Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet. / Härtill 5 uppsatser.

abstract: Legionella pneumophila is a waterborne pathogen that causes Legionnaires' disease, an infection which can lead to potentially fatal pneumonia. In a culture-based technique, Legionella is detected using buffered charcoal-yeast extract (BCYE) agar supplemented with L-cysteine, Iron salt and antibiotics. These supplements provide essential and complex nutrient requirements and help in the suppression of non-target bacteria in Legionella analysis. Legionella occurs naturally in freshwater environments and for their detection; a sample is plated on solid agar media and then incubated for several days. There are many challenges in the detection of Legionella in environmental waters and the built environments. A common challenge is that a variety of environmental bacteria can be presumptively identified as Legionella using the culture-based method. In addition, proper identification of Legionella requires long incubation period (3-9 days) while antibiotics used in BCYE agar have relatively short half-life time. In order to overcome some of the challenges, Legionella has been genetically modified to express reporter genes such Green Fluorescent Protein (GFP) that can facilitate its detection in process validation studies under controlled laboratory conditions. However, such studies had limited success due to the instability of genetically modified Legionella strains. The development of a genetically modified Legionella with a much rapid growth rate (1-2 days) in simulated environmental systems (tightly-controlled water distribution system) is achieved. The mutant Legionella is engineered by transforming with a specific plasmid encoding CymR, LacZ and TetR genes. The newly engineered Legionella can grow on conventional BCYE agar media without L-Cysteine, Iron salt and only require one antibiotic (Tetracycline) to suppress the growth of other microorganisms in media. To the best of our knowledge, this is the first report of L. pneumophila strain capable of growing without L-Cysteine. We believe that this discovery would not only facilitate the study of the fate and transport of this pathogen in environmental systems, but also further our understanding of the genetics and metabolic pathways of Legionella. / Dissertation/Thesis / Masters Thesis Civil, Environmental and Sustainable Engineering 2018

L’angioedème (AO) à kinine est caractérisé par la survenue spontanée et récurrente d’oedèmes des tissus sous cutanés et sous muqueux, conséquence de l’accumulation des kinines sur l’endothélium vasculaire. La sévérité des AO dépend de leur localisation, ils sont déformants sur les tissus sous cutanés, douloureux au niveau de la muqueuse digestive et peuvent mettre en jeu le pronostic vital s’ils affectent le larynx. L’AO à kinine a d’abord été associé au déficit pour C1 Inhibiteur (C1Inh) puis des formes sans déficit pour C1Inh ont été décrites. L’AO est décrit comme une maladie multifactorielle pour laquelle l’ensemble des facteurs décisionnels n’est pas encore identifié. Ce travail a permis (1) de définir des paramètres stratégiques de la production des kinines pour l’AO, (2) d’identifier les paramètres impliqués et décisionnels pour la survenue des crises et leur sévérité, (3) de repérer des phénotypes biologiques des sujets atteints d’AO. / Kinin mediated angioedema (AO) is characterized by spontaneous and recurrent oedema affecting subcutaneous and submucosal tissue. Oedemas develop subsequently to kinin accumulation on vascular endothelium. AO severity is depending on localisation, they are warping on subcutaneous tissue, painful on digestive mucous and life treating when affected the larynx. Kinin mediated AO was first associated with C1 Inhibitor (C1Inh) deficiency and thereafter AO without C1Inh deficiency has been described. AO is a multifactorial disease for which all the decision-making factor(s) is(are) not yet identified. As results of this work, have emerged (1) a characterization of strategic parameters of the kinin production for AO, (2) an identification of significant and decision-making parameters for the attack onset and severity, (3) a cover of biological phenotypes of the AO patients

