Escherichia coli patogênicas aviárias (APEC) causam infecções extraintestinais em frangos conhecidas como colibacilose. A APEC MT78, ao contrário de outras linhagens APEC, foi capaz de invadir células não-fagocitárias no modelo de fibroblastos aviários (CEC-32). Considerando que as interações patógeno-hospedeiro envolvem modificações na abundância de proteínas e padrões de expressão, principalmente nas proteínas de superfície, nosso objetivo foi comparar o proteoma da MT78 crescida em meio de cultura celular com o proteoma de MT78 isolada de fibroblastos aviários infectados (condição de co-cultura). Desenvolvemos aqui a padronização das etapas de extração de proteínas totais, isolamento de células bacterianas do co-cultivo e análise proteômica de modo a obtermos uma análise proteômica global reprodutível e de qualidade. A análise da interação APEC MT78 e células CEC-32 por microscopia óptica e eletrônica de varredura revelou que essa cepa se associa à célula-alvo em um padrão de adesão localizada. A internalização de APEC MT78 pareceu ocorrer como resultado de uma interação entre bactéria-célula que dispara rearranjos do citoesqueleto de actina da célula-alvo, formando estruturas filo e lamelipodiais que são dependentes da viabilidade bacteriana. O reisolamento de células bacterianas intactas, observadas por microscopia eletrônica de transmissão, após o co-cultivo com CEC-32 foi obtido através da técnica de solubilização diferencial de membranas. As células bacterianas foram sonicadas e as proteínas digeridas em solução seguida de uma etapa de purificação. Nós identificamos 69 proteínas, distribuídas em 9 classes funcionais, incluindo as proteínas de membrana FimA, OmpA and OmpC. A proteína OmpA já foi associada a invasão do patógeno humano NMEC (neonatal meningitis-associated E. coli) à células HBMEC. Esses experimentos representam a primeira investigação proteômica global em E. coli patogênica aviária. As proteínas identificadas representaram diferentes rotas metabólicas, funções fisiológicas e diferentes localizações subcelulares. / In poultry, Avian Pathogenic Escherichia coli (APEC) cause localized extra- intestinal infections that often become systemic. APEC strain MT78 was able to invade non-phagocytic avian fibroblasts in vitro, raising the possibility that some APEC strains may invade epithelial cells and gain systemic access. Using light microscopy and scanning electron microscopy, we observed that viable MT78 strain associated with CEC-32 fibroblasts cells in clusters, and following association, MT78 internalization appeared to result from cytoskeleton rearrangements, such as filopodia and lamellipodia, in the eukaryotic membrane. Considering that host-pathogen interactions involve modifications of protein abundance and expression, mainly in surface proteins, we compared the proteome of MT78 harvested from culture medium with the proteome of MT78 isolated from infected avian fibroblasts (co-culture condition). For this purpose, we developed standard analytical procedures for global protein extraction and isolation of bacterial cells from infected CEC-32. Judged by transmission electron microscopy, we successfully reisolated intact APEC MT78 cells from CEC-32 fibroblasts using the differential membrane solubilization method. Bacterial cells were then sonicated and proteins digested in solution following a clean up procedure. We identified 69 proteins, distributed in 9 functional classes, including the membrane proteins FimA, OmpA and OmpC. The OmpA protein was already associated to invasion of the human pathogen called NMEC (neonatal meningitis-associated E. coli) to endothelial cell line HBMEC. Our results represent the first global proteomic investigation in APEC. The proteome of MT78 infecting avian fibroblasts may allow us to identify key proteins linked to the successful adhesion and/or invasion of host cells by APEC and thus throw light into the pathogenesis of avian colibacillosis.

