No description available.

Introduction- Exercise-induced plasma volume (PV) expansion is typically associated with an increase in plasma albumin content. Increased hepatic albumin synthesis, a transcriptionally regulated process, is thought to contribute to the increase in albumin content. Objective- We tested the hypothesis that exercise training induces an increase in albumin gene expression in relationship to the increase in PV. Methods and Results- 40 adult male Sprague-Dawley rats weighing between 245-350 grams were randomly assigned to one of four groups: cage control (CC); sham exercise 10 min/day at 48% VO2max (NE); continuous exercise training, 60 min /day at 72% VO2max (LI); and high intensity, intermittent exercise training, 8 bouts of 4 min at 98% VO2max followed by 5 min at 48% VO2max (HI). The training period lasted for two weeks with 12 training sessions with equalized training volumes in the exercise groups. 24 hours after the last training session the rats were anesthetized and a jugular catheter was placed for collecting blood samples during PV determination by a dilution of a labeled-albumin molecule (Texas Red albumin). The liver and red quadriceps (RQ) muscle tissue was then removed, flash frozen, and stored for later analysis. The training protocol produced a significant increase in RQ citrate synthase activity (p < 0.05). PV increased in proportion to the exercise intensity (p < 0.05) averaging 23.6 ± 2.7 ml•kg-1 body weight in the CC group and 26.6 ± 1.3 ml•kg-1 body weight in the HI group. Albumin mRNA expression determined by real time polymerase chain reaction (PCR) increased 2.2 ± 0.1 and 2.9 ± 0.2 fold following LI and HI exercise training, respectively. Conclusion- These data support the hypothesis that, during exercise-induced PV expansion, albumin gene expression is increased and contributes to an increase in plasma albumin content and PV.

No description available.

<p>Cytochrome P450s (CYPs) are enzymes that are found throughout the three domains of life. They function in the metabolism of endogenous and exogenous compounds. CYPs are extensively studied in mammalian systems due to their importance in drug metabolism and are highly expressed in detoxification organs like the liver and intestine. Fish CYP3s are not well understood. CYP3s have diversified in fish and subfamilies A, B, C and D constitute the CYP3 clade in fish. In this study, CYP3C1, CYP3C2, CYP3C3 and CYP3C4 in zebrafish (<em>Danio rerio</em>) and CYP3B4, CYP3B5 and CYP3B6 in medaka (<em>Orzyias latipes</em>) were quantified in hepatic and extrahepatic organs. CYP3C genes were quantified throughout development. All CYP3B and 3C isoforms were detected in all organs except CYP3B4 in male organs and in female brain. CYP3C1-C3 were maternally acquired and expressed in all embryonic stages. Higher expression of some of the isoforms occurred in the liver and intestine of zebrafish and medaka. This is indicative of a possible role in xenobiotic metabolism. Differences in expression between males and females gonad was observed, suggesting a possible role for estrogen in gene regulation. Further research will contribute to characterizing the upstream response elements in order to understand whether estrogens or other compounds are responsible for CYP3 regulation in fish. This knowledge will contribute to understanding the potential function these unique families of CYPs serve for fish.</p> / Master of Science (MSc)

Einleitung: Kurzkettige Fettsäuren (SCFA) stellen das hauptsächliche Energiesubstrat für Wiederkäuer dar. In Anbetracht des - bedingt durch höhere Milch-, Mast und Reproduktionsleistung - steigenden Energiebedarfs von Hauswiederkäuern wie Milchkuh und Mastbulle ist es von zentraler Bedeutung, die Mechanismen zur Resorption dieser Energielieferanten bzw. Ansatzpunkte für die Beeinflussung dieser Transportprozesse genau zu kennen. Dieses Wissen kann möglicherweise dabei helfen, zukünftig die Energieaufnahme der Tiere zu unterstützen bzw. sogar effizienter zu gestalten. Ziele der Untersuchungen: Deshalb war es Ziel der vorliegenden Arbeit, die Mechanismen zur Resorption von SCFA zu charakterisieren, wobei