Dissertations / Theses on the topic 'Physiology (medical)'

The blood oxygen level dependent (BOLD) functional magnetic resonance imaging (fMRI) signal is one of the most recently developed imaging techniques used to identify localized changes in brain activity. The BOLD signal is a function of cerebral blood flow (CBF), the cerebral metabolic rate of oxygen consumption (CMRO2), and cerebral blood volume (CBV). Normally coupled changes in these physiological parameters during regional increases in neural activity will result in BOLD signal increases. This makes the BOLD signal useful as a tool for identifying areas of increased neural activity. Moreover, it can also be used to infer changes in neurophysiology as a response to certain stimuli in the healthy brain, in individuals with certain neurological conditions affecting the cerebrovasculature and under the effect of some types of drugs. There is a major challenge to be faced when using the BOLD signal to study neurophysiological changes that occur under the effect of certain drugs or in the case of neurovascular disease. Under these circumstances, the underlying physiology and normally coupled vascular/metabolic response to a stimulus will be altered. Consequently, changes seen in the BOLD signal during regional increases in neural activity may not be associated with the same neurovascular changes that would occur in the healthy brain. Caffeine is an example of a widely used drug which has been shown to elicit such changes in the neurophysiological state. Two experiments were performed on a group of healthy volunteers in order quantify the effects of caffeine on both baseline neurophysiology and the BOLD signal changes evoked during a visual stimulus. The first involved the measurement of the oxygen extraction fraction(OEF) by performing venous blood magnetic resonance (MR) relaxometry and the second, the measurement of the BOLD and CBF response to a visual stimulus. The results obtained demonstrate that after caffeine consumption there is an increase in baseline OEF (OEF0) and a decrease in baseline CBF (CBF0), but a non-significant change in baseline CMRO2 (CMRO2,0). The caffeine-induced change in the individual BOLD and CBF response to a visual stimulus was found to correlate negatively with individual caffeine-induced change in CBF0. However, the average percent change in visually evoked BOLD and CBF signals across all subjects remained unaltered following caffeine consumption, whereas the CMRO2 response to a visual stimulus was found to increase.
L'effet BOLD (blood oxygenation level dependent) est l'une des plus récentes techniques d'imagerie par résonance magnetique (IRM) utilisée pour identifier les changements localisés d'activité cérébrale. Le signal BOLD varie en fonction de la circulation sanguine cérébrale (CBF), du taux métabolique cérébral de la consommation d'oxygène (CMRO2), et du volume sanguin cérébral (CBV). Les changements normalement-couplés de ces paramètres physiologiques lors d'une augmentation régionale d'activité neurale causeront une augmentation du signal BOLD. Cela rend le signal BOLD utile comme outil pour identifier les régions d'augmentation de l'activité neuronale. Deplus, le signal BOLD permet de déterminer quels changements neurophysiologiques suivent certains stimulus dans les cas suivants : le cerveau sain; le cerveau de personnes atteintes d'affections neurologiques affectant la cérébrovasculature; et le cerveau sous l'effet de certains types de médicaments. Un défi majeur se présente lorsque le signal BOLD est utilisé pour étudier les changements neurophysiologiques se produisant sous l'effet de certains médicaments ou en cas de maladie neuro-vasculaire. Lors de ce type d'utilisation, la physiologie sous-jacente et la réponse normalement-couplée vasculaire/métabolique à un stimulus est altérée. Par conséquent, les changements observés dans le signal BOLD au cours des augmentations régionales d'activité neuronale ne correspondent pas aux changements neurovasculaires qui se produisent normalement dans le cas du cerveau sain. La caféine est un médicament couramment utilisé qui a suscité de tels changements dans l'état neurophysiologique. Deux expériences ont été réalisées sur un groupe de volontaires sains dans le but de quantifier les effets de la caféine sur la neurophysiologie de base et les changements évoqués sur le signal BOLD lorsd'un stimulus visuel. La première expérience mesure la fraction de l'extraction d'oxygène (OEF) en effectuant de la relaxométrie par résonance magnetique sur le sang veineux. La deuxième expérience mesure la réponse du signal BOLD et CBFà un stimulus visuel. Les résultats montrent que, après la consommation de caféine il y a une augmentation de l'OEF, une augmentation de la réponse du CMRO2 au stimulus visuel, une diminution de la CBF de base (CBF0) et un changement non-significatif du CMRO2 de base (CMRO2,0). On observe une corrélation négative entre les changements générés par la consommation de caféine sur les réponses du signal BOLD et de la CBF au stimulus visuel et les changements générés par la consommation de caféine sur la CBF0. Néanmoins, en moyenne, la consommation de caféine ne génére aucun changement sur la réponse du signal BOLD et de la CBF au stimulus visuel.

The experiments described in this thesis comprise a series of related, yet independent investigations examining the physiological and metabolic responses of well-trained amateur cyclists under conditions designed to mimic actual competitive situations, during individual and mass start races. In Section A the physiological responses to constant load and steady state exercise are determined. In Section B, the metabolic factors associated with constant and variable load cycling performance are examined.

