Made available in DSpace on 2014-12-17T14:10:29Z (GMT). No. of bitstreams: 1 KellyaFMB_DISSERT.pdf: 2697762 bytes, checksum: 1ffb7fc96fd841e9f2d4e4bbe9d99a4b (MD5) Previous issue date: 2013-12-17 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / The genus Saccharum belongs to Poaceae family. Sugarcane has become important monocultures in Brazil due to their products: ethanol and sugar. The production may change between different regions from Brazil. This difference is related to soil, climatic conditions and temperature that promotes oxidative stress that may induce an early flowering. The aim of this work was to identify the effects of oxidative stress. In order to analyse this, sugarcane plants were submitted to oxidative stress using hydrogen peroxide. After this treatment, the oxidative stress were analyzed Then, the plant responses were analyzed under different approaches, using morphophysiological, biochemical and molecular tools. Thus, sugarcane plants were grown under controlled conditions and until two months they were subjected first to a hydroponics condition for 24 hours in order to acclimation. After this period, these plants were submitted to oxidative stresse using 0 mM, 10 mM, 20 mM and 30 mM hydrogen peroxide during 8 hours. The histomorphometric analysis allowed us to verify that both root and leaf tissues had a structural changes as it was observed by the increased in cell volume, lignin accumulation in cell walls. Besides, this observation suggested that there was a change in redox balance. Also, it was analyzed the activity of the SOD, CAT and APX enzymes. It was observed an increase in the SOD activity in roots and it was also observed a lipid peroxidation in leaves and roots. Then, in order to identify proteins that were differently expressed in this conditions it was used the proteomic tool either by bidimensional gel or by direct sequencing using the Q-TOF EZI. The results obtained with this approach identified more than 3.000 proteins with the score ranging from 100-5000 ions. Some of the proteins identified were: light Harvesting; oxygenevolving; Thioredoxin; Ftsh-like protein Pftf precusor; Luminal-binding protein; 2 cys peroxiredoxin e Lipoxygenase. All these proteins are involved in oxidative stress response, photsynthetic pathways, and some were classified hypothetical proteins and/or unknown (30% of total). Thus, our data allows us to propose that this treatment induced an oxidative stress and the plant in response changed its physiological process, it made changes in tissue, changed the redox response in order to survival to this new condition / A cana-de-a??car ? uma das principais monoculturas no Brasil devido ? import?ncia dos seus produtos: etanol e a??car. A produtividade pode variar entre as diferentes regi?es do Brasil como sudeste e nordeste, por adapta??es destas gram?neas as diferentes condi??es edafo-clim?ticas. As condi??es do solo e da temperatura da regi?o Nordeste podem promover o estresse oxidativo, h?drico e consequentemente o florescimento precoce o que acarreta perdas consider?veis na produ??o. Neste trabalho, procurou-se averiguar as prov?veis altera??es morfofisiol?gicas, bioqu?micas e moleculares resultantes da resposta das plantas ao estresse oxidativo. Deste modo, plantas de cana-de-a??car foram cultivadas em condi??es controladas e quando atingiram dois meses foram submetidas a uma condi??o de hidrop?nia por 24 horas para sua aclimata??o e em seguida ao estresse oxidativo realizado por 8 horas utilizando as concentra??es de per?xido de hidrog?nio: 0 mM; 10 mM; 20 mM e 30 mM. Ap?s este per?odo, foram observadas as seguintes altera??es morfol?gicas para as folhas: o fechamento foliar parcial ou total de acordo com a concentra??o de per?xido de hidrog?nio utilizada. As an?lises histomorfol?gicas permitiram verificar para ambos os tecidos (foliar e radicular) que houve altera??es estruturais relacionadas ao