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Regular aerobic exercise has a wide range of positive effects on health and cognition. Exercise has been demonstrated to provide a particularly powerful and replicable method of triggering a wide range of structural changes within both human and animal brains. However, the details and mechanisms of these changes remain poorly understood. This thesis undertakes a comprehensive examination of the relationship between brain plasticity and aerobic exercise. A large, longitudinal experiment was conducted in which healthy but sedentary participants were scanned before and after six-weeks of monitored aerobic exercise. Increases in the volume of the anterior hippocampus were observed, as previously reported in an older cohort after a longer exercise intervention. Multimodal imaging methods allowed an in-depth exploration of the mechanisms underlying this volume change, which proved to be dominated by white matter changes rather than the vascular changes that have been previously reported. A surprising global change in the balance of CSF, blood, and brain tissue within the cranial cavity was also observed. Cross-sectional differences in memory and brain structure associated with fitness were also observed. The volume of the anterior hippocampus was shown to correlate with a measure of working memory. Higher cerebral blood volume throughout the brain was found to correlate with greater fitness and better working memory. Focal associations between fitness and magnetic susceptibility, a measure of iron content, were also observed in the basal ganglia. These findings demonstrate that aerobic fitness is associated with improved cognition and brain structure throughout the lifespan rather than simply acting to mitigate age related brain atrophy or accelerate brain development. Finally, a new pipeline was developed for analysing hippocampal morphometry using high-resolution, 7 Tesla scans. Striking variability in the convolution of the hippocampal surface is reported. This technique shows promise for imaging the precise nature of the change in hippocampal volume associated with aerobic exercise. This thesis adds to the evidence that aerobic exercise is a potent catalyst for behavioural and brain plasticity while also demonstrating that the mechanisms for those plastic changes are likely different than previously supposed. Future work will refine these measurement techniques, perhaps to a point where brain changes can be monitored on a single subject level. This work will provide an important tool to understand how best to utilize aerobic exercise to facilitate adaptive behavioural changes, mitigate the negative effects of ageing and disease on the brain, and maximize the benefits of active lifestyles.

In obesity, lipids stored in muscle as lipid droplets (LDs) lead to accumulation of fatty acid (FA) metabolites and insulin resistance. This research involves development of immunofluorescence microscopy methods to generate novel information on the subcellular content and distribution of key enzymes that play a role in the underlying mechanisms. Chapters 3 and 4 describe visualisation of two lipid synthesising enzymes. Both are more abundant in type I muscle fibres. Chapter 5 reveals no differences between these enzymes in non obese and obese elderly women. Chapter 6 reveals that a key lipolytic enzyme (ATGL) has a higher content in type I fibres, but its activator does not. Chapter 7 describes visualisation of SNAP23 and reveals a high content at the plasma membrane and mitochondria and low content in LDs. Chapter 8 fails to observe a difference between obese and non obese elderly women in plasma membrane SNAP23, and therefore fails to confirm the hypothesis that LDs hijack SNAP23. However, obese women have less SNAP23 in mitochondria and this may limit FA oxidation. In conclusion this thesis describes several novel mechanisms by which obesity leads to accumulation of FA metabolites and insulin resistance. The developed methods will be a valuable novel tool for future diabetes research.

