No description available.

No description available.

Hypoxia-inducible factors (HIFs) are a highly conserved group of transcriptional regulators responsible for cellular and systemic O2 homeostasis in animals. However, how HIFs are involved in basic adaptive ventilatory and metabolic responses to acute and chronic hypoxia remains incompletely characterized. Naked mole rats are among the most hypoxia tolerant mammals identified. As opposed to the typical hyperventilatory response of most adult mammals, naked mole rats exhibit a unique decline in ventilation, matching their substantial decrease in metabolic rate. Naked mole rats therefore provide an excellent model in which to investigate adaptations to hypoxic ventilatory and metabolic responses (HVR and HMR, respectively). Interestingly, naked mole rats possess a mutation within the von Hippel-Lindau (VHL) binding domain—a protein necessary for proteasomal degradation of HIF subunits in normal O2 concentrations—suggesting they retain elevated baseline expression of HIF and thus an upregulation of downstream gene targets. In designing our experiment, we focused on sustained hypoxia and HIF1, which is typically the first responder subunit upon exposure to low O2 stress. We sought to determine how increased HIF1 expression might contribute to the distinct HVR and HMR of naked mole rats, first by confirming the observed VHL mutation translates into increased HIF1 protein expression via immunoblotting. HIF1 protein expression was found to be 3-fold higher in naked mole rat brain than mouse brain and 4-fold higher than in mouse liver tissue (p < 0.05). We then investigated how elevated HIF1 levels might contribute to the HVR and HMR by treating naked mole rats with two different HIF1 inhibitors (either echinomycin; 0.5 and 1.0 mg kg-1, or PX-478; 80.0 mg kg-1) and subsequently examined changes in ventilatory and metabolic parameters in awake animals exposed to sustained hypoxia (7% O2; 1 hour). In control naked mole rats, minute ventilation (V̇E) reversibly decreased by 32% in hypoxia (1298.3 ± 188.5 to 882.6 ± 117.0 mL min-1 kg-1) because of changes in both breathing frequency (fR) and tidal volume (VT). Conversely, the HVR was not significantly affected in any of our three treatment groups however, normoxic ventilation increased in naked mole rats treated with low dose echinomycin (0.5 mg kg-1) by 72% (from 1298.3 ± 188.5 to 2239.5 ± 221.1 mL min-1 kg-1). Consistent with previous findings, metabolic rate in control naked mole rats decreased 70% (from 40.1 ± 5.0 to 11.9 ± 0.9 mL O2 min-1 kg-1). Again, treatment with our pharmacological agents did not significantly alter this response but did result in a 43% decrease in basal metabolic rate (V̇O2 and V̇CO2) in both high-dose echinomycin and PX-478 treated naked mole rats (40.1 ± 5.0 to 22.5 ± 3.6 and 23.0 ± 1.88 mL O2 min-1 kg-1 respectively, p < 0.05), dulling the magnitude of the HMR. As a result of unmatched changes in V̇E and V̇O2, HIF1 deficient naked mole rats treated with both low-dose echinomycin and PX-478 experienced an atypical increase in their air convection requirement (ACR; V̇E:V̇O2-1) in hypoxia (from 77.4 ± 11.3 to 159.2 ± 34.63 and 123.5 ± 35.5 respectively, p < 0.05), resembling a hyperventilation response closer to that of hypoxia-intolerant mammals. To further determine how increased HIF1 availability affects the HMR and HVR, we administered hypoxia-intolerant mice with a pharmacological HIF1 agonist (3,4- EDHB; 180 mg kg-1) and used identical experimental design to measure downstream ventilatory and metabolic responses. Mice exhibit similar reductions in metabolic rate during hypoxic exposure (from 60.3 ± 2.4 to 21.8 ± 1.8 mL O2 min-1 kg-1, p < 0.05) but experience a 30% increase in fR (from 157.5 ± 9.5 to 200.4 ± 10.8 breaths min-1, p < 0.05). In contrast, mice treated with EDHB and to exposed 7% O2 exhibited a 20% increase in fR (200.4 ± 10.8 to 236.5 ± 14.1 breaths min-1, p < 0.05) and a 30% reduction in the magnitude of their HMR (from 38.5 ± 2.8 to 27.8 ± 3.6 ΔV̇O2). No other significant trends were observed in any of the other parameters measured. We conclude metabolic and ventilatory control in naked mole rats and mice may partially depend on increased HIF1 expression.

