Um dos graves obstáculos para a manutenção e estabilidade da produção nacional de arroz (Oryza sativa) reside na susceptibilidade dos genótipos existentes a estresses abióticos. Tendo em vista a importância social e econômica do arroz e os efeitos extremamente danosos desses estresses sobre a agricultura, o conhecimento detalhado das interações entre os estresses abióticos e as respostas dos vegetais frente a esses estímulos ambientais faz-se necessário. O alumínio (Al) é considerado um dos principais fatores limitantes na produção agrícola, inibindo o crescimento das raízes e a absorção de minerais. A toxicidade do Al em plantas ocorre pela sua solubilização em solos com pH baixo ou solos ácidos. Os genes ASR (ABA, Stress and Ripening) são induzidos por estresse e ácido abscísico (ABA) em plantas, e seus níveis de expressão são rapidamente aumentados em resposta à salinidade e seca. Recentemente, foi demonstrado que o gene que codifica a proteína ASR5 é responsivo ao Al em raízes de arroz. Apesar do arroz ser considerado um dos cereais mais resistentes a Al, os mecanismos básicos de tolerância a este metal são pouco conhecidos no arroz em comparação a outros cereais. Por meio do presente trabalho objetivamos: i) a caracterização funcional dos membros da família gênica ASR de arroz em reposta ao Al; e ii) a construção de vetores binários de transformação de plantas visando o estudo da localização subcelular da proteína codificada pelo gene OsASR5, e o silenciamento gênico da família ASR de arroz. As análises dos transcritos por qRT-PCR mostraram que todos os genes da família ASR de arroz ssp Japonica respondem ao Al. Por outro lado, OsASR5 não sofre modulação de sua expressão em resposta ao Al em raízes de arroz ssp Indica. Essas diferenças de respostas dos genes OsASR5 em distintas variedades pode refletir diferenças no grau de tolerância ao Al de cada um desses genótipos. / One of the major obstacles to maintain the stability of the national production of rice (Oryza sativa) lies on the susceptibility of the different genotypes to abiotic stress. In view of the social and economic importance of rice and due to the extremely harmful effects of stress in agriculture, detailed knowledge of the interactions between these stresses and plant responses to environmental stimuli is necessary. Aluminum (Al) is considered one of the main limitation factors for agricultural productivity, inhibiting root growth and mineral absorption. Al toxicity occurs by its solubilization in soils with low pH or acid soils. The ASR (ABA, Stress and Ripening) genes are induced by stress and abscisic acid (ABA) in plants, and their expression levels are quickly increased in response to salinity and drought. Recently, it was demonstrated that the ASR5 gene is responsive to Al in rice roots. Despite the fact that rice is considered one of the most resistant crops to Al, the basic mechanisms of tolerance to this metal are poorly known when compared to other crops. This study aimed the functional characterization of the gene expression of rice ASR family members in response to Al, and the construction of binary vectors for the subcellular localization of the protein codified by the OsASR5 gene, and the construction of a gene silencing binary vector for the ASR family. Analyses of transcripts by qRT-PCR showed that in the ssp Japonica, all ASR genes responded to Al. In contrast, OsASR5 do not suffer expression modulation in response to Al in rice roots of ssp Indica. These differences in response of the OsASR5 gene in distinct varieties may reflect differences in the degree of Al tolerance in each genotype.