aumento do volume das c?lulas propiciando, assim, a quebra das paredes das membranas dos vasos condutores xilema e floema entre outras modifica??es anat?micas em diferentes regi?es, assim como a lignifica??o em algumas regi?es. Tamb?m foi analisada a atividade enzim?tica das enzimas Super?xido dismutase - SOD; Catalase - CAT e Ascorbato peroxidase - APX. Foi observado um aumento na atividade da enzima super?xido dismutase nas ra?zes. Nessa abordagem bioqu?mica, foi tamb?m analisada a peroxida??o de lip?deos que mostrou que ocorreram danos nas membranas das folhas e ra?zes principalmente para a concentra??o de 30 mM. Entretanto, para a enzima catalase foi observada uma baixa atividade em folhas e esta atividade n?o foi detectada nas ra?zes. Contudo, os resultados histomorfologicos juntamente com os resultados bioqu?micos fortalecem que o tratamento com per?xido de hidrog?nio pode ter alterado a homeostase redox e as vias de sinaliza??o promovendo assim as altera??es morfol?gicas (aumento de c?lulas, lignifica??o) e bioqu?micas (EROs e antioxidantes). Outra ferramenta utilizada nesse trabalho foi a prote?mica, onde foi utilizado o sequenciamento tanto por meio de g?is bidimensionais como tamb?m pelo sequenciamento direto utilizando o EZI Q TOF. Os resultados obtidos permitiram com essa abordagem identificar mais de 3.000 prote?nas com o score variando de 100-5000 ?ons. Algumas das prote?nas identificadas foram: light Harvesting; oxygen-evolving; Thioredoxin; Ftsh-like protein Pftf precusor; Luminal-binding protein; 2 cys peroxiredoxin e Lipoxygenase. Estas prote?nas est?o envolvidas no metabolismo em resposta ao estresse oxidativo, prote?nas do aparelho fotossint?tico, al?m de prote?nas hipot?ticas e/ou desconhecidas (30% do total). Desta forma, os dados obtidos nos permitem propor uma hip?tese, onde ?s prote?nas identificadas estariam envolvidas em processos fisiol?gicos, na remodela??o de tecidos, dano/degrada??o e s?ntese de antioxidante/ desintoxicante, correlacionada aos dados de resposta de defesa enzim?tica, ac?mulo de H2O2 nos tecidos ativando a peroxida??o lip?dica nas paredes das membranas das c?lulas. Desta forma ativando v?rias vias de cascata de sinaliza??o

Submitted by PPG Pediatria e Sa?de da Crian?a (pediatria-pg@pucrs.br) on 2018-02-16T19:08:17Z No. of bitstreams: 1 Tese Vers?o Final Tiago Fazolo 18_01_2018.pdf: 3827531 bytes, checksum: b081e8333d16cb49ba00d0df25dff485 (MD5) / Approved for entry into archive by Caroline Xavier (caroline.xavier@pucrs.br) on 2018-02-22T17:20:04Z (GMT) No. of bitstreams: 1 Tese Vers?o Final Tiago Fazolo 18_01_2018.pdf: 3827531 bytes, checksum: b081e8333d16cb49ba00d0df25dff485 (MD5) / Made available in DSpace on 2018-02-22T17:35:15Z (GMT). No. of bitstreams: 1 Tese Vers?o Final Tiago Fazolo 18_01_2018.pdf: 3827531 bytes, checksum: b081e8333d16cb49ba00d0df25dff485 (MD5) Previous issue date: 2017-03-20 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Respiratory syncytial virus (RSV) is the most common etiologic agent in severe lower respiratory tract infections (LRTI) in children. RSV-associated LRTI is the main cause of bronchiolitis, pneumonia and exacerbation of asthma. This infection is responsible for the high rates of hospitalizations related to respiratory diseases worldwide, especially in children younger than 2 years. Currently, annual mortality rate due to RSV infections is worrying worldwide and is estimated at approximately two hundred thousand cases. The treatment strategies to RSV infections are limited. Ribavirin is an approved drug for use in RSV infections, but its use is limited due to adverse side-effects and risks posed to health professionals who handle it. Palivizumab is a monoclonal antibody which targets RSV F glycoprotein and its use is only indicated as a prophylactic measure. This treatment is already accepted in several countries for groups of high risk children (premature children, with