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The objectives of this study were to characterize root-knot and lesion nematode populations of sugar cane fields of the Rio Grande do Sul state, and to evaluate the reaction of sugar cane genotypes to Meloidogyne javanica and Pratylenchus zeae. The Meloidogyne spp. populations were characterized biochemically by esterase (Est), and morphologically by the configuration of perineal region of females. The Pratylenchus populations were identified by morphological and morphometric characters. Subsequently, the reaction of ten sugar cane cultivars to M. javanica and P. zeae was evaluated at greenhouse conditions. Ninety Meloidogyne sp. populations were detected of in 67.17% of collected samples and 12 esterastic phenotypes (Est) were identified. Among the root-knot nematode populations purified and identified, 32 of them presented the Est J3 phenotype (Rm: 1.00; 1.24; 1.40), 51 populations, Est J2 (Rm: 1.00; 1.23), and four populations the phenotype Est J2a (Rm : 1.00; 1.40), typical of M. javanica, that corresponded to 19.75%, 31.48% and 2.47% of the samples, respectively. Four populations of M. incognita Est I1 (Rm: 1.02), and 25 of M. incognita Est I2 (Rm: 1.02; 1:09) phenotypes, corresponding to 2.47% and 15.43% of Meloidogyne spp. samples, respectively, were also detected and identified. In addition, 12 populations were identified as M. arenaria Est A2 (Rm: 1.26, 1.32), three populations of M. ethiopica Est E3 (Rm: Rm: 0.92; 1.15; 1.32), three of M. hapla Est. H1 (Rm: 1.17), and just one of M. luci (Rm: 1.00; 1.16; 1.34), corresponding to 7.41, 1.85, 1.85 and 0.62% of the root-knot nematode populations, respectively. Among the atypical species detected in this study, six populations of Meloidogyne sp.1 were identified with the phenotype Est Sc 1 (Rm: 0.86; 0.94), 12 Meloidogyne sp.2 as Est Sc 2 (Rm: 1.05; 1.29), and nine Meloidogyne sp.3 as Est A1 (Rm: 1.26), corresponding to 4.47, 9.05, and 6.71% of the root-knot nematodepopulations, respectively. However, by the perineal configurations, these populations could not be identified. Pratylenchus spp. was detectedin 98,8% of the collected samples. In the samples morphologica and morphometrically characterized to species of Pratylenchus, 51 populations were identified as P. zeae (84.61%) and 23 populations as P. brachyurus (35.38%). Evaluating the resistance reaction of sugar cane genotypes to both nematodes, although M. javanica and P. zeae have showed FR> 1.00 for all tested genetic materials, it was verified different levels of susceptibility. However, lower M. javanica reproduction was observed in RB008347, RB877935, RB975944, and 'RB987932; and either for P. zeae in RB987932 and RB966928 genotypes. / Teve-se por objetivo neste estudo caracterizar as populações do nematoide das galhas e das lesões em um levantamento realizado em lavouras de cana-de-açúcar do Estado do Rio Grande do Sul; e, avaliar a reação de genótipos da cultura as espécies Meloidogyne javanica e Pratylenchus zeae. As populaçõesde Meloidogyne spp. obtidas foram caracterizadas bioquimicamente através da isoenzima esterase (Est), e morfologicamente através da configuração da região perineal das fêmeas. As populações de Pratylenchus foram identificadas por meio de caracteres morfológicos e morfométricos. Posteriormente, avaliou-se em casa de vegetação a reação de 10 genótipos de cana-de-açúcar a M. javanica e P. zeae. Detectou-se 90 populações de Meloidogyne spp. em 67,17% das amostras coletadas e identificou-se 12 fenótipos esterásticos (Est). Entre as populações do nematoide das galhas purificadas e identificadas, 32 apresentaramo fenótipo Est J3 (Rm: 1.00; 1.24; 1.40), 51 populações, Est J2 (Rm: 1,00; 1,23), e, quatro com o fenótipo Est J2a (Rm: 1.00; 1.40), tipicas de M. javanica, as quais corresponderam a 19,75%, 31,48% e 2,47%, respectivamente. Também foram detectadas e identificadas quatro populações de M. incognita Est I1 (Rm: 1.02) e 25 com o fenótipo Est I2 (Rm: 1.02; 1.09), as quais corresponderam a 2,47 % e 15,43% das amostras de Meloidogyne spp., respectivamente. Além disso, foram identificadas 12 populações de M. arenaria Est A2 (Rm: 1.26; 1.32), três de M. ethiopica Est E3 (Rm: 0.92, 1.15, 1.32), três de M. hapla Est H1 (Rm: 1.17), e uma de M. luci (Rm: 1.00; 1.16; 1.34) que corresponderam a 7,41, 1,85, 1,85 e 0,62% das populações encontradas, respectivamente. Entre as populações atípicas, foram detectadas seis populações de Meloidogyne sp.1 com o fenótipo Est Sc 1 (Rm: 0.86; 0.94), 12 de Meloidogyne sp.2 Est Sc 2 (Rm: 1.05; 1.29) e nove de Meloidogyne sp.3 Est A1 (Rm: 1.26), correspondendo a 4,47, 9,05, e 6,71% das populações do nematoide das galhas, respectivamente; no entanto, pelas configurações perineais,essas populações não puderam ser identificadas. Detectou-se Pratylenchus spp. em 98,8% das amostras coletadas. Nas amostras caracterizadas morfologicamente quanto às espécies do gênero Pratylenchus, foram identificadas 51 populações de P.zeae (84,61%)e 23 populações de P. brachyurus (35,38%). Na avaliação da reação da cana-de-açúcar aos dois nematoides, embora M. javanica e P. zeae tenham apresentado FR>1,00 em todos os genótipos testados, observou-se diferentes níveis de suscetibilidade; porém, menor reprodução de M. javanicafoi observada nos genótipos RB008347, RB877935, RB975944 e RB987932; e de P. zeae em RB987932 e RB966928.