Submitted by FERNANDA TANAMATI null (fertanamati@gmail.com) on 2018-03-22T00:14:15Z No. of bitstreams: 1 Tese_Tanamati_versão final.pdf: 1118298 bytes, checksum: ad444e697e657dd704d25b099b8eeede (MD5) / Approved for entry into archive by Alexandra Maria Donadon Lusser Segali null (alexmar@fcav.unesp.br) on 2018-03-22T10:54:04Z (GMT) No. of bitstreams: 1 tanamati_f_dr_jabo.pdf: 1118298 bytes, checksum: ad444e697e657dd704d25b099b8eeede (MD5) / Made available in DSpace on 2018-03-22T10:54:04Z (GMT). No. of bitstreams: 1 tanamati_f_dr_jabo.pdf: 1118298 bytes, checksum: ad444e697e657dd704d25b099b8eeede (MD5) Previous issue date: 2018-02-27 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A mastite em búfalas leiteiras causa perdas econômicas devido à diminuição da produção de leite, além de ter um efeito negativo no bem-estar dos animais. Deste modo, foram realizados dois estudos: o objetivo do estudo I foi avaliar o nível de expressão dos genes da lactoferrina (LTF), fator de necrose tumoral alfa (TNF-α), interleucina-1 beta (IL-1β), interleucina-8 (IL-8), e toll-like receptors 2 (TLR-2) e 4 (TLR-4) em búfalas Murrah com e sem mastite subclínica. Amostras de leite de 12 búfalas com mastite e 12 de búfalas sem mastite foram coletadas, para a extração de RNA, síntese de cDNA e validação dos perfis de expressão por qRT-PCR. O ΔΔCt foi estimado utilizando contrastes ortogonais da expressão dos genes alvo corrigidos para a expressão dos genes housekeeping entre os tratamentos. A expressão dos genes TLR-2, TLR-4, TNF-α, IL-1β e IL-8 foi regulada positivamente em búfalos com mastite, enquanto que o gene LTF não apresentou expressão diferencial. O estudo desses genes da função imune que são ativos na glândula mamária é importante para o desenvolvimento de estratégias destinadas a preservar a saúde do úbere. O objetivo do estudo II foi avaliar as proteínas presentes no soro do leite de búfalas com e sem mastite subclínica, utilizando uma abordagem proteômica para identificar proteínas diferencialmente expressas e potencial biomarcador para a doença. Para isso, 16 amostras de leite de búfalas Murrah foram coletadas, sendo oito animais com e oito animais sem mastite subclínica. O método contagem espectral foi utilizado para quantificar a abundância relativa das proteínas individuais e os bancos de dados de proteínas anotadas para Bubalus bubalis e para Bos taurus foram utilizados na análise. Após integração das anotações genéticas das referências de búfalos e bovinos, foram identificadas 1.033 proteínas, das quais 156 proteínas foram diferencialmente reguladas entre animais sadios e infectados. Foram identificados 18 processos biológicos nos quais essas proteínas participam e a catelicidina-3 foi identificada como um potencial biomarcador da mastite subclínica. Os resultados são importantes para compreender melhor o comportamento da mastite na glândula mamária das búfalas e, portanto, podem auxiliar no diagnóstico precoce da doença. / Mastitis in dairy buffalo causes economic losses due to decreased milk production, along with having a negative effect on the animals’ welfare. Thus, two studies were performed: the objective of study I was to evaluate the expression levels of lactoferrin (LTF), tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), interleukin- 8 (IL-8) and toll-like receptors 2 (TLR-2) and 4 (TLR-4) in Murrah buffaloes with and without subclinical mastitis. Milk samples were collected from 12 buffaloes with mastitis and 12 buffaloes without mastitis for RNA extraction, cDNA synthesis, and validation of expression profiles by qRT-PCR. The ΔΔCt was estimated using orthogonal contrasts of the target genes expression adjusted for the expression of the housekeeping genes between both treatments. The expression of the TLR-2, TLR-4, TNF-α, IL-1β and IL-8 genes were upregulated in buffaloes with mastitis, while the LTF gene showed no differential expression. The study of these immune function genes that are active in the mammary gland is important for the development of strategies aimed at preserving the health of the udder. The objective of study II was to evaluate the proteins present in the milk whey from buffaloes with and without subclinical mastitis, using a proteomic approach to identify differentially expressed proteins and potential biomarkers for the disease. For this, 16 samples of Murrah buffalo milk were collected, comprised of eight animals with and eight animals without subclinical mastitis. The spectral counting method was used to quantify the relative abundance of the individual proteins, and the databases of proteins annotated for Bubalus bubalis and for Bos taurus were used in the analysis. After integrating the gene annotations from the buffalo and bovine references, a total of 1,033 proteins were identified, of which 156 proteins were differentially regulated between healthy and affected animals. 18 biological processes in which these proteins participate were identified, and cathelicidin-3 was identified as a potential biomarker of subclinical mastitis. These results are important to better understand the behavior of mastitis in the buffalo mammary gland, and in turn may aid in the early diagnosis. / FAPESP: 14/19321-4 / FAPESP: 14/25309-7 / FAPESP: 16/10526-8

