A cana-de-açúcar vem assumindo um papel de destaque na atual conjuntura nacional, impulsionada principalmente pela produção de etanol, que vai de encontro com a crescente preocupação mundial na busca por fontes de energias renováveis e menos impactantes ao ambiente. Por essa razão, é preciso assegurar o contínuo desenvolvimento técnico-científico do setor sucroalcooleiro nacional, mantendo o Brasil na posição de vanguarda na produção de biocombustíveis. Ante a disponibilidade de inúmeras ferramentas biotecnológicas, tornou-se possível avançar com maior celeridade na compreensão dos campos da genética e fisiologia da cana-de-açúcar. Neste trabalho é demonstrado a transformação genética via biobalística da cultivar RB835486. No processo foram usadas duas construções para silenciamento gênico via RNA de interferência (RNAi), com genes quiméricos do tipo shRNA (short harpin RNA) para silenciamento dos genes SspTIP1;1 e SspPIP1;4, em co-tranformação com o gene marcador npt- II. Os dois genes alvo selecionados codificam aquaporinas, proteínas transmembrana responsáveis pelo transporte de água na planta. Estes genes foram identificados anteriormente por seu possível envolvimento no processo de acúmulo de sacarose. A co-integração dos cassetes de silenciamento gênico e do gene marcador ocorreu em 13 plantas, sendo obtidas três linhagens para o gene SspTIP1;1 e 10 linhagens para o gene SspPIP1;4. Dentre elas, duas linhagens SspTIP1;1 e cinco linhagens SspPIP1;4 foram analisadas via RT-PCR, quanto a possíveis modificações nos níveis de expressão dos genes alvos. Nas duas linhagens transgênicas avaliadas para silenciamento do SspTIP1;1, não houve redução em sua expressão em relação ao controle não transformado, possivelmente devido a efeitos de posição. Nas outras cinco linhagens transgênicas avaliadas para silenciamento do SspPIP1;4, houve redução significativa em seus níveis de expressão em três linhagens em relação ao controle não transformado. Nestas plantas serão realizadas as análises fisiológicas a fim de validá-las funcionalmente quanto ao transporte de água e acúmulo de sacarose. / Sugarcane has taken a leading role in the current national economy, mainly boosted by ethanol production, which meet the growing global concern on searching for renewable energy and with low impact on the environment. Therefore, it is necessary to ensure the continuous technical and scientific development of the national sugar and ethanol sector, maintaining the leading position of Brazil in biofuel production. By the availability of numerous biotechnology tools, it became possible to advance more rapidly in understanding the fields of genetics and physiology of sugarcane. This work demonstrated the genetic transformation of the cultivar RB835486 via biolistic assay. In the process it was used two constructs for gene silencing via RNA interference (RNAi) with chimeric genes of the type shRNA (short harpin RNA) for silencing of the genes SspTIP1;1 and SspPIP1;4, co-transformed with the marker gene npt- II. The two selected target genes encode aquaporins, transmembrane proteins which are responsible for water transport in plants. These genes were previously identified for their possible involvement in the process of sucrose accumulation. The co-integration of both, the cassette gene silencing and gene marker was observed in 13 plants, three strains were obtained for the gene SspTIP1;1 and 10 strains for gene SspPIP1;4. Among them, two strains of SspTIP1;1 and five strains of SspPIP1;4 were analyzed by RT-PCR, searching for possible changes in the levels of target gene expression. In the two transgenic lines evaluated for silencing SspTIP1;1, no reduction in expression compared to control non-transformed was obtained, possibly due to effects of position insertion of the gene in the genome. The other five transgenic lines evaluated for silencing of SspPIP1;4, a significant reduction in their expression levels was obtained in three strains when compared to the control untransformed plants. These silenced plants will be physiologically analyzed to validate their function on water transport and sucrose accumulation.