der Schwerpunkt auf den Transport aus den Pansenepithelzellen ins Blut gelegt wurde, da hierzu im Gegensatz zu ihrer Aufnahme aus dem Pansenlumen in die Epithelzellen noch sehr wenig bekannt war. In einem zweiten Schritt sollte untersucht werden, inwiefern die nachgewiesenen Mechanismen einer Regulation unterliegen und über welche Signalwege diese vermittelt werden könnte. Materialien und Methoden: Zur Charakterisierung der beteiligten Resorptionsmechanismen wurden Epithelstücke aus dem ventralen Pansensack von Schafen in Ussing-Kammern eingespannt und mit Hilfe radioaktiv markierten Azetats, Butyrats und L-Laktats der Transport dieser Substrate unter verschiedenen Bedingungen sowie verschiedenen Hemmstoffeinflüssen untersucht. Zur Charakterisierung regulativer Einflüsse wurden die Epithelstücke über sechs bzw. 24 Stunden mit Butyrat inkubiert und anschließend RNA bzw. Totalprotein extrahiert. Hiermit konnten Veränderungen in mRNA- und Proteinexpression mittels quantitativer Echtzeit-PCR bzw. Western Blot nachgewiesen werden. Ergebnisse: Die Untersuchungen der vorliegenden Arbeit konnten zeigen, dass der Transport von SCFA über die basolaterale Membran des Pansenepithels hauptsächlich proteinvermittelt erfolgt. Eine signifikante Beteiligung lipophiler Diffusion, d.h. ein passiver Transport, kann weitgehend ausgeschlossen werden. Der aktive Transport wies eine bikarbonatabhängige und eine bikarbonatunabhängige Komponente auf. Der Einsatz von Hemmstoffen verschiedener Transportproteine ergab deutliche Hinweise darauf, dass der Monocarboxylattransporter (MCT) 1 eine Rolle beim bikarbonatgekoppelten Transport von Azetat bzw. allgemein unmetabolisierten SCFA spielt. Diese Hinweise wurden untersetzt durch die Beobachtung, dass MCT 1, aber auch der apikal bzw. intrazellulär lokalisierte MCT 4 durch langfristige Inkubation des Epithels mit Butyrat sowohl auf mRNA- als auch auf Proteinebene signifikant erhöht exprimiert wurden, was als Anpassungsreaktion an eine Substratakkumulation interpretiert werden kann. Außerdem wurde auch die mRNA-Expression des Putativen Anionentransporters (PAT) 1 durch Inkubation mit Butyrat erhöht, was für eine Beteiligung auch dieses Transportproteins am SCFA-Transport über das Pansenepithel spricht. Allerdings ist im Gegensatz zu MCT 1 die Lokalisation des PAT 1 in der basolateralen Membran noch fraglich. Die Expressionssteigerung von Zielgenen des Nukleären Faktors ĸB und des Peroxisomenproliferator-aktivierten Rezeptors α sowie des Hypoxie-induzierbaren Faktors selbst deuten weiterhin darauf hin, dass die Steigerung der Transportkapazitäten von MCT 1 und 4 und auch PAT 1 über diese Signalwege vermittelt wird. Schlussfolgerungen: Zusammenfassend konnte in dieser Arbeit erstmals der Transport von SCFA über die basolaterale Membran des Pansenepithels näher charakterisiert werden, sodass es nun möglich ist, zusammen mit den bereits vorliegenden Befunden für die apikale Membran ein komplettes Modell dafür zu erstellen. Auch wurden Erkenntnisse zu regulativen Einflüssen auf diesen Transport gewonnen, die es zukünftig ermöglichen könnten, die Resorption der SCFA aus dem Pansen nutritiv oder eventuell pharmakologisch zu beeinflussen. / Introduction: The main energy source for ruminants are short chain fatty acids (SCFA). Considering the ever increasing energy requirements of cattle due to increasing milk yield and meat production, it is crucial to identify the mechanisms for the resorption of these energy sources as well as possibilities to influence these transport mechanisms. This knowledge could help support the animals’ energy uptake or even making it more efficient. Aim: Thus, the aim of the present study was to characterise mechanisms for the resorption of SCFA focusing on their transport from the epithelial cells into the blood. In particular, since – compared to the research findings on the uptake of SCFA from ruminal lumen into the cells – so far only very little was known regarding this side of the epithelium. In a second step, the study aimed to elucidate whether the mechanisms observed are subject to regulatory processes and which signalling pathways are involved. Materials and