chronic lung disease and with congenital heart disease). However, palivizumab has a high cost for public health and is not available in all countries. The development of an effective RSV vaccine to generate a long-lasting immunological memory response that prevents infection may be the best alternative because it will reduce high public health expenditures with antiviral drugs and monoclonal antibodies. The first attempt in the search for a vaccine against RSV was in the 1960s. This vaccine produced high levels of serum antibodies but could not protect against infection. Children who were vaccinated developed a more serious disease when later infected with the same virus. To date, there is no licensed vaccine for RSV, so the search for effective vaccines is an important focus of research. Natural RSV infections do not induce lasting protective memory, and multiple reinfections can occur lifetime. Nasal secretions from infected infants presented a small number of regulatory CD4 T cells (Treg) in peripheral blood, an increase in interleukin 4 (IL-4) production and T helper type 2 (Th2) response. Treg cells are important for controlling an exacerbated increase in immune responses. A reduction of the Tregs caused by the RSV infection generates an exacerbation of the pulmonary disease due to a Th2 response. The M209-223 RSV peptide was identified to increase IFN-? production by peptide-specific CD4 T cells after challenge with the virus. The treatment with this peptide also induced an increase in pulmonary Treg frequency in infected mice. Recently, it has also been shown that Tregs aid in the development of a T CD8+ effector response, which is crucial for the control of RSV viral load. Our hypothesis is that the RSV M209-223 peptide impacts in the differentiation of CD4 T cells, increasing the population of specific Treg, reducing lung inflammation and modulating the anti-RSV immune response. This peptide in animal model induces the differentiation of specific Treg. Our findings suggest that vaccination with M209-223 peptide results in the differentiation of specific CD4 T cells into conventional effectors and Treg cells. Vaccination with this peptide decreased the expansion of a Th2 response in animals infected with RSV, protecting both the infection site and systemically. We believe that this approach could be an important component in vaccination strategies against this virus. / O v?rus sincicial respirat?rio (VSR) ? o agente etiol?gico mais comum nas infec??es graves do trato respirat?rio inferior (TRI) em crian?as. As infec??es do TRI associada com o VSR s?o a principal causa de bronquiolite, pneumonia e exacerba??o da asma. As TRI causadas pelo VSR s?o respons?veis pelas altas taxas das hospitaliza??es relacionadas ?s doen?as respirat?rias em todo o mundo, principalmente em crian?as menores de dois anos. Atualmente a taxa de mortalidade anual mundial devido ?s infec??es pelo VSR ? preocupante e ? estimada em aproximadamente duzentas mil crian?as. As estrat?gias de tratamento contra o VSR utilizadas s?o limitadas. A ribavirina ? um f?rmaco aprovado no uso para infec??es pelo VSR, por?m sua utiliza??o ? limitada devido aos efeitos secund?rios adversos e aos riscos que representam para os profissionais da sa?de que o manipulam. O palivizumabe ? um anticorpo monoclonal dirigido contra a glicoprote?na F do v?rus e sua utiliza??o ? apenas como medida profil?tica. Este tratamento j? ? aceito em v?rios pa?ses nos grupos de crian?as de alto risco (crian?as prematuras, com doen?a pulmonar cr?nica e com cardiopatia cong?nita). Entretanto o palivizumabe tem um alto custo para sa?de p?blica, n?o sendo disponibilizado em todos os pa?ses. O desenvolvimento de uma vacina eficaz contra o VSR pode ser a melhor alternativa, pois ao gerar resposta de mem?ria duradoura que previne a infec??o e reduz, desta forma, os altos gastos com a sa?de p?blica, com os f?rmacos antivirais e com os