Submitted by Jéssica Moraes Malheiros (jessicamalheiros@yahoo.com.br) on 2018-05-03T18:53:41Z No. of bitstreams: 1 TESE MALHEIROS, J. M..pdf: 1363679 bytes, checksum: 33291bc89815d196826e037eeda5b237 (MD5) / Approved for entry into archive by Alexandra Maria Donadon Lusser Segali null (alexmar@fcav.unesp.br) on 2018-05-04T18:11:25Z (GMT) No. of bitstreams: 1 malheiros_jm_dr_jabo.pdf: 1400936 bytes, checksum: bf9a223495b522a13300edf9c4026a2e (MD5) / Made available in DSpace on 2018-05-04T18:11:25Z (GMT). No. of bitstreams: 1 malheiros_jm_dr_jabo.pdf: 1400936 bytes, checksum: bf9a223495b522a13300edf9c4026a2e (MD5) Previous issue date: 2018-03-02 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O presente trabalho teve como objetivo avaliar a associação da expressão gênica e proteômica com a maciez da carne de bovinos da raça Nelore. A partir de uma população de 90 animais foram selecionados três grupos experimentais por meio da análise de força de cisalhamento (FC) e índice de fragmentação miofibrilar (MFI), sendo: carne moderadamente macia, carne moderadamente dura e carne muito dura. A expressão dos genes foi avaliada por meio da análise de PCR em tempo real e a análise proteômica foi realizada com base na separação de proteínas por meio da eletroforese bidimensional (2D-PAGE) e caracterizção por espectrometria de massas com ionização eletrospray (ESI-MS/MS). A expressão da isoforma da calpastatina (CAST2) mostrou-se up regulated (P<0,05) nos grupos de carne moderadamente dura e muito dura. Os genes HSP90AA1, DNAJA1 e HSPB1, os quais representam as proteínas de choque térmico Hsp90, Hsp40 e Hsp27, respectivamente, mostraram expressão down regulated (P<0,05) no grupo de carne moderadamente macia em relação ao grupo de carne muito dura. Na análise proteômica, a expressão do spot protéico das enzimas metabólicas TPI e PGM1, proteína estrutural PFN1 e aminiopeptidase LAP3 se mostraram up regulated (P<0,05) no grupo de carne moderadamente macia, enquanto que a expressão das proteínas estruturais (ACTA1, ACTB, ACTG1 e MLC1), estresse oxidativo (PRDX6, PRDX2, PRDX1 and PARK7), proteínas de choque térmico (HSP90AA1, HSP90AB1, HSPA1A, HSPA1B, HSPA1L, HSPD1 e HSPB1), e co-chaperonas e regulação celular (CD37, STIP1 e ARHGDIA) se mostraram down regulated (P>0,05) no mesmo grupo experimental. Estes resultados fornecem uma visão importante de novos possíveis marcadores biológicos atuantes no processo de amaciamento da carne, o que pode colaborar para melhor entender e gerar novas estratégias de seleção nos programas de melhoramento genético de bovinos Nelore. / The objective of this study was to evaluate the association of gene expression and proteomics with meat tenderness in Nellore cattle. From population of 90 animals three experimental groups were selected by shear force (SF) and/or myofibrillar fragmentation index (MFI): moderately tender meat, moderately tough meat and very tough meat. Gene expression was evaluated by real-time PCR and proteomics analysis was performed based on protein separation by two-dimensional gel electrophoresis (2D-PAGE) and characterisation by eletrospray ionisation mass spectrometry (ESI-MS/MS). Expression of the calpastatin isoform (CAST2) was up-regulated (P<0.05) in the moderately tough and very tough meat groups. Expression of the HSP90AA1, DNAJA1 and HSPB1 genes, wich represent the heat shock proteins Hsp90, Hsp40 and Hsp27, respectively, were down-regulated (P<0.05) in the moderately tender meat in relation to the very tough group. In the proteomics analysis, the expression of the protein spots of metabolism TPI1 and PGM1, structural protein PFN1, and aminopeptidase LAP3 were up regulated (P<0.05) in the moderately tender meat, while the expression of structural proteins (ACTA1, ACTB, ACTG1 and MLC1), oxidative stress (PRDX6, PRDX2, PRDX1 and PARK7), heat shock protein (HSP90AA1, HSP90AB1, HSPA1A, HSPA1B, HSPA1L, HSPD1 and HSPB1) and co-chaperones and cellular regulatory (CD37, STIP1 and ARHGDIA) were down regulated (P>0.05) in the same experimental group. The present results suggest an important view of possible new biological markers in the meat tenderization process, wich permit to unsderstand and generate new strategies for selection in Nellore cattle breeding programs. / FAPESP: 15/13021-1