Made available in DSpace on 2015-03-26T12:24:56Z (GMT). No. of bitstreams: 1 texto completo.pdf: 2107014 bytes, checksum: a646700aff1c3e6a44a344a0051aa176 (MD5) Previous issue date: 2009-12-09 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Polyphenols found in mango pulp, including gallotannins, flavonol glycosides, gallic acid, benzophenone derivatives and mangiferin have shown anticancer activity. Biological activities of polyphenols have been related to their bioavailability. Deglycosylation by β-glucosidases is a critical step in the metabolism and absorption of dietary polyphenols in humans, which might influence their anticancer properties. The objective of this study was to elucidate the anti-cancer effects of mango polyphenols of several varieties (Francis, Kent, Ataulfo, Tommy Atkins and Haden) in different types of cancer. The antiproliferative effects of mango polyphenols were studied in vitro using different cancer cell lines including Molt-4 leukemia, A-549 lung, MDA-MB-231 breast, LnCap prostate, SW-480 colon cancer cells and the non-cancer colon cell line CCD-18Co. Molecular mechanisms involved on the anti-cancer activities of mango polyphenols were assessed. The effect of mango polyphenols on gene expression, cell cycle regulation and reactive oxygen species production on colon cancer cells SW-480 were investigated by RT-PCR, flow cytometry and fluorescence intensity measurement, respectively. The effect of the hydrolysis of mango polyphenols with β-glucosidase on their antioxidant activity, cancer cell-growth suppression activity and in vitro intestinal absorption through human colon adenocarcinoma Caco-2 cell monolayers was evaluated. In addition, the antiproliferative effect of high and low molecular weight polyphenols rich-fractions on colon (SW-480) and breast (MDA-MB-231) cancer cells was studied. Cell lines were incubated with Ataulfo and Haden phenolic extracts, which were selected based on their superior antioxidant capacity compared to the other varieties. Ataulfo and Haden polyphenols inhibited the growth of all human cancer cell lines. SW-480 (colon cancer), MOLT-4 (leukemia) and MDA-MB-231 (breast-cancer) were statiscally equally most sensitive to Ataulfo, whereas SW-480 and MOLT-4 were the most sensitive cell lines to Haden, as determined by cell counting. The efficacy of phenolic extracts from all mango varieties in inhibiting cell growth was tested on SW- 480 colon carcinoma cells. Ataulfo and Haden demonstrated superior efficacy, followed by Kent, Francis and Tommy Atkins. At 5 mg GAE/L, Ataulfo inhibited the growth of colon SW-480 cancer cells by ~79% while the growth of non-cancer colonic myofibroblasts CCD-18Co cells was not inhibited. The growth inhibition exerted by Ataulfo and Haden polyphenols on SW-480 cells was associated with an increased mRNA expression of pro-apoptotic biomarkers (caspase 8, Bax and Bim) and cell cycle regulators (PKMYT1), cell cycle arrest and an alteration in the generation of reactive oxygen species. Phenolic extracts from mango pulp contained gallic acid, mangiferin, phenolic acid derivatives and gallotannins, which were characterized by HPLC-DAD and HPLC-ESI-MSn analysis before and after enzymatic hydrolysis (0.17 mg β- glucosidase 1000 KU/g mango pulp/ 4 h / 35°C). Phenolic acids including gallic, caffeic, ferulic, p-coumaric and p-hydroxybenzoic acids consisted the main compounds derived from enzymatic hydrolysis. Caco-2 cell monolayers were incubated for 2h on the apical side with hydrolyzed and non-hydrolyzed mango extracts. Gallic, caffeic, ferulic, p-coumaric, vanillic and p-hydroxybenzoic acids were detected on the basolateral side for hydrolyzed extract but only gallic acid was detected for the nonhydrolyzed extract. High molecular weight polyphenols, mangiferin and gallotannins, were not transported. Mango pulp polyphenols (control) from all varieties inhibited the proliferation of HT-29 colon (0-27 μg of gallic acid equiv/mL) and MDA-MB-231 breast (0-24 μg GAE/ mL) human cancer cells by up to 99.8 and 89.9 %, respectively. Despite enhanced absorption facilitated by enzymatic hydrolysis, a significant increase in antioxidant activity, phenolic content and antiproliferative effects on breast and colon cancer cells was not observed. Additionally, both high (422; 788-1852 Da) and low (138-194 Da) molecular weight polyphenols rich-fractions equally inhibited cell proliferation of colon and breast cancer cells at the same extent (0-20 μg of gallic acid equiv/mL), which may indicate that the anti-cancer efficacy of mango polyphenolics is not dependent on enzymatic hydrolysis. These results corroborate previous findings from in vivo studies, which suggest that the most of mango polyphenols are not absorbed intact through the small intestine, but may be hydrolyzed by intestinal enzymes into low molecular weight aromatic acids, which would be later absorbed; or when polyphenols are not absorbed, they likely reach the large intestine, modulating the gut microflora, and thus they contribute to reduce the risk of colon carcinogenesis. Overall, polyphenols from several mango varieties exerted anti-cancer effects, and these effects may not require enzymatic hydrolysis by β-glucosidase. / Polifenóis presentes em polpa de manga, incluindo galotaninos, glicosídeos de flavonóides, ácido gálico, derivados da benzofenona, e mangiferina, têm demonstrado propriedades anticarcinogênicas. A etapa de deglicosilação por β-glicosidases tem se