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Edwardsiella ictaluri is an intracellular Gram-negative pathogen, causing enteric septicemia of catfish (ESC). Universal stress proteins (USP) are important in bacterial virulence, but the role of USPs in E. ictaluri virulence is not explored yet. Our aim was to analyze gene expression of 13 usp (usp01-usp13) and 7 USP-interacting genes (groEL, groES, dnaK, dnaJ, clpB, grpE, and ppGpp) under low pH, H2O2, catfish serum, and in vivo stress conditions, construct USP mutants, and determine mutants’ role in E. ictaluri virulence. We found that usp05, usp07 and usp13 genes were highly expressed under all stress conditions, while groEL, groES, dnaK, grpE, and clpB were highly expressed in oxidative stress. Among the 10 E. ictaluri USP mutants, Eiusp05-07-08-09-10, and 13 were significantly attenuated in catfish and highly protective against wild type E. ictaluri infections in catfish. Eiusp05-07-08-09, and 13 were sensitive to oxidative stress, and all mutants were sensitive to pH exposure.

Synthesis of water stress proteins (Wsp) is induced upon repeated desiccation and rehydration of immobilized cells of the desiccation-tolerant cyanobacterium <i>Nostoc commune</i> UTEX 584 (<i>Nostoc</i> 584). The Wsp polypeptides synthesized and secreted by field material of N. <i>commune</i> have been extensively characterized and shown to exist as three isoforms with molecular masses of 33, 37 and 39 kDa. In order to understand the role of Wsp in the mechanism of tolerance to water stress an attempt was made to isolate the gene(s) that encodes Wsp in <i>Nostoc</i> 584. A polyclonal antibody raised against a mixture of the isoforms was used to screen expression libraries (phage and plasmid) of <i>Nostoc</i> 584 genomic DNA fragments. This work presents the analysis of clones, isolated from the expression libraries, which cross react with Wsp antiserum. Sequencing of the DNA from one of the cross reacting clones revealed an incomplete open reading frame (ORFA) that showed strong similarity to two fructose bisphosphate aldolases, CfxA and CfxB, from the photosynthetic purple non-sulfur bacterium, <i>Rhodobacter sphaeroides</i>. A promoter region present upstream of ORFA is recognized by RNA polymerase from <i>E. coli</i>. Further upstream of the promoter lies <i>trn</i>K encoding lysyl-tRNA, identified by evident similarity to the corresponding gene from the chloroplast of the liverwort, <i>Marchantia polymorpha</i>. The remainder of the aldolase gene (<i>fba</i>) was isolated using the colony hybridization technique. Sequence analysis of DNA from the second cross reactive clone revealed six ORFs (ORFs 1 through 6).The products of ORFI and ORF2 are overproduced in this clone. The polypeptide encoded by ORFI shows very strong cross-reactivity with the polyclonal Wsp antibody, whereas ORF2 does not. Database searches using the deduced amino acid sequences of the six ORFs have provided clues to the possible identities of these ORFs. ORF6 shows correspondence with a protein, in Arabidopsis thaliana, which is induced in response to cold, abscissic acid and water stress. The common feature shared by ORFs 1 to 5 is that the highest similarities are observed with enzymes involved in carbohydrate metabolism. ORFs 1 through 5 may possibly represent a novel cluster of genes that form all or part of an operon involved in the metabolism of carbohydrates in Nostoc 584. Fructose bisphosphate aldolase (EC 4.1.2.13) is a key enzyme in carbohydrate metabolism, playing a role in glycolysis as well in the Calvin cycle of carbon dioxide fixation. The potential roles played by aldolase and the products of ORFs 1 through 5 in overall carbon metabolism of <i>Nostoc commune</i> UTEX 584 are discussed. / Ph. D.