Orientador: Rogério de Castilho Jacinto / Banca: João Eduardo Gomes Filho / Banca: Paulo Carvalho Tobias Duarte / Resumo: Este estudo teve como objetivo comparar quantitativamente a diferença de expressão proteica na progressão da patogênese pulpar, bem como correlacionar as funções biológicas das proteínas identificadas no tecido pulpar normal, inflamado ou necrótico. As amostras foram obtidas de pacientes atendidos na Clínica Endodôntica da Faculdade de Odontologia de Araçatuba para tratamento endodôntico, sendo divididos em três grupos: grupo de polpa normal, com amostras do tecido pulpar obtidas a partir de dentes extraídos por indicação ortodôntica (n = 2); grupo de polpa inflamada, com amostras obtidas de pacientes com diagnóstico de pulpite irreversível (n = 2) e grupo de polpa necrótica, cujas amostras foram obtidas de pacientes com diagnóstico de periodontite apical crônica (n = 2). Após o preparo proteômico prévio, as amostras de polpa dentária foram processadas para análise proteômica quantitativa livre de marcadores em um sistema nanoACQUITY UPLC-Xevo QTof MS. A diferença na expressão entre os grupos de polpa normal e inflamada e grupos de polpa inflamada e necrótica foi calculada com o software Protein Lynx Global Service, usando o algoritmo Monte-Carlo, e expressa como p <0,05 para proteínas presentes em menor abundância e 1-p> 0,95 para proteínas presentes em maior abundância. Um total de 465 proteínas humanas foram identificadas em todos os grupos. Nos grupos normal, inflamado e necrótico, foram encontradas 241, 240 e 124 proteínas, respectivamente. Na análise quantitativa, as pr... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: This study aimed to quantitatively compare the difference in protein expression in the progression of pulp pathogenesis, as well as to correlate the biological functions of proteins identified in normal, inflamed or necrotic pulp tissue. The samples were obtained from patients treated at the Endodontic Clinic of the Araçatuba Dental School for endodontic treatment, and were divided into three groups: normal pulp group with pulp tissue samples obtained from orthodontic teeth (n = 2) ; inflamed pulp group, whose samples were obtained from patients diagnosed with irreversible pulpitis (n = 2) and necrotic pulp group, whose samples were obtained from patients diagnosed with chronic apical periodontitis (n = 2). After previous proteomic preparation, dental pulp samples were processed for label-free quantitative proteomic analysis in a nanoACQUITY UPLC-Xevo QTof MS system. The difference in expression between the normal and inflamed pulp groups and groups of inflamed and necrotic pulp was calculated using the Protein Lynx Global Service software using the Monte Carlo algorithm and expressed as p <0.05 for proteins present in lower abundance and 1-p> 0.95 for proteins present in greater abundance. A total of 465 human proteins were identified in all groups. In the normal, inflamed and necrotic groups, 241, 240 and 124 proteins were found, respectively. In the quantitative analysis, the most expressed proteins were hemoglobin, peroxiredoxins and immunoglobulins, whereas the less expressed were the tubulins in the inflamed pulp group in relation to the normal pulp group. Expression of albumins, immunoglobulins and alpha-2-macroglobulin were increased in the necrotic pulp group when compared to normal pulp, whereas hemoglobin and actin were less expressed. As for the qualitative analysis, the proteins identified in the normal... (Complete abstract electronic access below) / Mestre

Controlled Exposure Experiments revealed lesions in the statocysts of four cephalopod species of the Mediterranean Sea (Sepia officinalis, Loligo vulgaris, Illex coindetii and Octopus vulgaris), when exposed to relatively low intensity low frequency sounds. The analysis was performed through: scanning (SEM) and transmission (TEM) electron microscopy techniques of the whole inner structure of the cephalopod statocysts, especially on macula and crista; SEM of the epidermal lines of cephalopod hatchlings; and proteomic studies (2DE/MALDI –MS) of the statocyst’s endolymph. All exposed adult individuals presented the same lesions and the same incremental effects over time, consistent with a massive acoustic trauma observed in land species that were exposed to much higher intensities of sound. Immediately after exposure, the damage was observed in the macula statica princeps (msp) and in the crista sensory epithelium. Kinocilia on hair cells were either missing or were bent or flaccid. A number of hair cells showed protruding apical poles and ruptured lateral plasma membranes, most probably resulting from the extrusion of cytoplasmic material. Hair cells were also partially ejected from the sensory epithelium, and spherical holes corresponding to missing hair cells were visible in the epithelium. The cytoplasmic content of the damaged hair cells showed obvious changes, including the presence of numerous vacuoles and electron dense inclusions not seen in the control animals. The appearance of these lesions became gradually more pronounced in individuals after 12, 24, 48, 72, and 96 hours. Special attention was given to validate these findings with control animals that were