Repetitive stimulation of motor nerves causes an increase in the number of packets of transmitter ("quanta") that can be released in the ensuing period. This represents a type of conditioning, in which synaptic transmission may be enhanced by prior activity. Despite many studies of this phenomenon, there have been no investigations of the quantal mechanisms underlying these events, due to the rapid changes in transmitter output and the short time periods involved. To examine this problem, a method was developed in which estimates of the quantal release parameters could be obtained over very brief periods (3 s). Conventional microelectrode techniques were used to record miniature endplate potentials (MEPPs) from isolated frog (Rana pipiens) cutaneous pectoris muscles, before and after repetitive (40 sec at 80 Hz) nerve stimulation. Estimates were obtained of m (number of quanta released), n (number of functional release sites), p (mean probability of release) and var$\rm\sb{s}$p (spatial variance in p) using a method that employs counts of MEPPs per unit time. Fluctuations in the estimates were reduced using a moving bin technique (bin size = 3 s, $\Delta$bin = 1 s). Muscle contraction was prevented using low Ca$\sp{2+},$ high Mg$\sp{2+}$ Ringer or normal Ringer to which $\mu$-conotoxin GIIIA was added. These studies showed that: (1) the post-stimulation increase in transmitter release was dependent on stimulation frequency and not on the total number of stimulus impulses. When the total number of pulses was kept constant, the high frequency pattern produced a higher level of transmitter release than did the lower frequency patterns; (2) augmentation and potentiation were present in both low Ca$\sp{2+},$ high Mg$\sp{2+}$ and normal Ringer solutions, but potentiation, m, n, p and var$\rm\sb{s}$p were greater in normal Ringer solution than in low Ca$\sp{2+},$ high Mg$\sp{2+}$ solution. In low Ca$\sp{2+},$ high Mg$\sp{2+}$ solution, there was a larger decrease in n compared to p; (3) hypertonicity (addition of 100 mM sucrose) produced a marked increase in both basal and stimulation-induced values of m, n, and p. By contrast, there was a marked increase in the stimulation-induced but not the basal values of var$\rm\sb{s}$p; (4) hypertonicity produced a decrease in augmentation but had no effect on potentiation; (5) augmentation and potentiation appeared to involve mitochondrial uptake and efflux of cytoplasmic Ca$\sp{2+}.$ Tetraphenylphosphonium (which blocks mitochondrial Ca$\sp{2+}$ efflux and uptake) decreased augmentation and potentiation in low Ca$\sp{2+},$ high Mg$\sp{2+}$ solutions but increased potentiation in the same solution made hypertonic with 100 mM sucrose; (6) the overall findings suggest that this new method may be useful for investigating the subcellular dynamics of transmitter release following nerve stimulation.

The main objective of this PhD was to provide fundamental information regarding some metabolic aspects of selenium metabolism in cats and dogs. The total selenium content of a range of commercially available petfoods was analysed using a fluorometric method. The petfoods contained a wide range of selenium concentrations, with up to 6 µg Se/g DM found in cat foods. Mean concentrations of selenium in dog and cat foods were 0.40 and 1.14 µg Se/g DM respectively. All petfoods analysed met the recommended current minimum dietary selenium requirements. The use of blood parameters for the assessment of selenium status was investigated in a study in which cats were fed inorganic and organic selenium supplemented at concentrations of up to 2.0 µg Se/g DM for 32 days. Plasma selenium concentrations reflected dietary selenium intakes, however there were no differences between the different levels of supplementation. Whole blood selenium concentrations showed less distinct patterns and were thought to be a more useful indicator of longer term selenium status. Activities of glutathione peroxidase in plasma and whole blood showed no response and the response of cats to supplementation of the different forms of selenium were similar. In the same study, faecal and urinary excretion (µg/kg BW/d) were measured and apparent absorption and retention were estimated during the last seven days of the 32 day trial. Faecal excretion of selenium remained constant whereas urinary excretion of selenium increased with increased dietary intake. The form of selenium had no effect on excretion or apparent absorption however there was a trend in which more selenium was retained in cats fed organic selenium. A study was conducted with cats and dogs fed high levels (10 µg Se/g DM) of inorganic and organic selenium for 21 days to determine whether there were species differences in their metabolic response. Cats and dogs exhibited the same pattern of response, however cats showed higher plasma selenium levels, lower levels in liver and excreted more selenium compared to dogs. It was concluded from this data that cats and dogs differ in their metabolism of selenium. The effect of heat processing on the addition of inorganic and organic selenium to petfoods was investigated in cats fed 3.0 µg Se/g DM for 11 days. Apparent absorption was higher in cats fed inorganic selenium added after processing, whilst less selenium of organic origin was excreted in the urine when added after processing. These preliminary results suggest heat processing may decrease the apparent availability and utilisation of selenium in petfoods.

