Tesis sobre el tema "Metabolismo"

Orientador: Maria Terezinha Serrão Peraçoli
Resumo: O aumento da atividade dos radicais livres e da produção de citocinas pró-inflamatórias por monócitos pode estar envolvido na patogênese da pré-eclâmpsia. A silibinina é o componente mais ativo da silimarina (Silybum marianum), um flavonóide polifenólico que possui efeitos anti-oxidante e anti-inflamatório. O objetivo do presente estudo foi avaliar o efeito da silibinina sobre a produção de citocinas pró e anti-inflamatórias, bem como sobre a liberação de ânion superóxido (O2 -) por monócitos de gestantes com pré-eclâmpsia.Foram selecionadas 30 gestantes com préeclâmpsia (PE), 30 gestantes normais (GN) e 30 mulheres normais nãográvidas (NG). Monócitos de sangue periférico foram incubados na presença ou ausência de silibinina nas concentrações de 5 e 50 g/mL por 60min e, estimulados ou não com ester de forbol (PMA) para determinação de O2 -. Estes monócitos também foram estimulados ou não com lipopolissacáride (LPS) por 18h e, no sobrenadante das culturas foram determinadas, pela técnica de ELISA, as seguintes citocinas: fator de necrose tumoral-alfa (TNF- ), Interleucinas (IL-) IL-6, IL-10, IL-12, IL-15, fator estimulador de colônias de granulócitos e monócitos (GM-CSF) e fator de crescimento e transformação (TGF- . A atividade da enzima superóxido dismutase (SOD) foi avaliada em hemolisado de eritrócitos dos três grupos estudados.Níveis significativamente maiores de O2 - foram liberados por monócitos de gestantes com PE quando comparados com GN e NG, confirmando o estado ativado dessas células. A atividade da SOD foi significativamente maior no grupo de gestantes com PE. Os níveis endógenos de TNFforam significativamente mais elevados nas pacientes com PE, quando comparados com os grupos GN e NG, enquanto os níveis de IL-10 foram significativamente menores nas gestantes com PE. A capacidade de produção de citocinas pelos... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Increased free radical activity and high proinflammatory cytokine production by monocytes may be implicated in the pathogenesis of preeclampsia. Silibinin is a major active component of silymarin (Silybum marianum), a polyphenolic plant flavonoid that has antioxidant and anti-inflammatory effects. This study investigated the in vitro effect of silibinin on monocyte production of pro- and anti-inflammatory cytokines, as well as on superoxide anion (O2-) release in pregnant women with preeclampsia.Thirty preeclamptic (PE), 30 healthy pregnant (HP) and 30 healthy non-pregnant (NP) women were included. Peripheral blood monocytes were incubated with or without silibinin at 5 and 50ug/mL for 60 min, and stimulated with or without phorbol myristate acetate (PMA) for the assessment of O2-. These cells were also cultured with or without lipopolysaccharide (LPS) for 18h and tumor necrosis factor alpha (TNF-alpha), interleukin (IL)-6, IL-10, IL-12p40, IL-15, granulocyte-macrophage colony-stimulating factor (GM-CSF) and transforming growth factor-beta1 (TGF- 1) in monocyte culture supernatants were determined by ELISA. The activity of the enzyme superoxide dismutase (SOD) was evaluated in erythrocyte lisates of the three groups studied.Monocytes of preeclamptic patients release significantly higher levels of O2 - in comparison to HP and NP women confirming the activation state of these cells. SOD activity in erythrocytes was significantly higher in preeclamptic patients. The endogenous levels of TNFwere significantly higher in PE patients than in HP and NP groups, while IL-10 production was significantly lower in PE women. The levels of IL-6, IL-12 and GM-CSF spontaneously produced by monocytes were higher in HP and PE groups than in NP women. The capacity of cytokine production by LPS-stimulated monocytes was preserved in all the three groups studied... (Complete abstract click electronic access below)
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The analysis of the energetic and respiratory metabolism of Taenia crassiceps cysticerci innoculated into the encefalum of female BALB/c mice was performed. After 30 days of infection the mice were treated with low dosages (3.0 and 6.0 mg/kg) of albendazole and praziquantel aiming the observation of the alterations caused by a hostile environment on the parasite's metabolism. The aim of this study was to detect the influence of low dosages of anti-helminthic drugs on the production of organic acids related to the intermediary metabolism (oxaloacetate, malate, fumarate, succinate, citrate and α-ketoglutarate), carbohydrates metabolism (pyruvate, lactate, propionate, oxaloacetate and malate) and fatty acids and proteins catabolism (β-hydroxibutyrate, acetoacetate, acetate, α-ketoglutarate and oxalate) by T. crassiceps cysticerci innoculated into the CNS of BALB/c mice. Regarding the glycolisis, it was possible to detect lactate in all samples which shows that this parasite performs a lactic fermentation. As to the intermediary metabolism it was possible to detect oxaloacetate, citrate, malate, succinate, fumarate and α-ketoglutarate which indicates that the cysticerci may have used the tricarboxilic acid cycle (TCA) to produce energy. However facing the results found the evidences show that the cysticerci used a metabolic pathway denominated reversion of the TCA for energy production which presents as main end products propionate and acetate. There were no significant differences between the groups treated with the anti-helminthic and the control one except that the group treated with 6.0 mg/kg of albendazole presented acetate as main end product of the TCA reversion while the other groups presented propionate. In the groups treated with praziquantel, both concentrations, there was no production of acetate and in the 6.0 mg/kg of praziquntel there was no production of β-hydroxibutyrate wheter in the groups treated with both concentrations of albendazol there was an increase in the production of this organic acid which indicates the maximization of the energetic pathway of fatty acids oxidation and/or of the proteic catabolism. It was possible to conclude that T. crassiceps cysticerci when exposed to the conditions of this study preferred an anaerobic energy production pathway and probably performed the catabolism of carbohydrates, proteins and fatty acids.
Realizou-se a análise do metabolismo energético e respiratório de cisticercos de Taenia crassiceps inoculados no encéfalo de camundongos BALB/c fêmeas. Após 30 dias de infecção, esses camundongos foram tratados com baixas doses (3,0 e 6,0 mg/kg) de albendazol e praziquantel visando observação de alterações provocadas no metabolismo do parasito. O objetivo deste trabalho foi detectar a influência de baixas doses desses anti-helmínticos na produção dos ácidos orgânicos do metabolismo intermediário (citrato, α-cetoglutarato, succinato, fumarato, malato, oxaloacetato), de carboidratos (piruvato, lactato, oxaloacetato, malato, acetato e propionato), de ácidos graxos e proteínas (β-hidroxibutirato, acetoacetato, acetato, α-cetoglutarato e oxalato) realizados por cisticercos de T. crassiceps implantados no SNC de camundongos BALB/c fêmeas. Com relação a glicólise, detectou-se lactato em todas as amostras mostrando que esse parasito fez fermentação lática; em relação ao metabolismo intermediário foi possível detectar os ácidos orgânicos citrato, α-cetoglutarato, succinato, fumarato, malato e oxaloacetato, indicando que os cisticercos apresentaram elementos do ciclo do ácido cítrico para produzir energia. Porém diante dos resultados encontrados a principal evidência é de que esses cisticercos utilizaram uma via denominada reversão do ciclo do ácido cítrico para produzirem energia, obtendo como produtos finais propionato e acetato. Não se observou diferenças significativas entre os grupos que sofreram tratamentos e os grupos controle exceto pelo fato de que, o grupo tratado com albendazol 6,0 mg/kg de peso do camundongo teve como principal produto final da reversão do ciclo do ácido cítrico, o acetato, ao contrário dos demais grupos que tiveram o propionato; nos grupos tratados com praziquantel não houveram a produção de acetato como nos demais grupos e no grupo tratado com praziquantel 6,0 mg/kg não houve a produção de β-hidroxibutirato; nos grupos tratados com albendazol observou-se um aumento na produção de β-hidroxibutirato indicando que esse grupo maximizou a via de produção energética através da β-oxidação de ácidos graxos e/ou através do catabolismo proteico.Concluímos que os cisticercos de T. crassiceps, nas condições do presente trabalho, deram preferência para uma via de produção energética anaeróbia e provavelmente catabolizaram, além de carboidratos, proteínas e ácidos graxos para produzirem ATP.