caught, maintained and sequentially sacrificed following the same protocol as the exposed individuals. The statocyst ultrastructure was therefore revisited and a comparative analysis was carefully conducted to assess the lesions triggered by the exposure to noise This study also presents preliminary results of the sound effects on epidermal lines of cephalopod hatchlings. The lesions, consistent with an acoustic trauma, were identic in the three species that were exposed, but their evolution over time, in opposition with what was observed in the statocysts, were different, suggesting that the animal size and metabolic response might play a role in a possible recovery process. The analysis of noise effects in the statocyst endolymph by proteomic techniques was only conducted on Sepia officinalis. The presence of differential staining of gels from control and subjected to sound exposure individuals demonstrate that the injuries could be related to a possible physiological imbalance that would affect the protein levels of the endolymph. The lesions and findings described here are new to cephalopod pathology. Given that lowfrequency noise levels in the ocean are increasing (e.g. due to shipping, offshore industry, and naval maneuvers), that the role of cephalopods in marine ecosystems is only now beginning to be understood, and that reliable bioacoustic data on invertebrates are scarce, the present study and future investigations will bring an important contribution to the sustainable use of the marine environment. / Després de sotmetre'ls a experiments d'exposició controlada a sons de baixa intensitat i baixa freqüència es van observar lesions en els estatocists de quatre espècies de cefalòpodes de la mar Mediterrània (Sepia officinalis, Loligo vulgaris, Illex coindetii i Octopus vulgaris). L'anàlisi es va realitzar per mitjà de de microscòpia electronica d'escombratge (SEM) i de transmissió (TEM) de tota l'estructura interna de l'estatocist dels cefalòpodes, especialment en la màcula i en la crista, per SEM de les línees epidèrmiques de les larves dels cefalòpodes i per tècniques de proteòmica (2DE/MALDI-MS), de l'endolimfa de l'estatocist. Tots els estatocists d'individus adults de cefalòpodes exposats presentaven les mateixes lesions i aquests efectes eren més greus a mesura que passava el temps després de l'exposició als sons. Tots els animals exposats al soroll van mostrar lesions consistens amb trauma acústic massiu observat en altres espècies terrestres que havien estat exposades a intensitats molt més altes de so. Immediatament després de l'exposició, es van observar danys a la macula statica princeps (msp) i en l'epiteli sensorial de la crista. Els quinocilis de les cèl·lules ciliades desapareixien o es doblegaven i es tornaven flàccids. Un nombre important de cèl·lules ciliades mostraven els pols apicals sobresortint de l'epiteli sensorial, així com el trencament de les membranes plasmàtiques laterals, molt probablement com a resultat de l'extrusió de material citoplasmàtic. Les cèl·lules ciliades també van ser parcialment expulsades de l'epiteli sensorial deixant visibles forats esfèrics en el mateix. El contingut citoplasmàtic de les cèl·lules ciliades danyades va mostrar canvis obvis, com ara la presència de nombrosos vacúols i inclusions electrodenses que no es veien en els animals control. L'aparició d'aquestes lesions es va tornar gradualment més pronunciada en els individus analitzats després de 12, 24, 48, 72 i 96 hores. Es van validar curosament aquests resultats per mitjà de la comparació amb els animals control que van ser capturats, mantinguts i sacrificats de forma seqüencial seguint el mateix protocol que els individus exposats. La ultraestructura de l'estatocist va ser revisada i es va dur a terme un curós anàlisi comparatiu per tal d'avaluar les lesions provocades per l'exposició al soroll. Aquest estudi també presenta els resultats preliminars dels efectes del so en les línies epidèrmiques de cefalòpodes recent nascuts. Les lesions, consistens amb trauma acústic, eren idèntiques en les tres espècies que van ser exposades, però la seva evolució en el temps, en oposició amb el que es va observar en els estatocists, era diferent, cosa que suggereix que la grandària dels animals i la resposta metabòlica podria tenir influència en un possible procés de recuperació. L'avaluació dels efectes en l'endolimfa de l'estatocist per tècniques de proteòmica es va dur a terme només en Sepia officinalis. La presència de taques diferencials en els gels dels individus control i els sotmesos a exposició a so demostren que les lesions podrien estar relacionades amb un possible desequilibri fisiològic que tindria repercusions en els nivells proteics de l'endolimfa. Les lesions descrites aquí són noves pel que fa a la patologia dels cefalòpodes. Atès que els nivells de soroll de baixa freqüència a l'oceà estan augmentant (per exemple, a causa del transport, la indústria petrolera i les maniobres navals), que el paper dels cefalòpodes en els ecosistemes marins només ha començat a ser entès recentment, i que les dades bioacústiques fiables sobre els invertebrats són escasses, el present estudi i les investigacions futures aportaran una important contribució a l'ús sostenible del medi marí.

