Orientador: Everardo Magalhães Carneiro / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-21T18:31:05Z (GMT). No. of bitstreams: 1 Batista_ThiagoMartins_D.pdf: 2133170 bytes, checksum: 76495b990085ea3c1a065839c27b8bfe (MD5) Previous issue date: 2012 / Resumo: A resistência a insulina é um fator de risco para o diabetes mellitus tipo 2 (DM2) e é caracterizada pelo aumento da produção hepática de glicose, pela menor captação desse açúcar pelos músculos e tecido adiposo e pela hipersecreção de insulina pelas células beta pancreáticas. Estudos populacionais correlacionam o aporte insuficiente de nutrientes na fase gestacional e pós-natal com o desenvolvimento da resistência a insulina e do DM2 na vida adulta. Essas duas condições são frequentemente associadas a alterações no perfil de aminoácidos plasmáticos. Alguns estudos evidenciam menor concentração do aminoácido sulfurado, taurina, no plasma de humanos e camundongos diabéticos. Neste trabalho, investigamos o desenvolvimento da obesidade, da resistência a insulina e as alterações morfofisiológicas no pâncreas endócrino de camundongos submetidos a restrição protéica após o desmame e em seguida alimentados com dieta hiperlipídica. Também tivemos como objetivo verificar os efeitos da suplementação com taurina sobre o controle da homeostase glicêmica nesse modelo animal. Nossos resultados mostram que camundongos desnutridos respondem ao tratamento com dieta hiperlipídica de maneira semelhante aos camundongos alimentados com dieta normoprotéica. Ambos os grupos se tornaram obesos, hiperleptinêmicos e hipercolesterolêmicos; apresentou maior ingestão calórica, maior produção hepática de glicose e hipersecreção de insulina em ilhotas isoladas. A suplementação com taurina melhorou esses parâmetros com maior intensidade nos camundongos alimentados com dieta normoprotéica. Esses efeitos da taurina foram associados com maior fosforilação da proteína homóloga ao timoma viral (Akt) no fígado dos camundongos controles, e nos desnutridos houve maior fosforilação da proteína quinase ativada por AMP (AMPK). Em conclusão, a desnutrição pós desmame não acelera ou potencializa o desenvolvimento da resistência a insulina induzida por dieta hiperlipídica, porém confere resistência aos efeitos da suplementação com taurina / Abstract: Insulin resistance is a risk factor for type 2 diabetes mellitus (T2DM) and is characterized by increased hepatic glucose output, the lower uptake of this sugar by muscle and adipose tissue and by hypersecretion of insulin by pancreatic beta cells. Population studies correlate the insufficient nutrient intake during pregnancy and early life stages with the development of insulin resistance and T2DM in adulthood. These two conditions are often associated with changes in plasma amino acid profile. Some studies show lower concentration of the sulfur-containing amino acid, taurine, in plasma of diabetic humans and mice. In this project, we investigate the development of obesity; insulin resistance and morphophysiological changes in the endocrine pancreas of mice subjected to protein restriction after weaning and then fed a high-fat diet. We also aimed to investigate the effects of taurine supplementation on the control of glucose homeostasis in this animal model. Our results show that undernourished mice respond to treatment with high fat diet similarly to mice fed a normal protein diet. Both groups became obese, hypercholesterolemic, hyperleptinemic, had higher caloric intake, increased hepatic glucose output and insulin hypersecretion in isolated islets. The taurine supplementation improved these parameters with greater intensity in mice fed a normal protein diet. These effects of taurine were associated with increased phosphorylation of thymoma viral oncogene homolog (Akt) in the liver of control mice and in malnourished mice; the phosphorylation of the AMP-activated protein kinase (AMPK) was increased. In conclusion, malnutrition after weaning doesn't accelerate nor potentiate the development of insulin resistance induced by high fat diet, but confers resistance to the effects of taurine supplementation / Doutorado / Fisiologia / Doutor em Biologia Funcional e Molecular