O gene TrSNF1, homólogo aos membros da subfamília das proteínas serina-treonina quinases ativadas por AMP (AMPK) e relacionadas a SNF1, foi isolado do fungo filamentoso trichoderma reesei. A seqüência de aminoácidos putativa possui um domínio de quinase com 42% de identidade e 59% de similaridade com outras proteínas quinases da mesma subfamília. Em S. cerevisiae a SNFl é essencial para a expressão de genes reprimidos por glicose, em resposta a privação de glicose do meio de cultura. A expressão de TrSNFl em levedura mutante para SNF1, restaura a função de SNF1. A expressão de um antisense de TrSNFl em T. reesei causa um atraso na expressão do gene regulado por glicose, CBHI. Além disso, em experimento utilizando matrizes de DNA foi possível observar uma alteração da tendência global da expressão gênica entre a cepa selvagem e a cepa antisense. A observação da homologia estrutural com proteínas quinase da mesma subfamília, a similaridade funcional com SNFl de S. cerevisiae, e a alteração no padrão da expressão gênica in vivo, sugerem que TrSNFl pode estar envolvido na regulação do metabolismo de carboidratos em T. reesei.
A gene homologue to the members of the AMP-activated/SNFl protein kinase subfamily, TrSNF1, was isolated from the filamentous fungus Trichoderma reesei. The predicted protein of 692 amino acids has a kinase domain, that share 42 % identity and 59 % similarity to that of serine/threonine protein kinase of this family. In Saccharomyces cerevisiae, the SNFl protein kinase is required for expression of glucose repressed genes in response to withdrawal of glucose from the medium. Expression of the Trichoderma reesei SNF1-related sequence in yeast SNFl mutant restores SNFl function. The TrSNFl antisense expression in T. reesei causes a control alteration in the glucose-regulated gene CBHI. The observed structural identity with the AMP-activated/SNFl protein kinase subfamily, and the functional similarity to the yeast SNFl suggest that the TrSNFl may be involved in the regulation of sugar metabolism in Trichoderma reesei.

O melanoma é composto por células malignas e também por um estroma de sustentação que inclui fibroblastos, células imunológicas, endoteliais, matriz extracelular, dentre outros fatores. Assim, os tumores não são entidades independentes, eles interagem ativamente com o microambiente adjacente de forma bidirecional através de sinais moleculares que modulam o fenótipo maligno. Um dos sinais bioquímicos para desenvolvimento desse fenótipo se dá pelo catabolismo de Trp pela via das quinureninas, que gera compostos com diversas atividades biológicas, que no tumor estão envolvidas com tolerância e imunoescape e, logo, com prognóstico ruim para os pacientes. Até o presente momento apenas o consumo de Trp e a formação de um único metabólito, a quinurenina (KYN), tem sido associada a malignidade dos melanomas. A fim de ampliar e elucidar os mecanismos bioquímicos do metabolismo desse aminoácido em melanomas, estudamos mais de quinze compostos de todas as rotas catabólicas de Trp em células da pele, células imunológicas, linhagens tumorais e amostras clínicas de melanoma. De forma inédita pudemos observar que as células da pele tem maior habilidade de sintetizar KYN quando comparadas às linhagens tumorais, demonstrando que o catabolismo de Trp peritumoral pode ser responsável pelos fenômenos de imunotolerância e escape. Além disso, o metabolismo de Trp pode estar envolvido nos mecanismos de homeostasia da pele, já que especificamente essas células produzem compostos com atividade biológica nesse órgão. As células imunológicas possuem um perfil metabólico completamente diferente umas das outras: monócitos, macrófagos e dendríticas possuem maior ativação da via KYN enquanto linfócitos e neutrófilos possuem maior indução da rota que gera serotonina e melatonina. Mesmo nos diferentes fenótipos de macrófagos, M1 e M2a, foram observadas marcações especificas de metabolismo, que podem estar relacionadas às atividades anti- ou pró-tumoral dessas células no microambiente. Em amostras clínicas, apesar da principal diferença entre nevos e melanomas ser a concentração de KYN, diversas outras alterações no metabolismo de tiptofano foram observadas, o que mostra a complexa magnitude deste metabolismo na fisiopatologia da pele
Melanoma is composed of malignant cells and also by a stromal support that includes fibroblasts, immune cells, endothelial cells, extracellular matrix, among other factors. Thus, tumors are not separate entities; they actively interact with the surrounding microenvironment bi-directionally through molecular signals that modulate the malignant phenotype. One of biochemical signals for the development of this phenotype occurs by Trp catabolism through kynurenine pathway, that generates compounds with diverse biological activities, which in tumors are involved with tolerance and imunoescape and therefore with poor prognosis for patients. To date only the consumption of Trp and formation of a single metabolite, kynurenine (KYN), has been associated with malignant melanomas. In order to enlarge and clarify the biochemical mechanisms of this amino acid metabolism in melanomas, we have studied more than fifteen compounds of all catabolic routes of Trp in skin cells, immune cells, tumor cell lines and clinical samples of melanoma. In an unique way we could observe that the skin cells has superior ability to synthesize KYN when compared to tumor cell lines, demonstrating that the peritumoral catabolism of Trp may be responsible for the phenomena of immune tolerance and escape. Furthermore, the Trp metabolism may be involved in skin homeostasis mechanisms, since these cells produce specific compounds with biological activity in this organ. The immune cells have a completely different metabolic profile among them: monocytes, macrophages and dendritic cells have greater KYN pathway activation, and lymphocytes and neutrophils possess greater induction of the route that generates serotonin and melatonin. Even in different macrophages phenotypes, M1 and M2a, we observed specific metabolic marks, which may be related to the anti- or pro-tumoral activity of these cells in the tumor microenvironment. In clinical samples, although the main difference between nevi and melanomas is the concentration of KYN, a range of other changes in Trp metabolism were observed, which shows the complex magnitude of this metabolism in the skin pathophysiology