methods: To characterise the transport mechanisms involved, epithelial pieces from the ventral sac of ovine rumen were mounted in Ussing chambers. Using radioactively labelled acetate, butyrate and L-lactate, the transport of these substrates was investigated under different conditions and by applying different inhibitors for potential SCFA transport proteins. To characterise regulatory influences, epithelial pieces were incubated with butyrate for six and 24 hours, respectively. Subsequently, total RNA and protein were extracted to detect changes in mRNA and protein expression using quantitative real time PCR and western blot, respectively. Results: The present study could show that transport of SCFA across the basolateral membrane of rumen epithelium is mainly realised by protein-mediated mechanisms. A significant participation of lipophilic diffusion, i.e. a passive transport, can almost entirely be excluded. The active transport could be divided into a bicarbonate-dependent and a bicarbonate-independent part. The experiments with inhibitors of different transport proteins showed clear evidence of an involvement of monocarboxylate transporter (MCT) 1 in the bicarbonate-dependent transport of acetate and non-metabolised SCFA in general. This evidence was supported by the finding that the expression of MCT 1 but also of the apically and intracellularly localised MCT 4 was increased significantly on both mRNA- and protein-level after long-term incubation of the epithelium with butyrate. This can be interpreted as an adaptation to a substrate accumulation. Additionally, butyrate incubation led to an increased mRNA expression of putative anion transporter (PAT) 1, which makes an involvement of this transport protein in SCFA transport across ruminal epithelium likely as well. However, in contrast to MCT 1 the localisation of PAT 1 in the basolateral membrane is still questionable. The increased expression of target genes of nuclear factor ĸB and peroxisome-proliferator activated receptor α as well as of hypoxia inducible factor strongly point to an involvement of these pathways in the increased expression of MCT 1 and 4 as well as PAT 1. Conclusions: In summary, this study could characterise the transport of SCFA across the basolateral membrane of ruminal epithelium in detail for the first time. This enables us to draw a complete model of ruminal SCFA transport. Also, evidence for regulatory influence on this transport processes was found, perhaps making it possible to influence resorption of SCFA from rumen by nutritive or pharmacological means in the future.

A metionina é doadora de grupos metil para metilação do DNA, processo responsável por modificações da expressão gênica. Diante da importância da metionina para o crescimento e desenvolvimento normais, variações desse aminoácido na dieta podem alterar a estabilidade do DNA. O objetivo desse trabalho foi avaliar o efeito de dietas deficiente e suplementada em metionina sobre a instabilidade genômica e o estresse oxidativo em camundongos e suas mães tratadas durante gestação e lactação e destas também foi realizada a expressão de RNAm de Mat1a, Bhmt e Cbsdas vias de transmetilação, remetilação e transsulfuração da metionina, respectivamente, em fígado. As fêmeas foram divididas nos grupos de dietas de metionina (controle, 0,3% DL-metionina; suplementado, 2,0%; e deficiente 0%) até o fim da lactação (10 semanas) e, para cada grupo de fêmeas, os filhotes foram subdivididos e também receberam essas dietas durante 18 semanas após desmame. Foram avaliados o consumo de ração, massa corpórea e massa relativa de fígado e rinse a taxa de sobrevivência dos filhotes. Também foram realizadas a avaliação da peroxidação lipídica (quantificação das substâncias reativas ao ácido tiobarbitúrico, TBARS), da quantificação de glutationa (GSH)e da atividade da catalase, da instabilidade genômica (ensaio do cometa) e a análise do RNAm deMat1a, BhmteCbs somente em fígado das fêmeas. A dieta deficiente resultou em menor consumo de ração e massa corpórea em ambas fases e reduziu a sobrevivência dos filhotes que receberam essa dieta.A suplementação reduziu a concentração de TBARS