The main function of mesenchymal stem cells in the body is to facilitate the restoration and regeneration of damaged tissues. They are known for the ability to differentiate into tissue originating from the mesoderm, which among others includes connective tissues. Due to this feature are MSCs being intensively examined. Different directions of differentiation can be induced by treatment of specific polypeptides, so called growth factors. In the field of tissue engineering are growth factors used to induce and accelerate the healing processes. They may be incorporated into the nanofiber carrier which is inserted into the site of injury. Cells in this area would thus be stimulated by surrounding 3D microenvironment. At the same time the scaffold provides a supply of growth factors which are able to affect metabolism, motility and differentiation of present cells. In order to induce osteogenic differentiation of human MSCs the following bioactive substances were used: TGF-β, bFGF, HGF, IGF-1, VEGF and the BMP-2 and the organic acid taurine. During 21 days lasting experiments, were these molecules added to the medium in various combinations and in the case of taurine also at two different concentrations. Cells were cultured on plastic. The best effect on cellular metabolism of MSCs, evaluated by MTS...

Pre-eclampsia (PE) is a serious disease affecting approximately 5% of pregnancies per annum. The disease etiology is complex but its origin lies in abnormal placental development and function. PE is associated with inflammation, increased nitrative stress and abnormal renewal of syncytiotrophoblast (STB), the transporting epithelium of the placenta. STB is renewed by cytotrophoblasts (CTBs) that proliferate, differentiate and fuse with STB and this is balanced by apoptosis. The amino acid taurine facilitates proliferation, differentiation and apoptosis in non-placental tissues. Taurine is also cytoprotective, protecting cells from damage by inflammatory cytokines. Taurine is transported from maternal blood into STB by the amino acid transporter TauT. In isolated STB membranes, TauT activity is inhibited by agents that nitrate tyrosine residues. This thesis tested the hypothesis that STB TauT activity is down-regulated in PE due to post-translational modification of TauT through tyrosine nitration which lowers intracellular taurine and contributes to altered STB renewal. Placentas were collected from normal pregnancy (NP) and PE (blood pressure >140/90mmHg after 20 weeks gestation in previously normotensive women plus proteinuria >300 mg/L in a 24-hour collection). STB TauT activity, measured as Na+-dependent uptake of 3H-taurine into placental villous fragments, was significantly lower in PE (n=24) compared to NP (n=44). Western blotting of membrane enriched homogenates showed that TauT protein expression (normalised to β-actin) was significantly higher in placentas from PE (n=8) compared to NP (n=9). The presence of nitrotyrosine residues (marker of nitrative stress) in placentas of women with PE and NP was assessed by immunohistochemistry (IHC). The intensity of STB nitrotyrosine staining was greater in PE placentas that had reduced TauT activity (n=8) than in NP (n=7). To determine the effect of nitrative stress on TauT activity and STB renewal, placental villous explants from NP were cultured (7 days; n=6) and treated with SIN-1 (1mM; days 5,6) to induce nitrative stress. STB nitrotyrosine (IHC) and TauT activity (3H-taurine uptake) was determined on day 7 and STB renewal was assessed by IHC for apoptosis (M30), proliferation (dual staining for Ki67 and the CTB marker E-cadherin) and STB integrity (cytokeratin 7). SIN-1 increased STB nitrotyrosine staining intensity compared to controls, confirming induction of nitrative stress. SIN-1 reduced STB TauT activity, increased apoptosis, reduced CTB proliferation and altered STB regeneration compared to control. To determine the effect of reducing intracellular taurine on STB renewal, villous explants were cultured for 7 days with 2.5mM β-alanine to competitively inhibit taurine uptake (n=6). At day 7, intracellular taurine, measured as the steady-state accumulation of 3H-taurine, was 15% of normal. STB turnover was assessed at day 7 as described above. β-alanine significantly increased apoptosis and altered STB regeneration compared to controls. Following statistical analysis all p <0.05.In conclusion, STB TauT activity was lower, and protein expression higher, in PE compared to NP. STB nitrotyrosine was elevated in PE and nitrative stress inhibited STB TauT activity and disrupted STB renewal in vitro. Reducing intracellular taurine also disrupted STB renewal in vitro. Overall the data support the hypothesis that post-translational modification of TauT by nitration inhibits TauT activity in PE. This reduces intracellular taurine which contributes to abnormal renewal of STB. Further work is needed (a) to confirm that TauT is nitrated in PE and that reduced STB TauT activity lowers intracellular taurine and reduces taurine delivery to the fetus and (b) to determine the mechanism/s by which taurine regulates CTB apoptosis and facilitates renewal of STB.

