Дисертації з теми "Bioenergetics"

This research focuses on the development, evaluation, and application of a mercury (Hg) mass balance model for predicting the accumulation of Hg in fish. This model requires accurate estimates of Hg elimination rate by fish and feeding rates to adequately predict Hg concentration in fish. An empirical model was developed to estimate Hg elimination by fish using data obtained from published experiments. This analysis showed that Hg elimination rate was overestimated in short-term experiments, positively correlated to water temperature, negatively correlated to body size, and that the elimination rate of inorganic Hg was faster than that of methylmercury. This empirical model was then incorporated in a Hg mass balance model to predict the concentration of Hg in fish. The Hg mass balance model accurately predicted Hg concentration in fish when it was combined with food consumption rates that were determined using a radioisotopic method. This analysis suggested that the parameters of the Hg mass balance model were adequate for predicting Hg concentration in fish. I also showed that Hg concentration tended to be underestimated by the Hg mass balance model when it was combined with feeding rates determined with a laboratory-derived bioenergetic model, probably because activity costs derived in the laboratory do not reflect activity costs of fish in the field. Beside predicting Hg concentration in fish, I showed that this mass balance model could also be used to estimate feeding rates of fish in the field by measuring the concentration of Hg in fish. This approach was validated using data obtained from a published experiment. It was also successfully tested using independent estimates of feeding rates obtained with a radioisotopic method. I applied this Hg mass balance model to compare the energy budget of sympatric populations of dwarf and normal whitefish (Coregonus clupeaformis). This analysis showed that dwarf whitefish consumed 40--50% more food than normal whitefi

Non-canonical bioenergetics concerns with those physiological and pathophysiological situations under which ATP synthesis is suppressed. This thesis brings an outcome of three types of studies within the field of the non-canonical bioenergetics, investigating specific bioenergetic phenotypes of cancer cells, on one hand; and a role of mitochondrial uncoupling proteins as deduced from their transcript distribution in various tissues and organs; plus a role of a novel and likely pro-apoptotic factor CIDEa in mitochondria. Cancer cells generally present abnormal bioenergetic properties including an elevated glucose uptake, a high glycolysis and a poorly efficient oxidative phosphorylation system. However, the determinants of cancer cells metabolic reprogramming remain unknown. The main question in this project was how environmental conditions in vivo can influence functioning of mitochondrial OXPHOS, because details of mitochondrial bioenergetics of cancer cells is poorly documented. We have combined two conditions, namely glucose and oxygen deprivation, to measure their potential interaction. We examined the impact of glucose deprivation and oxygen deprivation on cell survival, overall bioenergetics and OXPHOS protein expression. As a model, we have chosen a human breast carcinoma (HTB-126) and appropriate control (HTB-125) cultured cells, as large fraction of breast malignancies exhibit hypoxic tumor regions with low oxygen concentrations and poor glucose delivery. The results demonstrate that glucose presence or absence largely influence functioning of mitochochondrial oxidative phosphorylation. The level of mitochondrial respiration capacity is regulated by glucose; by Crabtree effect, by energy substrate channeling towards anabolic pathways that support cell growth and by mitochondrial biogenesis pathways. Both oxygen deprivation and glucose deprivation can remodel the OXPHOS system, albeit in opposite directions. As an adaptative response to hypoxia, glucose inhibits mitochondrial oxidative phosphorylation to the larger extent than in normoxia. We concluded that the energy profile of cancer cells can be determined by specific balance between two main environmental stresses, glucose and oxygen deprivation. Thus, variability of intratumoral environment might explain the variability of cancer cells´ bioenergetic profile. Mitochondrial uncoupling proteins are proteins of inner mitochondrial membrane that uncouple respiration from ATP synthesis by their protonophoric activity. Originally determined tissue distribution seems to be invalid, since novel findings show that UCP1 is not restricted exclusively to brown fat and that originally considered brain-specific isoforms UCP4 and UCP5 might have wider tissue distribution. Hence, in second part of this thesis, I discuss consequences of findings of UCPn transcripts in the studied mouse and rat tissues. We have shown that mRNA of UCPn varies up to four orders of magnitude in rat and mouse tissues with highest expression in rat spleen, rat and mouse lung, and rat heart. Levels of the same order of magnitude were found for UCP3 mRNA in rat 100 and mouse skeletal muscle, for UCP4 and UCP5 mRNA in mouse brain, and for UCP2 and UCP5 mRNA in mouse white adipose tissue. Further, we have shown that expression pattern of UCPn varies between animal species, rat versus mouse, such as the dominance of UCP3/UCP5 vs. UCP2 transcript in mouse heart and vice versa in rat heart; or UCP2 (UCP5) dominance in rat brain contrary to 10-fold higher UCP4 and UCP5 dominance in mouse brain. spontaneous apoptosis due to CIDEa overexpression in HeLa cells, adapted for a tetracycline-inducible CIDEa expression, a portion of mitochondria-localized CIDEa molecules migrates to cytosol or nucleus
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While it is clear that thermodynamics plays a nontrivial role in biological processes, exactly how this affects the macroscopic structuring of living systems is not fully understood. Thus, the objective of this dissertation was to investigate how thermodynamic variables such as exergy, entropy, and information are involved in biological processes such as cellular metabolism, ecological succession, and evolution. To this end, I have used a combination of mathematical modelling, in silico simulation, and both laboratory- and field-based experimentation.

To begin the dissertation, I review the basic tenets of biological thermodynamics and synthesize them with modern fluctuation theory, information theory, and finite time thermodynamics. In this review, I develop hypotheses concerning how entropy production rate changes across various time scales and exergy inputs. To begin testing these hypotheses I utilized a stochastic, agent-based, mathematical model of ecological evolution, The Tangled Nature Model. This model allows one to observe the dynamics of entropy production over time scales that would not be possible in real biological systems (i.e., 10⁶ generations). The results of the model’s simulations demonstrate that the ecological communities generated by the model’s dynamics have increasing entropies, and that this leads to emergent order, organization, and complexity over time. To continue to examine the role of thermodynamics in biological processes I investigated the bioenergetics of marine microbes associated with benthic substrates on coral reefs. By utilizing both mesocosm and in situ experiments I have shown that these microbes change their power output, oxygen uptake, and community structure depending upon their available exergy.

Overall, the data presented herein demonstrates that ecological structuring and evolutionary change are, at least in part, determined by underlying thermodynamic mechanisms. Recognizing how physical processes affect biological dynamics allows for a more holistic understanding of biology at all scales from biochemistry, to ecological succession, and even long-term evolutionary change.

Each year the Organ Procurement and Transplantation Network (OPTN) reports an increase in patients requiring an organ transplant without an increase in available donor organs, leading to a transplant gap that continues to widen. Over 70% of donor hearts are deemed unsuitable for transplantation each year, and a large number of these organs (~50%) are discarded due to poor organ function, decreased ejection fraction, disease, or cardiac arrest (Scientific Registry of Transplant Recipients (SRTR) Annual Data Report 2011).We therefore set out to improve knowledge in the field of cardiac transplantation in terms of organogenesis, bioenergetics, and regeneration. The main goal through tissue bioengineering is to regenerate and salvage discarded hearts through organogenesis, or to lengthen the total organ preservation time such that organs would not be thrown away while a recipient was waiting to be found. Our first hypothesis was that an optimized acellular extracellular matrix scaffold would allow for cell adherence, growth and proliferation, and could potentially be grown into a clinically transplantable organ. To achieve these goals, an optimized protocol was developed for the total acellularization of a whole porcine heart, leaving behind a 3D bioscaffold. We showed that acellularized matrices could be successfully seeded using endothelial cells for acellular vasculature and stem cells for other acellular tissues, both as a 2D matrix and within a constantly perfused 3D Langendorff setup bioreactor. In order to best understand cell-cell and cell-matrix interactions, cellular bioenergetics were evaluated. We hypothesized that the bioenergetic demand of the type and anatomical origin of stem cells would affect the regeneration potential dependent on intrinsic metabolic demand. We therefore showed a differential of the bioenergetic profiles of human adipose-derived stem cells isolated from various adipose depots, concluding that the physiological microenvironment that supports stem cells in specific anatomic locations can regulate how stem cells participate in tissue regeneration, maintenance and repair, and also will vary based on donor-differences. During organ transplantation, organ preservation solutions are created for use at specific conditions, such as on ice or at room temperature. We hypothesized that hypothermia would slow down cellular metabolism, and that solutions containing a higher content of antioxidants and other protective substrates against ischemic reperfusion injury would create the best organ storage conditions. We tested three organ preservation solutions against control media and normal saline at 4 and 21 degrees C, for 4 to 8 hours, investigating the bioenergetics of organ preservation solution effects on cardiac cells. By simulating clinical conditions, we were able to determine that one of our solutions was ideal and had protective effects for cells for up to 8 hours at 4 degrees C. Finally, we believed that studying existing cardiac patches and optimizing cardiac matrix design would lead to improved cardiac physiological function and would aid in healing and repair during cardiac surgery. Following a clinical case report showing new cardiac tissue growth after implantation of an acellular porcine extracellular matrix, we devised a proof-of-concept study to show that clinical matrices could be easily cultured in vitro. We successfully seeded these clinical matrices using human amniotic stem cells, a commonly used cell type for regeneration and repair after surgery. Our preliminary studies suggest that preconditioned matrices can be potentially used clinically for greater efficacy and tissue regeneration.