mostrado necessária ao metabolismo e à absorção de polifenóis derivados da dieta pelo organismo humano, o que poderia influenciar suas propriedades anticarcinogênicas. O objetivo deste estudo foi elucidar os efeitos anticarcinogênicos de polifenóis extraídos da polpa de manga de diferentes variedades (Francis, Kent, Ataulfo, Tommy Atkins e Haden) em diferentes tipos de câncer. Os efeitos antiproliferativos de polifenóis de manga foram estudados utilizando modelos in vitro de cultura de células cancerosas, incluindo as linhagens celulares de câncer humano Molt-4 (leucemia), A-549 (câncer de pulmao), MDA-MB-231 (câncer de mama), LnCap (câncer de próstata), SW-480 (câncer de colón) e células de colón não cancerosas CCD-18Co. Os mecanismos moleculares envolvidos nas propriedades anticarcinogênicas de polifenóis de manga foram investigados. O efeito do tratamento com polifenóis na expressão gênica, na regulação do ciclo celular e na produção de espécies reativas de oxigênio em células cancerosas de colón humano SW-480 foi investigado por RT-PCR, citometria de fluxo e quantificação da intensidade de fluorescência, respectivamente. Além disso, o efeito da hidrólise de polifenóis de manga pela enzima β-glicosidase na atividade antioxidante, na supressão do crescimento tumoral e na absorção intestinal in vitro através da monocamada de células de adenocarcinoma de colón humano Caco-2 foi avaliado. Ademais, o efeito antiproliferativo de frações fenólicas enriquecidas com polifenóis de baixo e elevado peso molecular em células cancerosas de colón (SW-480) e mama (MDA-MB-231) foi estudado. Células cancerosas foram tratadas com extratos fenólicos das variedades Ataulfo e Haden, as quais foram selecionadas em razão da maior capacidade antioxidante quando comparada a outras variedades. Polifenóis de Ataulfo e Haden inibiram o crescimento de todas as linhagens celulares. SW-480 (câncer de cólon), MOLT-4 (leucemia) e MDA-MB-231 (câncer de mama) apresentaram igualmente maior sensibilidade ao tratamento com polifenóis de Ataulfo, enquanto SW- 480 e MOLT-4 mostraram-se mais sensíveis ao tratamento com polifenóis de Haden, segundo resultados obtidos por contagem de células. O efeito antiproliferativo dos extratos fenólicos de todas as variedades de manga foi avaliado em células cancerosas de colón humano (SW-480). As variedades Ataulfo e Haden demonstraram maior efeito supressor, seguidas de Kent, Francis e Tommy Atkins. Quando células cancerosas de colón SW-480 foram tratadas com 5 mg GAE/L de polifenóis de Ataulfo, o crescimento celular foi inibido em ~79%, enquanto a proliferação de miofibroblastos não cancerosos CCD-18Co não foi inibida. A supressão do crescimento celular pelo tratamento com polifenóis de Ataulfo e Haden em células de câncer de colón SW-480 foi associada com o aumento na expressão gênica de biomarcadores de apoptose (caspase 8, Bax e Bim) e reguladores do ciclo celular (PKMYT1), atraso do ciclo celular e alteração na produção de espécies reativas de oxigênio. Os extratos fenólicos da polpa de manga continham ácido gálico, mangiferina, derivados de ácidos fenólicos e galotaninos, os quais foram caracterizados por análises em HPLC-DAD e HPLC-ESI-MSn antes e após a hidrólise enzimática (0.17 mg β-glicosidase 1000 KU/g polpa de manga / 4 h / 35°C). Ácidos fenólicos incluindo ácido gálico, caféico, ferúlico, p-coumárico e p-hidroxibenzóico consistiram os principais compostos derivados da hidrólise enzimática. Monocamadas de células Caco-2 foram incubadas por 2h no compartimento apical com extratos controle e hidrolisado. Quando incubadas com o extrato hidrolisado, ácido gálico, caféico, ferúlico, p-coumárico, vanílico e p-hidroxibenzóico foram detectados no compartimento basolateral, enquanto apenas ácido gálico foi detectado quando as células foram tratadas com o extrato controle. Polifenóis de elevado peso molecular, incluindo mangiferina e galotaninos, não foram transportados. Polifenóis de polpa (controle) de todas as variedades inibiram a proliferação de células humanas de câncer de colón HT-29 (0-27 μg de ácido gálico equiv./mL) e de mama MDA-MB-231 (0-24 μg GAE/mL) em até 99.8 e 89.9 %, respectivamente. Apesar da hidrólise enzimática ter aumentado a absorção de polifenóis de manga, não houve aumento significativo na atividade antioxidante, no conteúdo fenólico e na supressão do crescimento de células cancerosas de colón e mama. Ademais, ambas as frações fenólicas enriquecidas com polifenóis de baixo (138-194 Da) e elevado (422; 788-1852 Da) peso molecular inibiram o crescimento de células cancerosas de colón e mama na mesma extensão (0- 20 μg GAE/mL), o que poderia indicar que a atividade anticarcinogênica de polifenóis de manga seria independente da hidrólise enzimática. Estes resultados corroboram resultados obtidos em estudos in vivo, que sugerem que a grande parte dos polifenóis não seriam absorvidos em sua forma intacta através do intestino delgado, mas poderiam ser hidrolisados por enzimas intestinais em ácidos aromáticos de baixo peso molecular, os quais seriam posteriormente absorvidos; ou ainda, quando não absorvidos, poderiam, alcançar o intestino grosso, modulando a microflora intestinal e, desta forma, contribuiriam para reduzir o risco de câncer de cólon. Desta forma, polifenóis de polpa de manga de diferentes variedades exibiram efeitos anticarcinogênicos em modelos de cultura de células, os quais poderiam não ser necessariamente dependentes da hidrólise enzimática pela β-glicosidase.