Pharmaceuticals which are used worldwide are designed to facilitate the life for the human society and have an important role in treatment and prevention of disease for both humans and animals. They are ubiquitous in the aquatic environment and are mainly derived from municipal wastewater treatment plants (WWTPs) due to their low removal rate. Therefore, their presence in the environment is directly linked to the human impact. Various biological and abiotic processes in the environment can transform them to transformation products (TPs). In many cases, transformation is already initiated in the human body by a variety of drug-metabolizing enzymes. The metabolites formed through human metabolism present some modifications in their chemical structures that can differ in physicochemical properties to their parent compound. Once they are excreted from the human body, both the unmetabolised parent drug and their metabolites enter WWTPs by means of the sewer system. Since the WWTPs are not designed to remove completely pharmaceutical residues, the fraction not removed after the treatment will eventually end up in the receiving water bodies. Consequently, due to pharmaceutical transformations in the human body, biotransformations in WWTPs and phototransformations in surface water, they can potentially produce a high number of TPs in real world samples which makes their identification a challenge. In this thesis, two different approaches (TPs profiling and suspect screening) based on high resolution mass spectrometry (HRMS) for the detection and identification of TPs of pharmaceuticals were investigated. TPs profiling approach was applied for the identification of phototransformation products of an antiviral zanamivir (ZAN) in batch reactors filled with surface water. On the other hand, suspect screening approach was applied for evaluation of transformation, prioritization and identification of photoTPs of six iodinated contrast media in surface water. Finally, a combination of suspect and TPs profiling approach was applied for the detection of TPs of an anticonvulsant lamotrigine and its main human metabolite lamotrigine N2-glucuronide which were formed as the result of their degradation in both activated sludge and pH dependent hydrolysis. The TPs profiling approach for evaluation of these transformations is illustrated in the example of photodegradation of an antiviral ZAN with identification of its TPs in surface water (Chapter 3.). Here a set of lab- scale experiments was performed in order to determine the susceptibility of ZAN towards photodegradation under simulated and natural sunlight. The identification of the TPs was performed using hydrophilic interaction liquid chromatography coupled to high resolution mass spectrometry (HILIC-HRMS) where four photoTPs were tentatively identified and their proposed structures were rationalized by photolysis mechanisms. Kinetic experiments showed that photodegradation kinetics of ZAN in surface waters would proceed with slow kinetics since upon exposure of aqueous solutions of surface water (20 μg L-1) to simulated sunlight, ZAN was degraded with t1/2 of 3.6 h. Under natural sunlight irradiating surface water, about 30 % of the initial concentration of the antiviral disappeared within 18 days. However, when ZAN and its TPs were retrospectively screened from surface water extracts, neither the parent nor the TPs were detected. The results of this TP profiling used for the identification of TPs of ZAN, although straightforward, suggests that it is not suitable when dealing with a considerably elevated number of TPs formed in batch experiments. However, time and effort needed to be optimised for the structure elucidation of 108 photoTPs of six iodinated contrast media (ICM) (Chapter 3.). Again, the photodegradation study was performed in surface water spiked with the ICMs using a sunlight lab-scale simulator. 108 TPs were generated and each photoTP was characterised by its unique exact mass of the molecular ion and retention time and added to a suspect list. Once the suspect list was generated, the photoTPs were searched in thirteen surface water samples which were extracted using a generic solid- phase extraction method (four cartridges of different chemistries in order to retain ample number of compounds with different chemical properties). Based on their detection frequency (those TPs with the frequency higher than 50 % were deemed important), eleven TPs were prioritized and their structures elucidated by HRMS and NMR (when possible). Out of the eleven prioritised TPs, ten were formed as the result of deiodination (either by deiodination, oxidative deiodination or intramolecular elimination). In the real surface water samples, median concentration of parent compounds was 110 ngL-1 reaching up to 6 µgL-1 for iomeprol while TPs were found at median concentration of 8 ngL-1, reaching up to 0.4 µgL-1 for iomeprol TP651-B. Here detection-based prioritization served as a crucial step to reduce the number of TPs to be identified and thereby reducing costs and time for the subsequent target analysis. This time-effective approach not only guaranties that the degradation products elucidated would be found, but also that they are environmentally relevant. In summary, the proposed screening approach facilitates the evaluation of the degradation of polar compounds at a real scale with a fast detection of TPs without prior availability of the standards. Approach used for detection and identification of TPs of ICM in Chapter 3 was an example of suspect screening where the suspect list of TPs was generated at lab-scale, In Chapter 4, the work started with a suspect screening of lamotrigine (LMG) and related compounds (its human metabolites, synthetic impurities and photoTPs) which were listed from the literature and searched in wastewater and surface water samples. As the result of suspect screening, LMG, three human metabolites and a LMG synthetic impurity (OXO-LMG) were detected in the screened samples. Preliminary results showed significantly higher concentrations of OXO-LMG in wastewater effluent, suggesting its formation in the WWTPs. However, biodegradation reactors amended with mixed liquor at neutral pH showed that LMG is resistant to biodegradation with only about 5 % elimination after 6 days. Since LMG is extensively and predominantly metabolised by phase II metabolism to its N2-glucuronide, this metabolite (LMG-N2-G) was degraded following the same experimental setup. Results showed that this metabolite was the actual source of the TP detected. Additionally, in batch experiments, LMG-N2-G was transformed, following pseudo-first kinetics, to three TPs as a result of i) deconjugation (to LMG), ii) oxidation of the glucuronic acid (to LMG-N2-G-TP430) and iii) amidine hydrolysis in combination with deconjugation (to OXO-LMG). In order to further rationalize the formation of the TP OXO-LMG, the stability of LMG-N2-G and related compounds was studied as a function of pH in the range of 4 – 9. Same as during biodegradation, LMG was stable across the entire pH range tested. However, LMG-N2-G was transformed to three TPs at neutral – basic pH. They were identified as TPs formed after hydrolysis of amidine and guanidine moieties. The third TP detected was an intermediated in the guanidine hydrolysis reaction. Kinetic experiments in wastewater samples at different concentration (20 and 200 nM) and pH (pH 6.5, 7, 8, 8.5 and 9) demonstrated that while the degradation constants were concentration independent, at higher pH, LMG-N2-G degraded at higher rate. The pH-dependent stability experiments of related compounds with different nitrogen N2-substituents on the 1,2,4-triazine ring showed that reaction of amidine and guanidine hydrolysis depends on imine tautomer equilibrium whose formation depends directly on the N2-supsitutent. LMG- N2-G major abiotic TP (amidine hydrolysis TP) was detected in hospital effluent and WWTP influent samples. Having in mind the concentrations of both biotic and abiotic TPs detected, a total mass balance at two- concentration levels batch reactors was closed at 86% and 102%, respectively. In three WWTPs total mass balance of LMG-N2-G ranged from 71-102%. Finally, LMG-N2-G and its TPs were detected in surface water samples with median concentration ranges of 23–186 ngL-1. The work presented in this chapter gives a new insight into the behaviour of glucuronides of pharmaceuticals, suggesting that they might also be sources of yet undiscovered, but environmentally relevant TPs.
Els productes farmacèutics, l'ús dels quals s'estén a nivell mundial, estan dissenyats per millorar la qualitat de vida de la societat i juguen un paper clau en el tractament i la prevenció de malalties, tant en homes com en animals. Aquests compostos químics es troben de forma ubíqua en el medi ambient. Això es deu principalment a les estacions depuradores d'aigua residual (EDARs), les quals no són capaces d'eliminar de manera eficient aquest tipus de compostos, ja que no estan dissenyades amb aquesta finalitat. Per tant, la presència de fàrmacs en el medi ambient està directament relacionada amb l'activitat humana. Un cop al medi ambient, l'estructura d'aquests compostos pot ser modificada per diferents processos biològics i abiòtics, generant-se així els que es coneixen com a productes de transformació (PTs). De fet, la transformació dels fàrmacs pot iniciar-se en alguns casos en el cos humà, després de la seva administració a causa de l'activitat metabòlica dels diferents enzims que posseeix l'home. Els metabòlits formats en aquests processos presenten algunes modificacions en les seves estructures químiques pel que fa al compost original, i, en conseqüència, unes propietats fisicoquímiques diferents. Un cop excretats, tant el fàrmac original no metabolitzat com els seus metabòlits arriben a les EDARs mitjançant la xarxa de sanejament municipal d'aigües residuals. La fracció d'aquests compostos que no s'elimina en els diferents tractaments realitzats en l'EDAR, es descarrega juntament amb l'efluent de la planta als aigües receptores. El gran nombre de transformacions que poden experimentar els fàrmacs en el seu cicle de vida a causa del seu metabolisme en el cos humà, la seva biotransformació per microorganismes i la seva fototransformació per llum solar, pot generar un nombre molt elevat de PTs en el medi ambient, i, per tant, la identificació dels mateixos, necessària per avaluar el destí dels fàrmacs en el medi ambient, és un desafiament. En el desenvolupament d'aquesta tesi es van aplicar dues aproximacions analítiques diferents: a)avaluació de perfils de PTs generats en experiments a escala de laboratori i b) anàlisi qualitativa dirigida suspect screening en mostres reals, tots dues basades en espectrometria de masses d'alta resolució (HRMS) per a la detecció i identificació de PTs de productes farmacèutics. L'aproximació d'avaluació de perfils de PTs en reactors a escala de laboratori es va aplicar per identificar productes de fototransformació (fotoPTs) de l'antiviral zanamivir (ZAN) en aigua superficial. L'aproximació de suspect screening es va aplicar per prioritzar i identificar fotoPTs de sis mitjans de contrast radiològics iodats (ICM) en aigua superficial. Finalment, una combinació de les dues aproximacions es va aplicar per detectar PTs de l’anticonvulsiu lamotrigina (LMG) i del seu principal metabòlit humà, el lamotrigina-N2-glucurònid (LMG- N2-G), resultants de la seva degradació tant en fangs activats com a reaccions d'hidròlisi a diferents valors de pH.