Hepatocyte growth factor (HGF) and its specific receptor MET play a role in many physiological functions including proliferation, migration and morphogenesis. Recently, research results in our laboratory showed that recombinant HGF variants (NK1, NK2, NK3 and NK4) became antagonists to HGF/MET pathway by suppressing proliferation, migration and invasion in human breast cancer cells (MDA-MB-435S, MDA). Similar results were achieved when HGF variants genes were introduced in MDA cells. To understand the molecular mechanism of breast cancer cells metastasis suppressed by HGF variants, MDA and five transfectants, including MDA-GFP, MDA-NK1, MDA-NK2, MDA-NK3 and MDA-NK4 cells were used for proteomic analysis using two-dimensional electrophoresis (2-DE). Differential analysis revealed that a total of 56 polypeptides were differentially expressed through five sets of comparison using wild-type MDA cells as a control. A total of 17 polypeptides were shown differential expression between MDA and MDA-GFP cells, with 11 down-regulated and 6 up-regulated. Eighteen polypeptides were differentially expressed between MDA and MDA-NK1 cells, with 15 down-regulated and 3 up-regulated. There were 22 differentially expressed polypeptides found between MDA and MDA-NK2 cells, in which 14 were down-regulated and 8 were up-regulated. Sixteen polypeptides were shown differentially expressed between MDA and MDA-NK3 cells, with 11 down-regulated and 5 up-regulated. A total of 18 polypeptides were shown differential expression between MDA and MDA-NK4 cells, with 15 down-regulated and 3 up-regulated. Proteomic analysis showed that a total of 43 polypeptides were differentially expressed through four sets of comparison (MDA-GFP and MDA-NK1, MDA-GFP and MDA-NK2, MDA-GFP and MDA-NK3, and MDA-GFP and MDA-NK4). To understand the differential expression among different HGF variants-transfected MDA cells, three sets of cross analysis were also carried out (MDA-NK1 and MDA-NK2, MDA-NK1 and MDA-NK3, and MDA-NK1 and MDA-NK4) and the results showed that a total of 37 differentially expressed polypeptides were found in the three sets of comparison. Similarly, when MDA-NK2 cells were used as a control to compare with MDA-NK3 and MDA-NK4 cells, 34 significantly differential expressed polypeptides were found. The last set of comparison between MDA-NK3 and MDA-NK4 cells, 19 polypeptides were found significantly differential expression. Therefore, our current results revealed that the differentially expressed polypeptides in MDA-MB-435S cells and HGF variants-transfected MDA cells could be related to the inhibition of proliferation and migration of human breast cancer cells by HGF variants.

Escherichia coli patogênicas aviárias (APEC) causam infecções extraintestinais em frangos conhecidas como colibacilose. A APEC MT78, ao contrário de outras linhagens APEC, foi capaz de invadir células não-fagocitárias no modelo de fibroblastos aviários (CEC-32). Considerando que as interações patógeno-hospedeiro envolvem modificações na abundância de proteínas e padrões de expressão, principalmente nas proteínas de superfície, nosso objetivo foi comparar o proteoma da MT78 crescida em meio de cultura celular com o proteoma de MT78 isolada de fibroblastos aviários infectados (condição de co-cultura). Desenvolvemos aqui a padronização das etapas de extração de proteínas totais, isolamento de células bacterianas do co-cultivo e análise proteômica de modo a obtermos uma análise proteômica global reprodutível e de qualidade. A análise da interação APEC MT78 e células CEC-32 por microscopia óptica e eletrônica de varredura revelou que essa cepa se associa à célula-alvo em um padrão de adesão localizada. A internalização de APEC MT78 pareceu ocorrer como resultado de uma interação entre bactéria-célula que dispara rearranjos do citoesqueleto de actina da célula-alvo, formando estruturas filo e lamelipodiais que são dependentes da viabilidade bacteriana. O reisolamento de células bacterianas intactas, observadas por microscopia eletrônica de transmissão, após o co-cultivo com CEC-32 foi obtido através da técnica de solubilização diferencial de membranas. As células bacterianas foram sonicadas e as proteínas digeridas em solução seguida de uma etapa de purificação. Nós identificamos 69 proteínas, distribuídas em 9 classes funcionais, incluindo as proteínas de membrana FimA, OmpA and OmpC. A proteína OmpA já foi associada a invasão do patógeno humano NMEC (neonatal meningitis-associated E. coli) à células HBMEC. Esses experimentos representam a primeira investigação proteômica global em E. coli patogênica aviária. As proteínas identificadas representaram diferentes rotas metabólicas, funções fisiológicas e diferentes localizações subcelulares. / In poultry, Avian Pathogenic Escherichia coli (APEC) cause localized extra- intestinal infections that often become systemic. APEC strain MT78 was able to invade non-phagocytic avian fibroblasts in vitro, raising the possibility that some APEC strains may invade epithelial cells and gain systemic access. Using light microscopy and scanning electron microscopy, we observed that viable MT78 strain associated with CEC-32 fibroblasts cells in clusters, and following association, MT78 internalization appeared to result from cytoskeleton rearrangements, such as filopodia and lamellipodia, in the eukaryotic membrane. Considering that host-pathogen interactions involve modifications of protein abundance and expression, mainly in surface proteins, we compared the proteome of MT78 harvested from culture medium with the proteome of MT78 isolated from infected avian fibroblasts (co-culture condition). For this purpose, we developed standard analytical procedures for global protein extraction and isolation of bacterial cells from infected CEC-32. Judged by transmission electron microscopy, we successfully reisolated intact APEC MT78 cells from CEC-32 fibroblasts using the differential membrane solubilization method. Bacterial cells were then sonicated and proteins digested in solution following a clean up procedure. We identified 69 proteins, distributed in 9 functional classes, including the membrane proteins FimA, OmpA and OmpC. The OmpA protein was already associated to invasion of the human pathogen called NMEC (neonatal meningitis-associated E. coli) to endothelial cell line HBMEC. Our results represent the first global proteomic investigation in APEC. The proteome of MT78 infecting avian fibroblasts may allow us to identify key proteins linked to the successful adhesion and/or invasion of host cells by APEC and thus throw light into the pathogenesis of avian colibacillosis.