The O’Brien lab has demonstrated that Heat shock protein 27 (HSP27)shows attenuated expression in human coronary arteries as the degree of atherosclerosis progresses. Moreover, over-expression of HSP27 reduces atherogenesis in mice. The precise mechanism(s) for HSP27-mediated "atheroprotection" are incompletely understood. Nuclear Factor-kappaB (NF-κB) is a key signaling modulator in atherogenesis. Hence, this project sought to determine if recombinant HSP27 (rHSP27) alters NF-κB signaling to affect atheroprotection. Treatment of THP1 macrophages with rHSP27 resulted in degradation of IκBα, coincided with nuclear translocation of the p65 subunit and produced transcriptional evidence of activation of NF-κB signaling. When the transcriptional profile of THP1 macrophages treated with rHSP27 was analyzed using NF-κB-pathway-specific qRT-PCR arrays, among the regulated genes, IL-10 and GM-CSF mRNA levels were markedly increased, as were parallel translational effects observed. These data provide new mechanistic insights into the atheroprotective effects of HSP27.

No description available.

Glutamate carboxypeptidase II (GCPII) usually called prostate specific membrane antigen (PSMA) is membrane bound metallopeptidase expressed mainly in prostate carcinoma (PCa). Agents targeting GCPII suitable for both imaging and treatment of PCa are in development and they show promising results in advanced clinical trials. Some studies showed that GCPII may serve also as PCa blood serum marker, but this has not been validated due to the lack of methods suitable for accurate detection of GCPII in human blood. Moreover, GCPII is also expressed in brain, where it cleaves inhibitory N-acetyl-α-L- aspartyl-L-glutamate (NAAG) to release excitatory L-glutamate and GCPII inhibition has been shown to be neuroprotective in animal models of several neuropathies. Tight binding inhibitors of GCPII have been identified by rational design, but all have poor bioavailability and thus cannot be used in clinics. Identifying new scaffolds by 'brute force' screening methods is thus essential; however, no such method for GCPII has been developed so far. Glutamate carboxypeptidase III (GCPIII) is also expressed in brain and cleaves NAAG. It is thus an important protein for understanding of GCPII function as well as GCPII targeting in medicine. Here, we focused on development of novel methods for quantification of both...

No description available.

No description available.