Thesis: Ph. D. in Biochemistry, Massachusetts Institute of Technology, Department of Biology, February, 2021
Cataloged from the official PDF of thesis. "February 2021." Vita. Page 179 blank.
Includes bibliographical references.
Cellular growth and proliferation necessitates the transformation of cell-external nutrients into biomass. Strategies of biomass accumulation across the kingdoms of life are diverse and range from carbon fixation by autotrophic organisms to direct biomass incorporation of consumed nutrients by heterotrophic organisms. The goal of this dissertation is to better understand the divergent and convergent modes of metabolism that support biomass accumulation and proliferation in eukaryotic cells. We first determined that the underlying mechanism behind why rapidly proliferating cells preferentially ferment the terminal glycolytic product pyruvate is due to an intrinsic deficiency of respiration to regenerate electron acceptors. We tested this model across an assorted array of proliferating cells and organisms ranging from human cancer cells to the baker's yeast Saccharomyces cerevesiae. We next determined that a major metabolic pathway of avid electron acceptor consumption in the context of biomass accumulation is the synthesis of lipids. Insights from this work has led to the realization that net-reductive pathways such as lipid synthesis may be rate-limited by oxidative reactions. Lastly, we established the green algae Chlorella vulgaris as a model system to study the comparative metabolism of photoautotrophic and heterotrophic growth. We determined that heterotrophic growth of plant cells is associated with aerobic glycolysis in a mechanism that may be suppressed by light. Collectively, these studies contribute to a more holistic understanding of the bioenergetics and metabolic pathways employed by eukaryotic cells to accumulate biomass and lay the foundation for future studies to understand proliferative metabolism.
by Zhaoqi Li.
Ph. D. in Biochemistry
Ph.D.inBiochemistry Massachusetts Institute of Technology, Department of Biology

It has been hypothesised that endogenous testosterone and AAS may predispose humans to premature CHD. However, there is no direct evidence to link these hormones with a greater prevalence of premature CHD. The aim of this thesis was to better describe atherosclerotic risk associated with these hormones by clarifying their effect on additional risk factors for premature atherosclerosis. Little is known about the effect of testosterone and AAS on 'atherogenic dyslipidaemia', a phenotype characterised by elevated postprandial triglyceridaemia, small dense LDL and a low HDLC concentration, which confers a high risk of CHD. Accordingly, the magnitude of postprandial triglyceridaemia, LDL and HDL particle size, and LDLC, HDLC and Lp(a) concentration were compared in male (n=9) and female (n=3) bodybuilders after self administration of AAS for 5-6 weeks (ON cycle) and again after a 4-6 week 'washout' period (OFF cycle), and in normal males (T) (n=10) before and during a reversible suppression of endogenous testosterone, induced using a GnRH agonist (triptorelin), and in a control group (C) (n=8). Lipoprotein size was assessed by gradient gel electrophoresis (GGE), lipoprotein concentrations by immuno and enzymatic assay, and postprandial triglyceridaemia by a standardised oral fat tolerance test (65g/m² ). HDLC decreased in male bodybuilders (0.94±0.30 vs 0.70±0.27 mmol/L, p=0.004; x ± SD) and female bodybuilders (1.3±0.5 vs 0.8±0.2 mmol/L) ON cycle. GGE studies suggested that mostly HDL₂ was reduced. There were no significant reductions in LDL particle size ON cycle. Two males had larger LDL species ON cycle. Lp(a) decreased in male bodybuilders (124.7±128.0 to 69.3±73.3 U/L, p=0.008). ON cycle postprandial triglyceride excursion was unchanged in female bodybuilders and reduced (11.6±10.0 vs 7.5±5.4 mmol/L.hr; p=0.027) in male bodybuilders. In the triptorelin study, HDLC was increased in T (1.07±0.18 vs 1.41±0.28 mmol/L, p=0.002) and not in C. GGE studies indicated an increase of HDL₂ in five T subjects and no increase in C. Total cholesterol increased in T (4.77±0.80 vs 5.24±1.04 mmol/L, p=0.039) but not in C. LDL size increased in four T subjects, and not in C. Lp(a) increased in T (277.9±149.l vs 376.5±222.2 U/L, p=0.004), but not in C. Postprandial triglyceridaemia was unchanged in both T and C. The results of these studies did not show any additional atherogenic effects of endogenous testosterone or AAS in humans. Rather, a suppression of Lp(a) may be an antiatherogenic effect of these hormones. A reduced postprandial triglyceridaemia and increased LDL size in individuals who are predisposed to 'atherogenic dyslipidaemia', may be further antiatherogenic effects of AAS use.

Bilirubin is a haem catabolite that is excreted through the hepatobiliary pathway and is therefore, commonly used as a biomarker of hepatic dysfunction and haemolysis in the clinical setting [1]. Although, bilirubin has been considered toxic [2], recent evidence suggests that mildly elevated circulating bilirubin concentrations may be protective against obesity, cardiovascular diseases (CVDs) and all-cause mortality [3–5]. Generally, the protective effects of bilirubin are attributed to its antioxidant potential [6–8], however, recent studies demonstrate that bilirubin modulates lipid metabolism and reduces adiposity, which could partly contribute to CVD protection [5,9–12]. However, a shortage of studies have examined the precise mechanisms of cholesterol metabolism and adiposity that could be affected by bilirubin. The main aims of this thesis were to: 1) determine whether hyperbilirubinaemia affects cholesterol synthesis, transport, and excretion; 2) explore bilirubin’s impact on body composition and bioenergetics including mitochondrial function in liver/skeletal muscle and changes in mitochondrial density and quality; 3) determine the effectiveness of oral Legalon® ingestion on circulating bilirubin concentrations, to investigate whether inducing mild hyperbilirubinaemia could impact circulating lipid concentrations in human participants. The first study measured the effect of hyperbilirubinaemia in mutant Gunn rats on circulating lipid concentrations, cholesterol synthesis, lipid excretion, and expression of hepatic genes/proteins involved in cholesterol metabolism. Female hyperbilirubinaemic (Gunn) rats had reduced serum cholesterol concentrations (0.60 ± 0.12 vs 1.56 ± 0.34 mM, P<0.001), elevated cholesterol synthesis (33.8 ± 3.77 vs 28.4 ± 5.73 % [13C]-cholesterol, P<0.05), enhanced LDL receptor (LDLr; P<0.01) expression, and increased biliary cholesterol excretion (232 ± 32.7 vs 141 ± 42.1 nmol hr-1 100g-1 bodyweight, P<0.001) compared to female normobilirubinaemic littermate (control) rats. These results indicate that female hyperbilirubinaemic Gunn rats have reduced circulating cholesterol in association with elevated LDLr expression. Increased LDLr expression and cholesterol synthesis is typical when hepatic cholesterol concentrations are decreased [13,14]. Therefore, increased cholesterol synthesis and LDLr expression observed in female Gunn rats may represent a counter-regulatory mechanism to maintain hepatic cholesterol content in the presence of elevated biliary cholesterol excretion [13,14]. The underlying mechanism explaining increased biliary lipid excretion in female Gunn rats remains unknown. However, this observation could be partly explained by greater relative biliary lipid (cholesterol+phospholipid) to bile acid excretion (0.33 ± 0.06 vs 0.24 ± 0.03 mol:mol, lipid:bile acids, P<0.01) in female Gunn rats. Previous studies have established that organic anions including bilirubin glucuronides disrupt the capacity of bile acid micelles to excrete lipids in the bile [15]. Biliary excretion of bilirubin conjugates was decreased in female (13.1 ± 2.92 vs 33.5 ± 5.09 nmol hr-1 100g-1 bodyweight, P<0.001) and male (11.0 ± 2.43 vs 43.2 ± 12.8 nmol hr-1 100g-1 bodyweight, P<0.001) Gunn rats compared to controls, due to UGT1A1 dysfunction and the inability to conjugate bilirubin. Therefore, decreased biliary excretion of bilirubin conjugates, as observed in Gunn rats, may potentially facilitate the greater coupled excretion of biliary lipids to bile acids as demonstrated in this study. It should be noted that this conclusion does not completely explain the results reported here because Gunn rats demonstrated significant sexual dimorphism in cholesterol metabolism. Male Gunn rats exhibited a non-significant reduction in circulating cholesterol concentrations (1.41 ± 0.15 vs 1.56 ± 0.23, P=0.14) and increased biliary lipid:bile acid excretion (0.31 ± 0.07 vs 0.25 ± 0.04 mol:mol, lipid:bile acid, P=0.08) compared to male normobilirubinaemic littermate (control) rats, indicating that additional mechanisms, beyond bilirubin excretion, are involved. For example, UGT1A1, which conjugates bilirubin also conjugates and facilitates the excretion of sex hormones including oestrogen. Therefore, oestrogen concentrations may be elevated in female hyperbilirubinaemic rats and synergistically impact lipid metabolism [16,17]. The second study examined the effect of hyperbilirubinaemia in vitro and in vivo on mitochondrial function and body composition. Dual X-ray absorptiometry (DEXA) analysis revealed that female Gunn rats had significantly reduced fat mass (9.94 ± 5.35 vs 16.1 ± 6.65 g, P<0.05) and lean mass (140 ± 12.1 vs 160 ± 16.0 g, P<0.05) compared to littermate controls. Female Gunn rats consumed fewer calories per day (54.1 ± 6.38 vs 63.3 ± 6.95 kcal day-1, P<0.01). However, weight gain relative to calories consumed was reduced (8.09 ± 5.75 vs 14.9 ± 5.10 mg kcal-1, P<0.05) in female Gunn rats indicating that they are less energetically efficient. This led to the analysis of mitochondrial function in liver and skeletal muscle using high-resolution respirometry to ascertain the cause of reduced energetic efficiency. This analysis revealed that female Gunn rats exhibited increased oxidative phosphorylation (OXPHOS) relative to maximal noncoupled mitochondrial respiration (ETS) in hepatic mitochondria (0.78 ± 0.16 vs 0.62 ± 0.09 OXPHOS:ETS, P<0.05). The above findings were consistent with the effect of exogenous addition of unconjugated bilirubin (UCB) to control hepatic mitochondria, with 31.3 and 62.5 μM UCB increasing the OXPHOS: ETS ratio. However, exogenous UCB addition produced this effect by inhibiting ETS without affecting OXPHOS, indicating that UCB induces mitochondrial dysfunction at high concentrations. Conversely, no change in ETS (1130 ± 217 vs 1290 ± 373 pmol s-1 ng-1 citrate synthase (CS), P=0.16) or OXPHOS (901 ± 222 vs 796 ± 259 pmol s-1 ng-1 CS, P=0.36) was observed between female Gunn rats and controls. These data indicate that the greater OXPHOS:ETS ratios are a combination of increased OXPHOS and decreased ETS in female Gunn rats. Analysis of mitochondrial respiratory complexes revealed greater hepatic mitochondrial complex IV (CIV; P<0.01) expression in female Gunn rats. These findings support a conclusion that hepatic mitochondria have increased quality in female Gunn rats [18,19]. At present it remains unknown how this change in mitochondrial quality relates to reduced fat mass and energetic efficiency, however, the changes observed in female Gunn rats could represent an adaptation to bilirubin mediated inhibition of CIV as reported in vitro [20,21]. Otherwise, alterations in reproductive hormone metabolism in Gunn rats could also partially explain altered energetic states, as speculated in study one. Considering that hyperbilirubinaemia induced perturbed lipid metabolism and body composition in chapters one and two, study three sought to determine whether increasing bilirubin could alter circulating lipid profile in humans. The effect of Legalon®, containing the active ingredient silymarin, supplementation on circulating bilirubin concentrations and lipid status was investigated in a placebo controlled, single blind crossover clinical trial in healthy individuals (ACTRN12619001296123). Legalon® capsules containing 140 mg of silymarin were supplemented thrice daily (total dose of 420 mg silymarin) in a cohort of healthy males for two weeks. Two weeks of Legalon® supplementation did not change UCB concentrations compared to baseline (Legalon®: 12.5 ± 7.63 vs Baseline: 11.4 ± 4.14 μM, P=0.79). Secondary outcomes including lipid concentrations, inflammation, and total antioxidant status were also reported. Two weeks of Legalon® supplementation did not change serum cholesterol (4.80 ± 1.00 vs 4.88 ± 1.00 mM, P=0.19), triglyceride (1.07 ± 0.63 vs 1.04 ± 0.54 mM, P=0.79), C-reactive protein concentrations (1.74 ± 1.88 vs 0.92 ± 0.87 mg L-1, P=0.23) or serum antioxidant capacity (1194 ± 182 vs 1183 ± 201 mmol Fe2+ L-1, P=0.19) compared to baseline. Several clinical trials evaluating the impact of silymarin have reported changes to bilirubin concentrations following treatment [22–24]. However, these studies were conducted in patients with hepatic disease, which confounded bilirubin results, and with greater doses or different formulations of silymarin to that reported in this thesis. Although the results of this study demonstrated a negative finding, they are important because they represent the first attempt to use an orally administered, commercially approved, nutraceutical compound to increase bilirubin. These results provide important guidance to future studies that could utilise different doses or commercial preparations to induce a transient unconjugated hyperbilirubinaemia and test the impact on circulating cholesterol concentrations. In conclusion, this thesis contains three novel investigations that aimed to determine the impact of unconjugated hyperbilirubinaemia on cholesterol metabolism, synthesis and hepatic excretion; in addition to its effect on mitochondrial metabolism and body composition in Gunn rats. To determine whether these effects could be induced in humans, a nutraceutical with documented effects on circulating bilirubin was administered in a clinical trial, utilising a randomised, single-blind, crossover design. The results of this thesis suggest that bilirubin has the potential to modulate lipid and whole-body metabolism, particularly in female animals and provides the groundwork for additional studies that seek to reveal the mechanisms responsible for bilirubin’s effects. In addition, this thesis will support the discovery of more effective orally administered compounds that can modulate circulating bilirubin and lipid profile for protection against CVD.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medical Science
Griffith Health
Full Text