Submitted by Marlene Santos (marlene.bc.ufg@gmail.com) on 2016-10-13T17:13:38Z No. of bitstreams: 2 Dissertação Victor de Melo Caixeta - 2015.pdf: 2750656 bytes, checksum: c7c2c97e0c6537b3d17aa54409152ad3 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Jaqueline Silva (jtas29@gmail.com) on 2016-10-14T20:03:21Z (GMT) No. of bitstreams: 2 Dissertação Victor de Melo Caixeta - 2015.pdf: 2750656 bytes, checksum: c7c2c97e0c6537b3d17aa54409152ad3 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-10-14T20:03:21Z (GMT). No. of bitstreams: 2 Dissertação Victor de Melo Caixeta - 2015.pdf: 2750656 bytes, checksum: c7c2c97e0c6537b3d17aa54409152ad3 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2015-10-15 / Background: Corticobasal Degeneration (CBD) is an rare and heterogeneous disease in its presentations, representing an great diagnostic challenge. There aren´t, in Brasil, large CBD case series, and there aren´t many in the world as well. Its cognitive and behavioural aspects have received little (despite growing) attention. Objectives: To describe sociodemographic, clinic, neuropsychiatric, neuropathological, and neuroradiologic aspects in a large brazilian DCB case series. To perform a literature review on CBD, with special focus on cognitive and behavioural aspects. Methods: 70 patients data was collected, with CBD diagnosis according to the Cambridge criteria modified by Bak and Hodges (2011). The patients underwent clinical, neuropsychiatric, neuroradiologic (structural and functional) and pathologic (in six cases) retrospective analysis. There were studied clinical, sociodemographic, neuropsychiatric, neuroanatomic variables and family history. Results and Discussion: The mean age at onset was 62.8 years (sd=9,5), and both sexes were equally affected (52,9% male). Hemispheric asymmetry was present in 97% of cases, and the left brain hemisphere was the most affected (68,2% of cases). The most frequent initial presentation was “psychiatric” (with changes in behaviour and/or mood), present in 68,1% of cases, followed by motor-extrapyramidal presentation (54.3%). In the course of the disease, the predominant clinical form (phenotype) was the extrapyramidal-motor with 61.4% of cases, followed by "psychiatric" with 51.4%. There were five cases with presentation of Posterior Cortical Atrophy (PCA) In 37.7% there were not found classic CBD syndromes (e.g., alien hand syndrome). We found 18.7% of cases with family DCB, with four families presenting a phenotype not yet described, of CBD with NPH (Normal Pressure Hydrocephalus). We also observed cognitive and functional impairments in the evaluated scales (MMSE, Pfeffer and CDR), and frequent medical and psychiatric comorbidities, especially diabetes mellitus (23.5%), hypothyroidism (22.1%) and Bipolar Spectrum Disorders (46, 4%), the last two significantly more frequent in the sample. There were six cases of rapidly progressive DCB and six confirmed by autopsy CBD cases in the sample. Conclusion: We observed, in agreement with the literature, a wide variety of CBD presentations, including new and little described phenotypes (NPH and PCA). In the characterization of the sample, we observed a great prevalence and importance of cognitive, affective and behavioural presentations. / Introdução: A Degeneração Corticobasal (DCB), é uma doença rara e heterogênea em suas apresentações, representando um grande desafio diagnóstico. Não há descrições de grandes amostras de DCB no Brasil, e poucas no mundo. Seus aspectos cognitivos e comportamentais têm recebido pouca (apesar de crescente) atenção. Objetivos: Descrever aspectos sociodemográficos, clínicos, neuropsiquiátricos, neuropatológicos e de neuroimagem em uma grande série de casos brasileiros com DCB. Realizar uma revisão da literatura sobre a DCB, com especial enfoque em aspectos cognitivos e comportamentais. Metodologia: Foram colhidos dados de 70 pacientes com DCB de acordo com critérios de Cambridge modificados por Bak e Hodges (2011). Os pacientes foram submetidos à avaliação retrospectiva clínica e neuropsiquiátrica, de neuroimagem estrutural e funcional e seis pacientes ao exame neuropatológico. Foram estudadas variáveis sociodemográficas, clínicas, neuropsiquiátricas, neuroanatômicas, e