Aspergillus fumigatus is an opportunistic fungal pathogen responsible for invasive aspergillosis in immunocompromised individuals. Current detection and treatment strategies for invasive aspergillosis, as well as other invasive fungal infections, are poor. Iron has been shown to be essential for Aspergillus fumigatus virulence. Furthermore, mechanisms in the iron regulatory network are believed to be potential drug targets since iron management in fungi is vastly different from that in mammals and other eukaryotes. Therefore a better understanding of iron homeostasis in Aspergillus fumigatus could help improve drug therapies for invasive aspergillosis. In this research a discrete model of iron uptake, storage and utilization in Aspergillus fumigatus with particular focus on siderophore-mediated iron acquisition is constructed. The model predicts oscillations in gene expression as the fungus adapts to a switch from an iron depleted to an iron replete environment. The model is validated via in vitro experiments. / Master of Science

Mycoplasma hominis est une bactérie opportuniste qui peut être responsable d’infections du tractus urogénital, d’infections néonatales ou d’infections disséminées notamment chez les patients immunodéprimés. La membrane des mycoplasmes constitue l’interface d’interaction directe avec le milieu extérieur en raison de l’absence de paroi. Cette membrane contient de nombreuses lipoprotéines qui ont le pouvoir d’activer des cellules dendritiques humaines (hDCs), d’induire la production de cytokines et de polariser le système immunitaire adaptatif. Nous avons étudié l’interaction de M. hominis PG21 avec les hDCs en nous penchant d’une part sur la fraction du mycoplasme qui active les hDCs et d’autre part sur la réponse immunitaire innée des hDCs. Apres avoir déterminé les lipoprotéines contenues dans un extrait TX-114 de M. hominis PG21, nous avons enrichi en lipoprotéines bioactives une fraction de vésicules membranaires du mycoplasme par une double extraction utilisant deux détergents non dénaturants, le Sarkosyl puis le Triton X-114. Apres séparation par SDS-PAGE, nous avons identifié vingt lipoprotéines qui pourraient entrainer la sécrétion d’IL-23 par les hDCs, notamment la lipoprotéine MHO_4720. Un lipopeptide synthétique correspondant à la fraction N-terminale de MHO_4720 est capable de stimuler les hDCs. En analysant les variations transcriptionnelles des gènes codant pour les 48 lipoprotéines de M. hominis PG21 par qRT-PCR, nous avons également déterminé que 21 lipoprotéines sont surexprimées après 4h ou 24h de contact entre le mycoplasme et les hDCs. Enfin, la réponse cellulaire a été évaluée par PCR array et ELISA. Nous avons observé l’activation d’inflammasome(s) par la mise en évidence de la production d’IL-1β dépendant de la caspase 5. / Mycoplasma hominis is involved in urogenital tract infections, neonatal infections or disseminated infections particularly in immunocompromised patients. Mycoplasmas have no cell wall and their membrane is the main interface mediating the interaction between the mycoplasma and its environment. Lipoproteins that are anchored to the extracellular side of the plasma membrane are known to induce the maturation of human dendritic cells (hDCs), to stimulate the pro-inflammatory cytokine production by hDCs and to polarize the adaptive immune system. We studied the interaction of M. hominis PG21 with hDCs in order to assess the lipoproteins that can induce the stimulation of hDCs, to determine the lipoproteins that are regulated upon interaction of the mycoplasma with the host cell and to evaluate the innate host cell response. Using a double extraction strategy with two non-denaturing detergents, Sarkosyl then Triton X-114, and separation by SDS-PAGE, we found that 20 lipoproteins may induce the secretion of IL-23 by the hDCs, especially the MHO_4720 lipoprotein. We showed that a synthetic lipopeptide corresponding to the N-terminus part of the MHO_4720 lipoprotein can stimulate the hDCs in a dose-dependent manner. Using qRT-PCR for the evaluation of the transcriptional regulation of the 48 lipoprotein-coding genes of M. hominis PG21, we also determined that 21 lipoproteins were upregulated upon 4h and 24h of contact of M. hominis with hDCs. Finally, the hDC innate immune response was evaluated by PCR array and ELISA. We observed a caspase 5-dependent production of IL- 1β corresponding to the activation of an inflammasome.