In mammals, the circadian clock coordinates multiple behavioural and physical processes, including energy homeostasis. At the centre of these rhythms lies the circadian clock machinery, a precisely coordinated transcription-translation feedback system required to maintain the correct time. Metabolic homeostasis requires accurate and coordinated circadian timing within individual cells and tissues of the body. Moreover, recent evidence has shown that the coupling of circadian and metabolic circuits involves reciprocal regulatory feedback. In line with this, mounting evidence suggests that disruption of the clock contributes to the development of obesity and its comorbidities. This is particularly concerning given that modern lifestyles often undermine our bodies’ clock. However, the casual mechanisms which link circadian disruption to metabolic disease are not well defined. This work aims to gain a further understanding of clock control of metabolic homeostasis and especially regulation of lipid metabolism. This work uses dietary challenge to determine which peripheral clocks and downstream metabolic pathways are particularly susceptible to diet induced obesity (DIO). We demonstrate that although behavioural rhythmicity was maintained in DIO, gene expression profiling revealed tissue-specific alteration to the phase and amplitude of the molecular clockwork. Clock function was most significantly attenuated in visceral white adipose tissue (WAT) of DIO mice, and was coincident with elevated tissue inflammation, and dysregulation of clock-coupled metabolic regulators PPARα/γ.The rhythmic expression of Rev-erbα, a nuclear receptor involved in the circadian clock, was particularly affected in DIO mice. This study uses the Rev-erbα-/- mouse to explore clock-metabolic coupling, specifically lipid metabolism. In line with published work, Rev-erbα-/- mice exhibit an obese phenotype with associated upregulation in gWAT of lipogenic (Dgat2, Fasn) and fatty acid liberation (Lpl) genes. Differences in fat mobilization are observed as Rev-erbα-/- mice show a heightened insulin stimulated lipogenic drive and an attenuation of the lipolytic drive in the fasted state, suggesting an increased propensity for fat accumulation. The role of the clock was further investigated in adipose tissue by deletion of Bmal1 (clock ablation) or Rev-erbα (clock manipulation) specifically in adipocytes using Cre-Lox methodology. AdipoCREBmal1flox/flox mice showed attenuated feeding rhythms, indicating a direct effect of the adipocyte circadian clock on hypothalamic feeding centres and severe dysregulation of metabolic genes. However, AdipoCRERev-erbαflox/flox displayed very little phenotypic difference compared to control littermates, suggesting that global loss of Rev-erbα may have reinforcing metabolic consequences. This work suggests a key role of the clock in lipid handling and the pathogenesis of obesity. Insights into this link may lead to novel targets for treating both obesity and metabolic complications.

Orientador: Carmen Silvia Passos Lima / Dissertação (mestrado) - Universidade Estadual de Campinas. Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-16T03:51:55Z (GMT). No. of bitstreams: 1 Scherr_MarcelaCustodio_M.pdf: 1746928 bytes, checksum: 1590f1bcf4c880aa0dbd063756c2c777 (MD5) Previous issue date: 2010 / Resumo: A habilidade de metabolizar xenobióticos é variável em humanos e em cães e está relacionada a genótipos distintos de genes polimórficos. Os portadores do genótipo CYP1A2 1117TT foram classificados como metabolizadores lentos de fármacos. Ainda, os alelos variantes T e C dos polimorfismos CYP1A2 C1117T e GSTA3 I71L foram descritos como menos eficazes na ativação e inativação de carcinógenos do que os selvagens, respectivamente. Assim, humanos e cães podem apresentar diferentes respostas a medicamentos e estar sob riscos distintos de ocorrência de tumores. As frequências dos genótipos dos polimorfismos CYP1A2 C1117T e GSTA3 I71L em cães de diferentes raças e sem raça definida (SRD) não foram ainda descritas. O objetivo deste estudo foi o de identificar as frequências dos genótipos em 105 cães da raça boxer, 84 cães da raça rottweiler, 110 cães da raça pastor alemão e 99 animais SRD, para verificar se ocorre distribuição distinta de genótipos em animais de diferentes grupos. O genótipos foram identificados por amplificação das regiões gênicas de interesse, em amostras de sangue periférico, por meio da reação em cadeia da polimerase seguida por sequenciamento ou digestão enzimática dos fragmentos amplificados. A frequência do genótipo homozigoto selvagem CC do polimorfismo CYP1A2 C1117T foi maior em cães de raça (96,4% versus 87,9%, P= 0,002) e boxers (98,0% versus 87,9%, P= 0,005) do que em cães SRD. Frequências genotípicas similares foram observadas em cães estratificados por aspectos clínicos. Apenas o genótipo variante CC do polimorfismo GSTA3 I71L foi identificado em nossos cães. Nossos resultados sugerem que cães de raça metabilizam fármacos e ativam carcinógenos de forma mais eficaz do que cães SRD e podem obter maior benefício medicamentoso e estar sob risco maior de tumores. Já o metabolismo de xenobióticos pela enzima GSTA3 parece similar em cães de diferentes raças e não influenciar diferentemente a resposta a medicamentos e o risco de tumores nesses animais / Abstract: The ability to metabolize xenobiotics is variable among humans and dogs, and is related to the distinct genotypes of polymorphic genes. Carriers of the CYP1A2 1117TT genotype were classified as poor metabolizers of drugs. In addition, the varying T and C alleles of CYP1A2 C1117T e GSTA3 I71L polymorphisms were described as less efficient in the activation and inactivation of carcinogens than the wild alleles, respectively. Thus, humans and dogs seem to obtain distinct benefits under drug treatments and also to be under different risks of tumours. The frequencies of genotypes of the refered genes in dogs of different breeds and mixed breed are yet not described. The objective of this study was to identify the frequencies of the genotypes in 105 boxers, 84 rottweilers, 110 german shepherds and 99 mixed breed dogs with the purpose of to verify if a distinct distribution of genotypes occurs in animals of different groups.The genotypes were evaluated by amplifying the area of interest of the genes, in peripheral blood samples, using a polymerase chain reaction followed by a sequencing or enzymatic digestion of the amplified fragments. The frequency of the homozygous wild CC genotype of the CYP1A2 C1117 polymorphism was higher in dogs of pure breed (96,4% versus 87,9%, P= 0.002) and boxers (98.0% versus 87,9%, P= 0.005) than in mixed breed dogs. Similar frequencies of the genotypes were seem in dogs stratified by clinical features. Just the CC varying genotype of the GSTA3 I71L polymorphism was identified in all dogs. The results of this study suggest that pure breed dogs might be more efficient in metabolizing drugs and in activating carcinogens than mixed breed dogs, and therefore, may obtain larger therapeutic benefits under treatment for diseases and may be under higher risk for tumors. In contrast, the metabolim of xenobiotics by the GSTA3 enzyme seems to be similar among dogs of different breeds and not to influence on a different way the drug metabolism or tumour risk in these animals / Mestrado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Mestre em Fisiopatologia Médica