antecedentes familiares. Resultados e Discussão: A idade média de início dos sintomas foi de 62,8 anos (dp=9,5), sendo os dois sexos igualmente afetados (52,9% masculino). Assimetria hemisférica esteve presente em 97% dos casos e o hemisfério esquerdo foi o mais acometido (68,2% dos casos). A apresentação inicial mais freqüente foi a “psiquiátrica” (com alterações do comportamento e/ou humor), presente em 68,1% dos casos, seguida da apresentação motora-extrapiramidal (54,3%). No decorrer da doença, a forma clínica (fenótipo) predominante foi a motora-extrapiramidal, com 61,4% dos casos, seguida da “psiquiátrica”, com 51,4%. Houve cinco casos com apresentação de Atrofia Cortical Posterior (ACP). Em 37,7% não ocorreram achados clássicos da DCB (por exemplo, síndrome da mão estrangeira). Encontramos 18,7% de casos com DCB familiar, com quatro famílias apresentando um fenótipo ainda não descrito, de DCB com HPN (Hidrocefalia de Pressão Normal). Observamos também prejuízos cognitivos e funcionais nas escalas avaliadas (MEEM, Pfeffer e CDR), e comorbidades clínicas e psiquiátricas frequentes, em especial Diabetes Mellitus (23,5%), hipotireoidismo (22,1%) e Transtornos do Espectro Bipolar (46,4%), os dois últimos com frequência na amostra significativamente maior que na população. Houve seis casos de DCB rapidamente progressiva e seis casos confirmados por necropsia na amostra. Conclusão: Observamos, em consenso com a literatura, uma grande variedade de apresentações da DCB, inclusive com formas novas e pouco descritas (HPN e ACP). Na caracterização da amostra, percebemos grande frequência e importância das apresentações comportamentais, afetivas e cognitivas.

A família Baculoviridae é um grande grupo de vírus com cerca de 700 espécies de insetos hospedeiros, com dois fenótipos: o ODV (occlusion derived virion), que faz a infecção primária do intestino médio; e o BV (budded virus), responsável pela infecção sistêmica. No Brasil, o nucleopoliedrovírus Anticarsia gemmatalis (AgMNPV) é utilizado como controle biológico da lagarta-da-soja Anticarsia gemmatalis. O genoma do AgMNPV-2D contém 152 ORFs, 26 das quais codificam proteínas estruturais. Entre elas, a glicoproteína GP64 é fundamental para infecção secundária. Este estudo visa identificar proteínas estruturais do AgMNPV-2D por duas abordagens de espectrometria de massas. Também comparar a variabilidade da gp64 de isolados geográficos por sequenciamento por Sanger e de alta cobertura. Assim, identificamos as substituições de gp64 e vimos que ela não suporta a separação geográfica dos isolados. Também identificamos 44 e 33 proteínas em ODV e BV, respectivamente. Seis novas proteínas foram identificadas no ODV e sete delas no BV. Além disso, 11 proteínas celulares foram identificadas no AgMNPV-2D, possivelmente necessárias para infecção. Este achado contribui para o entendimento da morfogênese do AgMNPV e fatores associados à multiplicação viral. / Baculoviridae are arthropod-specific viruses with more than 700 host insects, which produce two phenotypes: the budded virus (BV) and, the occlusion-derived virus (ODV) for intra and across host spread, respectively. Brazil uses the Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) as a biological control agent of the velvet bean caterpillar (A. gemmatalis). The genome of the AgMNPV-2D carries 152 ORFs, 26 of which code for structural proteins. Herein, the structural proteins of AgMNPV-2D were analyzed by two mass spectrometry techniques. The additional objective was to compare the gene gp64. of different geographical populations by Sanger and next generation sequencing. This analysis allowed us to observe the substitutions of gp64 and refuted the notion of a geographical isolation of the samples. We also observed a total of 44 proteins of the ODV and 33 of the BV. Six new proteins were found in the ODV and seven in the BV. Furthermore, 11 cellular proteins were also identified, which are possibly assorted during viral morphogenesis. These findings may provide novel insights into AgMNPV biology and its host interaction, leading us to a better understanding about morphogenesis and also the associated factors of the viral multiplication.