Escherichia coli patogênicas aviárias (APEC) causam infecções extraintestinais em frangos conhecidas como colibacilose. A APEC MT78, ao contrário de outras linhagens APEC, foi capaz de invadir células não-fagocitárias no modelo de fibroblastos aviários (CEC-32). Considerando que as interações patógeno-hospedeiro envolvem modificações na abundância de proteínas e padrões de expressão, principalmente nas proteínas de superfície, nosso objetivo foi comparar o proteoma da MT78 crescida em meio de cultura celular com o proteoma de MT78 isolada de fibroblastos aviários infectados (condição de co-cultura). Desenvolvemos aqui a padronização das etapas de extração de proteínas totais, isolamento de células bacterianas do co-cultivo e análise proteômica de modo a obtermos uma análise proteômica global reprodutível e de qualidade. A análise da interação APEC MT78 e células CEC-32 por microscopia óptica e eletrônica de varredura revelou que essa cepa se associa à célula-alvo em um padrão de adesão localizada. A internalização de APEC MT78 pareceu ocorrer como resultado de uma interação entre bactéria-célula que dispara rearranjos do citoesqueleto de actina da célula-alvo, formando estruturas filo e lamelipodiais que são dependentes da viabilidade bacteriana. O reisolamento de células bacterianas intactas, observadas por microscopia eletrônica de transmissão, após o co-cultivo com CEC-32 foi obtido através da técnica de solubilização diferencial de membranas. As células bacterianas foram sonicadas e as proteínas digeridas em solução seguida de uma etapa de purificação. Nós identificamos 69 proteínas, distribuídas em 9 classes funcionais, incluindo as proteínas de membrana FimA, OmpA and OmpC. A proteína OmpA já foi associada a invasão do patógeno humano NMEC (neonatal meningitis-associated E. coli) à células HBMEC. Esses experimentos representam a primeira investigação proteômica global em E. coli patogênica aviária. As proteínas identificadas representaram diferentes rotas metabólicas, funções fisiológicas e diferentes localizações subcelulares. / In poultry, Avian Pathogenic Escherichia coli (APEC) cause localized extra- intestinal infections that often become systemic. APEC strain MT78 was able to invade non-phagocytic avian fibroblasts in vitro, raising the possibility that some APEC strains may invade epithelial cells and gain systemic access. Using light microscopy and scanning electron microscopy, we observed that viable MT78 strain associated with CEC-32 fibroblasts cells in clusters, and following association, MT78 internalization appeared to result from cytoskeleton rearrangements, such as filopodia and lamellipodia, in the eukaryotic membrane. Considering that host-pathogen interactions involve modifications of protein abundance and expression, mainly in surface proteins, we compared the proteome of MT78 harvested from culture medium with the proteome of MT78 isolated from infected avian fibroblasts (co-culture condition). For this purpose, we developed standard analytical procedures for global protein extraction and isolation of bacterial cells from infected CEC-32. Judged by transmission electron microscopy, we successfully reisolated intact APEC MT78 cells from CEC-32 fibroblasts using the differential membrane solubilization method. Bacterial cells were then sonicated and proteins digested in solution following a clean up procedure. We identified 69 proteins, distributed in 9 functional classes, including the membrane proteins FimA, OmpA and OmpC. The OmpA protein was already associated to invasion of the human pathogen called NMEC (neonatal meningitis-associated E. coli) to endothelial cell line HBMEC. Our results represent the first global proteomic investigation in APEC. The proteome of MT78 infecting avian fibroblasts may allow us to identify key proteins linked to the successful adhesion and/or invasion of host cells by APEC and thus throw light into the pathogenesis of avian colibacillosis.

