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Concerns about the possible hazardous effects of electromagnetic fields (EMF ' s) has resulted in attempts to address this issue. Several authors have shown that EMF exposure affects biological systems and more specifically the pineal enzyme, serotonin N-acetyltransferase (SNAT), inhibiting the enzyme and thus melatonin production. The enzyme assay required, for performing this type of work is crucial, as the natural decay of the enzyme could bias experimental results. An assay for measuring SNAT activity was assessed and then improved. The Ca²⁺ ion chelator, EGTA (2mM), preserved enzyme activity during pineal homogenisation. The enzyme is heat sensitive and thus keeping the homogenates on ice facilitated enzyme preservation. Not only was preservation of the enzyme crucial, but optimum substrate concentrations were required to measure maximum levels of SNAT activity. Maximum levels of activity were measured when I/10th of a pineal gland was incubated with 12mM tryptamine. At the specified tryptamine concentration, SNAT activity increased as the concentration of Acetyl CoA increased. The enzyme efficiency, as determined from its Km (8.19x10⁻⁴M), and the level of activity exceeded those measured in other laboratories. This assay was used to determine SNAT activity following magnetic field exposure. The magnetic field studies investigated the effects of pulsed static and oscillating magnetic fields on SNAT activity. Male Long Evans rats were housed in short and long photoperiods and were exposed for either 60mins., 30mins. or 15mins. to magnetic fields at different times during the dark phase. Rats were exposed to regularly repeated inversion of either the vertical or horizontal component of the earth' s magnetic field or to regularly repeated horizontal or vertical lOOμT DC field inversions. Pulsed static magnetic field exposure in all conditions had no significant inhibitory effect on SNAT activity. Oscillating magnetic field studies investigated the effect of exposure of rats to magnetic fields tuned to ion parametric resonance conditions for Ca²⁺. The ion parametric resonance model of Blanchard and Blackman was used to determine the exposure parameters. Rats were thus exposed for an hour to a vertical AC field, of strength 14.3μT (rms), frequency was 17.2Hz while the strength of the vertical DC field was 22.55μT, the existing vertical geomagnetic field strength. Under these conditions oscillating magnetic field exposure also had no significant inhibitory effect on SNAT activity.

Includes bibliographical references. / A knowledge of the ionic and electrical gradients across the Malpighian tubules of Onymacris plana is fundamental to the understanding of the transport mechanisms of fluid across the basolateral and apical membranes. Until now no one has investigated the isolated perfused Malpighian tubule of Onymacris plana and simultaneously measured basolateral potentials. Malpighian tubule segments (about lmm length) were dissected out and perfused in vitro according to the method of Burg et. al. (1966), while the basolateral membrane potential (Vbl) was measured by means of an intracellular microelectrode. Using cable analysis, the effects of DNP, chloride free Ringers, BaCl² (a K⁺-channel blocker), low (25 mM) and high (130 mM) pntassium Ringers (bathing medium), cyclic-AMP and a corpora cardiaca homogenate (CCH) on. the transepithelial resistance and the fractional resistances of the basolateral and apical membranes were investigated.

Includes abstract. / Includes bibliographical references (leaves 159-179). / The aims of this dissertation were therefore (i) to follow a pathway-based approach investigating genes encoding proteins involved in the ECM degradation and apoptosis signalling cascade for associations with AT and (ii) to identify a polygenic risk model, comprised of several genetic markers within genes encoding proteins involved in the inflammatory pathway, to predict risk of AT.

Bibliography: pages 326-345. / Rats with unilateral 6-OHDA lesions of the nigrostriatal (NS) projection display motor asymmetry in the form of rotational behaviour. The rotation is in the direction ipsilateral with respect to the lesioned side (Ungerstedt 1979). The nett ipsilateral rotations decrease with time, from 1 week to about a month. This decrease has been interpreted as recovery from the lesion-induced motor asymmetry (Glick and Cox 1978). Pritzel et al. (1983) have ascribed the recovery from motor asymmetry to increased activity of a crossed NS projection, which is spared by the ipsilateral lesion. The present study has defined the size and anatomical path of this crossed projection, and has examined its involvement in the behavioural recovery of rats from lesion-induced motor asymmetry. The anatomy of the crossed projection was investigated in male Long-Evans rats using retrograde HRP tract tracing from deposition sites in the striatum.