Linhagens de camundongos AIRmax e AIRmin diferem quanto a reatividade inflamatória aguda às partículas de Biogel. A AIR divergente nessas linhagens está bem estabelecida, no entanto, diferenças na resposta inflamatória crônica ao Biogel ainda não foram descritas. Assim, nós decidimos verificar se o processo de seleção que modificou a resposta inflamatória aguda nessas linhagens, também afetou o desenvolvimento da resposta inflamatória crônica. A infiltração de células no exsudado da linhagem AIRmax foi maior que na linhagem AIRmin em ambos os períodos avaliados (48h e 30d), e no período de 48 horas, os animais AIRmax apresentaram alta produção de citocinas no exsudato inflamatório. Essa linhagem mostrou ainda maior número de genes ativados envolvidos na transdução de sinal, resposta imune e inflamatória. Alguns genes envolvidos com a resposta inflamatória aguda, também apresentaram diferenças após 30 dias. Esses resultados indicam que o processo de seleção para a inflamação aguda pode ter afetado também o desenvolvimento da resposta inflamatória crônica ao Biogel. / AIRmax and AIRmin mouse lines differ in terms of acute inflammatory response after Biogel injection. The distinct AIR in these lines is well established, however, differences in late or chronic inflammatory response to Biogel were not described yet. Thus, we decided to check if the genetic selection that modified the acute inflammatory response in these lines, also affected the development of a chronic inflammatory response. We found that AIRmax mice had higher cellular influx in the inflammatory exudate than AIRmin mice in both analyzed periods (48h and 30d) and that after 48 hours of Biogel injection, AIRmax mice showed higher cytokine levels in inflammatory exudates. This line also showed higher number of up regulated genes in AIRmax than in AIRmin mice involved with inflammatory response, immune response and signal transduction. Some acute inflammatory response genes also showed differences on day 30. Our results indicate that the genetic selection for acute inflammatory response may also have affected the chronic inflammatory response to Biogel.

Resistance to artemisinin combination therapies (ACTs) has emerged in southeast Asia threatening the most widely used treatment against antimalarial-resistant Plasmodium falciparum worldwide. Artemisinin resistance has been associated with a reduced rate of parasite clearance following treatment with an ACT and is attributed to increased survival of ring-stage parasites. Single nucleotide polymorphisms (SNPs) in kelch gene (K13) has been associated with delayed in vivo clearance half-life of artemisinin-resistant P. falciparum and is the only known molecular marker of resistance. The absence of reliable in vitro phenotypes for artemisinin resistance has limited our understanding of the resistance mechanism(s) and fitness costs, therefore we have culture adapted and cloned patient isolates from Thailand and Cambodia that had clinical resistant phenotypes. Stable reduced susceptibility to artemisinin derivatives and mefloquine was observed using a modified [3H]hypoxanthine drug susceptibility assay. In addition we devised an in vitro phenotype assay of artemisinin resistance, known as the delayed clearance assay (DCA), that was positively correlated with the in vivo delayed clearance and presence of K13 SNPs of artemisinin resistance. Remarkably we discovered for the first time altered patterns of intraerythrocytic development in artemisinin-resistant P. falciparum. In the absence of drug pressure most artemisinin-resistant clones have a prolonged ring phenotype with temporally compressed trophozoite stage, yet with the normal overall asexual life cycle period. Parasites remain in ring-stage up to 14 hours longer than wild-type, whereas time spent in trophozoite-stage is dramatically reduced. One parasite, PL08-09 (5C), exhibited an accelerated 36-hour life cycle in the absence of drug pressure and progressed through asexual development in equal time spent at each intraerythrocytic stage. These data support our hypothesis that parasite resistance to artemisinin results from reduced exposure to drug at the most susceptible stage of development (trophozoite). Interestingly, the most prevalent K13 mutation C580Y is associated with both cell cycle phenotypes. Another cell cycle phenotype, ring-stage dormancy, has been associated with artemisinin resistance in vitro reducing the dormant period of artemisinin-resistant parasites following dihydroartemisinin exposure allowing resistant parasite cultures recrudesce before wild-type. However, sensitive parasites have the ability to enter ring-stage dormancy causing recrudescence in vitro. It is possible that multiple cell cycle phenotypes enhance the survival and fitness of the resistant parasite population as a whole in the face of antimalarial exposure. We have demonstrated that there is a fitness cost associated with artemisinin resistance and remains an important component in the spread of genetic mutations associated with artemisinin resistance. Resistant parasites outcompeted sensitive in vitro only when exposed to dihydroartemisinin. Two mutations associated with artemisinin resistance, including a mutation in K13, were lost in artemisinin resistant parasite by the end of the study. Conversely, parasite cultures maintained artemisinin resistance phenotypes in vitro only if exposed to artemisinin drug pressure every 21-42 days. The mechanism of artemisinin resistance remains elusive and how the parasites alter their intraerythrocytic development is unknown. Therefore. we transfected green fluorescent protein (GFP) or luciferase into artemisinin-resistant P. falciparum clones from Thailand and Cambodia to aid the study the cell cycle phenotypes associated with artemisinin resistance. Artemisinin resistance phenotypes were maintained in stably transfected clones. Increased growth of artemisinin-resistant clones was observed following exposure to ACT partner drug. Low concentrations of antimalarials synchronized the luciferase expression of artemisinin-resistant parasites having different cell cycle phenotypes in the absence of drug pressure. Ring-stage dormancy was observed with many antimalarial drugs and contributes to recrudescence observed by antimalarials other than artemisinin. Our results show evidence that current ACT treatments are selecting multidrug resistant parasites in the field that are better able to tolerate all antimalarials through regulatory cell cycle mechanisms. These cell cycle phenotypes associated with artemisinin resistance contribute to reducing the fitness cost associated with genetic mutations causing artemisinin resistance. This leads to the survival of the most fit population of parasites that survive combination drug treatments, thus demonstrating the importance of discovering novel drugs to target ACT-resistant P. falciparum.