Made available in DSpace on 2014-10-09T12:23:13Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T13:57:23Z (GMT). No. of bitstreams: 1 00908.pdf: 447464 bytes, checksum: 2932d702c88b5e8d3f57989685245d3a (MD5) / Dissertacao (Mestrado) / IEA/D / Instituto de Biociencias, Universidade de Sao Paulo - IB/USP

Nutrient metabolism consists of a series of chemical processes concerned with supplying energy to the body. This enables the body to perform various physiological processes and to maintain homeostasis (Guyton and Hall, 2000). In healthy, non-diabetic subjects, plasma glucose concentrations are held within a narrow range throughout the day, despite wide fluctuations in nutritional intake and physical exercise, as well as other physiological, psychological and iatrogenic influences (Owens, 2002). The purpose of this research study was to determine the effect of the homoeopathic preparations, Saccharum officinale 9cH and 200cH, on glucose metabolism in healthy, non-diabetic humans. This research study was a double blind, placebo-controlled clinical trial. A group of thirty participants were required to undergo an oral glucose tolerance test for three hours. Timing started as soon as the participants started drinking the glucose solution. In addition, ten drops of the homoeopathically prepared medicine or a placebo were administered at each of the following times: -15 minutes, 27 minutes, 87 minutes and 147 minutes. Group one received the placebo (20% alcohol), Group two received Saccharum officinale 9cH and Group three received Saccharum officinale 200cH. Blood glucose concentrations were measured, using capillary blood samples and a glucose meter, at the following times: -30 minutes, 30 minutes, 60 minutes, 90 minutes, 120 minutes, 150 minutes, and 180 minutes. Vital signs were measured at: 10 minutes, 50 minutes, 110 minutes and 170 minutes in order to ascertain any detrimental changes in health. Data was expressed as mean ± standard error. Differences between the groups were determined using the one-way repeated measures analysis of variance method. The hypoglycaemic effect of Saccharum officinale 9cH and 200cH was not proven to be effective in reducing the rate of glucose disposal in the body. Even though a slight difference between the experimental groups and the control group was observed, these changes could not be attributed to the therapeutic effect of the remedy and was regarded as statistically insignificant. / Dr. Natasha Wolf Mr. Neil de Villiers

Impaired clearance of triacylglycerol-rich lipoproteins contributes to atherogenesis. It can be argued that exercise may decrease the risk of atherosclerotic diseases through its potential to improve the metabolic capacity for triacylgycerol and hence, clearance of triacylglycerol-rich lipoproteins. The investigations described in this thesis focused on the influence of exercise on postprandial lipid and lipoprotein metabolism.