Includes bibliographical references / Background: Previous evidence has demonstrated ethnic differences in the relationship between body fat distribution and metabolic risk between black and white women. However, the reasons for these differences are not known and may be explained in part by differences in sex hormones. The overall aim of this thesis was therefore to i) examine ethnic-specific associations between body fat and its distribution and cardio-metabolic outcomes (study 1) and ii) examine the associations between sex hormones and subcutaneous adipose tissue (SAT) expression of oestrogen receptors (ERα and ERβ) and aromatase (CYP19A), and body fat distribution and insulin resistance (IR) among black and white women (study 2).Methods: Study 1: In 288 black and 197 white premenopausal women, dual-energy X-ray absorptiometry (DXA) and computed tomography (CT) derived measures of body fat distribution and cardio-metabolic factors including IR (HOMAIR) and lipid levels were measured. Study 2: In a subsample consisting of 13 normal-weight and 15 obese black and 15normal-weight and 12 obese white women, HOMAIR and SI (frequently sampled intravenous glucose tolerance test) and ERα, ERβ and CYP19A gene expression were measured in abdominal and gluteal SAT. Results: Study 1: Compared to white women, black women had less central and greater lower body fat, but had similar IR and lower serum lipid concentrations. Despite these differences, the associations between body fat distribution and measures of IR, as well as TG and HDL-Concentrations were similar in black and white women. Notably, central and peripheral fat deposition was independently associated with IR in both the black and white women, and with TG in the black women. In contrast, the associations between body composition and fasting plasma glucose, TC and LDL-C concentrations differed between black and white women. Fasting glucose concentrations were associated with centralisation of body fat in black but not white women, whereas TC and LDL-C concentrations were associated with centralisation of body fat in white but not black women. In addition to body fat distribution, MVPA was associated with IR in the white women, and contraception use was associated with lipid levels in the black and white women. Study 2: CYP19A expression was positively associated with increased adiposity in black and white women in all three depots. Gluteal ERα was significantly higher and ERβ was significantly lower in the black compared to the white women, irrespective of BMI. Gluteal ERα was negatively associated with trunk fat mass (FM) and HOMAIR in the black women only. Gluteal ERα was significantly lower in obese white compared to normal weight white women. Additionally oestradiol (E2) levels were lower in obese compared to normal-weight white women, but did not differ by ethnicity. Conclusion: Our results indicate that it is important in both black and white populations, to decrease centralisation of body fat. Modifiable risk factors such as MVPA and contraception use should be used as therapeutic targets to prevent and manage CVD. Additionally, oestrogen receptors may be an important determinant of body fat distribution and risk in the black women.

Introduction: Sphingosine 1 phosphate (S1P) is a major constituent of high density lipoprotein (HDL) cholesterol. Both S1P preconditioning and ischaemic postconditioning reduce myocardial damage following an ischaemia-reperfusion insult but the mechanisms involved remain unclear. Janus kinase/Signal transducer and activator of transcription 3 (JAK/STAT-3) form part of a recently discovered powerful prosurvival path termed as the Survivor Activating Factor Enhancement (SAFE) pathway. The SAFE pathway plays a critical role in ischaemic preconditioning to promote cell survival but whether activation of STAT-3 is required for S1P preconditioning and ischaemic postconditioning induced cardioprotection is unknown. Hypothesis: Activation of the STAT-3 is required for S1P preconditioning and ischaemic postconditioning.