No description available.

In insects, the cuticle provides protection against physical injury and water loss, rigidness for muscle attachment and mechanical support, and flexibility in inter-segmental and joint areas for mobility. As most insects undergo metamorphosis, they need to shred off old cuticle and synthesize new cuticle to fit the body shape and size throughout their life cycles. The newly formed cuticle, mainly composed of cuticular proteins, chitin, and sclerotizing reagents, needs to be hardened through the crosslinks between cuticular proteins and sclerotizing reagents. This dissertation concerns the biochemical activities of several pyridoxal 5-phosphate (PLP)-dependent decarboxylases with most of them involved in insect cuticle hardening. Herein, we first present a detailed overview of topics in reactions and enzymes involved in insect cuticle hardening. Aspartate 1-decarboxylase (ADC) is at the center of this dissertation. beta-alanine, the product of ADC-catalyzed reaction from aspartate, is the component of an important sclerotizing reagent, N-beta-alanyldopamine; the levels of beta-alanine in insects regulate the concentrations of dopamine, therefore affecting insect sclerotization and tanning (collectively referred as cuticle hardening in this dissertation). Biochemical characterization of insect ADC has revealed that this enzyme has typical mammalian cysteine sulfinic acid decarboxylase (CSADC) activity, able to generate hypotaurine and taurine. The result throws lights on research in the physiological roles of insect ADC and the pathway of insect taurine biosynthesis. Cysteine was found to be  an inactivator of several PLP-dependent decarboxylases, such as ADC, glutamate decarboxylase (GAD) and CSADC. This study helps to understand symptoms associated with the abnormal cysteine concentrations in several neurodegenerative diseases. A mammalian enzyme, glutamate decarboxylase like-1 (GADL1), has been shown to have the same substrate usage as insect ADC does, potentially contributing to the biosynthesis of taurine and/or beta-alanine in mammalian species. Finally, the metabolic engineering work of L-3, 4-dihydroxyphenylalanine decarboxylase (DDC) and 3, 4-dihydroxylphenylacetaldehyde (DHPAA) synthase has revealed that the reactions of these enzymes could be determined by a few conserved residues at their active site. As both enzymes have been implicated in the biosynthesis of sclerotizing reagents, it is of great scientific and practical importance to understand the similarity and difference in their reaction mechanisms. The results of this dissertation provide valuable biochemical information of ADC, DDC, DHPAA synthase, and GADL1, all of which are PLP-dependent decarboxylases. ADC, DDC, DHPAA synthase are important enzymes in insect cuticle hardening by contributing to the biosynthesis of sclerotizing reagents. Knowledge towards understanding of these enzymes will promote the comprehension of insect cuticle hardening and help scientists to search for ideal insecticide targets. The characterization of GADL1 lays groundwork for future research of its potential role in taurine and beta-alanine metabolism. / Ph. D.