O genoma de Xanthomonas citri (Xac) possui mais de 20 tipos de transportadores do tipo ABC incluindo o operon ssuABC associado ao transporte de alcano sulfonatos. Deste operon, escolhemos a proteína periplasmática ligadora de alcano sulfonatos SsuA2, para caracterização e análises espectroscópicas e estruturais. A rSsuA2 foi expressa no citoplasma de células de Escherichia coli e utilizada para a preparação de anticorpos em camundongos, que foram capazes de reconhecer a proteína recombinante, mas não a nativa no extrato de células de Xac. A rSssuA2 apresenta estrutura característica de proteínas alfa-beta, maior estabilidade em pH neutro (7.0), como também foi evidenciado pela obtenção de cristais somente nesta faixa, e pouca flexibilidade ao desenovelamento térmico. Os cristais difratam com resolução de 1.8 Å e pertencem ao grupo espacial de simetria P21. Além do o operon ssu (ssu2) altamente conservado, Xac apresenta o operon tau (ssu1) para captação de taurina. O papel do operon para Xac é discutido. / Xanthomonas citri (Xac) genome has more than 20 different ABC transporters, including the ssuABC operon. In this work, the alkanosulphonate-periplasmic binding protein SsuA2 was chosen for spectroscopic and structural analysis. The rSsuA2 protein was expressed as a soluble form and purified by immobilized metal affinity chromatography. Antibodies produced from the recombinant protein were able to recognize the rSsuA2, but not the native protein in the Xac extract samples. The protein presents secondary structure defined by alfa helices and beta-sheets, high stability in neutral pH and low flexibility to the thermal denaturation. The determination of the optimal pH range was important to produce crystals of high quality diffracting at 1.8 Å with symmetry of the P21 spatial group. Besides the highly conserved operon ssu (ssu2), Xac has the tau operon (ssu1) for taurine uptake.

In eukaryotic cells, membrane-bound vesicles carry cargo between intracellular compartments, to and from the cell surface, and to the extracellular environment. Many conserved families of proteins are required for properly localized vesicle fusion, including the multi-subunit tethering complexes and the SNARE complexes. These protein complexes work together to promote proper vesicle fusion in other trafficking pathways. Contrary to these other pathways, our lab previously suggested that the exocyst subunit Sec6, a component of the exocytosis-specific tethering complex, inhibited Sec9:Sso1 SNARE complex assembly due to interactions in vitro with the SNARE protein Sec9 (Sivaram et al., 2005). My goal for this project was to test the hypothesis that Sec6 inhibited SNARE complex assembly in vivo. I therefore chose to generate Sec6:Sec9 loss-of-binding mutants, and study their effect both in vitro and in vivo. I identified a patch of residues on Sec9 that, when mutated, are sufficient to disrupt the novel Sec6-SNARE interaction. Additionally, I found that the previous inhibitory role for Sec6 in SNARE assembly was due to a data mis-interpretation; my re-interpretation of the data shows that Sec6 has a mild, if any, inhibitory effect on SNARE assembly. My results suggest a potential positive role for Sec6 in SNARE complex assembly, similar to the role observed for other tether-SNARE interactions.

Vesicular trafficking, the movement of vesicles between organelles and the plasma membrane for secretion, consists of multiple highly regulated processes. Many protein families function as specificity and regulatory determinants to ensure correct vesicle targeting and timing of trafficking events. The SNARE proteins dock and fuse vesicles to their target membranes. Sec1/Munc18 (SM) proteins regulate membrane fusion through interactions with the SNAREs—SM proteins have been shown to act as both inhibitors and stimulators of SNARE assembly and membrane fusion. However, the details of these SM protein functions are not understood. Constructing a model of SM protein function has been challenging due to the various modes of interactions reported between SM proteins and their SNAREs. SM proteins interact with their cognate SNAREs and SNARE complexes through several distinct modes. The most conserved mode is an interaction with the syntaxin N-peptide; other modes of binding, such as the syntaxin closed conformation, are hypothesized to be specific for specialized cell types. In order to elucidate the general function of SM proteins, I investigated the function of the endosomal SM protein Vps45p by analyzing its interactions with its cognate syntaxin Tlg2p and its role in SNARE assembly. I had two main hypotheses: that the Tlg2p N-peptide does not solely mediate the interaction between Vps45p and Tlg2p; and that Vps45p functions to stimulate SNARE complex assembly. I systematically mapped the interaction between Vps45p and Tlg2p using various Tlg2p truncations containing the different domains of Tlg2p and discovered a second binding site on Tlg2p that corresponds to the closed conformation. The neuronal SM-syntaxin pair interacts in a similar manner, indicating that this interaction mode is conserved. To characterize the closed conformation binding mode further, and determine its relationship to the N-peptide binding mode, I developed a quantitative fluorescent electrophoretic mobility shift assay. Results indicate that these two sites do not bind simultaneously and that the N-peptide binding modulates the closed conformation affinity. Furthermore, I monitored the effect of Vps45p on SNARE complex assembly using size exclusion chromatography. Under the conditions tested, Vps45p did not appear to stimulate SNARE complex assembly. The work presented here addresses several puzzling issues in the field and significantly contributes to the construction of a new mechanistic model for SM protein function. In this new model, the SM protein is recruited to the membrane by its interaction with the syntaxin N-peptide. The SM protein then binds the syntaxin closed conformation thus inhibiting SNARE complex assembly. Upon dissociation of the SM protein from the closed conformation, an event perhaps regulated by the SM protein, syntaxin opens and interacts with the other SNAREs to form a SNARE complex. Fusion ensues, stimulated by the SM protein.

