inhibieren.

Non-thermal emission in High Frequency Peaked blazars towards the Square Kilometer Array era

In this thesis work we will explore and discuss the properties of the gamma-ray sources included in the first Fermi-LAT catalog of sources above 10 GeV (1FHL), by considering both blazars and the non negligible fraction of still unassociated gamma-ray sources (UGS, 13%). We perform a statistical analysis of a complete sample of hard gamma-ray sources, included in the 1FHL catalog, mostly composed of HSP blazars, and we present new VLBI observations of the faintest members of the sample. The new VLBI data, complemented by an extensive search of the archives for brighter sources, are essential to gather a sample as large as possible for the assessment of the significance of the correlation between radio and very high energy (E>100 GeV) emission bands. After the characterization of the statistical properties of HSP blazars and UGS, we use a complementary approach, by focusing on an intensive multi-frequency observing VLBI and gamma-ray campaign carried out for one of the most remarkable and closest HSP blazar Markarian 421.

Evidence of prognostic relevant expression profiles of heat-shock proteins and glucose-regulated proteins in oesophageal adenocarcinomas

A high percentage of oesophageal adenocarcinomas show an aggressive clinical behaviour with a significant resistance to chemotherapy. Heat-shock proteins (HSPs) and glucose-regulated proteins (GRPs) are molecular chaperones that play an important role in tumour biology. Recently, novel therapeutic approaches targeting HSP90/GRP94 have been introduced for treating cancer. We performed a comprehensive investigation of HSP and GRP expression including HSP27, phosphorylated (p)-HSP27((Ser15)), p-HSP27((Ser78)), p-HSP27((Ser82)), HSP60, HSP70, HSP90, GRP78 and GRP94 in 92 primary resected oesophageal adenocarcinomas by using reverse phase protein arrays (RPPA), immunohistochemistry (IHC) and real-time quantitative RT-PCR (qPCR). Results were correlated with pathologic features and survival. HSP/GRP protein and mRNA expression was detected in all tumours at various levels. Unsupervised hierarchical clustering showed two distinct groups of tumours with specific protein expression patterns: The hallmark of the first group was a high expression of p-HSP27((Ser15, Ser78, Ser82)) and low expression of GRP78, GRP94 and HSP60. The second group showed the inverse pattern with low p-HSP27 and high GRP78, GRP94 and HSP60 expression. The clinical outcome for patients from the first group was significantly improved compared to patients from the second group, both in univariate analysis (p = 0.015) and multivariate analysis (p = 0.029). Interestingly, these two groups could not be distinguished by immunohistochemistry or qPCR analysis. In summary, two distinct and prognostic relevant HSP/GRP protein expression patterns in adenocarcinomas of the oesophagus were detected by RPPA. Our approach may be helpful for identifying candidates for specific HSP/GRP-targeted therapies.

Proteomic changes in the rat brain induced by homogenous irradiation and by the bystander effect resulting from high energy synchrotron X-ray microbeams

Abstract Purpose: To further evaluate the use of microbeam irradiation (MBI) as a potential means of non-invasive brain tumor treatment by investigating the induction of a bystander effect in non-irradiated tissue. Methods: Adult rats were irradiated with 35 or 350 Gy at the European Synchotron Research Facility (ESRF), using homogenous (broad beam) irradiation (HI) or a high energy microbeam delivered to the right brain hemisphere only. The proteome of the frontal lobes were then analyzed using two-dimensional electrophoresis (2-DE) and mass spectrometry. Results: HI resulted in proteomic responses indicative of tumourigenesis; increased albumin, aconitase and triosphosphate isomerase (TPI), and decreased dihydrolipoyldehydrogenase (DLD). The MBI bystander effect proteomic changes were indicative of reactive oxygen species mediated apoptosis; reduced TPI, prohibitin and tubulin and increased glial fibrillary acidic protein (GFAP). These potentially anti-tumourigenic apoptotic proteomic changes are also associated with neurodegeneration. However the bystander effect also increased heat shock protein (HSP) 71 turnover. HSP 71 is known to protect against all of the neurological disorders characterized by the bystander effect proteome changes. Conclusions: These results indicate that the collective interaction of these MBI-induced bystander effect proteins and their mediation by HSP 71, may confer a protective effect which now warrants additional experimental attention.