anticorpos monoclonais. A primeira tentativa na busca de uma vacina contra o VSR foi na d?cada de 60. A vacina produzida estimulou n?veis moderadamente elevados de anticorpos no soro, mas n?o conseguiu proteger contra ? infec??o. As crian?as que foram vacinadas desenvolveram uma doen?a mais grave quando mais tarde infectados com o v?rus. At? o presente momento n?o existe nenhuma vacina licenciada para o VSR. Desta forma, a busca de vacinas eficazes constitui um importante foco de pesquisa em todo mundo. As infec??es naturais pelo VSR n?o induzem mem?ria protetora duradoura, ocorrendo m?ltiplas reinfec??es ao longo da vida. Em crian?as infectadas, observou-se um n?mero reduzido de c?lulas T CD4+ regulat?rias (Treg) no sangue perif?rico, um aumento na produ??o de interleucina 4 (IL-4) e uma resposta T helper do tipo 2 (Th2) nas secre??es nasais. As c?lulas Treg s?o importantes para controlar um aumento exagerado da resposta imunol?gica. Por este fato acredita-se que quando h? uma redu??o das Tregs causada pela infec??o do VSR ocorre uma exacerba??o da doen?a pulmonar devido uma resposta Th2. Foi identificado que o pept?deo M209-223 do VSR aumenta a produ??o de IFN-? nas c?lulas T CD4+ ap?s o desafio com VSR. O tratamento com este mesmo pept?deo tamb?m apresentou um aumento na frequencia de c?lulas Treg ap?s infec??o prim?ria pelo VSR. Recentemente tamb?m foi demonstrado que as Tregs auxiliam no desenvolvimento de uma resposta efetora T CD8+, que ? crucial para o controle da carga viral do VSR. Nossa hip?tese ? que o pept?deo M209-223 do VSR influencia na diferencia??o das c?lulas T CD4+, aumentando a popula??o de c?lulas T efetoras e regulat?rias espec?ficas, reduzindo a inflama??o pulmonar e modulando a resposta imune. Os nossos resultados sugerem que a vacina??o com pept?deo M209-223 resulta na diferencia??o de c?lulas T CD4+ espec?ficas em efetoras convencionais, que produzem mais IFN-? e em c?lulas Treg. A vacina??o com este pept?deo diminuiu a expans?o de uma resposta Th2 nos animais infectados com o VSR, protegendo da inflama??o exacerbada tanto no local da infec??o como sistemicamente. Acreditamos que esta abordagem pode constituir um componente importante nas estrat?gias de vacina??o contra este v?rus.

Previous nutritional physiology research using L-histidine and zinc in American lobster intestine (Homarus americanus) has suggested that these solutes can be co-transported as complexes (Histidine-Zinc-Histidine) across the intestine using a peptide transporter. Furthermore, transport of L-leucine was shown to be inhibited by high calcium concentrations. Dipeptide and bis-complex transport and the role of calcium were investigated in the perfused intestines of lobster and Atlantic white shrimp (Litopenaeus setiferus). Following trans-intestinal transport, serosal medium was analyzed for amino acid composition by gas chromatography. In lobster, the transport of glycylsarcosine (Gly-Sar) from mucosa to serosa was stimulated two-fold with luminal pH 8.5, compared to the pH 5.5 control. Mucosa to serosa and serosa to mucosa fluxes of Gly-Sar were measured; the dipeptide was transported intact in both directions, but the net flux was from mucosa to serosa. The use of 0.5mM calcium chloride stimulated Gly-Sar transport two-fold, compared to 25 mM. In shrimp, the addition of 50 µM zinc chloride increased the rate of L-histidine transport, while Gly-Sar inhibited histidine transport in the presence of zinc. The rate of histidine transport was significantly higher with 1mM calcium chloride than with 25mM. These results suggest that shrimp transport bis-complexes in a manner similar to lobster. High calcium concentration had an inhibitory effect on both amino acid and dipeptide transport. Proposed mechanisms accounting for the effects of metals and calcium on trans-intestinal transports of both amino acids and dipeptides by lobster and shrimp digestive tracts are discussed.