Bile is a biofluid synthesized by liver and concentrated in the gallbladder. Interference with the bile flow may cause cholestasis. Primary sclerosing cholangitis (PSC) is an inflammatory cholestatic disorder which eventually may result in liver cirrhosis and failure. The management of PSC is controversial. The only effective treatment for end stage disease is orthotopic liver transplantation (OLT). However, cholangiocarcinoma (CC), which is the major complication of this long-lasting disease, is an absolute contraindication for the surgery. Therefore, early diagnosis of the disease can not only improve the outcome of PSC, but also facilitate the allocation of donated livers to those who can benefit from transplantation. Unfortunately, the diagnosis of CC is challenging. Endoscopic retrograde cholangiopancreatography (ERCP), the gold standard technique, is highly invasive. Non-invasive alternatives such as magnetic resonance cholangiopancreatography (MRCP) have lower accuracy. Therefore, it is essential to develop more accurate and less invasive diagnostic techniques. Magnetic resonance spectroscopy (MRS) is an evolving technique with potential to detect disease-related metabolic changes. In vitro studies have proven the capacity of MRS in the early detection of hepatopancreatobiliary (HPB) disorders based on the metabolic analysis of bile obtained invasively. An in vivo alternative has been attempted by others on human bile within the gallbladder. However, due to the poor quality of the acquired spectra, quantification of most major bile metabolites was not possible, except for choline-containing phospholipids (chol-PLs). In the current study, the quality of the in vivo 1D spectra has been greatly improved, and we have obtained the first 2D L-COSY spectra from bile within the gallbladder. Spectral data from healthy controls and PSC patients were compared. Statistically significant differences in the concentrations of chol-PLs, and glycine- and taurine-conjugated bile acids were revealed in the 1D analysis. Our 2D spectra also demonstrated potential for the detection of metabolic differences between the two groups. The success of these studies indicates a strong potential of in vivo bile MRS techniques to characterize and diagnose a wide variety of HPB disorders. / May 2014

Stroke is the third leading cause of mortality in the United States, and so far, no clinical interventions have been proved truly effective in stroke treatment. Stroke my result in hypoxia, glutamate release and oxidative stress, etc. The purpose of this dissertation study is to evaluate the neuroprotective effects of four drugs (taurine, G-CSF sulindac and DETC-MeSO) on PC12 cell line or primary cortical neuronal cell culture, and to understand the protective mechanisms underlying in three stroke-related models : hypoxia, excessive glutamtate and oxidative stress. In the first part of this dissertation, we studied the neuroprotection of taurine against oxidative stress induced by H2O2 in PC12 cells. Our results show that extracellular taurine exerts a neuroprotective function by restoring the expression of Bcl-2 and downregulation of the three Endoplasmic Reticulum (ER) stress markers : GRP78, Bim and CHOP/GADD153, suggesting that ER stress can be provoked by oxidative stress and can be suppressed by taurine. In the second part, glutamate excitotoxicity-induced ER stress was studied with dose and time as variables in primary cortical neurons. The results demonstrate that glutamate excitotoxicity leads to the activation of three ER stress pathways (PERK, ATF6 and IRE1) by initiating PERK first, ATF6 second and IRE1 pathway last. The third part of this dissertation studied the robust and beneficial protection of taurine in cortical neurons under hypoxia/reoxygenation or glutamate toxicity condition. We found that taurine suppresses the up-regulation of GRP778, Bim, caspase-12 and GADD153/CHOP induced by excessive glutamate or hypoxia/reoxygenation, suggesting that taurine may exert a protective function against hypoxia/regeneration by reducing the ER stress. / Moreover, taurine can down-regulate the ratio of cleaved ATF6 and full length ATF6, and p-IRE1 expresssion, indicating that taurine inhibits the ER stress induced by hypoxia/reoxygenation or glutamate through suppressing ATF6 and IRE1 pathways. In the fourth part, the synergistic benefits of the combination of taurine and G-CSF, and the neuroprotective effects of G-CSF, sulindac or DETC-MeSO are studied in cortical neurons. Our results show that G-CSF, sulindac or DETC-MeSO can highly increase the neuron visibility by inhibiting ER stress induced by hypoxia/reoxygenation or glutamate toxicity. Furthermore, we proved that G-CSF or sulindac can significantly inhibit the activation of ATF6 or IRE1 pathway stimulated by hypoxia/reoxygenation, and DETC-MeSO can suppress the activation of both PERK and IRE1 pathways in primary neuron cultures. These findings provide promising and rational strategies for stroke therapy. / by Chunliu Pan. / Thesis (Ph.D.)--Florida Atlantic University, 2012. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2012. Mode of access: World Wide Web.