Hydrodynamic analysis and electron microscopy of GLUT1/lipid/detergent micelles and freeze fracture electron microscopy of GLUT1 proteoliposomes support the hypothesis that the glucose transporter is a multimeric (probably tetrameric) complex of GLUT1 proteins. Some detergents (e.g. octylglucoside) maintain the multimeric complex while other detergents (e.g. CHAPS and dodecylmaltoside) promote the dissociation of GLUT1 oligomers into smaller aggregation states (dimers or monomers). GLUT1 does not appear to exchange rapidly between protein/lipid/detergent micelles but is able to self-associate in the plane of the lipid bilayer. Quantitatively deglycosylated GLUT1 displays aberrant electrophoretic mobility, but each protein band contains full-length GLUT1 and the less mobile species, when treated with additional detergent and reductant, converts to the more mobile species. Preliminary structural analysis suggests that denaturing detergent- and thiol chemistry-related changes of α-helical content may mirror mobility shifts. Limited proteolysis of membrane-resident GLUT1 (± ligands) releases membrane-spanning α-helical domains suggesting that (i) some bilayer-resident helices are highly solvent exposed; (ii) membrane-spanning domains 1, 2, & 4 and 7, 8, & 10 are destabilized upon ligand binding; and (iii) helix packing compares well with high-resolution structures of prokaryotic transporters from the same superfamily. Results are consistent with a central, hydrophilic, translocation pathway comprised of amphipathic, membrane-spanning domains that alter associations upon ligand/substrate binding. We have resolved technical difficulties (heterogeneity, lipid/detergent removal, glycosylation, small molecule contamination) associated with GLUT1 analysis by mass spectrometry; and we map global conformational changes between sugar uptake and sugar efflux.

<p>Interindividualne razlike u sastavu i aktivnosti crevne mikroflore mogu uticati na metabolizam lekova kao i na njihov konačan terapijski odgovor. Simvastatin je lek iz grupe statina i karakteri&scaron;e ga izuzetno mala rastvorljivost u vodi, mala bioraspoloživost (&lt;5%) i velike interindividualne razlike u terapijskom odgovoru čiji uzroci nisu u potpunosti obja&scaron;njeni. Poslednjih godina velika pažnja se posvećuje ispitivanjima žučnih kiselina u razvoju novih farmaceutskih formulacija zbog svoje uloge u solubilizaciji i modifikaciji prodora lekova kroz biolo&scaron;ke membrane. Zbog svega navedenog, u fokusu na&scaron;eg istraživanja su bile potencijalne interakcije između simvastatina, probiotskih bakterija i žučnih kiselina o kojima se vrlo malo zna, a od izuzetne su važnosti, zbog mogućeg uticaja na farmakokinetske i farmakodinamske osobine simvastatina, pa samim tim i na konačan terapijski odgovor kod pacijenta.Cilj istraživanja je bio da se ispita prodor i metabolizam simvastatina u probiotskim bakterijama kao i uticaj različitih žučnih kiselina na transport ovog leka u bakterijske ćelije. Takođe, cilj je bio da se ispita uticaj soli žučnih kiselina na distribucioni koeficijent simvastatina, kao i interakcije žučnih kiselina sa simvastatinom na nivou transportnih proteina probiotskih bakterija kako bi se objasnila priroda očekivanih interakcija.Identifikacija i kvantifikacija uzoraka vr&scaron;ena je metodom tečne hromatografije sa masenom spektrometrijom (LC-MS/MS). Kori&scaron;ćenjem programskih paketa VolSurf+ i Molinspiration, za identifikovane metabolite su izračunati molekulski deskriptori koji opisuju fizičko-hemijske i farmakokinetske osobine molekula. Određivanje distribucionog koeficijenta vr&scaron;eno je Shake-flask metodom. Interakcije žučnih kiselina sa simvastatinom na nivou transportnih proteina probiotskih bakterija ispitane su doking studijama pomoću SwissDock programa. Prilikom dvadesetčetvoročasovne inkubacije sa probiotskim bakterijama uočen je statistički značajan pad koncentracije simvastatina u ekstracelularnom sadržaju. Ukupan sadržaj simvastatina, kao zbir ekstracelulamog i intracelularnog sadržaja, je tokom čitavog ispitivanog perioda bio statistički značajno niži u odnosu na kontrolnu grupu bez probiotika navodeći na zaključak da se deo simvastatina tokom vremena metabolisao pod dejstvom enzima ispitivanih bakterija. Detektovano je i identifikovano 8 metaboličkih produkata simvastatina. Na osnovu izračunatih vrednosti molekulskih deskriptora, očekuje se da će metabolit M-452, koji predstavlja hidroksilovani produkt simvastatinske kiseline, pokazati najbolje rezultate u pogledu fizičko-hemijskih