Introduction: Diabetes mellitus (DM) is a leading cause of morbidity and mortality all over the world, and the main cause of the mortality is cardiovascular complications. Such diabetic cardiovascular complications include coronary heart disease, cardiac autonomic neuropathy and ventricular dysfunction. Furthermore, DM is associated with electrolyte disturbances such as those involving potassium, calcium and magnesium (Mg2+). Among these electrolyte disturbances hypomagnesemia is common in diabetes and is associated with increased cardiovascular risk. Recent evidence has shown that Mg2+ supplementation can prevent cardiac autonomic dysfunction and improve ventricular compliance in acute DM. However, the underlying mechanisms of Mg2+ action and Mg2+ effects in chronic DM are unknown. Therefore, the present study explored the effects of Mg2+ administration and its possible mechanisms of action in chronic streptozotocin (STZ) induced diabetic rats. Methods: Adult male Wistar rats were injected intraperitoneally (i.p) once with either STZ (50 mg/Kg body weight) or the STZ vehicle (citrate buffer). The rats were then injected i.p once daily with either magnesium sulphate (MgSO4; 270 mg/Kg body weight) or the MgSO4 vehicle (normal saline) for 28 consecutive days. Blood glucose and body weight were measured throughout the period of the study. On day 28 of the experiments, in-vivo heart rate variability (HRV) parameters were measured to assess cardiac autonomic function using tail pulse plethysmography. Orthostatic stress was induced by tilting the animals from flat position to 70° head-up position. Ex-vivo hemodynamic and electrocardiograph (ECG) measurements were performed on a Langendorff perfusion system. Histological studies of ventricular tissue were performed using haematoxylin-eosin and Masson’s trichrome staining. Western blot analyses of the cardiac autonomic presynaptic marker (synaptophysin) and of the mitochondrial marker of oxidative stress (ATP5A) were performed on right atrial tissue. Plasma Mg2+ concentration was measured using automated photometric assays. Results: STZ treatment significantly increased the blood glucose level and decreased the body weight, and these STZ effects were not prevented by Mg2+ treatment. Diabetes decreased the root mean square differences of successive normal-to-normal intervals (RMSSD) and increased the low frequency (LF) /high frequency (HF) power ratio, which are both indicative of abnormal HRV. These diabetes effects on HRV parameters were significantly prevented by Mg2+ treatments (P < 0.05, STZ+Mg vs. STZ). DM also reduced both the heart rate and orthostatic stress-induced tachycardia, and these effects were reversed by Mg2+ treatment (P < 0.05, STZ+Mg vs. STZ). DM also decreased the left ventricular (LV) developed pressure and the maximal rate of LV pressure increase (+dP/dt), and these diabetic effects were prevented by Mg2+ treatment (P < 0.05, STZ+Mg vs. STZ). DM also decreased the maximal rate of LV pressure decline (-dP/dt) and the rate pressure product, but these parameters were not improved by Mg2+ treatment. DM and Mg2+ treatment did not affect the ECG waveforms and the coronary flow rate in all groups. Histologically, there were no differences in ventricular cardiomyocyte width or in the extent of interstitial fibrosis in all groups. Western blot analysis qualitatively showed a decrease in the expression of synaptophysin in DM that was prevented by Mg2+ treatment. Neither DM nor Mg2+ treatment altered ATP5A expression. The plasma Mg2+ concentration was not altered by DM or Mg2+ treatment. Conclusion: This study showed that Mg2+ treatment prevented cardiac autonomic dysfunction and improved hemodynamic function impairment in chronic DM. Based on the expression of synaptophysin, the mechanism through which Mg2+ improved cardiac autonomic function could involve the prevention of synaptic degradation in diabetes. The effects of Mg2+ on hemodynamic impairment in diabetes seemed to be unrelated to the Mg2+ effects on the cardiac histological structure or on the changes in coronary perfusion. Moreover, the overall effects of Mg2+ in diabetes were independent of its effects on the blood glucose level or the alteration of plasma Mg2+ level. Thus, Mg2+ treatment may have long-lasting therapeutic effects on ventricular dysfunction and cardiac autonomic impairment in chronic diabetes, but further studies are needed to explore the precise underlying mechanisms.