nas fêmeas em ambos tecidos e a deficiência não diferiu. Nos filhotes, a suplementação reduziu a concentração de TBARS em fígado, enquanto que a deficiência aumentou. A suplementação aumentou a concentração de GSH em fígado das fêmeas, assim como a deficiência em rins. Nos filhotes, houve uma inversão de respostas, ou seja, a suplementação reduziu a concentração de GSH em fígado, assim como a deficiência, também observada em rins. Não houve diferença na catalase das fêmeas, mas houve redução em ambos tecidos dos filhotes. A suplementação e a deficiência reduziram os danos ao DNA hepático das fêmeas, mas em rins a suplementação aumentou e a deficiência reduziu. Nos filhotes, a suplementação e a deficiência aumentaram os danos ao DNA em ambos tecidos. A suplementação de metionina em fêmeas não alterou a expressão dos RNAm avaliados e a deficiência reduziu em Cbs somente. Concluiu-se que na fase materna, a suplementação ou a deficiência de metionina não resultou em estresse oxidativo, mas a suplementação reduziu a instabilidade genômica no fígado e aumentou no rim. A deficiência resultou em menor instabilidade nos dois tecidos. Na fase descendente, a suplementação e a deficiência de metionina apresentaram variação de estresse oxidativo em ambos tecidos e também resultaram em maior instabilidade genômica. / Methionine is the main methyl donor for the DNA methylation, a process responsible for gene expression modifications. Since this essential amino acid is required for normal growth and development, variations of this compound in the diet may lead to alterations on DNA stability. Thus, this study aimed to evaluate the effect of deficient and supplemented methionine diets on oxidative stress and genomic instability in mice and their dams treated during pregnancy and lactation, and the expression of Mat1a, Bhmt and Cbs mRNA of transmethylation, remetthylation, and transulfuration pathways, respectively, in dams livers. The dams were divided into three methionine diets groups (control, 0,3% DL-methionine; supplemented, 2,0%; and deficient, 0%) until the end of lactation (10 weeks). For each dams groups, the offspring were subdivided and were also treated with the same diets during 18 weeks after weaning. The parameters evaluated were food intake, body weight, relative liver and kidney weights, and survival of the offspring. Also, it was carried out theevaluation of lipid peroxidation (thiobarbituric acid reactive substances, TBARS), quantification of glutathione (GSH) and catalase activity, and genomic instability (comet assay) in liver and kidneys; andMat1a, Bhmt, and CbsmRNA analysis only in dams liver. The deficient diet resulted in lower food intake and body weights in both phases and reduced the survival of the offspring that were treated with this diet. The supplemented diet reduced the TBARS concentration in both tissues of dams and the deficient diet did not differ. In the offspring, the supplementation reduced liver TBARS, whereas the deficiency raised. The supplementation increased the liver GSH concentration of dams, as well as the deficiency in kidneys. In the offspring, the responses were different; the supplementation reduced the liver GSH, as well as the deficiency, also observed in kidneys. There were no differences of catalase parameter of dams, but there was a reduction in both tissues of the offspring. Both supplementation and deficiency reduced the liver DNA damage of dams, however the supplementation increased and the deficiency reduced the DNA damage of kidneys. In the offspring, both diets increased the DNA damage in both tissues. The methionine supplementation did not differ the mRNA expression and the deficiency only reduced the Cbs, mRNA expression. It was concluded that the methionine supplementation or deficiency did not resulted in oxidative stress in dams, but the supplementation reduced the genomic instability of liver and raised the kidney one. The deficiency resulted in lower genomic instability in both tissues in dams. In the offspring, both methionine supplementation and deficiency presented variation of oxidative stress in both tissues and resulted more genomic instability.