Mitochondria have a central role in energy supply in cells, ROS production and apoptosis and have been implicated in several human disease and mitochondrial dysfunctions in hypoxia have been related with disorders like Type II Diabetes, Alzheimer Disease, inflammation, cancer and ischemia/reperfusion in heart. When oxygen availability becomes limiting in cells, mitochondrial functions are modulated to allow biologic adaptation. Cells exposed to a reduced oxygen concentration readily respond by adaptive mechanisms to maintain the physiological ATP/ADP ratio, essential for their functions and survival. In the beginning, the AMP-activated protein kinase (AMPK) pathway is activated, but the responsiveness to prolonged hypoxia requires the stimulation of hypoxia-inducible factors (HIFs). In this work we report a study of the mitochondrial bioenergetics of primary cells exposed to a prolonged hypoxic period . To shine light on this issue we examined the bioenergetics of fibroblast mitochondria cultured in hypoxic atmospheres (1% O2) for 72 hours. Here we report on the mitochondrial organization in cells and on their contribution to the cellular energy state. Our results indicate that prolonged hypoxia cause a significant reduction of mitochondrial mass and of the quantity of the oxidative phosphorylation complexes. Hypoxia is also responsible to damage mitochondrial complexes as shown after normalization versus citrate synthase activity. HIF-1α plays a pivotal role in wound healing, and its expression in the multistage process of normal wound healing has been well characterized, it is necessary for cell motility, expression of angiogenic growth factor and recruitment of endothelial progenitor cells. We studied hypoxia in the pathological status of diabetes and complications of diabetes and we evaluated the combined effect of hyperglycemia and hypoxia on human dermal fibroblasts (HDFs) and human dermal micro-vascular endothelial cells (HDMECs) that were grown in high glucose, low glucose concentrations and mannitol as control for the osmotic challenge.
I mitocondri hanno un ruolo fondamentale nella produzione di energia nella cellula, ma sono coinvolti anche in altri processi tra cui la produzione di ROS e l’apoptosi. Disfunzioni del metabolismo mitocondriale sono state associate a diversi disordini, tra cui: diabete di tipo II, malattia si Alzheimer, infiammazione, cancro ed ischemia cardiaca. Quando i livelli di ossigeno nella cellula diventano limitanti, la funzione mitocondriale viene modulata per consentire l’adattamento biologico. La via dell’AMP- activated protein kinase (AMPK) ha il compito di monitorare lo stato energetico della cellula mantenendo i livelli fisioligici di ATP/ADP. In seguito all’esposizione prolungata in ambiente ipossico, l’attivazione di HIF-1 e’ in grado di upregolare diversi geni coinvolti nella sopravvivenza cellulare a basse concentrazioni di ossigeno. In questo lavoro, e’ stata valutata la bioenergetica mitocondriale in fibroblasti primari coltivati a basse concentrazioni di ossigeno (1 % O2) per 72 ore; in particolare, abbiamo preso in considerazione l’organizzazione mitocondriale nella cellula e il loro contributo nel mantenere lo stato energetico cellulare. I nostri risultati indicano che l’esposizione prolungata all’ipossia causa una significativa riduzione della massa mitocondriale e della quantita’ dei complessi della fosforilazione ossidativa, nonostante le cellule siano in grado di mantenere i livelli intracellulari di ATP. Inoltre abbiamo studiato l’ipossia nel contesto patologico del diabete ed in particolare delle complicanze del diabete. E’ noto che l’iperglicemia e l’ipossia, dovuta ad ischemia a danni vascolari, hanno un ruolo importante nell’insorgenza delle complicanze del diabete. HIF-1α rappresenta uno stimolo nella rigenerazione delle ferite, in quanto stimola la vascolarizzazione e la migrazione dei cheranociti ed e’ stato ipotizzato che le cellule perdano la capacita’ di adattarsi e rispondere all’ipossia quando sono coltivate in presenza di elevate concentrazioni di glucosio (>25 mM). Abbiamo valutato il ruolo della destabilizzazione di HIF-1α nella produzione di ROS, considerati i principali responsabili della progressione del diabete.

Mitochondria have a central role in energy supply in cells, ROS production and apoptosis and have been implicated in several human disease and mitochondrial dysfunctions in hypoxia have been related with disorders like Type II Diabetes, Alzheimer Disease, inflammation, cancer and ischemia/reperfusion in heart. When oxygen availability becomes limiting in cells, mitochondrial functions are modulated to allow biologic adaptation. Cells exposed to a reduced oxygen concentration readily respond by adaptive mechanisms to maintain the physiological ATP/ADP ratio, essential for their functions and survival. In the beginning, the AMP-activated protein kinase (AMPK) pathway is activated, but the responsiveness to prolonged hypoxia requires the stimulation of hypoxia-inducible factors (HIFs). In this work we report a study of the mitochondrial bioenergetics of primary cells exposed to a prolonged hypoxic period . To shine light on this issue we examined the bioenergetics of fibroblast mitochondria cultured in hypoxic atmospheres (1% O2) for 72 hours. Here we report on the mitochondrial organization in cells and on their contribution to the cellular energy state. Our results indicate that prolonged hypoxia cause a significant reduction of mitochondrial mass and of the quantity of the oxidative phosphorylation complexes. Hypoxia is also responsible to damage mitochondrial complexes as shown after normalization versus citrate synthase activity. HIF-1α plays a pivotal role in wound healing, and its expression in the multistage process of normal wound healing has been well characterized, it is necessary for cell motility, expression of angiogenic growth factor and recruitment of endothelial progenitor cells. We studied hypoxia in the pathological status of diabetes and complications of diabetes and we evaluated the combined effect of hyperglycemia and hypoxia on human dermal fibroblasts (HDFs) and human dermal micro-vascular endothelial cells (HDMECs) that were grown in high glucose, low glucose concentrations and mannitol as control for the osmotic challenge.
I mitocondri hanno un ruolo fondamentale nella produzione di energia nella cellula, ma sono coinvolti anche in altri processi tra cui la produzione di ROS e l’apoptosi. Disfunzioni del metabolismo mitocondriale sono state associate a diversi disordini, tra cui: diabete di tipo II, malattia si Alzheimer, infiammazione, cancro ed ischemia cardiaca. Quando i livelli di ossigeno nella cellula diventano limitanti, la funzione mitocondriale viene modulata per consentire l’adattamento biologico. La via dell’AMP- activated protein kinase (AMPK) ha il compito di monitorare lo stato energetico della cellula mantenendo i livelli fisioligici di ATP/ADP. In seguito all’esposizione prolungata in ambiente ipossico, l’attivazione di HIF-1 e’ in grado di upregolare diversi geni coinvolti nella sopravvivenza cellulare a basse concentrazioni di ossigeno. In questo lavoro, e’ stata valutata la bioenergetica mitocondriale in fibroblasti primari coltivati a basse concentrazioni di ossigeno (1 % O2) per 72 ore; in particolare, abbiamo preso in considerazione l’organizzazione mitocondriale nella cellula e il loro contributo nel mantenere lo stato energetico cellulare. I nostri risultati indicano che l’esposizione prolungata all’ipossia causa una significativa riduzione della massa mitocondriale e della quantita’ dei complessi della fosforilazione ossidativa, nonostante le cellule siano in grado di mantenere i livelli intracellulari di ATP. Inoltre abbiamo studiato l’ipossia nel contesto patologico del diabete ed in particolare delle complicanze del diabete. E’ noto che l’iperglicemia e l’ipossia, dovuta ad ischemia a danni vascolari, hanno un ruolo importante nell’insorgenza delle complicanze del diabete. HIF-1α rappresenta uno stimolo nella rigenerazione delle ferite, in quanto stimola la vascolarizzazione e la migrazione dei cheranociti ed e’ stato ipotizzato che le cellule perdano la capacita’ di adattarsi e rispondere all’ipossia quando sono coltivate in presenza di elevate concentrazioni di glucosio (>25 mM). Abbiamo valutato il ruolo della destabilizzazione di HIF-1α nella produzione di ROS, considerati i principali responsabili della progressione del diabete.