Patients with type 2 diabetes have reduced gene expression of Heat Shock Protein (HSP) 72 which correlates with reduced insulin sensitivity. Heat therapy, which activates HSP72, improves clinical parameters in these patients. Activation of several inflammatory signalling proteins such as c-jun amino terminal kinase (JNK) can induce insulin resistance but HSP72 can block the induction of these molecules in vitro. Whether up-regulation of HSP72 can protect against insulin resistance is not known. In experiments reported in this thesis we show that HSP72 protects against insulin resistance and blocks the activation of JNK in vivo. We first show that mice that underwent weekly heat shock therapy to increase intramuscular HSP72 protein expression were protected from high fat diet (HFD)-induced hyperinsulinemia, hyperglycemia and glucose intolerance, factors associated with reduced JNK phosphorylation. To determine whether the elevation in intramuscular HSP72 expressio n and protection from insulin resistance are causally linked, we studied muscle specific HSP72 overexpression mice (HSP72+/+). Compared with wild-type mice, HSP72+/+ mice were protected from hyperglycemia, hyperinsulinemia, glucose intolerance and insulin resistance when placed on a HFD, factors associated with a complete inhibition of HFD-induced JNK phosphorylation in skeletal muscle. Finally, we show that HSP72+/+ mice display greater mitochondrial enzyme activity in the liver, adipose tissue and skeletal muscle, corresponding to reduced plasma free fatty acid levels, white adipose tissue mass and alterations in circulating adipokines. These data identify HSP72 as being pivotal in protecting against obesity-induced insulin resistance possibly by blocking JNK and/or by up-regulation of mitochondrial oxidative capacity.

Abstract Previous studies have examined stress-induced heme oxygenase-1 (HO-1) expression primarily in mammalian systems. The present study examines, for the first time in amphibians, the effect of heat shock, sodium arsenite, cadmium chloride, and the proteasomal inhibitor MG132 on HO-1 accumulation in Xenopus laevis A6 kidney epithelial cells. Western blot analysis revealed that exposure of A6 cells to a range of heat shock temperatures (30-35 °C), which induced HSP30 accumulation, did not induce HO-1 accumulation. In contrast, cells treated with sodium arsenite (5-50 μM), cadmium chloride (50-200 μM) or MG132 (5-30 μM) exhibited a dose- and time-dependent accumulation of HO-1. Additionally, immunocytochemical analysis revealed that HO-1 and HSP30 accumulation occurred in a granular pattern primarily in the cytoplasm in cells treated with sodium arsenite, cadmium chloride, or MG132. In cells recovering from sodium arsenite or cadmium chloride treatment, HO-1 and HSP30 accumulation initially increased to a maximum at 12 h and 24 h recovery, respectively, followed by a 50% reduction at 48 h. This initial increase in the relative levels of stress proteins was likely the result of new synthesis as it was inhibited by cycloheximide. In comparison, cells recovering from MG132 treatment displayed reduced but prolonged accumulation of HO-1 and HSP30. Interestingly, cells treated with low concentrations (10 μM) of sodium arsenite or MG132 but not cadmium chloride in combination with a mild 30 °C heat shock had enhanced accumulation of HO-1 and HSP30 accumulation compared to either of the stressors individually. This study has shown for the first time in amphibians that HO-1 accumulation is induced in response to metals and proteasomal inhibitors, suggesting that it may play a role in mediating the cellular stress response in X. laevis.

Organisms synthesize stress proteins in response to a variety of stressors. The 68/70-kDa proteins (synonymous to the 72/73-kDa proteins) have shown to be the most promising stress proteins, and have been proposed as a biomarker of general organismal stress. The 68/70-kDa proteins were used in an antigen/antibody based approach to determine the duration of the stress protein response of Pimephales promelas following an acute exposure to copper sulphate.