osobina i bioraspoloživosti u biolo&scaron;kom sistemu. Žučne kiseline nisu dovele do statistički značajne modifikacije transporta simvastatina u/iz probiotskih bakterija. Ipak, u nekim vremenskim tačkama primećena je ne&scaron;to veća koncentracija leka u ekstracelulamom prostoru u grupama sa žučnim kiselinama. Ove razlike se mogu delimično objasniti rezultatima određivanja distribucionog koeficijenta koji su pokazali da ispitivane žučne kiseline dovode do statistički značajnog smanjenja distribucionog koeficijenta simvastatina usled povećanja rastvorljivosti u vodenoj fazi. Rezultatima doking studija procenjeno je da ispitivane žučne kiseline imaju veći afinitet prema čak 80% multidrug transportera ispitivanih bakterija u odnosu na simvastatin &scaron;to govori o mogućnosti ostvarivanja interakcija žučnih kiselina sa ovim lekom na nivou transportnih proteina probiotskih bakterija. Na osnovu dobijenih rezultata možemo zaključiti da probiotske bakterije imaju ogroman uticaj na sudbinu simvastatina u biolo&scaron;kom sistemu. Uzimajući u obzir činjenicu da probiotske bakterije ulaze u sastav normalne crevne flore i da svaki organizam poseduje specifičan bakterijski sastav, trebalo bi posvetiti vi&scaron;e pažnje ispitivanju njegovog uticaja na farmakokinetiku lekova. Neophodna su dalja in vivo ispitivanja kako bi se utvrdila potencijalna farmakolo&scaron;ka aktivnost identifikovanih metabolita simvastatina nastalih pod dejstvom enzimske aktivnosti probiotskih bakterija. Povećanje rastvorljivosti simvastatina pomoću žučnih kiselina otvara mogućnost za dalja istraživanja u cilju razvoja novih farmaceutskih formulacija sa pobolj&scaron;anom bioraspoloživosti i farmakokinetskim osobinama.</p> / <p>Interindividual differences in the composition and activity of the gut microflora may affect the metabolism of drugs as well as their final therapeutic response. Simvastatin is drug from the group of statins and has extremely low water solubility, low bioavailability (&lt;5%) and high interindividual differences in therapeutic response whose causes are not fully understood. In recent years, great attention has been paid to studies of bile acids in the development of new pharmaceutical formulations because of their role in the drug solubilization and modification of drug transport through biological membranes. Accordingly, interactions between simvastatin, probiotic bacteria and bile acids were the focus of our research due to great importance and potential influence on the pharmacokinetic and pharmacodynamic properties of simvastatin, and therefore the final therapeutic response in the patients. The aim of the study was to investigate the simvastatin transport and metabolism in probiotic bacteria as well as the effect of various bile acids on drug transport into the bacterial cell. Additonally, the aim was to investigate the influence of bile salts on the distribution coefficient of simvastatin, and the interactions of bile acids with simvastatin at the level of probiotic transport proteins in order to elucidate the nature of expected interactions. Identification and quantification of samples were performed with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Molecular descriptors that describe the physico-chemical and pharmacokinetic properties of identified metabolites were calculated using the software packages VolSurf+ and Molinspiration. Determination of the distribution coefficient was performed using Shake-flask method. Interaction of bile acids with simvastatin at the level of bacterial transport proteins were studied using docking studies with SwissDock program. During the twenty-four hours of incubation with probiotic bacteria, simvastatin concentrations in the extracellular contet showed a statistically significant decrease. The total amount of simvastatin, as the sum of the extracellular and intracellular amount, during the whole study period, was significantly lower in comparison with control group without probiotics, suggesting that the part of simvastatin was metabolized by the enzymatic activity of studied bacteria. Accordingly, eight metabolic products of simvastatin were detected and identified. Based on the calculated values of molecular descriptors, it is expected that the metabolite M-452, which is the hydroxylated product of simvastatin acid, will show the best results in terms of physico-chemical properties and bioavailability in biological system. Bile acids did not show a significant influence on simvastatin transport into probiotic bacteria. However, in some time