A metionina é doadora de grupos metil para metilação do DNA, processo responsável por modificações da expressão gênica. Diante da importância da metionina para o crescimento e desenvolvimento normais, variações desse aminoácido na dieta podem alterar a estabilidade do DNA. O objetivo desse trabalho foi avaliar o efeito de dietas deficiente e suplementada em metionina sobre a instabilidade genômica e o estresse oxidativo em camundongos e suas mães tratadas durante gestação e lactação e destas também foi realizada a expressão de RNAm de Mat1a, Bhmt e Cbsdas vias de transmetilação, remetilação e transsulfuração da metionina, respectivamente, em fígado. As fêmeas foram divididas nos grupos de dietas de metionina (controle, 0,3% DL-metionina; suplementado, 2,0%; e deficiente 0%) até o fim da lactação (10 semanas) e, para cada grupo de fêmeas, os filhotes foram subdivididos e também receberam essas dietas durante 18 semanas após desmame. Foram avaliados o consumo de ração, massa corpórea e massa relativa de fígado e rinse a taxa de sobrevivência dos filhotes. Também foram realizadas a avaliação da peroxidação lipídica (quantificação das substâncias reativas ao ácido tiobarbitúrico, TBARS), da quantificação de glutationa (GSH)e da atividade da catalase, da instabilidade genômica (ensaio do cometa) e a análise do RNAm deMat1a, BhmteCbs somente em fígado das fêmeas. A dieta deficiente resultou em menor consumo de ração e massa corpórea em ambas fases e reduziu a sobrevivência dos filhotes que receberam essa dieta.A suplementação reduziu a concentração de TBARS nas fêmeas em ambos tecidos e a deficiência não diferiu. Nos filhotes, a suplementação reduziu a concentração de TBARS em fígado, enquanto que a deficiência aumentou. A suplementação aumentou a concentração de GSH em fígado das fêmeas, assim como a deficiência em rins. Nos filhotes, houve uma inversão de respostas, ou seja, a suplementação reduziu a concentração de GSH em fígado, assim como a deficiência, também observada em rins. Não houve diferença na catalase das fêmeas, mas houve redução em ambos tecidos dos filhotes. A suplementação e a deficiência reduziram os danos ao DNA hepático das fêmeas, mas em rins a suplementação aumentou e a deficiência reduziu. Nos filhotes, a suplementação e a deficiência aumentaram os danos ao DNA em ambos tecidos. A suplementação de metionina em fêmeas não alterou a expressão dos RNAm avaliados e a deficiência reduziu em Cbs somente. Concluiu-se que na fase materna, a suplementação ou a deficiência de metionina não resultou em estresse oxidativo, mas a suplementação reduziu a instabilidade genômica no fígado e aumentou no rim. A deficiência resultou em menor instabilidade nos dois tecidos. Na fase descendente, a suplementação e a deficiência de metionina apresentaram variação de estresse oxidativo em ambos tecidos e também resultaram em maior instabilidade genômica. / Methionine is the main methyl donor for the DNA methylation, a process responsible for gene expression modifications. Since this essential amino acid is required for normal growth and development, variations of this compound in the diet may lead to alterations on DNA stability. Thus, this study aimed to evaluate the effect of deficient and supplemented methionine diets on oxidative stress and genomic instability in mice and their dams treated during pregnancy and lactation, and the expression of Mat1a, Bhmt and Cbs mRNA of transmethylation, remetthylation, and transulfuration pathways, respectively, in dams livers. The dams were divided into three methionine diets groups (control, 0,3% DL-methionine; supplemented, 2,0%; and deficient, 0%) until the end of lactation (10 weeks). For each dams groups, the offspring were subdivided and were also treated with the same diets during 18 weeks after weaning. The parameters evaluated were food intake, body weight, relative liver and kidney weights, and survival of the offspring. Also, it was carried out theevaluation of lipid peroxidation (thiobarbituric acid reactive substances, TBARS), quantification of glutathione (GSH) and catalase activity, and genomic instability (comet assay) in liver and kidneys; andMat1a, Bhmt, and CbsmRNA analysis only in dams liver. The deficient diet resulted in lower food intake and body weights in both phases and reduced the survival of the offspring that were treated with this diet. The supplemented diet reduced the TBARS concentration in both tissues of dams and the deficient diet did not differ. In the offspring, the supplementation reduced liver TBARS, whereas the deficiency raised. The supplementation increased the liver GSH concentration of dams, as well as the deficiency in kidneys. In the offspring, the responses were different; the supplementation reduced the liver GSH, as well as the deficiency, also observed in kidneys. There were no differences of catalase parameter of dams, but there was a reduction in both tissues of the offspring. Both supplementation and deficiency reduced the liver DNA damage of dams, however the supplementation increased and the deficiency reduced the DNA damage of kidneys. In the offspring, both diets increased the DNA damage in both tissues. The methionine supplementation did not differ the mRNA expression and the deficiency only reduced the Cbs, mRNA expression. It was concluded that the methionine supplementation or deficiency did not resulted in oxidative stress in dams, but the supplementation reduced the genomic instability of liver and raised the kidney one. The deficiency resulted in lower genomic instability in both tissues in dams. In the offspring, both methionine supplementation and deficiency presented variation of oxidative stress in both tissues and resulted more genomic instability.