Estudos têm relatado o potencial de aplicação cosmética de classes de proteínas que conferem resistência aos diferentes tipos de estresse sobre a pele. Nesse contexto, as proteínas de estresse - proteínas de choque térmico, metalotioneínas e sirtuínas - enquadram-se entre as proteínas de grande potencial no combate ao fotoenvelhecimento. Diversos extratos vegetais tem mostrado potencial de induzir uma ou mais proteínas de estresse. Dessa forma, o objetivo do trabalho foi avaliar os efeitos in vitro da aplicação isolada dos extratos de Anadenanthera colubrina (Angico branco), Rhodiola rosea (Rodiola), Pfaffia paniculata (Pfaffia), sobre a expressão gênica das proteínas do choque térmico (Heat shock proteins, HSP32 e HSP72), metalotioneína (MT-I) e da sirtuína isoforma SIRT1, em condição basal e mediante exposição aguda das culturas celulares à radiação UV. Como resultados de maior relevância o extrato de Anadenanthera colubrina foi capaz de modular positivamente as expressões gênicas de HSP72, SIRT-1 e MT-I, em fibroblastos sob condição basal. Já o extrato de Pfaffia paniculata foi capaz de elevar a expressão gênica de SIRT-1, em fibroblastos irradiados por UVA após incubação com o extrato vegetal e sob condição basal, além de induzir MT-I em culturas não irradiadas. Por fim, o extrato de Rhodiola rosea elevou as expressões de HSP32 e MT-I, em fibroblastos sob condição basal. Devido à capacidade indutora das proteínas de estresse pelos extratos vegetais avaliados, sugere-se que os mesmos apresentam potencial para utilização em produtos cosméticos, especialmente, com finalidade antienvelhecimento. / Studies have reported the potential for cosmetic application of proteins classes that confer resistance to different types of stress on the skin. In this context, stress proteins - heat shock proteins, metallothioneins and sirtuins - fall between the proteins of great potential in the fight against photoaging. Several plant extracts have been shown potential to induce one or more stress proteins. Thus, the objective of this study was to evaluate the in vitro effects from the isolated application of the extracts Anadenanthera colubrina (Angico-branco), Rhodiola rosea (Rodiola), Pfaffia paniculata (Fáfia) on the gene expression of heat shock proteins, HSP32 and HSP72, metallothionein (MT-I) and the sirtuin, isoform SIRT1, in basal conditions and upon acute exposure of cell cultures to UV radiation. As most relevant results, Anadenanthera colubrina extract was able to positively modulate the gene expression of HSP72, SIRT-1 and MT-I in fibroblasts under basal conditions. The Pfaffia paniculata extract was able to increase the gene expression of SIRT-1 in fibroblasts irradiated by UV after incubation with plant extract and in basal conditions, in addition to induce MT-I in non-irradiated cultures. Finally, the extract of Rhodiola rosea increased HSP32 and MT-I gene expressions in fibroblasts under basal conditions. Considering the plant extracts ability to induce stress proteins, it is suggested that they have potential for use in cosmetic products, especially, for anti-aging purposes.

Estudos têm relatado o potencial de aplicação cosmética de classes de proteínas que conferem resistência aos diferentes tipos de estresse sobre a pele. Nesse contexto, as proteínas de estresse - proteínas de choque térmico, metalotioneínas e sirtuínas - enquadram-se entre as proteínas de grande potencial no combate ao fotoenvelhecimento. Diversos extratos vegetais tem mostrado potencial de induzir uma ou mais proteínas de estresse. Dessa forma, o objetivo do trabalho foi avaliar os efeitos in vitro da aplicação isolada dos extratos de Anadenanthera colubrina (Angico branco), Rhodiola rosea (Rodiola), Pfaffia paniculata (Pfaffia), sobre a expressão gênica das proteínas do choque térmico (Heat shock proteins, HSP32 e HSP72), metalotioneína (MT-I) e da sirtuína isoforma SIRT1, em condição basal e mediante exposição aguda das culturas celulares à radiação UV. Como resultados de maior relevância o extrato de Anadenanthera colubrina foi capaz de modular positivamente as expressões gênicas de HSP72, SIRT-1 e MT-I, em fibroblastos sob condição basal. Já o extrato de Pfaffia paniculata foi capaz de elevar a expressão gênica de SIRT-1, em fibroblastos irradiados por UVA após incubação com o extrato vegetal e sob condição basal, além de induzir MT-I em culturas não irradiadas. Por fim, o extrato de Rhodiola rosea elevou as expressões de HSP32 e MT-I, em fibroblastos sob condição basal. Devido à capacidade indutora das proteínas de estresse pelos extratos vegetais avaliados, sugere-se que os mesmos apresentam potencial para utilização em produtos cosméticos, especialmente, com finalidade antienvelhecimento. / Studies have reported the potential for cosmetic application of proteins classes that confer resistance to different types of stress on the skin. In this context, stress proteins - heat shock proteins, metallothioneins and sirtuins - fall between the proteins of great potential in the fight against photoaging. Several plant extracts have been shown potential to induce one or more stress proteins. Thus, the objective of this study was to evaluate the in vitro effects from the isolated application of the extracts Anadenanthera colubrina (Angico-branco), Rhodiola rosea (Rodiola), Pfaffia paniculata (Fáfia) on the gene expression of heat shock proteins, HSP32 and HSP72, metallothionein (MT-I) and the sirtuin, isoform SIRT1, in basal conditions and upon acute exposure of cell cultures to UV radiation. As most relevant results, Anadenanthera colubrina extract was able to positively modulate the gene expression of HSP72, SIRT-1 and MT-I in fibroblasts under basal conditions. The Pfaffia paniculata extract was able to increase the gene expression of SIRT-1 in fibroblasts irradiated by UV after incubation with plant extract and in basal conditions, in addition to induce MT-I in non-irradiated cultures. Finally, the extract of Rhodiola rosea increased HSP32 and MT-I gene expressions in fibroblasts under basal conditions. Considering the plant extracts ability to induce stress proteins, it is suggested that they have potential for use in cosmetic products, especially, for anti-aging purposes.