El canvi en l’estil de vida que s’ha produït en les societats desenvolupades els darrers anys ha tingut com a contrapartida l’aparició de conductes sedentàries i modificacions en la dieta. Com a conseqüència d’aquests factors s’han produït diverses alteracions metabòliques que han causat un augment de la prevalença de l’obesitat. Aquesta obesitat té una sèrie d’efectes adversos sobre la fisiologia cardiovascular i és considerada un important factor de risc pel desenvolupament de la insuficiència cardíaca. De fet, el consum de dietes amb un elevat contingut en greixos (HFD) s’ha relacionat amb una sèrie d’alteracions cardíaques directes com són la inflamació, la hipertròfia i la disfunció contràctil. Durant el procés inflamatori que es produeix en les esmentades malalties cardiovasculars, les cèl•lules cardíaques humanes secreten citocines i quimiocines proinflamatòries com el TNF-α, MCP-1, i IL-6, molècules que es troben sota el control transcripcional del factor de transcripció ubic i induïble anomenat NF-κB. Aquestes citocines exerceixen diversos efectes pleiotròpics autocrins en les cèl•lules cardíaques, tot produint un efecte de retroalimentació positiva del procés inflamatori que contribueix al desenvolupament d’aquestes malalties. En condicions normals, el cor de mamífers adults obté energia en forma d’ATP principalment a partir de la β-oxidació d’àcids grassos en el mitocondri, encara que aquest orgànul és capaç de catabolitzar altres substrats com la glucosa o el lactat per tal d’assegurar una font constant d’energia. Ara bé, en determinades circumstàncies, com és el cas de la hipertròfia i la insuficiència cardíaques, aquesta flexibilitat de substrat es veu compromesa i la β-oxidació d’àcids grassos es redueix degut a que la font principal d’energia passa a ser la glucosa. Aquests canvis metabòlics comporten una desregulació del control transcripcional de gens relacionats amb el transport, captació i catabolisme dels àcids grassos i la glucosa. En el miocardi, els factors de transcripció implicats en el control d’aquests gens inclouen ERRα i PPARβ/δ. Ambdós factors de transcripció, participen en l’activació de la PDK4, enzim clau en la modulació homeostàtica de la glucosa. Aquesta cinasa regula l’activitat de la PDC, enzim que catalitza la reacció de descarboxilació del piruvat a acetil-CoA, tot limitant l’ús de carbohidrats com a font d’energia en mitocondris i afavorint així la β-oxidació d’àcids grassos. En l’activació de la transcripció de PDK4 també hi participa PGC-1α, que interacciona amb ERRα i PPARβ/δ, tot incrementant-ne la seva activitat transcripcional. Estudis recents però, semblen indicar que no només aquests dos factors de transcripció participen en la regulació de PDK4. És el cas d’E2F1, un factor de transcripció clau en la regulació de la transició de la fase G1 a la fase S del cicle cel•lular i del qual la regió promotora del gen que codifica per PDK4 en presenta dos llocs d’unió. Estudis recents suggereixen que PPARβ/δ, que és la forma predominant en les cèl•lules cardíaques, pot atenuar les vies de senyalització inflamatòries i, per tant, interferir en la remodelació cardíaca. Aquesta funció és en gran mesura deguda a la capacitat dels PPAR, un cop han estat activats per agonistes, de formar complexos amb altres factors de transcripció activats, com ara NF-κB i STAT resultant així en la inhibició de l’activitat transcripcional d’aquests últims. Així l’ús d’agonistes PPARβ/δ podria ser un camí força interessant de cara a trobar potencials fàrmacs per tal de pal•liar les afeccions cardíaques derivades d’alteracions metabòliques i amb un rerefons inflamatori. En conjunt en aquest treball es presenten una sèrie de resultats destinats a conèixer de forma més detallada els mecanismes moleculars que relacionen les alteracions metabòliques i els processos inflamatoris en cor, per tal de poder buscar potencials dianes farmacològiques amb l’objectiu de prevenir i tractar aquests estats patològics.
The change in lifestyle that has occurred in developed societies in recent years has been accompanied by the rise of sedentary behavior and changes in diet that have caused an increasing obesity prevalence. Obesity has a huge number of adverse effects on cardiovascular physiology and is considered an important risk factor for heart failure developement. In fact, high fat diets have been linked with direct cardiac abnormalities such as inflammation, hypertrophy and contractile dysfunction. During the inflammatory process that occurs in these diseases, human cardiac cells secrete proinflammatory cytokines and chemokines such as TNF-α, MCP-1, and IL-6, molecules that are under the control of the ubiquitous and inducible transcription factor NF-κB. In certain circumstances, such in hypertrophy and heart failure, the substrate flexibility in heart is compromised and the fatty acids β-oxidation is reduced because the main source of energy becomes the glucose. These metabolic changes lead to a deregulation on the transcriptional control of genes associated with transport, uptake and catabolism of fatty acids and glucose. In the myocardium, among the transcription factors involved in the control of these genes we found ERRα and PPARβ/δ. Both transcription factors, are involved in PDK4 activation, an important enzyme in the homeostatic modulation of glucose. This kinase regulates PDC activity, an enzyme that catalyzes the decarboxylation from pyruvate to acetyl-CoA, limiting the use of carbohydrates as energy source in mitochondria and thus favoring the fatty acid β-oxidation. In the PDK4 transcription activation also participates PGC-1α, which interacts with ERRα and PPARβ/δ, increasing its transcriptional activity. However recent studies, suggest that not only these two transcription factors are involved in PDK4 regulation. Other transcription factors as E2F1, which is crucial for cell cycle control, may regulate PDK4 expression. Overall, the results shown in this work are aimed to learn in more detail the molecular mechanisms linking the metabolic disorders and inflammatory processes in heart, in order to find potential drug targets to prevent and treat these pathological states.

Orientador: Douglas Silva Domingues
Resumo: As plantas em seu ambiente respondem a uma infinidade complexa de fatores que podem romper sua homeostase fisiológica. Com relação aos estresses abióticos, a capacidade de organismos vegetais responderem a esses estresses é flexível e depende do balanceamento da interação entre moléculas de sinalização. Destacam-se entre as moleculas sinalizadora em plantas as espécies reativas de oxigênio (EROs) e espécies reativas de nitrogênio (ERNs), que estão envolvidos na aclimatação a estresses abióticos. O transporte, a biossíntese e o metabolismo de EROs e ERNs influenciam os mecanismos de resposta das plantas ao ambiente. Um elemento chave na geração de EROs em plantas são a NADPH oxidases, que são enzimas codificadas por uma família gênica em plantas, pouco estudada do ponto de vista evolutivo. Já um elemento regulador importante da homeostase de ERNs em plantas são as S-nitrosoglutationa redutases, enzimas também pouco estudadas do ponto de vista evolutivo. Nos últimos anos, diversos trabalhos sugerem que as plantas podem ser elicitadas por compostos químicos para melhor tolerar estresses abióticos. No entanto, os mecanismos de reprogramação fisiológica e transcricional por trás destes sinalizadores ainda são pouco conhecidos. O ácido hexanoico é um ácido carboxílico que, em baixas concentrações, possui um efeito indutor de resistência e modulador do metabolismo em diversos sistemas vegetais. No entanto, o impacto do ácido hexanoico na geração de EROs e de ERNs nunca foi avaliado.... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Plants in their environment respond to a complex infinity of factors that can interfere their physiological homeostasis. Regarding abiotic stresses, the ability of plants to respond to these stresses is flexible and depends on balancing the interaction among signaling molecules. Among the signaling molecules in plants, we highlight reactive oxygen species (ROS) and reactive nitrogen species (RNS), which are involved in acclimatization to abiotic stresses. The transport, biosynthesis and metabolism of ROS and ERNs influence plant response mechanisms to the environment. A key element in the generation of EROs in plants are NADPH oxidases, which are enzymes encoded by a gene family in plants, that is scarcely studied in an evolutionary approach. An important regulator of the homeostasis of plant ERNs is S-nitrosoglutathione reductase, which is also a poorly addressed enzyme in terms of evolutionary studies. Recently, several studies have suggested that plants can be elicited by chemical compounds, to better tolerate abiotic stresses. However, the mechanisms of physiological and transcriptional reprogramming behind these signaling molecules are still poorly understood. Hexanoic acid is a carboxylic acid, which, at low concentrations, can induce resistance to stresses and it can also modulate metabolism on numerous plant systems. However, the impact of hexanoic acid on the generation of ROS and ERNs has never been evaluated. In this project, we tested the hypothesis if hexanoic ac... (Complete abstract click electronic access below)
Mestre