points, slightly higher drug concentrations in the extracellular medium in groups with bile acids were observed. These differences can be partly explained by the results of the determination of the distribution coefficients which showed that investigated bile acids lead to a statistically significant decrease in simvastatin distribution coefficient due to increased solubility in the aqueous phase. The results of docking studies estimated that studied bile acids have stronger affinities for the 80% of bacterial multidrug transporters compared to simvastatin indicating the possibility of achieving the interactions of bile acids with simvastatin at the level of transport proteins of probiotic bacteria. Based on the obtained results it could be concluded that probiotic bacteria have great influence on the fate of simvastatin in a biological system. Taking into account the fact that probiotic bacteria are the normal part of gut microflora and that each individual has specific bacterial fingerprint, more attention should be paid on studying its influence on drug pharmakocinetics. Further in vivo studies are required in order to determine potential pharmacological activity of identified simvastatin metabolites. Increased water solubility of simvastatin with bile acids may open the possibility for further investigations with the aim of development of new pharmaceutical formulation with improved bioavailability and pharmacokinetic properties.</p>

Author: Nikola Jozefčeková Title: Endoglin and liver Form: Diploma Thesis University: Charles University, Faculty of Pharmacy in Hradec Králové Degree: Pharmacy This diploma thesis concludes the available information about endoglin, its isoforms in the liver and its impact on the liver during various pathological conditions. In the first part of the thesis describes morphology and physiology of the liver, its structure, histology and metabolic functions. Second part contains an information about endoglin, its isoforms and role in TGF-β signaling complex, expression of endoglin and regulation of the expression. In the third part are described liver diseases during which is expression of endoglin changed. This part deals with the significance of endoglin as a diagnostic and prognostic marker. Endoglin (CD105, TGF-β receptor III) is a homodimeric transmembrane glycoprotein and a membrane co-receptor TGF-β with high expression in endothelial cells. Endoglin plays an important role in the vascular development. Endoglin is involved in the processes of angiogenesis, vascular homeostasis and TGF-β signalization. It affects activity of TGF-βRII, ALK1 and ALK5 receptors. Due to the interactions with TGF-β complex, modulation of activity of ALK receptors and Smads, endoglin controls fibrotic and anti-...

Introdução: O enfisema pulmonar é o maior componente da doença pulmonar obstrutiva crônica (DPOC), é caracterizado pelo alargamento, destruição alveolar e inflamação do parênquima e vias aéreas pulmonares. A recente descrição do sistema colinérgico anti-inflamatório, um mecanismo neural que controla a inflamação por inibição de citocinas pró-inflamatórias, sugere uma importante participação deste sistema na fisiopatologia das doenças pulmonares. A acetilcolina (ACh), principal mediador deste sistema, é estocada em vesículas sinápticas pelo transportador vesicular de ACh (VAChT), proteína essencial para sua liberação na fenda sináptica. Objetivos: Avaliar se os efeitos da hipofunção colinérgica, por redução da expressão do VAChT, interferem com as alterações pulmonares em modelo experimental de enfisema pulmonar. Metodologia: Camundongos machos selvagens e mutantes, estes últimos com redução da função colinérgica por modificação genética nos níveis do VAChT, foram submetidos ao protocolo de elastase (PPE instilação nasal) ou salina. No dia 28, foi avaliado a função pulmonar, a inflamação e o remodelamento pulmonar. Por imunohistoquímica, avaliou-se a expressão de macrófago, NF-kB e isoprostano no pulmão. Algumas citocinas pró-inflamatórias foram avaliadas no homogenato pulmonar pelo Bioplex. Resultados: Animais selvagem que receberam elastase tiveram redução de elastância de tecido, aumento da inflamação no LBA e no tecido, aumento de citocinas pró-inflamatórias e IL-10, aumento do remodelamento pulmonar, e da expressão de NF-kB e de isoprostano. A deficiência colinérgica nestes animais submetidos ao mesmo protocolo de elastase amplificou a resposta inflamatória (macrófago e neutrófilo) no pulmão, níveis de MCP-1 e também aumento células positivas para NF-kB e