Aquest és el primer informe que demostra que l'autofàgia està mecànicament vinculat al manteniment de les cèl•lules tumorals que expressen alts nivells de CD44 i baixos nivells de CD24, que són típics de les cèl•lules mare del càncer de mama. Els nostres resultats actuals proporcionen una nova visió de com la divisió mitocondrial s'integra a la xarxa de la transcripció impulsada per factors de reprogramació, especifica de la pluripotència única de les cèl•lules mare. L'autofàgia pot controlar la refractarietat de novo de carcinomes de mama amb el gen HER2 amplificat per l'anticòs monoclonal trastuzumab (Herceptin). Per tant, el tractament de combinació amb trastuzumab i cloroquina, com a fàrmac anti-malàric i inhibidor de l’autofàgia, suprimeix radicalment el creixement del tumor en un xenoempelt de tumor completament refractari a trastuzumab en un model murí. L’addició de cloroquina amb els règims amb trastuzumab pot, per tant, millorar els resultats en les dones amb càncer de mama HER2. Aquesta és una àrea molt emocionant i molt prometedora de la investigació del càncer, com la modulació farmacològica de l'autofàgia sembla augmentar l'eficàcia dels règims contra el càncer disponibles en l'actualitat i s'obre el camí per al desenvolupament de noves estratègies terapèutiques combinatòries que s'espera que contribueixin a l'eradicació del càncer.
This is the first report demonstrating that autophagy is mechanistically linked to the maintenance of tumor cells expressing high levels of CD44 and low levels of CD24, which are typical of breast cancer stem cells. Our current findings provide new insight into how mitochondrial division is integrated into the reprogramming of the factors-driven transcriptional network that specifies the unique pluripotency of stem cells. Autophagy may control the de novo refractoriness of HER2 gene-amplified breast carcinomas to the monoclonal antibody trastuzumab (Herceptin). Accordingly, treatment with trastuzumab and chloroquine, as antimalarial drug and inhibitor of autophagy, radically suppresses tumor growth in a tumor xenograft completely refractory to trastuzumab in a mouse model. Adding chloroquine to trastuzumab-based regimens may therefore improve outcomes among women with autophagy-addicted HER2-positive breast cancer. This is a very exciting and highly promising area of cancer research, as pharmacologic modulation of autophagy appears to augment the efficacy of currently available anticancer regimens and opens the way to the development of new combinatorial therapeutic strategies that will hopefully contribute to cancer eradication.

Ultraendurance mountain bike racing is a relatively new sport and has received scant research attention. The practical difficulty of field-testing during competition has played a role in this dearth of knowledge. The purpose of this thesis was to investigate the physiology and bioenergetics of cross-country marathon (XCM) and 24 hour team relay (24XCT) mountain bike racing. Study One analysed the physiological characteristics of XCM competitors and compared them to data from studies in the literature for Olympic-distance cross-country (XCO) mountain bike competitors. The XCM participants had lower mean peak aerobic capacity (58.4 ± 6.3 mL•kg-1•min-1), greater body mass (72.8 ± 6.7 kg) and estimated percentage body fat (10.4 ± 2.4%) compared to values reported for XCO competitors in the literature. Stature (1.77 ± 6.0 m) and normalised peak power output (5.5 ± 0.7 W•kg-1) were comparable. These data suggest that specific physiological characteristics of XCM competitors differ from those of XCO competitors. Study Two quantified and described the exercise intensity during a XCM race by monitoring heart rate responses. The mean heart rate (150 ± 10 beats•min-1) for the duration of the race equated to 82 percent of maximum heart rate and did not differ significantly throughout the race (p = 0.33). The data indicated that the XCM race was of a high aerobic intensity. Prior to the competition the relationship between heart rate and O2peak for each participant was established during an incremental laboratory test. Energy expenditure was estimated by assigning 20.2 kJ to each litre of oxygen consumed. The mean rate of energy expenditure during the race was estimated to be 59.9 kJ•min-1. Furthermore, no anthropometric or physiological measures were correlated to race speed, indicating that other factors contribute to race performance. The third study was a laboratory-based investigation to determine whether physiological factors relevant to 24XCT racing change with time of day. On separate days participants cycled on an ergometer for 20 min at 82 percent of maximum heart rate at 06:00, 12:00, 18:00, and 00:00 h. Significant differences (p < 0.05) were observed for several physiological responses (heart rate, oxygen uptake, salivary cortisol concentrations and intra-aural temperature) but not for performance variables (power output and self-selected cadence). It was concluded that the laboratory protocol lacked ecological validity and that it was necessary to test within a race using authentic 24XCT competitors. In order to measure in-race performance, Study Four examined the agreement between a bottom-bracket ambulatory ergometer (Ergomo®Pro) and the criterion SRM power meter in a field-based setting. Analysis of absolute limits of agreement found that the Ergomo®Pro had a systematic bias (± random error) of 4.9 W (± 6.12). Based on tolerances recommended in the literature the unit was considered fit for purpose for measuring power output during 24XCT racing. Study Five was a multiple case-study design that examined the physiological and performance parameters of a team during a 24XCT race. It was reported that mean work-shift speed (18.3 ± 2.6 km•h-1), power output (219 ± 50.9 W) and cadence (64.1 ± 9.3 rpm) were variable between participants and between work-shifts. A commonality amongst the participants was an increase in speed during the final work-shift compared to the penultimate one. A decline in work-shift heart rate was observed throughout the race. For the majority of participants an increase in gross efficiency (1.7 ± 1.4 %) was reported from the penultimate to the final work-shift. It was concluded that pacing strategies were employed and that the improved efficiency was caused, in part, by an increased familiarity with the course during the race. Study Six examined the nutritional practices and energy expenditure of the same team during the same 24XCT race. Energy expenditure during the work-shifts was estimated in accordance with Study Two. Resting energy expenditure during the recovery periods was estimated using the Harris and Benedict formula (1919). Food and fluid consumption were determined via food diaries and hydration status was assessed by measuring the refractive index of urine. Energy consumption (17.3 ± 2.2 MJ) was considerably less than energy expenditure (30.4 ± 6.1 MJ) with the former accounting for only 57 percent of the latter. The energy cost during the work-shifts was estimated to be 74.5 kJ•min-1. Mean fluid intake (6.3 ± 0.9 L) for the 24 h was sufficient to maintain hydration status. Based on these studies an integrated model of the factors that influence ultraendurance mountain bike performance was developed. The domains that influence race speed are physiological factors, technical and tactical factors, and nutritional strategies. The sub domain that influences these is environmental factors. Collectively this information is of practical importance to sport scientists, coaches and athletes involved with designing nutritional and tactical preparation strategies and training programmes for this sport.

Thesis (Sc. D.)--Harvard-MIT Division of Health Sciences and Technology, 2005.
Includes bibliographical references (p. 185-195).
Space-suited activity is critical for human spaceflight, and is synonymous with human planetary exploration. Space suits impose kinematic and kinetic boundary conditions that affect movement and locomotion, and in doing so modify the metabolic cost of physical activity. Metabolic requirements, found to be significantly elevated in space-suited activity, are a major driver of the allowable duration and intensity of extravehicular activity. To investigate how space suited locomotion impacts the energetics of walking and running, I developed a framework for analyzing energetics data, derived from basic thermodynamics, that clearly differentiates between muscle efficiency and energy recovery. The framework, when applied to unsuited locomotion, revealed that the human run-walk transition in Earth gravity occurs when energy recovery for walking and running are approximately equal. The dependence of muscle efficiency on gravity -during locomotion and under a particular set of assumptions- was derived as part of the framework. Next, I collected and transformed data from prior studies of suited and unsuited locomotion into a common format, and performed regression analysis. This analysis revealed that in reduced gravity environments, running in space suits is likely to be more efficient, per unit mass and per unit distance, than walking in space suits. Second, the results suggested that space suits may behave like springs during running. To investigate the spring-like nature of space suit legs, I built a lower-body exoskeleton to simulate aspects of the current NASA spacesuit, the Extravehicular Mobility Unit (EMU).
(cont.) Evaluation of the exoskeleton legs revealed that they produce knee torques similar to the EMU in both form and magnitude. Therefore, space suit joints such as the EMU knee joint behave like non-linear springs, with the effect of these springs most pronounced when locomotion requires large changes in knee flexion such as during running. To characterize the impact of space suit legs on the energetics of walking and running, I measured the energetic cost of locomotion with and without the lower-body exoskeleton in a variety of simulated gravitational environments at specific and self- selected Froude numbers, non-dimensional parameters used to characterize the run-walk transition. Exoskeleton locomotion increased energy recovery and significantly improved the efficiency of locomotion, per unit mass and per unit distance, in reduced gravity but not in Earth gravity. The framework was used to predict, based on Earth gravity data, the metabolic cost of unsuited locomotion in reduced gravity; there were no statistical differences between the predictions and the observed values. The results suggest that the optimal space-suit knee-joint torque may be non-zero: it may be possible to build a 'tuned space suit' that minimizes the energy cost of locomotion. Furthermore, the observed lowering of the self-selected run-walk transition Froude number during exoskeleton locomotion is consistent with the hypothesis that the run-walk transition is mediated by energy recovery. The major contributions of the dissertation include: 1. A model that predicts metabolic cost in non-dimensional form for unsuited locomotion across running and walking and across gravity levels, 2.
(cont.) An assessment of historical data that reveals the effect of pressure suits on work output and the metabolic cost of locomotion, 3. A method of simulating a space suit using a lower-body exoskeleton, and methods for designing and characterizing the exoskeleton, 4. An explanation for the differences in the energetic costs of walking and running in space suits, 5. Evidence that there is an optimal space suit leg stiffness, perhaps an optimal space suit leg stiffness for a given gravity environment, 6. Evidence, mostly indirect, that energy recovery plays a role in gait switching.
by Christopher Edward Carr.
Sc.D.

Bibliography: leaves 363-421. This thesis presents studies relating to the regulation of appetite, body fat stores and energy balance in the marsupial Sminthopsis crassicaudata. All of the studies presented have been published in international journals, accepted for publication, or submitted for publication. These studies have provided novel data on the regulation of food intake, body fat stores and energy balance in the marsupail Sminthopsis crassicaudata, representing fundamental advances in marsupial biology.