La economía ecológica y la ecología política necesariamente requieren enmarcarse en paradigmas conceptuales diferentes, más contemporáneos e integradores. En ese contexto, las ciencias de la complejidad y las ciencias post-normales, se constituyen en marcos teóricos y de aplicación muy interesantes, se podría decir incluso, que ideales para cumplir con ese objetivo. Dichos paradigmas, permiten a su vez, romper con la tradicional dicotomía sociedad-naturaleza, integrándolas en un solo sistema: el sistema socio-ecológico. Por otro lado, en el Ecuador surge el paradigma andino del Sumak Kawsay, el que puede ser traducido como la vida plena, el buen vivir o el saber vivir. Este paradigma, al igual que las ciencias de la complejidad, concibe a la sociedad y a la naturaleza como una misma cosa. Es así que, el Ecuador ha adoptado al Sumak Kawsay como política de estado, al haberlo incluido en su última constitución como el último objetivo de las políticas públicas. De esta manera, este trabajo ha pretendido, bajo el marco conceptual de la economía ecológica, de la ecología política, y del Sumak Kawsay, estudiar el sistema socio-ecológico de la ciudad de Cuenca, en Ecuador. Esta ciudad fue escogida, por ser considerada como un referente tanto a nivel internacional, como nacional, debido a su gestión del agua y sus esfuerzos para la conservación de la biodiversidad. Por esas razones, el trabajo se enfocó en el estudio del metabolismo del agua. Dicho estudio se hizo en dos fases. Primero, se realizó una investigación de las percepciones sobre la naturaleza y el territorio, a nivel institucional, es decir, que se indagó en los discursos que dirigen las instituciones que toman decisiones sobre la naturaleza y el territorio en Cuenca. La segunda fase, consistió en un estudio del metabolismo del agua de la ciudad de Cuenca, evaluando cuánta agua se extraé del sistema ecológico, y cómo se usa por los diferentes niveles de la sociedad cuencana. Para estudiar las percepciones se aplicó el método Q para el estudio de la subjetividad humana. Se identificaron cuatro discursos, nombrados sólo por fines didácticos como: conservacionista, tecnocrático, desarrollista y sistémico. Los cuatro discursos serían incompatibles con el Sumak Kawsay. Para estudio del metabolismo social del agua, en cambio, se aplicó un Análisis Multiescalar del Metabolismo Social y Ecológico (MuSIASEM). Se encontró, que aunque en apariencia existe agua suficiente para abastecer a la ciudad de Cuenca, los ríos de los que toma el agua podrían estar sometidos a un estrés ecológico severo. Por otro lado, la mayor demanda de agua es para el sector agrícola, seguida del sector industrial, y del sector doméstico. En los tres sectores los niveles de consumo son muy altos, y parecerían tener un altísimo grado de desperdicio. De esta manera, el metabolismo del agua de la ciudad de Cuenca sería ecológicamente insustentable, y por lo tanto, también incompatible con el Sumak Kawsay. En resumen, esta tesis aplica el marco teórico de las ciencias de la complejidad a la economía ecológica y a la ecología política, para el análisis de un sistema socio-ecológico particular. Esto ha permitido profundizar en el funcionamiento del metabolismo social de Cuenca, y por lo tanto, identificar los procesos donde se podría intervenir para buscar la sustentabilidad, y hacerlo compatible con el paradigma del Sumak Kawsay.
Ecological economics and political ecology necessarily have to be framed in different theoretical paradigms, which have to be contemporary and more integrating conceptual frameworks. In this context, the complexity sciences and post-normal science, constitute a very interesting theoretical and application frameworks, one could even say, ideal to meet that goal. Such paradigms allow in turn, break the traditional dichotomy between society and nature, integrating them into one system: the socio-ecological system. Additionally, within Ecuador emerged the ancestral and Andean paradigm of Sumak Kawsay, which can be translated as good living, as living wisely or as to know how to live. This paradigm, like the sciences of complexity, conceives society and nature as the same thing. In this context, Ecuador adopted the Sumak Kawsay as a state policy, by including it in their last constitution, as the ultimate goal of public policy. Thus, this work has attempted to study the socio-ecological system of the city of Cuenca in Ecuador. The research was framed within the conceptual framework of ecological economics, political ecology, and Sumak Kawsay. This city was chosen because it is considered as an example to follow, both internationally and nationally, due to its water management system and its conservation of biodiversity efforts. For these reasons, the work was focused on the study of water metabolism. This thesis was done in two phases. First, we did an investigation of the perceptions about nature and territory, at the institutional level. That implied an inquire of the main discourses found within institutions that make decisions about nature and territory. The second phase consisted in studding the water societal metabolism of the city of Cuenca. It was assessed the quantity of the water extracted from the ecological system, and how it is used by the different levels of Cuenca’s society. On the one hand, to study the perceptions, Q method for the study of human subjectivity was applied. Four discourses were identified, named only for identifying purposes as: conservationist, technocratic, developmentalist and systemic. The four discourses happen to be inconsistent with Sumak Kawsay. On the other hand, in order to study the societal metabolism of water, we applied the Multiscale Integrated Analysis of Societal and Ecological Metabolism (MuSIASEM). It was found that, despite an apparently enough water supply, the rivers from Cuenca extracts its water have risk of severe ecological stress. The most important pressure comes from the increasing demand of water for agriculture, followed by the demand of the industrial sector, and finalley, the demand from the home sector. In all three sectors consumption levels are very high, and seem to have a high degree of waste. Thus, water metabolism of Cuenca would be ecologically unsustainable, and thus also inconsistent with Sumak Kawsay. In summary, this thesis applies the theoretical framework of the complexity sciences to ecological economics and political ecology for the analysis of a specific socio-ecological system. This has deepened in the functioning of the societal metabolism of Cuenca, and therefore, identify processes where they could intervene to seek sustainability, and make it compatible with the paradigm of Sumak Kawsay.

Las células tumorales presentan alteraciones metabólicas. Una de las diferencias con las células no transformadas es que los tumores usan más glucosa incluso en condiciones de normoxia, lo que se utiliza actualmente para visualizar tumores mediante la técnica de PET. Ultimamente se están desarrollando terapias basadas en estas particularidades metabólicas de las células tumorales, que las hacen más sensibles a inhibición del metabolismo glicolítico. Aquí estudiamos la muerte producida por la falta de glucosa en sarcomas y otros tipos tumorales. La privación de glucosa induce apoptosis o necrosis dependiendo de la línea celular. Mostramos cómo la privación de glucosa induce actividad caspasa en células deficientes de Bax/Bak, mientras que en las líneas de rabdomiosarcoma induce necrosis. Además la caspasa-8 está implicada en la muerte por ausencia de glucosa en estas células deficientes en Bax/Bak y también en células humanas HeLa. Investigamos el uso de 2-deoxiglucosa como inductor de muerte celular frente a líneas de rabdomiosarcoma alveolar y embrional. La 2-deoxiglucosa promueve muerte celular en líneas de rabdomiosarcoma alveolar. También induce diferenciación acompañada por una bajada de PAX3/FOXO1a, una proteína surgida de la translocación cromosómica en las células de rabdomiosarcoma alveolar y crítica en el desarrollo oncogénico de las mismas. Caracterizamos la muerte celular apoptótica inducida por 2-deoxiglucosa en líneas de sarcoma, in vitro. Estudiamos las moléculas de las rutas de apoptosis que determinan la sensibilidad de estas células a la 2-deoxiglucosa, con especial interés en las proteínas de la familia del oncogén Bcl-2. La muerte celular inducida por el tratamiento está asociada a la activación de Bax y Bak. Observamos que la sobre-expresión de proteínas anti-apoptóticas de la familia Bcl-2 como Bcl-xL y de Mcl-1 previene la apoptosis, indicando que la muerte sucede a través de la ruta mitocondrial. Enseñamos que la bajada de Mcl-1 y la subida de Noxa son críticos en la muerte por 2-DG. Además, la 2-DG promueve estrés reticular acompañado de la inducción de ATF4 y chaperonas. Al bajar los niveles de ATF4, protegemos a las células de la muerte inducida por 2-DG y prevenimos la pérdida de Mcl-1. Incubamos células en presencia de manosa, que revierte el estrés inducido por la 2-DG y previnimos la muerte sin subir los niveles de ATP. Así, el estrés energético causado por la 2-DG no es la principal causa de muerte celular. También la 2-DG promueve la fosforilación de eIF2α, un inductor de ATF4, y la inactivación de mTOR. Nuestros resultados sugieren que el uso de inhibidores glicolíticos como la 2-DG pueden ser efectivos en el tratamiento de los rabdomiosarcoma alveolares y que Noxa podría ser un marcador pronóstico de la eficiencia de estas drogas. Por otro lado, caracterizamos las respuestas a la falta de glucosa y 2-DG, en particular la respuesta autofágica que puede determinar que las células mueran o no en respuesta a la falta de nutrientes. Observamos que las células están sensibilizadas al tratamiento con 2-DG tras el uso de cloroquina. Sin embargo, la presencia de cloroquina no afecta a la privación de glucosa. Intentamos clarificar si la falta de glucosa está induciendo macroautofagia. El flujo de LC3 por western blot nos indica que no hay más macroautofagia ni en células DKO ni en las Rh4 en ausencia de glucosa. Por microscopía confocal y analizando células HeLa y Rh4, tampoco vemos mayores acúmulos de GFP-LC3 tras la retirada de glucosa comparando con tratamientos clásicos de inducción de autofagía como la retirada de aminoácidos o con rapamicina. Estos datos sugieren que la combinación de 2-DG con inhibidores de la autofagia podría ser útil en el tratamiento contra rabdomiosarcomas y que la falta de glucosa no induce autofagia.
Characterization of cell death induced by inhibition of glucose metabolism Rhabdomyosarcoma (RMS) is the most common soft-tissue tumor of childhood characterized by high malignancy, local invasiveness and a marked predisposition to metastasize. Recently, a number of therapies targeting tumor cell metabolism are being developed, since oncogenic changes make tumors more sensitive to inhibition of glycolytic metabolism. We observed that the glycolytic inhibitor 2-deoxyglucose (2-DG) can efficiently promote cell death in p53-deficient alveolar rhabdomyosarcoma (the rhabdomyosarcoma subtype with poorer prognosis), but not in embryonal rhabdomyosarcoma. Differential expression of HIF-1 could not explain the different sensitivity of tumor subtypes. 2-deoxyglucose induced nuclear chromatin condensation, cleavage of caspase substrates and DNA degradation which was inhibited by caspase inhibitors. Cell death triggered by 2-DG was associated with its ability to activate Bax and Bak and was prevented by overexpression of the anti-apoptotic Bcl-2 homologs Bcl-xL and Mcl-1. Conversely, siRNA against Bcl-xL or Mcl-1 accelerated death. 2-DG promoted downregulation of the anti-apoptotic protein Mcl-1 and accumulation of two BH3-only proteins, Noxa and Bim. siRNA against these proteins indicated that Noxa mediates 2-DG-induced cell death. Addition of different carbon/energy sources indicated that apoptosis is not associated with loss of ATP but rather with endoplasmic reticulum stress and the eIF2-alpha-ATF4 pathway. 2-DG promoted Mcl-1 loss, probably due to general inhibition of translation, since both eIF2-alpha phosphorylation and inactivation of the mTOR pathway were observed. All these events (ER stress, energetic stress and inhibition of the mTOR pathway) are known to induce autophagy. Indeed, 2-DG induces autophagy, and inhibition of autophagy at different levels sensitizes cells to 2-DG. Together, our findings suggest that glycolysis inhibitors such as 2-DG may be effective in treating alveolar rhabdomyosarcoma.