Background: Ischemic heart diseases (IHD) are a leading cause of death among cardiovascular diseases. Unfortunately, the myocardial damage due to ischemia in IHD may be worsened by reperfusion therapy, a phenomenon called ischemic-reperfusion (I/R) injury. Coronary vascular damage is a key feature of I/R injury. Among the coronary vascular structures, the endothelial glycocalyx is a delicate polysaccharide and protein-rich layer that plays an important role in the regulation of vascular permeability, and is easily damaged during I/R. Sphingosine-1-phosphate (S1P) is a membrane phospholipid metabolite that has been shown to protect the heart against I/R. It has also been shown to regulate the synthesis of glycocalyx, but its effects on coronary endothelial glycocalyx damage and possible mechanism during I/R are unknown. Therefore, we hypothesized that S1P-induced cardioprotection is mediated by modulation of the glycocalyx during I/R in the isolated rat heart. Methods: Isolated male Wistar hearts were perfused on a Langendorff system with Krebs-Henseleit buffer via retrograde perfusion at constant temperature and pressure. The hearts were stabilized and pre-treated with S1P (10 nM for 7 minutes) before inducing 20 minutes of global ischemia, followed by 60 minutes reperfusion. Functional parameters were recorded throughout the protocol, including left ventricular developed pressure (LVDP), left ventricular end diastolic pressure (LVEDP), heart rate (HR) and coronary flow (CF). Ventricular infarct size was measured by using triphenyltetrazolium chloride stain. Coronary net filtration rate (NFR) was calculated as a ratio of the amount of transudate to CF. Cardiac edema was assessed by calculating the heart wet/dry weight ratio and histologically quantifying size of the interstitial compartment. The shedding of the glycocalyx was estimated by measuring the release of the glycocalyx component syndecan-1 in the coronary effluent using enzyme-linked immunosorbent assay (ELISA) and determining relative syndecan-1 staining intensity between groups in immuno-stained wax sections of perfusion-fixed hearts. In addition, the histo-morphology of the myocardium was assessed using hematoxylin and eosin staining. Results: The cardiac performance was depressed after I/R, as was reflected by decreased LVDP (P=0.02 vs. control), and an increased LVEDP (P<0.0001 vs. control). I/R also significantly increased infarct size (P=0.04 vs. control). Treatment with S1P before I/R significantly decreased infarct size (P=0.01 vs. I/R), but did not improve the post-ischemic decrease in LVDP or stabilize the LVEDP, and had no effect on CF. I/R significantly increased release of syndecan-1 in the coronary effluent (P=0.0002 vs. control). Immunohistochemically-stained imaging also revealed syndecan-1 staining intensity was significantly decreased or absent in ischemic hearts (P≤0.001 vs. control). Pretreatment with S1P had neither effect on syndecan-1 level in the coronary effluent nor on the intensity of syndecan-1 signal in immuno-stained sections (P=n.s vs. I/R). Histological analysis of cardiac edema revealed an increase in the extracellular area in ischemic hearts compared to the control hearts (P≤0.001 vs. control), and S1P treatment decreased the extracellular area (P≤0.01 I/R+S1P vs. I/R). The NFR, and heart wet/dry ratio were not significantly different post-reperfusion between the groups and S1P had no effect on these parameters. Conclusion: This study showed that pretreatment with S1P protects the heart against I/R injury, as was indicated by the decreased infarct size, and decreased extracellular cardiac edema. S1P had no effect on hemodynamic performance or the shedding of syndecan-1. These results suggest that S1P-induced cardioprotection is not mediated by protection of the glycocalyx via stabilization of syndecan-1. However, it is possible that S1P may stabilize other minor glycocalyx components which were not measured in this study, such as heparan sulphate and hyaluronic acid. This is the first study that evaluated syndecan-1 in the cardiac effluent of the isolated heart of rats with global ischemia, and the study opens up prospects for further investigation of the role of the glycocalyx in other models of I/R injury, such as the more clinically-relevant regional ischemia disease model.

INTRODUCTION: A dysfunction in fatty acid beta-oxidation (β-oxidation), particularly medium chain acyl-CoA dehydrogenase (MCAD) dysfunction is a major cause of mortality and its diagnosis is usually achieved by measuring specific protein activities or metabolites in blood and/or urine samples. However, these methods do not account for secondary defects that accompany primary deficiency; such as where measures of disruption in fatty acid metabolism do not account for defects in the TCA cycle and oxidative phosphorylation. These metabolic pathways are connected and dysfunction in one pathway (primary) could lead to dysfunction in the other (secondary). We propose the use of methods that combines all aspects of the bioenergetics module (enzyme activity in substrate oxidation within each individual pathway, transfer of electrons through the electron transport system (ETS) and oxidative phosphorylation for ATP generation) may be a more effective assessment technique. High resolution respirometry (HRR) is a recently developed technique that accounts for substrate oxidation, electron transfer via the ETS and oxidative phosphorylation. It measures the rate of oxygen consumption or flux at different respiratory states when appropriate substrates, uncouplers and inhibitors (SUIT protocols) are used. With this method, two substrate combinations are commonly used to assess medium-chain fatty acid β-oxidation; a) Octanoylcarnitine and carnitine, which is