COORDENAÇÃO DE APERFEIÇOAMENTO DO PESSOAL DE ENSINO SUPERIOR
O mercúrio usado no garimpo do ouro na região amazônica é liberado para a atmosfera, solo e rios. Uma vez na atmosfera, o metal é oxidado e imediatamente se deposita. Na água, ocorre a transformação para o metilmercúrio principalmente pela ação de microrganismos. A formação do metilmercúrio aumenta a dispersão e biodisponibilidade do elemento no ambiente aquático. O metilmercúrio pode ser incorporado pelo plancton entrando, assim, na cadeia alimentar. A concentração do metal aumenta a medida que se ascende nos níveis tróficos da cadeia, atingindo os valores mais elevados em peixes carnívoros como o tucunaré. Dessa forma, as emissões de mercúrio provocam a contaminação dos recursos naturais e aumentam os riscos para a saúde dos consumidores habituais de pescado. O objetivo deste trabalho é testar um modelo de bioacumulação de mercúrio em peixes para estimar concentrações em predadores de topo da cadeia alimentar. O modelo pode ser usado como ferramenta para a gestão ambiental de ecossistemas aquáticos potencialmente contaminados com mercúrio. Este objetivo foi atingido através da combinação dos modelos de balanço de massa de Trudel e bioenergético de Wisconsin, aplicados em espécies tropicais do gênero Cichla (tucunaré) da bacia do rio Tapajós. O modelo bioenergético de Wisconsin foi usado para determinar as taxas de consumo de alimento a partir de dados de crescimento estimados. Os parâmetros usados nos modelos foram obtidos na literatura. A habilidade da modelagem na predição dos teores de mercúrio em tucunarés foi avaliada através da comparação com dados de campo obtidos nos anos 1992 e 2001, no rio Tapajós e no sistema de lagos Maicá. Os melhores resultados foram alcançados para os espécimes coletados no ano 1992, o que parece estar relacionado com uma melhor estimativa da concentração de mercúrio no alimento neste ano.
The mercury used in the artisanal mining (garimpo) of gold in the Amazon region is emitted to the atmosphere, soil and rivers. Once in the atmosphere, the metal is oxidized and immediately deposited. In the water, the transformation to methylmercury takes place mostly due to the action of microorganisms. The formation of methylmercury increases the dispersion and bioavailability of the element in the aquatic environment. The methylmercury can be assimilated by the plankton and enters the food chain. The concentration of the metal increases further up in the trophic levels of the chain and reaches the highest values in carnivorous fishes like tucunaré. Thus, the mercury emissions cause the contamination of natural resources and enhance the risks to the health of regular fish consumers. The objective of this work is to test a model for bioaccumulation of mercury in fishes. The model calculates concentrations in top predators of the food chain and can be used as a tool for the management of aquatic ecosystems potentially contaminated with mercury. This objective was accomplished through the combination of the Trudel mass balance and Wisconsin bioenergetics models, applied to tropical species of the Cichla (tucunaré) genre of the Tapajós river basin. The Wisconsin bioenergetics model was utilized to determine the rates of food consumption from estimated fish growth data. The parameters used in the models were found in the literature. The ability of the models to predict mercury contents in tucunarés was evaluated by comparison with the field data, obtained in 1992 and 2001, in the Tapajós River and in the Maicá lakes. The best results were attain ed for specimens collected in 1992 what seems to be related to a better estimate of mercury concentration in the food in this year.

The rampant growth of obesity worldwide has stimulated explosive research into human metabolism. Metabolic rate has been shown to be altered by diets differing in macronutrient composition, with low-carbohydrate, ketogenic diets eliciting a significant increase over other interventions. The purpose of this study was to determine the effects of the ketone β-hydroxybutyrate (βHB) on mitochondrial respiration and coupling status in adipose tissue. To explore this, we employed three distinct systems, namely cell, rodent, and human models. In every model, βHB robustly increased mitochondrial respiration. Furthermore, in cultured adipocytes and rodent adipose, we quantified the expression of genes involved in mitochondrial biogenesis and coupling status. We observed that genes involved in mitochondrial biogenesis and uncoupling were significantly higher in models exposed to ketone treatments. In conclusion, ketones increase mitochondrial respiration in cells and mammalian adipose tissue, but not ATP production, indicating greater mitochondrial uncoupling. These findings may partly explain the increased metabolic rate evident in states of elevated ketones and may facilitate the development of novel obesity interventions in the future.