The glycine receptor (GlyR) is a ligand-gated ion channel member of the cys-loop receptor superfamily, responsible for inhibitory neurotransmission in the brain and spinal cord. Both glycine and the partial agonist taurine act as endogenous ligands of the GlyR. Taurine-activated GlyR may have a role in the rewarding effects of drugs of abuse, such as ethanol. As a partial agonist, taurine has a decreased efficacy relative to glycine, resulting in a decreased maximum response. We investigated the effects of ethanol, anesthetics, inhalants, and zinc to determine if these allosteric modulators could increase the efficacy of the taurine-activated GlyR. Whole cell recordings of wild type GlyR revealed that each of the allosteric modulators potentiated currents generated by saturating concentrations of taurine but not glycine, implying an increase in efficacy. Zinc is found at GlyR-potentiating concentrations throughout the nervous system, so we examined the combinatorial effects of these allosteric modulators with zinc to mimic in vivo conditions. Whole cell recordings revealed that zinc potentiation of saturating taurine-generated currents decreased further potentiation by another allosteric modulator, indicating no synergistic effects on efficacy. We next investigated the actions of ethanol and isoflurane on the taurine-activated GlyR at the single channel level, finding that both allosteric modulators stabilized the channel open state, increasing the efficacy of the taurine-activated GlyR. We previously identified a mutation in the ligand-binding domain of the GlyR (D97R) that produces spontaneously activating channels, on which taurine has increased efficacy. We identified a residue, R131, as a possible binding partner of D97 in forming an electrostatic interaction that holds the channel in the closed state. We found that disruption of this interaction results in greatly increased taurine efficacy, indicating that efficacy for partial agonists may be determined by agonist ability to break this bond early in the activation process following binding. Thus we find differential mechanisms of allosteric modulation and efficacy determinations for the GlyR when activated by taurine vs. glycine. / text

The glycine receptor (GlyR) is a ligand-gated ion channel member of the cys-loop receptor superfamily, responsible for inhibitory neurotransmission in the brain and spinal cord. Both glycine and the partial agonist taurine act as endogenous ligands of the GlyR. Taurine-activated GlyR may have a role in the rewarding effects of drugs of abuse, such as ethanol. As a partial agonist, taurine has a decreased efficacy relative to glycine, resulting in a decreased maximum response. We investigated the effects of ethanol, anesthetics, inhalants, and zinc to determine if these allosteric modulators could increase the efficacy of the taurine-activated GlyR. Whole cell recordings of wild type GlyR revealed that each of the allosteric modulators potentiated currents generated by saturating concentrations of taurine but not glycine, implying an increase in efficacy. Zinc is found at GlyR-potentiating concentrations throughout the nervous system, so we examined the combinatorial effects of these allosteric modulators with zinc to mimic in vivo conditions. Whole cell recordings revealed that zinc potentiation of saturating taurine-generated currents decreased further potentiation by another allosteric modulator, indicating no synergistic effects on efficacy. We next investigated the actions of ethanol and isoflurane on the taurine-activated GlyR at the single channel level, finding that both allosteric modulators stabilized the channel open state, increasing the efficacy of the taurine-activated GlyR. We previously identified a mutation in the ligand-binding domain of the GlyR (D97R) that produces spontaneously activating channels, on which taurine has increased efficacy. We identified a residue, R131, as a possible binding partner of D97 in forming an electrostatic interaction that holds the channel in the closed state. We found that disruption of this interaction results in greatly increased taurine efficacy, indicating that efficacy for partial agonists may be determined by agonist ability to break this bond early in the activation process following binding. Thus we find differential mechanisms of allosteric modulation and efficacy determinations for the GlyR when activated by taurine vs. glycine. / text

The thesis deals with the consumption of energy and stimulative drinks by adolescents in the Czech Republic. This current topic is treated in the theoretical part of the thesis, in its two sections. In the first section, the important terms, such as ?adolescence? or ?digesting drinks?, are explained. The second theoretical section deals with the daily fluid intake and describes energy and stimulative drinks, their composition and their impact on human body. There is also information about the use of these drinks in combination with alcohol. The practical part of the thesis contains analysis of the consumption of energy and stimulative drinks by adolescents from 15 to 26 years of age. More than 1,200 respondents took part in the electronic research. To compare the results, there are two age groups: 15-20 years of age and 21-26 years of age. The results of the research confirm that energy drinks are consumed at the usual rate, while stimulative drinks are included in the daily fluid income of Czech adolescents very often. The main reason for consumption of these drinks is to eliminate the feeling of tiredness.