partial to the β-oxidation cycle alone, and b) Octanoylcarnitine and malate, which assesses the influence of the TCA cycle. Additionally, a combination of pyruvate, glutamate and malate is used to assess oxidation within the TCA cycle. We investigated the sensitivity of commonly used substrate combinations in HRR assessment to detect changes in mitochondrial respiration and dysfunction induced by the inhibition of either β-oxidation or the TCA cycle in C2C12 myotubes. Furthermore, we assessed MCAD, citrate synthase and aconitase enzyme activities when β-oxidation or TCA cycle was inhibited in C2C12 myotubes. METHODS: C2C12 myotubes were differentiated for 6 days and treated for 12 hours with a high or a low concentration of one of two inhibitors as follows; a) 2 mM or 8 mM 2-mercaptoacetate to inhibit medium chain acyl-CoA dehydrogenase (MCAD); b) 6 mM or 9 mM fluorocitrate to inhibit aconitase. Each treatment was compared to control myotubes grown for the same length of time without the addition of inhibitors. The activities of MCAD, aconitase and citrate synthase were determined. In addition, mitochondrial respiration measured as O2 flux at Routine, Leak, OXPHOS and ETS respiratory states were assessed in an Oxygraph-2K after inhibition or in control treatments using; i) Octanoylcarnitine and carnitine ii) Octanoylcarnitine and malate iii) pyruvate, malate and glutamate substrate combinations. For each assessment we corrected O2 flux recorded at each state to; a) approximate number of cells (pmol O2/s/million cells) b) protein concentration (pmol O2/s/mg protein) c) Flux control ratio (FCR) of each state to the maximum ETS capacity; ETSFAO+CI+CII (convergent electron flow from Fatty acid oxidation (FAO), Complex I (CI) and CII) d) FCR to either FAO-linked ETS capacity; (ETSFAO) or CI-linked ETS capacity (ETSCI). RESULTS: Treatment of cells with either a low or high concentration of 2-mercaptoacetate to inhibit MCAD resulted in no significant difference in MCAD activity. Fluorocitrate treatment decreased aconitase activity with low treatment (p = 0.011) compared to control, and conversely it increased MCAD activity in high treatment compared to control (p = 0.024). Both 2-mercaptoacetate (p = 0.03) and fluorocitrate (p < 0.01) treatment at high concentrations resulted in increased citrate synthase activity, compared to low concentration and control. Mitochondrial respiration with octanoylcarnitine and carnitine substrate combination was not altered with MCAD or aconitase inhibition. Octanoylcarnitine and malate substrate combination showed a decrease in mitochondrial respiration at the following respiratory states with both MCAD and aconitase inhibition; Routine (p = 0.01), LeakFAO (p = 0.029), OXPHOSFAO (p = 0.006), ETSFAO (p = 0.008), ETSFAO+CI (p = 0.017). FCR of each state to the maximum capacity (ETSFAO+CI+CII) revealed a decrease with both MCAD and aconitase inhibition at the following states; routine (p = 0.001), OXPHOSFAO (p = 0.003), ETSFAO (p = 0.018), ETSFAO+CAR (p = 0.008) and ETSFAO+CI (p = 0.027). Pyruvate, malate and glutamate substrate combination showed decreased mitochondrial respiration with MCAD inhibition at the following respiratory states; Routine (p = 0.004), LeakCI (p = 0.007), OXPHOSCI (p = 0.003), ETSCI (p = 0.003), ETSCI+FAO (p = 0.01) and ETSCI+FAO+CII (p = 0.003). FCR of each state to the maximum capacity (ETSCI+FAO+CII) decreased with both MCAD and aconitase inhibition at Routine (p = 0.024), OXPHOSCI (p = 0.024) and ETSCI (p = 0.035) states. DISCUSSION: The main finding of this study was related to two of the SUIT protocols 1) octanoylcarnitine and malate, and 2) pyruvate, malate and glutamate. These protocols were sensitive in showing decreased respiratory capacity and coupling control ratios and may be appropriate for assessing changes in oxidative metabolism when there is a defect in β-oxidation and/ or the TCA cycle. On the other hand, octanoylcarnitine and carnitine substrate combination is not sensitive to detect dysfunction induced by inhibition of either β-oxidation or TCA cycle. Irrespective of the enzyme inhibited, HRR detected dysfunction in complex I (CI), although, when aconitase was inhibited, reduced CI-linked respiration was more pronounced compared to MCAD inhibition. Furthermore, primary inhibition of MCAD to inhibit β-oxidation may have caused secondary inhibition of TCA cycle via aconitase, shown in decreased TCA cycle CI-linked respiration where MCAD was inhibited. In contrast, primary inhibition of aconitase seemed to be compensated for by increased MCAD activity and mitochondrial respiration related to β-oxidation. Lastly, enzyme assaysshould not be used as standalone techniques for assessing metabolic dysfunction at the level of TCA, β-oxidation and the mitochondria since they are not sensitive to low level defects, nor do they account for secondary interactions that influence either TCA or betaoxidation. HRR is useful to assess mitochondrial respiration and dysfunction, when using an appropriate substrate combination and should be used in combination with the more traditional enzyme activity assays.