O recente desenvolvimento de técnicas proteômicas tem permitido a análise do perfil proteico completo dos sistemas biológicos, permitindo um melhor entendimento da fisiologia normal do organismo, bem como dos mecanismos de doenças, descoberta de biomarcadores para detecção precoce de doenças, identificação de novas terapias e descobertas de fármacos. No presente trabalho, a análise proteômica foi usada para auxiliar no entendimento dos mecanismos moleculares envolvidos na intoxicação induzida pelo fluoretono fígado de ratos e definir biomarcadores potenciais de toxicidade. Três grupos de ratos Wistar machos recém-desmamados (21 dias de vida) foram tratados ad libitum com água de beber contendo 0 (controle), 5 ou 50 mg/L de fluoreto, por 60 dias (n=6/grupo). Os animais foram eutanasiados e o fígado e o soro foram coletados. O fígado foi dividido em lobos, sendo que o esquerdo foi destinado à dosagem de fluoreto (eletrodo íon-sensível, após difusão facilitada por hexametildisiloxano), o direito à análise histológica (hematoxilina e eosina) e o mediano, à análise proteômica (eletroforese bidimensional associada a LC-MS/MS). Os dados foram analisados por ANOVA e teste de Tukey (p<0,05). Foi possível detectar uma dose-resposta em relação à ingestão para os níveis de fluoreto presentes no soro e no fígado houve diferença significativa entre os grupos que receberam 5 e 50 mg/L de fluoreto. A análise morfométrica histológica não revelou alterações nas estruturas celulares e exame o morfológico indicou inclusões lipídicas nos grupos 5 mg/L e 50 mg/L de fluoreto, mais intensas no último. A análise quantitativa de intensidade (software ImageMaster 2D-Platinum v 7.0), comparando a porcentagem de alteração de volume do spot protéico, revelou que 33, 44 e 29 spots aumentaram ou diminuíram sua expressão nos grupos controle X 5 mg/L, controle X 50 mg/L e 5 mg/L X 50 mg/L de fluoreto, respectivamente. Além disto, foram encontradas 18, 1 e 5 spots exclusivos nos grupos controle, 5 mg/L e 50 mg/L de fluoreto, respectivamente. Dentre os spots proteínas com expressão diferencial, foram identificadas 94 proteínas (72,3%). Em síntese, a exposição ao fluoreto alterou a expressão a nível hepático de proteínas pertencentes a todas as categorias funcionais, com predominância daquelas relacionadas ao metabolismo, sendo que as alterações mais pronunciadas foram observadas no grupo de 50 mg/L de fluoreto. Proteínas que tiveram sua expressão ausente mediante exposição ao fluoreto ou que tiveram sua expressão induzida por este elemento são potenciais biomarcadores para este tipo de exposição e devem ser melhor investigadas. Uma vez que se trata do primeiro trabalho envolvendo análise proteômica de fígado de animais expostos a diferentes doses de fluoreto, estes achados apontam importantes vias e processos celulares afetados no fígado pela exposição ao fluoreto, os quais devem ser melhor analisados em estudos futuros. / The recent development of proteomic techniques allowed the analysis of the whole proteomic profile of the biological systems, allowing the comprehension of the normal physiology, as well as of the mechanisms underlying diseases. It has also made easier the discovery of biomarkers of diseases, as well as the identification of new therapies and drugs. In the present study, proteomic analysis was used as a tool to help understanding the molecular mechanisms underlying chronic fluoride toxicity in rat liver and also to define potential biomarkers of toxicity. Three groups of weanling male Wistarrats (21 days) were treated ad libitum with drinking water containing 0 (control), 5 or 50 mg/L fluoride for 60 days (n=6/group). After euthanasia, liver and serum were collected. The liver was divided into lobes. The left lobe was used for fluoride analysis (ion-sensitive electrode, after hexamethyldisiloxane-facilitated diffusion). The right lobe was used for histological analysis (hematoxylin and eosine) while the median was used for proteomic analysis (2D-PAGE associated with LC-MS/MS). Data were analysed by ANOVA and Tukeys test, (p<0.05). A dose-response relationship was observed for serum fluoride levels. In liver, a significant increase in fluoride levels was observed in the 50 mg/L group when compared with the 5 mg/L group. Morphometric analysis did not reveal alterations in the cellular structures. The morphological exam revealed macrovesicular lipid droplets in the 5 and 50 mg/L groups which were more intense in the later. Quantitative intensity analysis (software ImageMaster 2D-Platinum v 7.0) comparing the percentage of alteration of volume of protein spots revealed 33, 44 and 29 protein spots that had increase or decrease in expression in the groups control X 5 mg/L, control X 50 mg/L and 5 mg/L X 50 mg/L, respectively. In addition, 18, 1 and 5 protein spots were detected exclusively in groups control, 5 mg/L and 50 mg/L, respectively. From the differentially expressed spots, 94 proteins were satisfactorily identified (72.3%). Briefly, exposure to fluoride altered the expression of liver proteins belonging to all the functional categories, especially those related to metabolism. More pronounced alterations were seen for the 50 mg/L group. Proteins that were not detected upon exposure to fluoride or that had their expression induced in the presence of fluoride are potential biomarkers of toxicity and should be better investigated. Since this is the first study involving proteomic analysis of liver in rats exposed to different doses of fluoride, these findings indicate important pathways and cellular processes affected by exposure to this element that should be addressed in details in future studies.