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
O objetivo deste estudo foi avaliar o efeito da restrição alimentar prévia ou exposição anterior a ingredientes concentrados sobre o tempo para adaptação à dietas de alto concentrado, sob os parâmetros da fermentação ruminal e o perfil de microrganismos ruminais de bovinos Nelore canulados confinados. Foram utilizados 6 bovinos da raça Nelore, castrados, com peso vivo inicial aproximado de 236 ± 23 kg, 15 meses de idade e canulados no rúmen, os quais foram divididos em dois quadrados latinos 3 x 3. Os tratamentos diferiram somente sobre o tipo de alimentação estabelecida previamente ao período de adaptação: Controle (forragem ad libitum + suplemento mineral); Restrição (forragem restrita a 1,4% do peso vivo + suplemento mineral) e Concentrado (forragem ad libitum + 0,5% do peso vivo de ingredientes concentrados e suplemento mineral). A duração deste estudo foi de 119 dias, sendo compostos por 3 períodos experimentais (33 dias cada período) e dois intervalos de washout (10 dias) entre os períodos 1 e 2, e 2 e 3. Os períodos foram divididos em: 14 dias de pré- adaptação, 6 dias de adaptação 1 (72% de concentrado), 6 dias de adaptação 2 (79% de concentrado) e 7 dias de dieta de terminação (86% de concentrado). O pH ruminal foi monitorado por meio da utilização de data loggers a cada 10 minutos. Foram avaliados a concentração de ácidos graxos de cadeia curta e amônia ruminal; a quantificação relativa das bactérias celulolíticas e utilizadoras de lactato por meio da técnica de PCR e a contabilidade total e diferencial de protozoários (0,4,8 e 12 horas após o trato). Os animais restritos, na fase de adaptação, tiveram maior concentração de AGCC (P< 0,01), maior duração de pH abaixo de 6,2 (P<0,01) e menor pH máximo nos dias 15 e 16 (P≤ 0,10) em relação ao tratamento controle. Na fase de terminação, esses animais tiveram menor CMS (P=0,02), maior pH médio e menor área de pH abaixo de 6,2 (P≤0,10) em relação ao tratamento controle. Os animais expostos previamente ao concentrado, na fase de adaptação, tiveram menor concentração de butirato e NH3 ruminal (P≤0,10) em relação ao tratamento controle. Apresentaram também menor quantidade relativa de Fibrobacter succinogenes (P=0,10) e maior CMS (P<0,01) em relação ao controle. Já na fase de terminação o CMS não teve diferença significativa em relação ao tratamento controle. Os animais que passaram por restrição alimentar ou por exposição de concentrado na fase de pré-adaptação apresentam características ruminais semelhantes durante a fase a terminação aos animais em forragem ad libitum.
The objective of this study was to evaluate the effect of diet restriction or prior exposure to concentrate ingredientes prior to the adaption period to high concentrate diet on parameters ruminal fermentation patterns and ruminal microorganisms profile of cannulated Nellore cattle. Six Nelore steers was used, with initial body weight about 350 kg, 20 months and cannulated in the rumen, which will be divided into two Latin squares 3 x 3. The treatments differed only with respect to the type of diets prior to the adaptation period: Control (forage ad libitum + mineral supplement); Restriction (forage restricted to 1.4% of body weight + mineral supplement) and concentrate (forage ad libitum + 0.5% of the body weight of concentrated ingredients + mineral supplement). The study was last 119 days, in which animals was submitted to three experimental periods (33 days each one) and two washout intervals (10 days) between the periods 1 and 2, 2 and 3; each period was divided as follows: 14 days of pre-adaptation, 6 days of adaptation 1 (72% concentrate), 6 days of adaptation 2 (79% concentrate) and 7 days of finishing diet (88% concentrate). The rumen pH and temperature was be monitored through the use of data loggers it was evaluated the rumen production of short chain fatty acids and ammonia concentration; the relative quantification of cellulolytic, amylolytic and lactate bacteria utilizing PCR and total and differential quantification of protozoa. Thus, the hypothesis is to test if Nelore cattle exposed to feed restriction or comsuption of concentrate ingredientes prior to the adaptation period present same rumen fermentation patters and microrganism profile when compared to animals of control treatment in the finish diet. Restricted animals, in the adaptation phase, had a higher concentration of SCFA (P<0.01), a higher duration of pH below 6.2 (P <0.01) and lower maximum pH on days 15 and 16 (P≤ 0.10) in relation to the control treatment. In the finish diet, these animals had lower DMI (P = 0.02), higher mean pH and lower pH area below 6.2 (P≤0.10) in relation to the control treatment. The animals previously exposed to the concentrate in the adaptation diet had a lower concentration of butyrate and ruminal NH3 (P≤0.10) in relation to the control treatment. They also presented lower amount of Fibrobacter succinogenes (P = 0.10) and higher DMI (P <0.01) in relation to the control. In the termination diet the DMI did not affect in relation to the control treatment. Cattle previously submitted to either nutricional restriction or intake of concentrate have similar ruminal characteristics during the finishing diet of the animals in control treatment.
2016/04262-8

Los cuerpos lipídicos (CLs) son los principales orgánulos encargados del almacén de lípidos, siendo el mayor reservorio energético de las células. Están formados por un núcleo de lípidos neutros hidrófobos separados del citoplasma por una monocapa de fosfolípidos en la que se anclan determinadas proteínas. Fueron descritos a finales del siglo XIX por Hanstein, Altmann y Wilson en diferentes publicaciones y originalmente fueron llamados liposomas o microsomas. Desde entonces estos orgánulos han sido denominados de múltiples maneras: cuerpos lipídicos, gotas lipídicas, adiposomas, gránulos, inclusiones lipídicas, triglicéridos intramiocelulares, partículas lipídicas, oleosomas o cuerpos de aceite. Inicialmente fueron considerados como simples orgánulos estáticos únicamente implicados en el almacén lipídico y básicamente importantes en tejidos especializados como el tejido adiposo. No obstante, la presencia de CLs no es exclusiva de tejidos especializados como el adiposo, sino que sucede en todas las células eucariotas y en muchos procariotas. En la última década el número de estudios sobre estos orgánulos ha incrementado exponencialmente especialmente desde el descubrimiento de la perilipina, la primera proteína residente en CLs descrita, en el laboratorio de C. Londos en 1991. Poniendo de manifiesto que se trata de orgánulos complejos, recientemente se ha descrito la implicación de los CLs en múltiples procesos como el metabolismo energético, la síntesis de lípidos y hormonas, la señalización celular, el almacén y la degradación de proteínas, la secreción de lipoproteínas, la respuesta inflamatoria, el ciclo vital de algunos patógenos, el cáncer o la longevidad. Además, la acumulación excesiva de CLs es una característica común en múltiples patologías asociadas con la obesidad como el síndrome metabólico, la esteatosis hepática y la ateroesclerosis, subrayando la relevancia del estudio de la biología de estos orgánulos.