Les maladies chroniques constituent l'un des défis du 21ème siècle. La Broncho-pneumopathie chronique obstructive est une maladie respiratoire caractéristique de ces maladies, en raison de son caractère hétérogène et de ses répercussions systémiques. Parmi celles-ci, la dysfonction musculaire périphérique est cruciale, mais ses liens avec l'atteinte pulmonaire restent mal expliqués. La réduction d'activité physique a été le premier lien proposé, mais le remodelage musculaire dans la BPCO est bien différent à celui observé chez des sujets déconditionnés (possiblement en raison d'une exposition à la sédentarité plus ancienne et importante), et d'autres facteurs tels le stress oxydant ont été incriminés. La comparaison directe de la dysfonction musculaire périphérique de BPCO à celle de sujets sains sédentaires est limitée par l'hétérogénéité de l'atteinte musculaire. Enfin, chez les patients BPCO, le réentrainement n'a jamais fait la preuve d'adaptations musculaires similaires à celles de sujets sains sédentaires. L'objectif de cette thèse est donc la mise en évidence du rôle exact de la réduction d'activité physique et de l'exercice dans la dysfonction musculaire périphérique hétérogène de la BPCO. Nous montrons que l'exposition à la l'inactivité au cours de la vie n'est pas plus importante dans la BPCO que chez des sujets sains sédentaires. Parallèlement, il existe des phénotypes de dysfonction musculaire dans la BPCO. Cependant, quel que soit le phénotype considéré, il persiste des anomalies ultrastructurales entre patients BPCO et sujets sains de même niveau d'activité physique. Finalement, un même programme de réentrainement à l'effort n'a pas entrainé les mêmes adaptations fonctionnelles, morphologiques et angiogéniques que chez les sujets sains sédentaires.En conclusion, ces différents travaux remettent en cause le paradigme classique de la spirale du déconditionnement dans la BPCO et ouvrent des pistes pour l'optimisation de la réhabilitation respiratoire. / Chronic diseases are one of the medical challenges of the 21st century. The chronic obstructive pulmonary disease is paradigmatic of this type of diseases, because of its heterogeneity, and its systemic repercussions. The peripheral muscle dysfunction constitutes a key-repercussion in COPD. However, the links between this muscle dysfunction and the pulmonary impairment remain poorly understood.The physical activity reduction has been the first link proposed. However, the magnitude of structural muscle remodeling in COPD differs to the one of deconditioned sedentary subjects (though, this could be the consequence of greater and older inactivity in COPD), and other factors like the oxidative stress have been incriminated. The peripheral muscle dysfunction in COPD patients has never been directly compared to the one of healthy subjects of the same physical activity level, and is limited by the heterogeneity of the muscle dysfunction in COPD patients. Last, the exercise training has never shown similar muscle response in COPD patients as compared to healthy sedentary subjects. The aim of this PhD Thesis was to understand the exact contribution the physical inactivity and the exercise training in the heterogeneous peripheral muscle, dysfunction in COPD patients.First, we observed that the lifetime physical activity was not greater in COPD patients as compared to lifetime sedentary healthy subjects. In another hand, we showed phenotypes of peripheral muscle dysfunction in COPD patients. However, and whatever the phenotype considered, there was significantly more ultra-structural damage in COPD patients vs. healthy sedentary subjects. Last, a similar exercise training program did not induce similar functional, histo-morphological and angiogenic muscle responses in COPD patients vs. healthy sedentary subjects.Altogether, our work challenges the classical paradigm of the COPD spiral of decline and open doors to research on other specific pathways of the field of muscle dysfunction in COPD in order to optimize the pulmonary rehabilitation.