O flúor é comprovadamente um agente terapêutico contra a cárie dentária, porém, quando consumido de maneira inadequada pode causar reações indesejáveis, tanto crônicas como agudas. A proteômica é uma ferramenta que permite analisar o perfil proteico completo dos sistemas biológicos, permitindo um melhor entendimento da fisiologia normal do organismo, bem como dos mecanismos de doenças, descoberta de biomarcadores para detecção precoce de doenças, identificação de novas terapias e descobertas de fármacos. Assim, o presente estudo teve como trabalho realizar um estudo proteômico diferencial em amostras de tecido renal de ratos submetidos à intoxicação aguda por fluoreto. Para isso, 27 animais, receberam desde o desmame água deionizada e ração AIN-93 por 50 dias. Ao completarem 75 dias de vida, e, após jejum por 12 horas, os animais receberam via gástrica as seguintes doses agudas de F: 0 (controle), 50 e 100 mgF/Kg peso corporal. Após 2 horas foram, os animais foram anestesiados e tiveram o sangue e os rins coletados. Inicialmente foi realizada a dosagem de flúor no plasma e tecidos renais, após difusão facilitada por hexamethyldisiloxano. Em um segundo momento, as proteínas do tecido renal foram extraídas e submetidas à eletroforese bidimensional. Os géis obtidos foram analisados através do software ImageMaster 2D-platinum v. 7.0. Os spots que se apresentaram diferencialmente expressos foram submetidos à identificação por espectrometria de massas. As proteínas identificadas foram classificadas em 5 categorias funcionais. A categoria metabolismo e energia reuniu a maior parte das proteínas (40%), seguida pelas categorias de transporte e processos celulares com 20% e 13% respectivamente. Na categoria estrutura e organização estrutural, que reúne proteínas com funções relacionadas ao citoesqueleto e membrana celular, foram identificadas 17% das proteínas. Por fim, os 10% restantes das proteínas pertenciam à categoria vias de informação, que abriga proteínas envolvidas nos processos de síntese/degradação de DNA e RNA. Com base nos resultados obtidos pode se concluir que doses agudas de fluoreto alteram a expressão a nível renal de diversas proteínas relacionadas a distintos processos celulares. / Fluoride has been widely used in dentistry as a caries prophylactic agent. However, if consumed in high concentrations, it can cause side effects as dental fluorosis, skeletal fluorosis and, in extreme situations, death. The recent development of proteomic techniques has allowed the analysis of the entire protein profile of biological systems, contributing to the understanding of the normal physiology of the organism, as well as the mechanisms of diseases and the investigation of biomarkers for their early detection, identification of new therapies and drugs. This study used proteomic techniques to analyze the differential protein expression in kidney of rats submitted to acute fluoride treatments. Three groups of five 75-day-old Wistar rats received, by gastric gavage, the following single doses of fluoride: 0 (control), 50 and 100 mgF/Kg body weight. After the treatments, the animals were killed and the left kidney and plasma were collected for fluoride analysis. For proteomic analysis, the right kidney was collected. Fluoride in plasma and renal tissues was analyzed with the electrode, after hexamethyldisiloxanefacilitated diffusion. Proteins from kidney were profiled by two-dimensional gel electrophoresis. Gels from control and treated groups were digitalized using the ImageScanner III and analyzed with ImageMaster 2D Platinum version 7.0 for statistical differences (ANOVA, p <0.05). The proteins identified were classified into five functional categories. The category of metabolism and energy had the majority of the proteins (40%), followed by the categories of \"transport\" and cell processes, with 20% and 13% of the proteins, respectively. In the category \"structure and cell organization, which brings together proteins with functions related to the cytoskeleton and plasma membrane were identified 17% of the protein. Finally, 10% of the proteins belonged to the category \"information pathways\", which comprises the proteins involved in the processes of synthesis and degradation of DNA and RNA. Based on the results obtained, it can be concluded that acute doses of fluoride alters the expression of several proteins related to different cellular processes in the kidneys.