An unknown Saccharopolyspora species which displayed fungicidal activity was selected as a representative of the genus. The antifungal activity could not be repeated in liquid cultures after the initial screen despite several different approaches. A defined medium was developed. However, biomass yields were low (< lg/1) and antifungal activity was not detected. Good growth was observed in nutrient broth (NB) and on nutrient agar (NA). The maximum specific growth rate in NB was 0.25h-1. Growth on NA (measured using image analysis) conformed to the linear growth law with a mean radial growth rate of 0.0327mm/h. Fungicidal activity was measured on solid media, this was presumably due to antibiotic production, although an active compound could not be isolated. A mutation program yielded only non producing mutants despite the mutants having a range of growth rates. A known Saccharopolyspora species, Saccharopolyspora erythraea, was then adopted as a representative of the genus for the remainder of the study. Defined media were developed to permit growth limitation by a range of nutrients. A comparison of several approaches to calculate the maximum specific growth rate was made (ranging from 0.119 to 0.148h-1 in carbon limited media, 0.093 to 0.130h-1 in nitrogen limited, and 0.143 to 0.165h-1 in phosphate limited). Curve fitting provided an accurate series of specific growth rate estimates. The control mechanisms involved in the biosynthesis of erythromycin by Saccharopolyspora erythraea were investigated. By culturing this organism in a range of conditions, and analysing the effects on various growth parameters, nutrient specific patterns of control were observed. Increasing biomass and increasing antibiotic titres occurred concurrently in media when growth was carbon (C), nitrogen (N), and phosphate (P) limited. Generally, when antibiotic titre parallels growth, it is assumed that the product is growth linked. However, further analysis contradicted this. The peaks of specific growth rates and specific erythromycin production rates, obtained by curve fitting, were clearly temporally separated in carbon and phosphate limited conditions indicating non-growth linked production of erythromycin. Growth associated production was confirmed in nitrogen limited conditions. Phosphate limitation supported higher antibiotic yields than the other limitations (Yp/x max mg/g = 6.4 in CL1, 18.5 in NL1, and 28.8 in PL1). Erythromycin production was sensitive to ammonium and to glucose during phosphate limitation. However, this sensitivity to glucose was not obvious in the nitrate limited medium, suggesting different control mechanisms may affect the growth linked and non-growth linked production of erythromycin. Investigations into the regulation of the initiation of erythromycin production indicated that energy charge was not obviously involved, as it appeared to be related to the specific growth rate rather than antibiotic production. The rate of protein synthesis appeared to be a strong candidate for the initiator of antibiotic biosynthesis. Chemostat culture confirmed that erythromycin production was growth linked in nitrate limited conditions. However, the kinetics were more complicated as both biomass and antibiotic decreased with increasing dilution rates. The data conformed to the model proposed by Pirt (1975) for non-competitive inhibition by a growth linked product. A modelling package generated estimates of parameters involved (ki = 1.2mg/1, ks = 0.017g/l, micro max = 0.062h-1 and Yp/s = 0.0019).

Aerobactin in human and animal disease Mark Roberts The aerobactin iron uptake system is specified by a cluster of five genes, four for the synthesis of the siderophore aerobactin and the fifth for its cognate outer membrane protein receptor. The aerobactin genes have been cloned and subjected to molecular analysis. The role of the product of one of these genes (the 50,000 dalton protein) was in dispute and had been reported to be involved in both the transport and the biosynthesis of aerobactin. Complementation studies showed that the protein is intimately involved in aerobactin biosynthesis. Also, strains carrying plasmids with transposon insertions that abolished production of the 50 K protein were still able to transport aerobactin, showing that the outer membrane receptor is the only protein specified by the aerobactin system necessary for the uptake of ferri-aerobactin. E. coli strains isolated from domestic animals were screened for aerobactin and colicin V production by bioassay and by colony hybridization using DNA probes derived from the aerobactin biosynthesis and colicin V activity regions of ColV-K30. There was a high incidence of aerobactin production among septicaemic strains compared with a very low incidence among faecal strains. Colicin V production correlated with aerobactin production but the association was not absolute. Southern hybridization experiments indicated that the aerobactin genes of animal strains almost always reside on large (usually ColV) plasmids. Diarrheagenic strains of E. coli were also screened for aerobactin production. No enterotoxigenic strains produced aerobactin but strains belonging to the enteropathogenic, enteroinvasive and facultatively enteropathogenic E. coli groups contained members that produced aerobactin. The aerobactin biosynthesis probe hybridized to small plasmids of enteropathogenic E. coli strains in Southern blots. Antisera were raised in rabbits to both native and denatured aerobactin receptor protein (IutA). The antisera raised to denatured protein reacted with denatured protein in Western blots (WB) and immunoprecipitation (IP) but did not inhibit aerobactin or cloacin binding to IutA. The serum raised against the native protein inhibited cloacin binding but did not react in WB or IP. The cloned aerobactin genes restored the virulence in a murine peritonitis model of a wild type animal strain of E. coli cured of its CoIV plasmid. Prior growth in iron-restricted medium enhanced the virulence of the infecting strain. None of the anti-IutA sera passively protected mice from infection with a wild type iutA+ strain.