Made available in DSpace on 2015-03-26T13:47:09Z (GMT). No. of bitstreams: 1 texto completo.pdf: 226846 bytes, checksum: fab823cf7946385dda72c2e97936a8ec (MD5) Previous issue date: 2006-09-28 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Mammary tumor is the most frequent neoplasms in female dogs and the similarity of those tumors with the mammary tumors in the woman determines an interest in the study of the compared pathology. The expression of Heat Shock Proteins (HSP) have been observed in many neoplasms, including breast cancer. The aim of this work was to classify histopatologically and to investigate the expression of HSP 72 in normal and neoplastic canine mammary glands of female dogs assisted in the service of the Veterinary Hospital of the Federal University of Viçosa. 34 samples of mammary tissue were collected, including 23 neoplastics and 11 normal tissue, for histopatological analysis and investigation of expression of HSP 72 using Western blotting. Furthermore, the variables age to the diagnosis, breed, attacked mammary glands and histopatological classification of the tissue were investigated. Statistical analysis was carried out using Chi-square test (&#967;²). The result were considered statistically significant in the P < 0,05. The major frequency of the neoplasm diagnostic was at 8 to 13 years old, at the inguinal and caudal abdominal mammary glands and no statistical difference was observed between malignants and benign tumors nor at histopatological classifications. The expression of HSP 72 was determined in normal and neoplastic mammary gland tissue, this last one presenting an accentuated expression of this protein. The neoplastic tissue presented a larger proportion of expression of the protein HSP 72 when compared to the normal tissue, being observed equivalence among benign and malignants tumors as for the expression of that protein. / Tumor de mama é a neoplasia mais freqüente em cadelas e a semelhança com os tumores da mama na mulher determina um interesse crescente no estudo da patologia comparada. A expressão de proteínas de choque térmico (HSP) tem sido observada em muitas neoplasias, incluindo o câncer de mama. O objetivo desse trabalho foi classificar histopatologicamente e investigar a expressão de HSP 72 em glândula mamária normal e neoplásica de cadelas atendidas no serviço ambulatorial do Hospital Veterinário da Universidade Federal de Viçosa. Foram utilizadas 34 amostras de tecido mamário, sendo 23 neoplásicas e 11 normais, submetidas à análise histopatológica e pesquisa de HSP 72 através de Western blotting. Além disso, foram investigadas as variáveis: idade ao diagnóstico, raça, glândulas mamárias acometidas e classificação histopatológica da amostra. Os dados foram analisados por meio de tabelas de freqüência e a comparação entre as proporções das variáveis pelo teste de Qui-quadrado (&#967;²), utilizando nível de significância de 5%. Observou-se maior frequência de diagnóstico da neoplasia mamária em cadelas entre 8 e 13 anos de idade, nas glândulas mamárias abdominais caudais e inguinais, sem diferença estatística entre malignas e benignas e classificação histopatológica. Os tecidos neoplásicos apresentaram uma maior proporção de expressão da proteína HSP 72 quando comparados ao tecido normal, sendo observado equivalência entre tumores benignos e malignos quanto à expressão dessa proteína.