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第20440号 / 農博第2225号 / 新制||農||1049(附属図書館) / 学位論文||H29||N5061(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 植田 充美, 教授 小川 順, 教授 渡邊 隆司 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Grâce à la spectrométrie de masse de type MALDI-TOF, l'identification des bactéries est maintenant possible en quelques minutes. Mais le taux de mortalité des patients augmente lorsqu'une antibiothérapie inappropriée est utilisée et les instruments MALDI-TOF ne sont pas capables d'analyser rapidement et exhaustivement la résistance bactérienne. Actuellement, 6 à 24 heures sont nécessaires pour déterminer le phénotype de résistance. En couplant une chromatographie liquide et un spectromètre de masse à ionisation électrospray (LC-MS/MS), nous avons identifié les marqueurs de résistance en 1 à 2 heures. En 30 min, nous avons pu détecter les mécanismes de résistance aux β-lactamines, aux glycopeptides, à la méthicilline et aux fluoroquinolones, à l'aide de méthodes de type "Suivi de Réactions Sélectionnées", ou "Selected Reaction Monitoring" (SRM). Au cours de la même analyse multiplexée, des dizaines de protéines peuvent être détectées de façon hautement spécifique et sensible. Comme l'illustre l'étude de la résistance multifactorielle chez Acinetobacter baumannii, cette approche permet en outre une analyse quantitative d'un grand intérêt pour certains mécanismes de résistance. Cependant, malgré ces perspectives attrayantes, la LC-MS/MS reste, aujourd'hui, loin d'une possible implantation en routine dans les laboratoires de microbiologie. Les instruments sont trop coûteux et la technologie trop complexe pour un usage pour du diagnostic in vitro. La spectrométrie de masse pourrait déjà avantageusement compléter les technologies actuelles de biologie moléculaire. Aujourd'hui, le séquençage de nouvelle génération est la méthode de référence pour la caractérisation moléculaire des bactéries. Mais, comme démontré dans ce travail, l'annotation des gènes est perfectible. Pour quelques dizaines d'euros et quelques heures d'analyse, les peptides identifies par spectrométrie de masse facilitent l'assemblage des séquences (« scaffolds ») et la détection des gènes. De surcroît, la spectrométrie de masse permet une quantification précise des protéines. Elle apporte ainsi une nouvelle dimension analytique et une vision moléculaire plus proche du phénotype. En conclusion, la spectrométrie de masse LC-MS/MS peut être une technologie complémentaire attractive, voir une future alternative, à la biologie moléculaire pour la caractérisation des bactéries / Thanks to MALDI-TOF mass spectrometry, identification of isolated bacteria is now possible within a few minutes. But doctors also need to rapidly know the phenotype of resistance of the bacteria. Indeed, the patient mortality rate increases when the antibiotherapy is not appropriate. However, MALDI-TOF instruments are not able to analyze antibacterial resistance rapidly and comprehensively.Today, 6 to 24 hours are nedded for antibiotic susceptibility testing. When combining a liquid chromatography and a mass spectrometer with electrospray ionization (LC-MSMS), the detection of resistance biomarkers was possible within 1 to 2 hours. Using a Selected Reaction Monitoring (SRM) method, resistance mechanisms to beta-lactams, methicillin, glycopeptides and fluoroquinolones were detected in strains within 30 minutes. Tens of resistance determinants can be analyzed in a single multiplexed assay, with high specificity and sensitivity. Illustrated by the study of multifactorial resistance in Acinetobacter baumannii, the technology allows furthermore a quantitative analysis, which is of great value for some resistance mechanisms. Similarly, we identified virulent strains of enterohemorrhagic Escherichia coli by targeting toxins and serotype biomakers in the same assay. Mass spectrometry offered deeper bacterial characterization than conventional serotyping using polyclonal antibodies. However, despite all these favorable prospects, LC-MS/MS remains today far from reaching a routine use in microbiological hospital laboratories. Instruments are too expensive and the technology is too cumbersome for a daily in vitro diagnostic use. Waiting for a more suitable use, mass spectrometry could yet advantageously complement current molecular technologies. Today, the gold standard to study bacteria at molecular level is next generation sequencing. However, as demonstrated during this work, gene annotation remains imperfect. For tens of euros and few hours of analysis, peptides identified by mass spectrometry analysis of a bacteria might improve scaffold assembly and gene detection. Moreover, mass spectrometry gives an accurate protein quantitation and brings a new analytical dimension, potentially closer to the phenotype than molecular techniques. In conclusion, LC-MS/MS mass spectrometry could be an attractive complementary, or alternative technology in a near future, to conventional molecular biology techniques for deep characterization of bacteria