Made available in DSpace on 2015-03-26T13:39:42Z (GMT). No. of bitstreams: 1 texto completo.pdf: 2024686 bytes, checksum: 927a0cecb4bcec1327a0e0c20f95445e (MD5) Previous issue date: 2011-07-08 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Somatic embryogenesis is the process of development of embryos from somatic cells. The aim of this study was to evaluate the efficiency of growth regulators at different stages of somatic embryogenesis of three cultivars of sugarcane, RB 867515, RB928064 e RB925345, and characterize the effects of in vitro treatments of the anatomical development of somatic embryos. Embryogenic calluses were obtained after inoculation of immature leaves arising from lateral buds on MS medium plus 30g.L sucrose, 2.5 g.L agar and different concentrations of 2,4-D, Dicamba and CPA (5,10,15 and 20 mM). After 30 days of induction it showed that the dose of 20μM 2, 4-D was the concentration that induced the highest percentage of callus, 36.95%, and callus were compact and whitish. The calluses were subcultured in the same medium for 30 days and after this period, the callus were transferred to medium with low concentration of 2,4-D (0.5 mg.L-1) to obtain globular pro-embryos. The globular pro-embryos were transferred to multiplication medium and after 30 days were transferred to maturation medium. osmotic regulators were tested, mannitol and sucrose (30, 60 and 90 gL) and ABA (0 and 5 mM). The embryos were transferred to regeneration medium containing 1 mg. L-1 BAP and GA3. The embryos that were in the medium with 30g. L-1, with ABA when transferred to regeneration medium formed healthy and vigorous plants. The histochemical Blue Evans test and Carmine Acetic confirmed the embryogenic of the material. The anatomical study of the process of somatic embryogenesis in leaf explants of sugar cane showed the emergence of callus from the epidermis, which in more advanced stages (globular embryos) easily detached from the explant. It was evident the presence of a well-defined protoderms, isodiametric cells with prominent nuclei and nucleoli and high nucleus / cytoplasm ratio. In the later stages were observed the basal and apical meristem well defined, bounded by a protoderm and immersed in a mass of vacuolated cells, assuming one is in the process of somatic embryo formation. At the end of 30 days in maturation, it was observed a bipolar structure, consisting of stem and root apices, which had a closed vascular system, without connection to the adjacent tissue, confirming the presence of the somatic embryo. / A embriogênese somática é o processo de desenvolvimento de embriões a partir de células somáticas. O objetivo desse trabalho foi avaliar a eficiência de reguladores de crescimento nas diferentes fases da embriogênese somática de três cultivares de cana-de- açúcar, RB 867515, RB928064 e RB925345, e caracterizar os efeitos dos tratamentos in vitro no desenvolvimento anatômico dos embriões somáticos. Foram obtidos calos embriogênicos após inoculação de folhas imaturas advindas de gemas laterais em meio MS acrescido de 30g.L de sacarose, 2,5g.L de ágar e diferentes concentrações de 2,4-D, Dicamba e CPA (5,10,15 e 20 μM). Aos 30 dias após indução verificou-se que a dose de 20μM de 2,4-D foi a dose que induziu maior porcentagem de calos embriogênicos, 36,95%, sendo que estes calos tinham aspecto compacto e eram de coloração esbranquiçada. Os calos foram subcultivados no mesmo meio por mais 30 dias e após esse período, os calos embriogênicos obtidos foram transferidos para meio com baixa concentração de 2,4-D (2,26μM) para obtenção de embriões globulares. Os embriões globulares foram transferidos para meio de multiplicação composto do memso meio de indução, acrescido de três diferentes doses de AIA (6, 12 e 24 μL/placa) e foi observada diferença significativa entre as variedades testadas, sendo que na maior dose ocorreu maior multiplicação de calos embriogênicos. Após 30 dias foram transferidos para meio de maturação, onde foram testados os reguladores osmóticos, manitol e sacarose (30, 60 e 90 g.L) e o ABA (0 e 5 μM). Os embriões viáveis foram transferidos para meio de regeneração, contendo 1mg. L-1 de BAP e GA3. Os embriões somáticos que estavam em meio com 30g. L-1, com ABA quando transferidos para meio de regeneração formaram plântulas sadias e vigorosas. O teste histoquímico azul de Evans e carmim acético confirmaram a natureza embriogênica do material. O estudo anatômico do processo de embriogênese somática em explantes foliares de cana-de-açúcar evidenciou o surgimento de calos embriogênicos a partir da epiderme, que em estágios mais avançados (embriões globulares) se desprendiam facilmente do explante. Era evidente a presença de uma protoderme bem definida, células isodiamétricas, com núcleo e nucléolo proeminentes e alta relação núcleo/citoplasma. Nos estágios posteriores foi possível observar uma estrutura apresentando meristema apical e basal bem definido, delimitada por uma protoderme e imersa em uma massa de células vacuolizadas, supondo ser um embrião somático em processo de formação. Ao final dos 30 dias em meio de maturação foi observada uma estrutura bipolar, constituída de ápices caulinar e radicular, que apresentava um sistema vascular fechado, sem conexão com os tecidos adjacentes, confirmando ser um embrião somático.

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