Solute carriers (SLCs) constitute the largest family of membrane-bound transporter proteins in humans, and they convey transport of nutrients, ions, drugs and waste over cellular membranes via facilitative diffusion, co-transport or exchange. Several SLCs are associated with diseases and their location in membranes and specific substrate transport makes them excellent as drug targets. However, as 30 % of the 430 identified SLCs are still orphans, there are yet numerous opportunities to explain diseases and discover potential drug targets. Among the novel proteins are 29 atypical SLCs of major facilitator superfamily (MFS) type. These share evolutionary history with the remaining SLCs, but are orphans regarding expression, structure and/or function. They are not classified into any of the existing 52 SLC families. The overall aim in this thesis was to study the atypical SLCs with a focus on their phylogenetic clustering, evolutionary conservation, structure, protein expression in mouse brains and if and how their gene expressions were affected upon changed food intake. In Papers I-III, the focus was on specific proteins, MFSD5 and MFSD11 (Paper I), MFSD1 and MFSD3 (Paper II), and MFSD4A and MFSD9 (Paper III). They all shared neuronal expression, and their transcription levels were altered in several brain areas after subjecting mice to food deprivation or a high-fat diet. In Paper IV, the 29 atypical SLCs of MFS type were examined. They were divided into 15 families, based on phylogenetic analyses and sequence identities, to facilitate functional studies. Their sequence relationships with other SLCs were also established. Some of the proteins were found to be well conserved with orthologues down to nematodes and insects, whereas others emerged at first in vertebrates. The atypical SLCs of MFS type were predicted to have the common MFS structure, composed of 12 transmembrane segments. With single-cell RNA sequencing and in situ proximity ligation assay, co-expression of atypical SLCs was analysed to get a comprehensive understanding of how membrane-bound transporters interact.   In conclusion, the atypical SLCs of MFS type are suggested to be novel SLC transporters, involved in maintaining nutrient homeostasis through substrate transport.

No description available.

Pulmonary arterial hypertension (PAH) can be caused by pathogenic variants in the gene bone morphogenetic protein receptor 2 (BMPR2). While BMPR2 protein expression levels are known to be reduced in the lung tissue of heritable PAH (HPAH) patients, a systematic study evaluating expression in more easily accessible blood samples and its clinical relevance is lacking. Thus, we analyzed the BMPR2 mRNA expression in idiopathic/HPAH patients and healthy controls in blood by quantitative polymerase chain reaction and protein expression by enzyme-linked immunosorbent assay. Clinical parameters included right heart catherization, echocardiography, six-minute walking test and laboratory tests. BMPR2 variant-carriers (n = 23) showed significantly lower BMPR2 mRNA expression in comparison to non-carriers (n = 56) and healthy controls (n = 30; p < 0.0001). No difference in BMPR2 protein expression was detected. Lower BMPR2 mRNA expression correlated significantly with greater systolic pulmonary artery pressure and pulmonary vascular resistance. Higher BMPR2 mRNA expression correlated with greater glomerular filtration rate, cardiac index and six-minute walking distance. We demonstrated the feasibility to assess BMPR2 expression in blood and, for the first time, that BMPR2 mRNA expression levels are significantly reduced in variant carriers and correlated with clinical parameters. Further studies may evaluate the usefulness of BMPR2 mRNA expression in blood as a new marker for disease severity.