isoprostano na região do eixo broncovascular. Conclusão: A ACh parece ter um papel protetor da inflamação neste modelo de enfisema pulmonar, pelo menos em parte pelo controle do NF-kB e do estresse oxidativo. Estes resultados sugerem ainda que o remodelamento e a função pulmonar no enfisema experimental não dependem totalmente do grau de inflamação pulmonar / Banckground: Pulmonary emphysema is a major component of chronic obstructive pulmonary disease (COPD), is characterized by enlargement, alveolar destruction and inflammation of the airways and lung tissue. The recent description of the cholinergic anti-inflammatory, a neural mechanism that controls inflammation by inhibition of proinflammatory cytokines, suggests an important role of this system in the pathophysiology of lung disease. The main mediator of this system is acetylcholine (ACh), which is stored in synaptic vesicles by vesicular acetylcholine transporter (VAChT) protein, which is essential for ACh release into the synaptic cleft. Aim: To evaluate whether the effects of cholinergic hypofunction by reduction on VAChT expression, interferes with pulmonary alterations in an experimental model of pulmonary emphysema. Methods: Male mice wild-type and mutant, the last one with reduced cholinergic function by genetic modification in the levels of VAChT, were submitted to the protocol of elastase (PPE intranasally) or saline. On day 28, pulmonary mechanics, inflammation in bronchoalveolar lavage fluid and tissue remodeling were analyzed. By immunohistochemistry, the expression of macrophage, NF-kB and isoprostane in lung was evaluated. Some proinflammatory cytokines were measured in lung homogenate by Bio Plex. Results: Wild-Type animals that received elastase presented a reduction in tissue elastance, an increase in BALF and tissue inflammation as well as in proinflammatory cytokines, IL-10, pulmonary remodeling, and expression of NF-kB and isoprostane. Cholinergic deficient in these animals submitted to the same elastase-induced emphysema protocol amplified the inflammatory response (macrophage and neutrophils) in the lungs, the levels of MCP-1 and the number of positive cells to NF-kB and isoprostane in bronchovascular axis. Conclusions: The ACh seems to have a protective role inflammation in this experimental model of emphysema, at least in part by controlling NF-kB and oxidative stress. These results further suggest that the remodeling and lung function in experimental emphysema does not depend entirely on the degree of lung inflammation

Introdução: O enfisema pulmonar é o maior componente da doença pulmonar obstrutiva crônica (DPOC), é caracterizado pelo alargamento, destruição alveolar e inflamação do parênquima e vias aéreas pulmonares. A recente descrição do sistema colinérgico anti-inflamatório, um mecanismo neural que controla a inflamação por inibição de citocinas pró-inflamatórias, sugere uma importante participação deste sistema na fisiopatologia das doenças pulmonares. A acetilcolina (ACh), principal mediador deste sistema, é estocada em vesículas sinápticas pelo transportador vesicular de ACh (VAChT), proteína essencial para sua liberação na fenda sináptica. Objetivos: Avaliar se os efeitos da hipofunção colinérgica, por redução da expressão do VAChT, interferem com as alterações pulmonares em modelo experimental de enfisema pulmonar. Metodologia: Camundongos machos selvagens e mutantes, estes últimos com redução da função colinérgica por modificação genética nos níveis do VAChT, foram submetidos ao protocolo de elastase (PPE instilação nasal) ou salina. No dia 28, foi avaliado a função pulmonar, a inflamação e o remodelamento pulmonar. Por imunohistoquímica, avaliou-se a expressão de macrófago, NF-kB e isoprostano no pulmão. Algumas citocinas pró-inflamatórias foram avaliadas no homogenato pulmonar pelo Bioplex. Resultados: Animais selvagem que receberam elastase tiveram redução de elastância de tecido, aumento da inflamação no LBA e no tecido, aumento de citocinas pró-inflamatórias e IL-10, aumento do remodelamento pulmonar, e da expressão de NF-kB e de isoprostano. A deficiência colinérgica nestes animais submetidos ao mesmo protocolo de elastase amplificou a resposta inflamatória (macrófago e neutrófilo) no pulmão, níveis de MCP-1 e também aumento células positivas para NF-kB e isoprostano na região do eixo broncovascular. Conclusão: A ACh parece ter um papel protetor da inflamação neste modelo de enfisema pulmonar, pelo menos em parte pelo controle do NF-kB e do estresse oxidativo. Estes resultados sugerem ainda