Alzheimer’s Disease (AD) is a neurodegenerative disorder of the central nervous system. It is mainly sporadic, however, a little percentage of cases is inherited (Familial AD, FAD) and due to autosomal dominant mutations on three different genes, coding for Amyloid Precursor Protein (APP), Presenilin 1 (PS1) and Presenilin 2 (PS2). Presenilins, mainly localized at Endoplasmic Reticulum (ER) membranes, are the catalytic core of the ɣ-secretase complex, although several ɣ-secretase-independent activities of PSs, such as modulation of neurites outgrowth, apoptosis, autophagy, synaptic functions and regulation of Ca2+ homeostasis, have been described. Ca2+, a key intracellular second messenger, is involved in multiple cellular functionalities. Interestingly, alterations in Ca2+ homeostasis have been proposed as an early event in different neurodegenerative diseases, including AD. Notably, FAD-PS mutants have been reported to be directly involved in these dysregulations. In our lab, it has been previously showed that PS2 expression, both WT and, more potently, FAD mutants (such as PS2-T122R), but not PS1, decreases the ER Ca2+ content, mainly by inhibiting SERCA pump activity. Moreover, PS2 increases ER-mitochondria physical and functional coupling, favouring the process of ER to mitochondria Ca2+ transfer. However, due to its effect on ER [Ca2+], which results in a lower amount of available Ca2+ within the ER, its expression dampens mitochondrial Ca2+ rises upon cell stimulation. Based on the well-established role of Ca2+ on mitochondrial metabolism, here we investigate the possible effects on mitochondrial functionalities of the complex balance between alterations in ER Ca2+ content and increased ER-mitochondria coupling, induced by FAD-PS2 mutants expression. A neuroblastoma cell line (SH-SY5Y) grown in a medium containing galactose, as a substitute of glucose, has been used. This growth condition enhances mitochondrial metabolism and results in an excellent experimental protocol to visualize possible mitochondrial defects. Lower total cellular ATP levels were measured in FAD-PS2-T122R expressing cells, grown either in glucose- or galactose-containing medium, with the reduction more evident in the latter condition, thus suggesting possible mitochondrial defects induced by PS2 expression. In order to investigate how Ca2+ dysregulation induced by PS2 could influence mitochondrial metabolism, we stimulated mitochondrial ATP production inducing ER Ca2+ release, followed by mitochondria Ca2+ uptake, using both bradykinin, as a maximal IP3R stimulation, and Fetal Calf Serum (FCS), as a more physiological stimulus. In both conditions, a reduction in mitochondrial ATP production, measured by a mitochondrial luciferase-based ATP probe, has been observed in cells expressing FAD-PS2, but not PS1. The defects in ATP synthesis were observed in SH-SY5Y, MEF, HT22 cells and in cortical neurons from PS2-N141I transgenic (Tg) mice (PS2.30H), by employing FRET-based ATP probes (ATeam 1.03) specifically targeted to the mitochondrial matrix or the nucleus. We also evaluated the glycolytic flux in these cells, by both employing a cytosolic luciferase-based ATP probe and measuring the extracellular medium acidification, but we did not observed any difference in these two parameters in FAD-PS2 expressing cells, compared to controls. In order to understand the mechanism through which PS2 causes the observed mitochondrial dysfunction, we firstly considered the marked Ca2+ dysregulation induced by PS2 expression. We thus decided to modulate Ca 2+ handling in control cells, to mimic the ER Ca2+ depletion caused by PS2 expression. We used two different approaches: i) treating control cells with a SERCA pump inhibitor (Cyclopiazonic acid, CPA), to partially reduce the ER Ca2+ content, or ii) overexpressing a mutated-MICU1 (MICU1mut), a component of the mitochondrial Ca2+ uniporter complex. Although both approaches were able to reduce the capacity of control cells to produce ATP, for similar mitochondrial Ca2+ uptake in control and PS2-expressing cells, a lower mitochondrial ATP production in FAD-PS2 expressing-cells compared to CPA-treated or MICU1mut expressing controls was still observed. Taken together, these results suggest that part of the FAD-PS2-induced defects in mitochondrial metabolism is due to a reduced ER Ca2+ content and, consequently, mitochondrial Ca2+ uptake, negatively regulating the Ca2+-dependent mitochondrial metabolism. However, additional mechanisms, induced by FAD-PS2, are likely involved in mitochondrial dysfunctions. We thus evaluated the respiratory chain activity measuring the oxygen consumption rate (OCR): both basal and maximal OCR were reduced in FAD-PS2, but not in FAD-PS1, expressing cells. Moreover, a reduced mitochondrial ATP-linked respiration was measured in PS2-T122R expressing cells, while no difference was found in the proton leak. Since the expression levels of the ATP synthase and the respiratory chain complexes were not affected by FAD-PS2 expression, and isolated mitochondria from WT and PS2-N141I Tg mice did not reveal substantial differences in mitochondrial respiratory activity, we reasoned that the impairment in ATP production observed in intact cells is not due to defective mitochondria per se, but likely depends on the cellular environment. Importantly, for a proper mitochondrial metabolism, the right amount of substrates produced through glycolysis in the cytosol has to reach the mitochondrial matrix to support the TCA cycle and the respiratory chain activity. Hexokinase1 (HK1), the enzyme that catalyses the first step of glycolysis converting glucose to glucose 6-phosphate, seems to be involved in the modulation of the mitochondrial substrates import, since HK1 interaction/detachment with/from mitochondria can modulate mitochondrial substrates permeability. Firstly, we measured a reduced HK1-mitochondria co-localization in FAD-PS2 expressing SH-SY5Y cells, in FAD-PS2 patient-derived fibroblasts and in primary cortical neurons from FAD-PS2-N141I Tg mice, compared to controls. By mimicking the FAD-PS2 effect on HK1-mitochondria interaction treating control cells with Clotrimazole, a drug capable to detach HK1 from mitochondria, a reduced mitochondrial ATP production was measured; however, the impairment on ATP production induced by clotrimazole was less marked than that caused by FAD-PS2 expression. These results indicate that, although the detachment of HK1 from mitochondria plays a pivotal role in causing mitochondrial defects upon FAD-PS2 expression, the PS2-induced Ca2+ dysregulation, described above, may additionally contribute to the overall mitochondrial impairment. These results have been confirmed also by a genetic approach. We down-regulated the expression of endogenous HK1, by specific siRNAs, and we rescued HK1 protein level by over-expressing siRNA-resistant full-length- (FL-HK1) or truncated- (Tr-HK1) HK1. This latter protein lacks the mitochondrial binding domain, but still conserves the catalytic activity. We found that, upon endogenous HK1 silencing, mitochondrial ATP production is strongly reduced. Interestingly, while the re-expression of FL-HK1 was able to completely rescue the reduced ATP production, the Tr-HK1 was unable to do it, again confirming that the detachment of HK1 from mitochondria is involved in the mitochondrial impairment caused by FAD-PS2. Related to HK1 and its role in the regulation of mitochondrial substrates permeability, an increase in the cytosolic amount of pyruvate was measured in FAD-PS2 expressing cells, compared to controls, employing a cytosolic FRET-based pyruvate probe, Pyronic. Importantly, by pharmacologically blocking mitochondrial pyruvate carrier (MPC), the protein responsible for mitochondrial pyruvate uptake, with two different drugs, UK5099 and Pioglitazone, no differences were anymore detected between control and FAD-PS2 expressing cells, suggesting that FAD-PS2 is acting on this pathway. Overall, we have showed that FAD-PS2 mutants decrease cellular ATP levels, in particular mitochondrial ATP production, by two different mechanisms: 1) causing Ca2+ dysregulation, mainly decreasing the ER Ca2+ content, and thus the amount of Ca2+ available for mitochondrial Ca2+ uptake; 2) inducing the detachment of HK1 from mitochondria, likely affecting the availability of substrates (i.e., pyruvate) for mitochondria. Further experiments will be aimed at: i) evaluate the impact of the PS2-dependent strengthened ER-mitochondria coupling on the reported mitochondrial defects; ii) defining the molecular mechanism through which FAD- PS2 mutants affect HK1 intracellular distribution; iii) evaluate the impact of these alterations on the onset/progression of the AD phenotype.
La malattia di Alzheimer è un disturbo neurodegenerativo del sistema nervoso centrale. È, principalmente, una malattia sporadica; tuttavia in una piccola percentuale di casi è ereditata e dovuta a mutazioni autosomiche dominanti in tre diversi geni, che codificano per la Proteina Precursore dell’Amiloide (APP), per Presenilina1 (PS1) e per Presenilina2 (PS2). Le preseniline, principalmente localizzate nella membrana del reticolo endoplasmatico (RE), costituiscono la porzione catalitica del complesso enzimatico della ɣ-secretasi. Le stesse, oltre ad essere fondamentali per l’attività di questo complesso enzimatico, hanno molte funzioni che sono indipendenti dalla ɣ-secretasi; tra queste, la modulazione della crescita dei neuriti, dell’apoptosi, dell’autofagia, delle funzioni sinaptiche e dell’omeostasi del Ca2+. Il Ca2+ è un secondo messaggero intracellulare fondamentale, coinvolto in molteplici funzionalità cellulari; alterazioni dell'omeostasi del Ca2+ sono state proposte come eventi precoci in diverse malattie neurodegenerative, tra cui la malattia di Alzheimer. In particolare, è stato dimostrato che mutazioni in PS2 associate a forme familiari di Alzheimer (FAD) sono direttamente coinvolte in queste alterazioni. Nel nostro laboratorio è stato precedentemente dimostrato che l'espressione di PS2, sia della forma WT ma soprattutto delle forme mutate associate a FAD (come PS2-T122R), ma non di PS1, riduce il contenuto di Ca2 + nel RE principalmente inibendo l'attività della pompa SERCA. PS2, inoltre, aumenta la vicinanza fisica e funzionale di RE e mitocondri, favorendo il processo di trasferimento di Ca2+ tra i due organelli; tuttavia, a causa del suo effetto sulla [Ca2+] nel RE, che ha come conseguenza una minore quantità di Ca2 + disponibile per il rilascio nel citosol, la quantità di Ca2 + che entra nei mitocondri, dopo stimolazione, è ridotta. Sulla base del ruolo fondamentale svolto dal Ca2 + nella regolazione del metabolismo mitocondriale, nel lavoro presentato in questa tesi abbiamo esaminato i possibili effetti sulla funzionalità mitocondriale del complesso equilibrio tra alterazioni del contenuto di Ca2+ nel RE e l’aumento della vicinanza tra RE e mitocondri, indotti dall’espressione di forme mutate di PS2 legate a FAD. Per svolgere questo studio abbiamo utilizzato una linea cellulare di neuroblastoma (SH-SY5Y), cresciuta in un terreno contenente galattosio, invece di glucosio. Infatti, le cellule cresciute in un terreno che contiene galattosio aumentano il metabolismo mitocondriale, rendendo così questo protocollo sperimentale ottimale per evidenziare eventuali difetti mitocondriali. In cellule esprimenti FAD-PS2-T122R, cresciute in un terreno contenente glucosio o galattosio, sono stati misurati livelli totali di ATP cellulare minori rispetto a quelli di cellule di controllo. La riduzione di questo parametro era più evidente in cellule cresciute in terreno contenente galattosio, suggerendo possibili difetti mitocondriali indotti da PS2. Per studiare come la deregolazione del Ca2+, causata dall'espressione di PS2, possa influenzare il metabolismo mitocondriale, abbiamo indotto il rilascio di Ca2+ dal RE, a cui segue un aumento di Ca2+ nei mitocondri che conseguentemente stimola la produzione di ATP mitocondriale. A tal fine abbiamo utilizzato sia bradichinina, come stimolo massimale del recettore IP3, sia siero fetale di vitello (FCS), contenente fattori che inducono una stimolazione più fisiologica dello stesso recettore. In entrambe le condizioni, è stata osservata una riduzione nella produzione di ATP mitocondriale, misurata utilizzando luciferasi (in particolare la sonda mitocondriale), in cellule esprimenti FAD-PS2, ma non in cellule che esprimevano FAD-PS1. I difetti nella sintesi di ATP sono stati osservati in cellule SH-SY5Y, MEF, HT22 e in neuroni corticali di topi FAD-PS2-N141I (Tg, PS2.30H), utilizzando anche sonde per l’ATP basate su FRET (ATeam 1.03), contemporaneamente espresse nella matrice mitocondriale e nel nucleo. Abbiamo anche valutato se l’espressione di FAD-PS2 potesse influenzare la glicolisi; per fare questo, abbiamo espresso in cellule una luciferasi citosolica, per valutare l’ATP prodotta nel citoplasma, e abbiamo misurato l’acidificazione del mezzo extracellulare, come indice di glicolisi. Per entrambe i parametri, non abbiamo osservato alcuna differenza tra cellule esprimenti FAD-PS2 o di controllo. Per comprendere il meccanismo attraverso il quale PS2 causa la disfunzione mitocondriale osservata, data la nota deregolazione dell’omeostasi del Ca2+ indotta da PS2, abbiamo innanzitutto deciso di simulare la deplezione di Ca2+ nel RE causata dall’espressione di PS2 nelle cellule di controllo. Abbiamo usato due approcci diversi: da un lato abbiamo trattato le cellule di controllo con un inibitore della pompa SERCA (acido ciclopiazonico, CPA) per ridurre il contenuto di Ca2+ nel RE, dall’altro abbiamo sovraespresso una forma mutata di MICU1 (MICU1mut). In entrambi i casi abbiamo ottenuto una riduzione nell’entrata di Ca2+ nel mitocondrio, mimando perfettamente il difetto causato dall’espressione di FAD-PS2. Come atteso, il trattamento con CPA e l'overepressione di MICU1mut riducono notevolmente la produzione di ATP rispetto alle cellule di controllo non trattate. Ciononostante, a parità di Ca2+ che entra nel mitocondrio in cellule esprimenti o meno FAD-PS2, abbiamo misurato una minore produzione di ATP mitocondriale in cellule esprimenti forme mutate di PS2, rispetto ai controlli trattati con CPA o esprimenti MICU1mut. Tali risultati suggeriscono che i difetti nel metabolismo mitocondriale indotti dall’espressione di FAD-PS2 solo almeno in parte riconducibili alla riduzione del contenuto di Ca2+ nel RE, e quindi al suo ingresso nei mitocondri. Tuttavia, sono probabilmente coinvolti meccanismi aggiuntivi nelle disfunzioni mitocondriali osservate. Abbiamo, quindi, valutato l'attività della catena respiratoria misurando la velocità nel consumo di ossigeno (OCR). E’ stato così possibile osservare che sia il consumo di ossigeno a basale che il massimo consumo di ossigeno sono ridotti in cellule esprimenti FAD-PS2, ma non FAD-PS1. Inoltre, in cellule esprimenti PS2-T122R è stata misurata una riduzione della respirazione mitocondriale legata alla produzione di ATP. Tuttavia, poiché i livelli di espressione dell’ATP sintasi e dei complessi della catena respiratoria non variano, in seguito all’espressione di PS2, e dato che misure di respirazione in mitocondri isolati da topi WT e PS2-N141I Tg non hanno rivelato differenze sostanziali, la riduzione nella produzione di ATP osservata in cellule intatta non è verosimilmente dovuta ad un’alterazione intrinseca nell'attività della catena respiratoria. Questo suggerisce che i difetti riscontrati possano dipendere dall'ambiente cellulare, piuttosto che da un difetto intrinseco degli stessi mitocondri. Per un corretto metabolismo mitocondriale, la giusta quantità di substrati prodotti nel citoplasma attraverso la glicolisi deve raggiungere la matrice mitocondriale per supportare il ciclo di Krebs e l'attività della catena respiratoria. L’esochinasi 1 (HK1), enzima che catalizza la prima reazione della glicolisi, convertendo il glucosio in glucosio 6-fosfato, sembra anche modulare l’ingresso dei substrati nei mitocondri, poiché l'interazione/distacco di HK1 con/dai mitocondri può modulare la permeabilità mitocondriale ai substrati. Abbiamo misurato una riduzione nella co-localizzazione tra HK1 e mitocondri in cellule SH-SY5Y esprimenti FAD-PS2, in fibroblasti da pazienti FAD con mutazioni in -PS2 e in neuroni corticali da topi transgenici FAD-PS2. Il trattamento di cellule di controllo con clotrimazolo, una sostanza nota per avere la capacità di indurre il distacco di HK1 dai mitocondri, si è rivelato capace di ridurre la colocalizzazione tra HK1 e mitocondri a un livello simile a quello causato da PS2, mimandone così l'effetto. In seguito a questo trattamento, cellule di controllo mostravano una ridotta produzione di ATP mitocondriale, rispetto a cellule non trattate; tuttavia, l'effetto del clotrimazolo sulla produzione di ATP era meno evidente rispetto alla diminuzione causata dall'espressione di FAD-PS2. Questo significa che, anche se il distacco di HK1 dai mitocondri svolge un ruolo importante nel determinare i difetti mitocondriali osservati in seguito a espressione di FAD-PS2, la disfunzione nell’omeostasi del Ca2+, descritta in precedenza, contribuisce anch’essa alla diminuzione complessiva dell’attività mitocondriale. Questi risultati sono stati confermati anche con un approccio genetico. Abbiamo abbattuto l'espressione di HK1 endogena, mediante specifici siRNAs e abbiamo sovra-espresso la forma intera di HK1 (FL-HK1) o la forma tronca di HK1 (Tr-HK1), proteina ques’ultima che manca del dominio di legame mitocondriale ma che presenta ancora l'attività catalitica. Il silenziamento della proteina endogena causa una notevole riduzione nella produzione di ATP mitocondriale; la ri-espressione di FL-HK1 è in grado di recuperare completamente il difetto nella produzione di ATP, mentre quella di Tr-HK1 no. Questi risultati confermano nuovamente che il distacco di HK1 dai mitocondri è coinvolto nella manifestazione dei difetti mitocondriali osservati in seguito all’espressione di FAD-PS2. Relativamente a HK1 e al suo ruolo nella regolazione della permeabilità mitocondriale ai substrati, in cellule esprimenti PS2 è stato misurato un aumento nella quantità di piruvato nel citoplasma . È importante notare come il blocco farmacologico della proteina responsabile del trasporto del piruvato all’interno del mitocondrio (MPC) con due diversi farmaci, UK5099 e Pioglitazone, annulli le differenze tra le cellule esprimenti FAD-PS2 e i controlli, indicando che l'espressione di FAD-PS2 agisce anche su questa via metabolica. In questo lavoro, abbiamo mostrato che forme mutate di PS2 legate a FAD diminuiscono i livelli cellulari di ATP, in particolare la produzione di ATP mitocondriale, con due diversi meccanismi: 1) causando una deregolazione dell’omeostasi del Ca2+, principalmente diminuendo il contenuto di Ca2+ nel RE, e quindi il conseguente ingresso di Ca2+ nel mitocondrio; 2) inducendo il distacco di HK1 dai mitocondri, influenzando così la disponibilità di substrati (per es., piruvato) per i mitocondri. Ulteriori esperimenti saranno finalizzati a: i) valutare l'impatto dell’aumento della vicinanza tra RE e mitocondri causato dall’espressione di PS2 sui difetti mitocondriali riportati; ii) definire il meccanismo molecolare attraverso il quale FAD-PS2 induce il distacco di HK1 dai mitocondri; iii) valutare l’eventuale impatto di queste alterazioni nella progressione del fenotipo AD.