Orientador: Ricardo Della Coletta
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
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Resumo: A fissura labial e/ou palatina (FL/P) não-sindrômica é uma malformação congênita do lábio e/ou palato com alta frequência na população brasileira. A etiologia das fissuras é complexa e conta com a participação de fatores genéticos e ambientais. Inúmeros estudos demonstraram que variantes polimórficas das enzimas relacionadas ao metabolismo do ácido fólico podem ser importantes fatores de risco materno para o nascimento de uma criança FL/P não-sindrômica. O objetivo deste estudo foi estudar a influência do consumo de suplementos vitamínicos durante o primeiro trimestre de gravidez e comparar a frequência alélica e genotípica de 4 genes (MTHFR, MTHFD1, MTR e RFC1) que codificam enzimas da via metabólica do ácido fólico entre mães de indivíduos portadores de FL/P não-sindrômicas (grupo experimental) e mães de indivíduos clinicamente normais (grupo controle). Amostras de DNA de 184 mães do grupo controle e de 106 mães do grupo experimental foram genotipadas por reação em cadeia da polimerase associada à análise de polimorfismo de fragmentos de restrição enzimática (PCR-RFLP). A ausência de suplemento vitamínico durante o primeiro trimestre de gravidez aumentou de forma discreta (aproximadamente em 0,4 vezes) o risco de uma mulher ter um filho com FL/P não-sindrômica. Dos 15 polimorfismos analisados neste estudo, 2 apresentaram diferenças entre os grupos. No polimorfismo rs2274976 do gene MTHFR, o alelo A e o genótipo GA ocorreram em uma frequência significantemente maior no grupo experimental que no grupo controle (p<0,000001), aumentando em aproximadamente 6 vezes o risco de uma mãe ter um filho com FL/P não-sindrômica. O genótipo AA no lócus polimórfico rs2236225 do gene MTHFD1 foi significantemente mais prevalente no grupo experimental comparado com o grupo controle (p=0,02). A presença deste genótipo aumentou em aproximadamente 2 vezes o risco de uma mãe ter um filho com FL/P não-sindrômica. Análise multivariada demonstrou que estes fatores contribuíram de maneira independente para a etiologia das FL/P não-sindrômicas. O presente estudo demonstra que os polimorfismos rs2274976 do gene MTHFR e rs2236225 do gene MTHFD1 e a suplementação vitamínica durante o primeiro trimestre de gravidez estão associados ao desenvolvimento de FL/P não-sindrômicas na população brasileira. Este estudo corrobora com evidências prévias que demonstraram a influência de fatores ambientais e genéticos na etiopatogenia das FL/P não-sindrômicas.
Abstract: Nonsyndromic cleft lip with or without cleft palate (CL/P) is a congenital malformation of the lip and/or palate with elevating frequency in the Brazilian population. The etiology of the nonsyndromic CL/P is complex and both environmental and genetic factors play important roles. Several studies demonstrated that polymorphisms in the folic acid metabolic enzymes may be important maternal risk factor for the birth of a child with nonsyndromic CL/P. The aim of this study was to determine the influence of the multivitamin supplements during the first trimester of pregnancy and to compare the allele and genotypic frequencies of 4 genes (MTHFR, MTHFD1, MTR and RFC1) that encode enzymes of the acid folic metabolic pathway between mothers of nonsyndromic CL/P patients (experimental group) and mothers of clinically normal children (control group). DNA samples from 184 mothers of the control group and from 106 mothers of the experimental group were genotyped by polymerase chain reaction associated with reaction fragment length polymorphism (PCR-RFLP). The lack of multivitamin supplementation during the pregnancy first trimester increased in approximately 0.4-fold the maternal risk of a nonsyndromic CL/P child. Two out of 15 polymorphisms showed differences between groups. In rs2274976 MTHFR polymorphism, allele A and genotype GA occurred in a significantly higher frequency on experimental group when compared to control group (p<0.000001), rising in approximately 6 times the risk of a mother giving birth to a nonsyndromic CL/P child. Genotype AA in the rs2236225 MTHFD1 polymorphic locus was significantly more prevalent in experimental group than in control group (p=0.02). This genotype raised in approximately twice the risk of a mother giving birth to a nonsyndromic CL/P child. Multivariate analysis demonstrated that those factors contributed in an independent manner to nonsyndromic CL/P etiology. The present study shows that rs2274976 MTHFR and rs2236225 MTHFD1 polymorphisms, as well as the multivitamin supplementation during the first trimester of pregnancy, are associated with the development of nonsyndromic CL/P in the Brazilian population. This study corroborates with previous evidences demonstrating the influence of environmental and genetic factor on etiopathogenesis of the nonsyndromic CL/P.
Mestrado
Patologia
Mestre em Estomatopatologia

A regulação dependente de Ca2+ da atividade ATPásica da acto-miosina em concentrações fisiológicas de actina, tropomiosina e troponina ocorre exclusivamente na presença de troponina T (TnT). Nosso grupo demonstrou que um polipeptídeo correspondente aos primeiros 191 aminoácidos da TnT ativa a atividade ATPásica da acto-miosina na presença de tropomiosina e na ausência das outras duas subunidades do complexo troponina (TnI/TnC). Com o objetivo de mapear e caracterizar esse domínio ativatório da TnT, construímos fragmentos de TnT correspondentes às regiões compreendidas entre os resíduos de aminoácidos: 1-157 (TnTl-157), 1-76 (TnTl-76), 77-157 (TnT77-57), 77-191 (TnT77-191) e 158-191 (TnT158-191). Estudos das interações desses fragmentos com actina e tropomiosina demonstraram que: i) o fragmento TnTl-76 não se liga à tropomiosina ou a actina; ii) a região da TnT correspondente aos resíduos 158-191 liga-se à actina cooperativamente, mas não se liga à tropomiosina; iii) a região correspondente seqüência de aminoácidos 77-157 é necessária para a interação da TnT com o resíduo de aminoácido 263 da tropomiosina; iv) TnT77-191 ativa a atividade ATPásica da acto-miosina com a mesma intensidade que TnTl-191. Também observamos que TnTl-157, TnTl-76, TnT77-157, TnT158-91 e combinações de TnT158-191 com TnTl-157 e TnT77-157 não afetam a atividade ATPásica da acto-miosina. Concluímos que a região da TnT delimitada pelos aminoácidos 77 e 191 é essencial para a ativação da atividade ATPásica da actomiosina e que essa ativação é mediada pelas interações dessa região da TnT com a tropomiosina e a actina.
The Ca2+-regulation of the actomyosin ATPase activity at physiological ratios of actin, tropomyosin and troponin occurs only in the presence of troponin T. Our group has previously demonstrated that a recombinant polypeptide corresponding to the first 191 amino acids of TnT (TnTl-191) activates the aetomyosin Mg2+-ATPase activity in the presence of tropomyosin and in the absence of TnI/TnC. In order to further map and characterize this activation domain, we constructed a set of recombinant or synthetic TnT fragments, corresponding to amino acids 1-157 (TnTl-157), 1-76 (TnTl-76), 77-57 (TnT77-157), 77-191 (TnT77-191) and 158-191 (TnT158-191). Binding assays using these fragments demonstrated that: i) amino acids 1-76 of TnT do not bind to tropomyosin or actin; ii) amino acids 158-191 bind to actin cooperatively, but not to tropomyosin; iii) the sequence 77-157 is necessary for TnT\'s interaction with residue 263 of tropomyosin; iv) TnT77-191 on its own activates de actomyosin ATPase activity to the same extent as previously described for TnTl-191. TnT1-157; TnTl-76; TnT77-157; TnT158-191 and combinations of TnT158-191 with TnTl-157 or TnT77-157 showed no effect on the ATPase activity. We conclude that interactions of amino acids 77-191 of TnT with tropomyosin and actin are essential for the activation of actomyosin ATPase activity, and that this activation may be mediated in part by a direct interaction between TnT residues 158-191 and actin.