que o remodelamento e a função pulmonar no enfisema experimental não dependem totalmente do grau de inflamação pulmonar / Banckground: Pulmonary emphysema is a major component of chronic obstructive pulmonary disease (COPD), is characterized by enlargement, alveolar destruction and inflammation of the airways and lung tissue. The recent description of the cholinergic anti-inflammatory, a neural mechanism that controls inflammation by inhibition of proinflammatory cytokines, suggests an important role of this system in the pathophysiology of lung disease. The main mediator of this system is acetylcholine (ACh), which is stored in synaptic vesicles by vesicular acetylcholine transporter (VAChT) protein, which is essential for ACh release into the synaptic cleft. Aim: To evaluate whether the effects of cholinergic hypofunction by reduction on VAChT expression, interferes with pulmonary alterations in an experimental model of pulmonary emphysema. Methods: Male mice wild-type and mutant, the last one with reduced cholinergic function by genetic modification in the levels of VAChT, were submitted to the protocol of elastase (PPE intranasally) or saline. On day 28, pulmonary mechanics, inflammation in bronchoalveolar lavage fluid and tissue remodeling were analyzed. By immunohistochemistry, the expression of macrophage, NF-kB and isoprostane in lung was evaluated. Some proinflammatory cytokines were measured in lung homogenate by Bio Plex. Results: Wild-Type animals that received elastase presented a reduction in tissue elastance, an increase in BALF and tissue inflammation as well as in proinflammatory cytokines, IL-10, pulmonary remodeling, and expression of NF-kB and isoprostane. Cholinergic deficient in these animals submitted to the same elastase-induced emphysema protocol amplified the inflammatory response (macrophage and neutrophils) in the lungs, the levels of MCP-1 and the number of positive cells to NF-kB and isoprostane in bronchovascular axis. Conclusions: The ACh seems to have a protective role inflammation in this experimental model of emphysema, at least in part by controlling NF-kB and oxidative stress. These results further suggest that the remodeling and lung function in experimental emphysema does not depend entirely on the degree of lung inflammation

<p>The appearance of adverse drug reactions is a common reason for hospitalization in Western countries. Research on underlying biological mechanisms for interindividual variability in drug response aims to better identify patients with exceptional genetic traits, disease conditions or risk of drug-drug interactions and thereby help to prevent adverse drug reactions. </p><p>Active transport mechanisms are involved in the absorption and disposition of several therapeutic agents. The main objective of this thesis was to investigate factors potentially affecting transport proteins and thus contributing to variability in drug absorption and disposition. Studies of physiological, genetic, environmental, and pathological factors were included. The main focus was the two ATP-binding cassette (ABC) transporters: P-glycoprotein 170 (Pgp) and Breast Cancer Resistance Protein (BCRP). </p><p>Quantification of transport protein mRNAs along the human intestine indicated that eight of the nine investigated drug transporters were expressed in a region-dependent manner. Effects of drug-drug interactions may therefore vary depending on the site of absorption. The genetic aspect was illustrated by identification of sequence variation in the gene encoding BCRP, the most highly expressed ABC transporter along the human intestine. Drug-drug interactions are important environmental causes of interindividual variability. An evaluation of the effects of Pgp-mediated drug-drug interactions showed that patients receiving Pgp inhibitors had elevated serum concentrations of the Pgp substrate digoxin and that digoxin concentrations were positively correlated with the number of co-administered Pgp inhibitors. The final topic in this thesis was that of drug-disease interactions. BCRP and Pgp were down-regulated during active inflammation in patients with ulcerative colitis. This may contribute to altered concentrations of drug in the intestinal mucosa during periods of inflammation and possibly to changes in drug absorption.</p><p>To summarize, results of this thesis emphasize the complex background to the interindividual variability of drug transport proteins, where physiological, genetic, environmental and pathological factors all can contribute.</p>