Blood flow distribution during forced and voluntary diving in ducks, and the energetic cost of diving was investigated. It has been suggested that in order for the leg muscles to generate enough power for ducks to dive, blood flow to those tissues must be maintained. A technique to determine blood flow distribution which could be used during voluntary diving was first developed and tested during forced laboratory dives of ducks. This technique was then used to determine the blood flow distribution during voluntary diving. Regional blood flow distribution was visualized by utilizing a radioactive tracer technique (macro aggregated albumin labelled with ⁹⁹ⅿ technetium). The tracer when injected into an animal is trapped and held by capillaries. During forced dives in dabbling (Anas platyrhynchos) and diving (Aythya affinis) ducks the blood flow distribution was found to be restricted to the thoracic and head areas. Whereas during a voluntary dive in A. affinis blood flow distribution was shown to be preferentially directed towards three tissue areas, the heart, brain, and active leg muscles. The work required to dive was determined from the measurement of subsurface drag forces and buoyancy in A. affinis. Subsurface drag increased as a nonlinear function of swimming velocity. At a velocity of 1 m•s⁻¹, the drag force was approximately 1.067 N. The average measured buoyant force of 11 ducks was 0.953 N. The calculated mechanical work done by ducks during a 14.4 s unrestrained dive was 9.34 J. The power output during voluntary was estimated to be 0.751 W (0.0374 ml 0₂•s⁻¹). During diving buoyancy is clearly the dominant force (8.8 J) against which ducks have to work while drag (0.54 J) adds little (~6%) to the energetic cost of diving.
Science, Faculty of
Zoology, Department of
Graduate

In this thesis, thermodynamic-based mathematical modelling is combined with experimental in vitro extracellular flux analysis in order to assess drug redox cycling and cellular bioenergetics. It is widely accepted that pharmacological activity of certain classes of drugs (e.g. anticancer, antimalarial) is related to their ability to accept one or two electrons. However, pharmacological activity via redox cycling is an understated mechanism of toxicity associated with many classes of drugs. In particular, oxidative stress as a result of redox cycling plays a pivotal role in the cause of cardiac toxicity. For example, doxorubicin is an anti-neoplastic drug used to treat cancer. It has strong links to redox cycling-induced cardiac toxicity associated directly with elevated levels of reactive oxygen species (ROS) and oxidative stress within the mitochondria. The underlying mechanisms of redox cycling is very difficult to elucidate, due to the fleeting existence of the radical species. However, assessment of such cellular bioenergetics can be ameliorated with the aid of computational assistance. In chapter 2 the development of a novel thermodynamic-based in silico model of doxorubicin redox cycling is described, which is parameterized using data from in vitro extracellular flux analysis. The model is used to simulate mitochondrial-specific ROS, with its outputs confirmed against in vitro data. Chapter 3 describes construction of a pH-dependent thermodynamic model of hepatic glycolytic flux, used to determine the role of the monocarboxylate transporter 1 flux during extracellular acidification. Finally, chapter 4 describes a thermodynamic-based in silico model of mitochondrial bioenergetics, capable of simulating oxygen consumption rates of a cohort of in vitro human primary hepatocyte data. The model is finally used to simulate perturbations in key bioenergetic variables and reaction fluxes, illustrating the resulting changes on mitochondrial pH, membrane potential and subsequent oxygen consumption rates.

The movement patterns, range areas and energetics of canvasbacks (Aythya valisineria) wintering in the upper Chesapeake Bay, Maryland, were investigated. Eighty-seven juvenile female canvasbacks were radio-tracked between 30 December 1988 and 25 March 1989. Diurnal time and energy budgets were constructed for a time of day-season matrix for canvasbacks using riverine and main bay habitats. Canvasbacks were very active at night, making regular and often lengthy crepuscular movements (x = 11.7 km) from near shore habitats during the day to off shore habitats at night. Movement patterns were similar for birds using habitats on the eastern and western shores of the Bay. Canvasbacks had extensive home ranges averaging 14,286 ha, and used an average of 1.97 core areas. Sleeping was the predominant diurnal behavior. Telemetry indicated that canvasbacks actively fed at night. Canvasbacks spent more time in active behaviors (e.g. swimming, alert) on the eastern shore than on the the western shore. Similarly, canvasbacks were more active during daytime hours at locations where artificial feeding occurred. Behavioral patterns were only weakly correlated with weather patterns. Canvasbacks appeared to reduce energy expenditure in mid-winter by reducing distances moved, reducing feeding activities and increasing the amount of time spent sleeping. This pattern was observed even though 1988-89 mid-winter weather conditions were very mild.
Master of Science

Bibliography: pages 95-109.
The bioenergetics and growth of white steenbras, Lithognathus lithognathus, under culture conditions were determined to assess its suitability for mariculture. Fish were captured by seine netting, from nearby estuaries and transported to the laboratory where they were successfully kept for the duration of the experimental period. The fish were initially fed on white mussels and then weaned onto a diet of commercial trout pellets. The bioenergetics of L.lithognathus was examined using a large, simple closed system respirometer in which nitrogen excretion and oxygen consumption were measured simultaneously. This system permitted the use of groups of fish for experiment which simulated aquaculture conditions. The effects of fish size, ration size and temperature on faecal and nonfaecal excretion, and on oxygen consumption of L.lithognathus were examined.

This thesis investigates the role of vitamin D metabolites 1,25-dihydroxyvitamin D and 25-hydroxyvitamin D in human bioenergetics and inflammation. The evidence presented comes from a narrative review, two systematic reviews, two cross-sectional studies and an observational longitudinal study design. Collectively, the thesis supports a modulatory role for vitamin D in whole body and cellular bioenergetics and inflammation. Future studies could build on these findings; establishing a causal role for vitamin D in inflammation and energy metabolism.

Primena biomarkera ćelijske energetike, uključujući indikatore metabolizma kreatina u krvi, je relativno novijeg datuma, gde se ovi indikatori koriste kao mogući pokazatelji stanja organizma pri maksimalno intenzivnim fizičkim aktivnostima. Cilj istraživanja je obuhvatao utvrđivanje efekata pojedinačnih epizoda aerobnog i anaerobnog vežbanja maksimalnog intenziteta na biomarkere perifernog zamora i ćelijske bioenergetike kod mladih muškaraca i žena. Istraživanje je dizajnirano tako da obuhvati populaciju fizički aktivnih muškaraca i žena, kao i populaciju aktivnih sportista. U prvom eksperimentalnom tretmanu fizički aktivni ispitanici muškog (n =12) i ženskog pola (n = 11) podvrgnuti su test protokolima aerobnog i anaerobnog opterećenja maksimalnog intenzivnog i kratkog trajanja. Tokom aerobnog test protokola ispitanici su trčali do maksimalnog voljnog otkaza na tredmil traci sa progresivnim povećanjem opterećenja. Pri anaerobnom test protokolu ispitanici su izvršili testiranje snažne izdržljivosti gornjih ekstremiteta do otkaza potiskom sa ravne klupe, uz opterećenje od 25% od njihove telesne težine. Drugi eksperimentalni tretman je sačinjen iz pre-eksperimentalnog testiranja kardiorespiratorne forme i eksperimentalne protokol sesije trčanja do maksimalnog voljnog otkaza na pokretnoj traci, pri konstantnoj individualnoj brzina trčanja na anaerobnom pragu. U ovom eksperimentalnom tretmanu bila je uključena populacija aktivnih sportista     (n = 10). Pre, tokom i nakon eksperimentalnih sesija praćena je koncentracija različitih biohemijskih i hematoloških markera: guanidinosirćetna kiselina (GAA); kreatin (Cr); kreatinin (Crn); laktat (Lac); interleukin-6 (IL-6); kreatin kinaza (CK); kortizol (Cor). Rezultati prvog eksperimentalnog tretmana su utvrdili statistički značajne promene u koncentraciji GAA, Cr i Crn u ktvi pre i nakon pojedinačne epizode aerobnog i anaerobnog vežbanja maksimalnim intenzitetom. Utvrđena je i statistički značajna povezanost između vežbanjem-indukovanih promena u cirkulatornim vrednostima GAA, Cr, Crn za vreme pre, tokom i nakon drugog eksperimentalnog tretmana. Uočena je statistički značajna povezanost između promena koncentracije GAA, Cr, Crn u serumu sa tradicionalnim biomarkerima perifernog zamora (IL6, Cor, Lac, CK). Dobijeni rezultati ukazuju na mogućnost primene biomarkera metabolizma kreatina u krvi prilikom praćenja i evaluacije stanja organizma tokom maksimalnih intenzivnih fizičkih aktivnosti kod mladih muškaraca i žena.
The use of biomarkers of cellular bioenergetics in exercise science appears more prevalent in recent years, where these outcomes perhaps describe changes in creatine metabolism during strenuous exercise. The aim of this study was to determine the effects of individual episodes of strenuous aerobic and anaerobic exercise on several biomarkers of peripheral fatigue and cellular bioenergetics in young men and women. The study recruited physically active men and women, and active athletes. In the first experiment, physically active men (n = 12) and women (n = 11) were subjected to strenuous aerobic and anaerobic exercise. During the aerobic test, subjects ran to exhaustion while during the anaerobic test, subjects performed repetitive bench press exercise. The second experimental treatment consisted of a pre-experimental testing of cardiorespiratory fitness, and an experimental protocol of a strenuous running session to exhaustion at constant individual running speed at the anaerobic threshold; active athletes (n = 10) were included in this experimental treatment. The blood levels of various biochemical and hematological markers were monitored before, during and after the experimental sessions, including guanidinoacetic acid (GAA); creatine (Cr); creatinine (Crn); lactate (Lac); interleukin-6 (IL-6); creatine kinase (CK); cortisol (Cor), and plethora of other physiological outcomes. We found statistically significant changes in serum GAA, Cr and Crn before and after a single session of strenuous aerobic and anaerobic exercise. A significant correlation was found between exercise-induced changes in serum GAA, Cr and Crn before, during and after the second experimental intervention. A statistically significant association was observed between changes in serum GAA, Cr, Crn and traditional biomarkers of peripheral fatigue (IL6, Cor, Lac, CK). The results of the present study suggest that biomarkers of creatine metabolism might be used as innovative tools in monitoring strenuous exercise in young men and women.