Na maioria dos estudos de digestibilidade de aminoácidos em suínos usam-se animais de peso igual ou superior a 20 kg, isso se deve a menor dificuldade na implantação de cânulas íleo-cecais e a melhor recuperação pós-cirúrgica na fase do crescimento. No entanto, avaliar ingredientes com leitões mais jovens torna-se importante, visto que há limitações fisiológicas do trato gastrointestinal nesse período que podem afetar o seu desenvolvimento. Assim, 175 leitões desmamados, divididos em 7 ensaios experimentais com 25 animais cada, foram usados para determinar o balanço de nutrientes e da energia, a digestibilidade ileal aparente (AID) e estandardizada (SID) dos aminoácidos (AA) de sete ingredientes usualmente utilizados em dietas de leitões, com ou sem suplementação de multi-carboidrase (MC) e fitase (F). Os leitões foram desmamados aos 23 dias de idade e alojados em gaiolas para estudo de digestibilidade e metabolismo, permanecendo no experimento até os 45 dias de idade. Adaptação, coleta total de fezes e urina ocorreram do 10° ao 20° dia do período experimental e as amostragens do conteúdo ileal se deu ao abate, no 22° dia (45 dias de idade). Foi utilizado o delineamento experimental inteiramente casualisado com 4 tratamentos e cinco repetições. O leitão foi considerado como unidade experimental. As dietas experimentais consistiram de ingrediente teste sem enzimas e combinado a MC, F ou MC + F. Dois tipos de dieta referência foram usados como base para os cálculos do balanço nutricional e os coeficientes de digestibilidades aparente e estandardizada dos aminoácidos. Óxido de cromo (0,3%) foi usado como marcador indigestível nas avaliações dos aminoácidos. A enzima MC apresentava 10% de galactomananase, 10% de xilanase, 10% de β-glucanase, 60% de cevada maltada e 10% de α-galactosidase. A F era proveniente da fermentação de Saccharomyces cerevisiae com atividade de 10.000 FTU/g. Os ingredientes testados foram: farelo de arroz integral, soja integral micronizada, farelo de trigo, quirera de arroz, sorgo, milho e farinha de soja texturizada. Os resultados indicaram efeitos da combinação ingrediente e enzima exógena e as evidencias se deram de acordo com as características bromatológicas de cada um
Most of the studies, involving AA digestibility in pigs are used animals with 20 kg minimal weight or higher than. This is due difficulty to implant a simple T-cannula on distal ileum of younger pigs, besides a better post-surgical recovery in growing phase. However, ingredient evaluations with young pigs become important because there are physiological limitations in gastrointestinal tract that may affect its performance. Thus, 175 weaned pigs, divided into seven experimental trials with 25 animals each, were used to determine the nutrients and energy balance, the apparent (AID) and standardized (SID) ileal digestibility of amino acids (AA) from seven ingredients usually used in pig diets, with or without multi-carbohydrase (MC) and phytase (Phy) supplementation. Weaned piglets at 23 d old were housed in cages to studies digestibility and metabolism, remaining in the experiment until 45 d of age. Pig adaptation to feces and urine collection was 10 to 20 d experimental period and ileal content sampling at slaughter at 22 d (45 d old). A completely randomized experimental design was used with 4 treatments and 5 replicates. The pig was considered as an experimental unit. The experimental diets consisted of test ingredient as the sole source of protein with or without MC, Phy or MC+Phy. Two kind of reference diet were used to calculate the nutrient balance or AID and SID of AA. Titanium dioxide (0.3%) was used as indigestible marker. The MC enzyme had: galactomannanase, 10%; xylanase, 10%; beta-glucanase, 10%; malted barley, 60% and α-galactosidase, 10%. The Phy was obtained from the Saccharomyces cerevisiae fermentation with 10,000 FTU/g activity. The ingredients used were: rice bran, micronized full fat soybean meal, wheat bran, broken rice, sorghum, corn and texturized soybean meal. The results indicated effects from ingredients and enzymes combinations and the evidences occurred according to bromatological characteristics of each one

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas
Os erros inatos do metabolismo consistem numa condição genética em que ocorre uma mutação ao nível dos genes que codificam enzimas envolvidas num determinado processo metabólico; tal mutação origina a deficiência de uma enzima, o que faz com que o processo metabólico não ocorra. A consequência pode passar pela acumulação do substrato duma reação no organismo, pela deficiência de produto dessa mesma reação, pela acumulação de metabolitos intermediários da reação, pela falta de feedback negativo e ainda pela falha nos mecanismos de transporte. Os erros inatos do metabolismo podem ocorrer na via dos hidratos de carbono, dos aminoácidos, dos ácidos orgânicos, das purinas e pirimidinas, dos lípidos e ácidos biliares. Neste momento em Portugal, a deteção de alguns erros inatos de metabolismo está inserida num programa de triagem neonatal, programa este que se baseia no diagnóstico de 25 doenças utilizando amostras de sangue. Neste trabalho serão abordados os erros inatos do metabolismo associados aos aminoácidos, nomeadamente, os mais frequentes em Portugal: fenilcetonúria, hiperfenilalaninemia, tirosinemia tipo I, tirosinemia tipo II, homocistinúria, deficiência na metionina adenosiltransferase e doença da urina de xarope de bordo. Para além de se caracterizar cada uma destas desordens, faz-se uma referência à via metabólica envolvida, assim como ao diagnóstico e tratamento de cada uma delas. Inborn errors of metabolism consist of a genetic condition which a mutation occurs at the level of genes encoding enzymes involved in a specific metabolic process; the mutation causes a deficiency in an enzyme, which does not enable the metabolic process occur. The consequence can pass by the accumulation of the substrate or by the product deficiency of a certain reaction, by the accumulation of intermediate metabolites of the reaction, by the lack of negative feedback and even failure in the transport mechanisms. Inborn errors of metabolism may occur in the path of carbohydrates, amino acids, organic acids, purines and pyrimidines, lipids and bile acids. At this point in Portugal the detection of some inborn errors of metabolism is inserted in a neonatal screening program, which is based on the diagnosis of 25 diseases using blood samples. In this work the inborn errors of metabolism associated with amino acids are discussed, including the most frequently in Portugal: phenylketonuria, hyperphenylalaninemia, tyrosinemia type I, tyrosinemia type II, homocystinuria, methionine adenosyl transferase deficiency and maple syrup urine disease. Each of these disorders are characterized and the metabolic pathway involved, as well as the diagnosis and treatment are referred.

La tiamina o vitamina B1 es un cofactor involucrado en el metabolismo energético y en la síntesis de ácidos nucleicos, antioxidantes, lípidos y neurotransmisores. Se han reconocido fenotipos clínicos bien definidos en los siguientes defectos del transporte y metabolismo de la tiamina: 1) SLC19A2, (transportador de tiamina-1); 2) SLC19A3 (transportador de tiamina-2); 3) TPK1 (tiamina pirofosfoquinasa) y 4) SLC25A19 (transportador mitocondrial de tiamina pirofosfato). Las deficiencias primarias y secundarias de tiamina tienen características superponibles en pediatría, incluyendo episodios agudos de encefalopatía, lesiones cerebrales particulares y un aumento de la excreción de ácidos orgánicos específicos de las enzimas mitocondriales dependientes de tiamina, principalmente lactato, alfa-cetoglutarato y cetoácidos de cadena ramificada. Las deficiencias de tiamina no diagnosticadas y no tratadas son a menudo fatales o conducen a secuelas graves. Por lo tanto, la suplementación de tiamina tiene un fuerte impacto en el pronóstico de los pacientes con deficiencia de SLC19A3, conduciendo a la estabilidad metabólica, la reducción de la discapacidad de estos niños y la supervivencia mejorada en comparación con los pacientes no tratados. La suplementación con tiamina restaura los niveles intracelulares y en LCR, probablemente a través del transporte alternativo de la vitamina. Este trabajo demuestra que la cuantificación de tiamina por el método de HPLC en muestras de sangre entera es un método útil para la evaluación de la adherencia al tratamiento de estos pacientes y más significativamente, que el déficit de tiamina-libre en LCR puede emplearse como método diagnóstico de pacientes con déficit de SLC19A3. Esta tesis describe la historia natural de 79 pacientes con defectos heredados en el transporte de la tiamina y el metabolismo, la mayor cohorte de pacientes con estos defectos genéticos publicados hasta la fecha. Se han identificado de mal factores pronóstico incluyendo niños con lesiones cerebrales difusas al debut, sobretodo si existe compromiso del tronco cerebral y los núcleos pálidos. En esta tesis, también se presentan criterios diagnósticos para defectos heredados de tiamina que ayudan a diferenciarlos de deficiencias secundarias.
Thiamine or vitamin B1 is a critical cofactor involved in energy metabolism and in the synthesis of nucleic acids, antioxidants, lipids, and neurotransmitters. Well-defined clinical phenotypes have been recognized in the following thiamine defects: 1) SLC19A2 (thiamine transporter-1); 2) SLC19A3 (thiamine transporter-2); 3) TPK1 (thiamine pyrophosphokinase) and 4) SLC25A19 (mitochondrial thiamine pyrophosphate carrier). Primary and secondary conditions leading to thiamine deficiency have overlapping features in children, presenting with acute episodes of encephalopathy, bilateral symmetric brain lesions, and high excretion of organic acids that are specific of thiamine-dependent mitochondrial enzymes, mainly lactate, alpha-ketoglutarate, and branched chain keto-acids. Undiagnosed and untreated thiamine deficiencies are often fatal or lead to severe sequelae. Therefore, thiamine supplementation has a strong impact on the prognosis of SLC19A3-deficient patients, leading to metabolic stability, reduced disability and improved survival compared to untreated patients. Thiamine supplementation restores intracellular and CSF levels, probably through alternative transport of the vitamin. This work also demonstrates that the quantification of thiamine by the HPLC method in whole blood samples is a useful method for evaluating the adherence to treatment of these patients and more significantly, that decreased free-thiamine in CSF can be used as a diagnostic method for SLC19A3-deficient patients. This thesis describes the natural history of 79 patients with inherited defects in thiamine transport and metabolism. This study indicates a better prognosis than other causes of LS, encouraging clinicians to suspect the disease and to make an early diagnosis and accurately prescribe treatment. This thesis also contributes with diagnostic criteria for inherited thiamine defects and helps differentiate them from secondary causes of thiamine deficiency.