Recent advances in technology permitted the first simultaneous digital recording of intranarial pressure and on-axis acoustic data from bottlenose dolphins during a biosonar target recognition task. Analysis of pressurization events in the intranarial space quantifies and supports earlier work, confirming that intranarial pressure is increased when whistle vocalizations are emitted. The results show complex relationships between various properties of the biosonar click to the intranarial pressure difference at the time it was generated. The intranarial pressure that drives the production of clicks is not the primary determinant of many of the acoustic properties of those clicks. A simple piston-cylinder physical model coupled with a sound production model of clicks produced at the monkey-lips/dorsal bursae complex yields an estimate of mechanical work for individual pressurization events. Individual pressurization events are typically associated with a single click train. Mechanical work for an average pressurization event is estimated at 10 Joules.

Trypanosoma brucei is the vector of African trypanosomiasis in both domestic animals (nagana) and sleeping sickness in humans (Human African Trypanosomiasis). These protozoan parasites are transmitted by the bite of infected tsetse flies (Glossina sp.). African trypanosome infections cause parasite-induced anorexia (PIA) and cachexia in livestock, experimental animals and in humans, and are of economic, veterinary and medical importance in sub-Saharan Africa. The overall aim of this project was to characterise the phenomenon of inappetence in relation to overall energy budget in African trypanosome infection and to then identify potential causal factors and mechanisms. A mouse model of T.b. brucei infection was established with a reproducible time course for the development of inappetence and bodyweight loss. Following an initial parasitaemic peak on day 6 post-infection, a profound period of inappetence was observed from days 7 to 11, accompanied by a 10% loss of body mass. Metabolisable energy intake was reduced, while assimilation efficiency increased significantly but not enough to compensate for the severe reduction in food intake. During the course of T.b. brucei infection, both total energy expenditure and physical activity were reduced. Although physical activity was markedly declined in both light and dark phases, trypanosome infected mice maintained their circadian rhythm albeit at a lower amplitude, with most of the activity occurring at the start of the dark phase. Resting metabolic rate was unchanged in infection. Plasma concentrations of the inflammatory cytokines, IL-6 and TNF-α were increased in infected mice and were associated with inappetence. Reductions of leptin and insulin concentration corresponded to a loss in fat mass. The hypothalamic control of appetite appeared to be normal with increases in appetite stimulating AgRP, decreases in the appetite inhibiting POMC and MC4R. There has been no previous data published on the control of appetite and energy expenditure in African trypanosome infections thus the data presented here provides a novel insight into the pathophysiology of this serious disease, and may lead to new therapies to manage the clinical and veterinary consequences of trypanosome infection.

Obesity, through a persistent positive energy and fat balance is of major public health significance due to its detrimental health, social and financial costs. Increasing physical activity levels through recreational exercise and decreasing energy intake have been implicated with obesity prevention. However, the addition of exercise to normally sedentary routines will only prevent positive energy balance if it is not tracked b a compensatory response in energy intake and non-exercise physical activity [also termed non-exercise activity thermogenesis (NEAT)]. The current series of studies set out to examine the quantitative and temporal relationship between exercise and energy balance with specific reference to appetite, energy intake and NEAT. These studies were designed using similar methodologies that could be compared and related to existing studies. The results showed that in younger motivated individuals, moderate-high intensity mandatory exercise increased daily energy expenditure leading to a marked negative energy balance. However for periods of up to two weeks, analysis of temporal trends revealed evidence of compensatory changes to re-establish energy balance (re-equilibrium phase). This re-equilibrium was a result of not only increases in energy intake, but also (and to a greater extent) decreases in NEAT. Inter-individual variability in the extent of compensation was evident and independent of age, sex, BMI and restraint status. Decreases in physical activity did not lead to a compensatory reduction in energy intake and lead to a marked positive energy balance. Using an exercise intervention, in line with government guidelines, in a group venerable to becoming obese showed that body mass was largely unaffected since overall energy expenditure was not significantly elevated, primarily due to a lack of motivation to reach the required exercise prescription. The results have public health significance in the formation of policy to increase physical activity in the population.

Caleosins were originally described as one of the two major protein components of storage lipid bodies in the seeds of higher plants, the other being oleosins. In contrast with oleosins, caleosins have a single calcium-binding EF-hand domain plus several potential phosphorylation sites and have been hypothesised as playing a role in lipid-body formation and possibly mobilisation in seeds. In Arabidopsis, there are six functional caleosin genes, two of which encode seed-specific proteins while the other isoforms are expressed in a variety of vegetative and reproductive tissues. More recently, seed caleosins have been shown to have a peroxygenase activity but the function of this was uncertain. This study describes the characterisation of a specific membrane-bound caleosin isoform, termed Clo-3 in Arabidopsis and Brassica spp, that appears to be present in all plant tissues and is responsive to a variety of biotic and abiotic stresses. Bioinformatic analysis reveals that similar caleosin-like genes/proteins are present in all vascular plants, as well as in nonvascular plants such as mosses, and even in single-celled algae. Intriguingly, caleosin-like genes are also present in the genomes of most fungi described to date, with the surprising exception of the yeasts. In order to understand the function of caleosins in plants, a detailed structural and functional analysis of this novel class of protein is reported here. Biochemical studies demonstrate that the Clo-3 isoform binds calcium (one atom per molecule), can be phosphorylated most likely, by a casein kinase 2 (CK2) protein kinase, and has putative peroxygenase activity. In addition to biochemical data, microscopy analysis shows that Clo-3 may be located both on the endoplasmic reticulum and chloroplastid envelope membranes. specifically the chloroplast envelope. Biochemical evidence of cell membrane localisation is also presented. Protease digestion experiments show that the membrane bound Clo-3 has a Type I transmembrane orientation, where its N-terminal domain faces the lumen of microsomes while the C-terminal is on the cytosolic face. Such an orientation is common for receptors or proteins that may be activated by signalling molecules. The Clo-3 gene and its encoded protein are each upregulated by salt and drought stresses and by abscisic acid (ABA) treatment. Reverse genetics using RNAi knockdown mutants demonstrate specific transcription factors involved in regulating Clo-3 during different stresses. Peroxygenase activities of Clo-3 enriched microsomes were higher following salt stress. Although the data is representative of potentially many peroxygenases, it does provide indirect evidence that Clo-3 abundance increases and/or catalytic activity is induced during stress. The study also presents evidence of the response of Clo-3 to biotic stress and related signalling molecules. Arabidopsis Clo-3 is highly responsive to the phytohormone salicylic acid, to the salicylic acid synthetic analogue DCINA, the biotic signalling molecule hydrogen peroxide, and to infection by the common fungal pathogen of Brassicas, Leptosphaeria maculans (Phoma), while experiments utilising the non-expressor of pathogenesis related protein 1 (npr1) knockout mutant plant demonstrates Clo-3 response to salicylic acid (SA) is chiefly via npr1 translocation to the nucleus. The type of peroxygenase epitomised by Clo-3 is similar to those involved in the formation of epoxy alcohols from fatty acid hydroperoxides. The latter are a class of oxylipins that are seen in fungal infection, and also play a role in various aspects of fungal spore development including sporulation and a role in cuticle synthesis. As such, Clo-3 in Arabidopsis and possibly similar caleosins in other species might play roles in oxylipin signalling pathways that are involved in a protective role during both biotic and abiotic stress responses.

Ingestion, assimilation and egestion rates of different sized filter-feeding blackfly larvae Simulium medusaeforme/hargreavesi (Diptera: Simuliidae) were determined. The effects of food type, food concentration and larval size on ingestion rates were tested. These bioenergetic parameters were used to obtain an indication of the importance of blackfly larvae in processing of particulate organic matter in the Buffalo River. Mean ingestion rates for S. medusaeforme/hargreavesi larvae feeding in the field ranged from 900 - 1600 μg/mg/h compared with those for larvae feeding in the laboratory on the same food type (260 - 680 μg/mg/h) and on algae, Chlorella sp. (590 - 1110 μg/mg/h) and Selenastrum sp. (340 - 1140 μg/mg/h). Ingestion rates obtained in this study were much higher than those reported by previous workers. These high ingestion rates are thought to be related to the low organic fraction of the suspended solids in transport in the Buffalo River. Larger larvae were found to ingest between three and six times as much food as smaller larvae. Ingestion rates were affected by the presence of nematodes and by imminent pupation. Assimilation rates on algae ranged from 2.3 - 49.0 μg/mg/h and were comparable with results of previous workers. The low assimilation efficiencies obtained for larvae feeding on algae (0.4 - 15.1 %) are due to the high ingestion rates. From a survey of the Buffalo River larvae were found to ingest between 0.00011 - 0.15 % of the suspended solids per metre of stream bed, about 1 - 7 times more than what has been reported by other workers. In the upper reaches of the Buffalo River larvae were potentially able to ingest all the suspended solids in the size class 0 - 250μ.m within a distance of 3.2km. Blackfly larvae play an important qualitative role in the functioning of a river system as they remove seston from transport and facilitate the action of gut microflora. Blackfly larvae in association with micro-organisms and other invertebrates are responsible for the majority of the retention and mineralisation of organic matter in the river