Bibliography
Thesis (MScMedSc (Biomedical Sciences. Medical Physiology))--University of Stellenbosch, 2010.
ENGLISH ABSTRACT: Male infertility is often associated with impaired sperm motility and morphology (asthenoteratozoospermia) for which there is no specific therapeutic treatment. It has come to light that the modification and expression of human sperm proteins play a crucial role in sperm function. In the present study, we present proteomic data of human spermatozoa in the context of sperm dysfunction. Novel techniques have been used to successfully isolate and identify differences in protein expression on a cellular level associated with asthenoteratozoospermia. In the first part of the study, differences in protein expression within the total sperm proteome were investigated between immature and mature sperm populations. Semen was collected from healthy donors (n=23) and separated into mature and immature sperm populations by 3-layer Percoll gradient centrifugation. Cells were washed and motility and morphology were measured by computer assisted sperm analysis (CASA). For the proteomic investigation cells were lysed and proteins separated by means of two-dimensional gel electrophoresis (2D electrophoresis). PD-Quest was used to identify the differentially expressed proteins. The protein spots of interest were excised and subjected to in-gel digestion. Peptides were separated by High Pressure Liquid Chromatography (HPLC) analysis and amino acid sequences determined by mass spectrophotometry. Proteins were identified by Mascot, using the Swiss Prot database. The results show that the motility (immature; 26.1±1.75% total motile cells vs. mature; 60.93±3.24% total motile cells; p<0.001) and morphology parameters (immature; 64.1±2.75% normal head morphology vs. mature; 87.63±3.24% normal head morphology; p<0.001) of the two populations differed significantly. After 2D electrophoresis, 16 differentially expressed protein spots were identified within the total sperm proteome between the immature and mature sperm populations. 56% of the differentially expressed proteins were more abundant in the immature sperm population compared to the mature sperm population. Functions have been ascribed to these proteins of which only four proteins, namely Tubulin -3C/D chain, Tubulin -2C chain, Outer dense fibre protein 2 and A-Kinase anchoring protein 4 precursor, were directly related to sperm motility and morphology. In the second part of the study the expression of nuclear proteins in human spermatozoa was investigated between immature and mature sperm populations. Semen was collected from healthy donors (n=156) and further separated from the seminal plasma by PureSperm® gradient centrifugation. The immature and mature sperm populations were retrieved and used during further analysis. For the proteomic analysis of nuclear proteins, cells were fractionated into four different subcellular protein fractions, instead of analyzing the whole sperm proteome. The results show that the motility (immature; 32.33±0.51% total motile cells vs. mature; 88.67±0.85% total motile cells; p<0.0001) and morphology parameters (immature; 13.51±0.87% normal head morphology vs. mature; 20.89±1.20% normal head morphology; p<0.0001) of the two populations differ significantly. After 2D electrophoresis, 21 differentially expressed nuclear proteins were identified between the immature and mature sperm populations. 95% of the differentially expressed nuclear proteins were less abundant in the immature population compared to the mature population. Only one nuclear protein namely 78kDa Glucose regulated protein was more abundant in the immature population compared to the mature population. Functions ascribed to these individual proteins were directly related to sperm motility, morphology and energy metabolism. In conclusion,In conclusion, in the current study novel techniques have been employed to investigate protein differences between immature and mature sperm populations. From these results it is evident that protein expression in the total sperm proteome and nuclear protein fraction is significantly different and incomplete in the immature population, compared to mature population. Based on these findings, it is recommended that further studies should be done on human spermatozoa to validate the role of the individual proteins in sperm function. Proteomics is an ideal tool to identify idiopathic causes of male infertility, as it can help to identify novel receptors (and signal transduction pathways) that can be used in the screening of drugs to alleviate sperm dysfunction.
AFRIKAANSE OPSOMMING: Manlike infertiliteit word dikwels geassosieer met verlaagde sperm motiliteit en morfologie (asthenoteratozoospermia) waarvoor daar tot dusver nog geen spesifieke terapeutiese behandeling is nie. Dit het aan die lig gekom dat die modifisering en uitdrukking van menslike sperm proteïene ‘n belangrike rol speel in spermfunksie. In die huidige studie stel ons data voor van proteiene in menslike sperme in die konteks van abnormale spermfunksie. Unieke tegnieke was gebruik om verskille in proteïen uitdrukking op sellulêre vlak suksesvol te isoleer en identifiseer wat verband hou met asthenoteratozoospermia. Tydens die eerste deel van die studie was verskille in proteïen uitdrukking binne die totale spermproteoom tussen onvolwasse en volwasse spermpopulasies ondersoek. Sperme van gesonde skenkers (n=23) is geskei in twee spermpopulasies (onvolwasse en volwasse sperme) deur middel van ‘n 3-laag Percoll gradiënt sentrifugasie tegniek. Selle is gewas en sperm motiliteit en morfologie is gemeet deur rekenaar geassisteerde sperm analise (CASA). Vir proteomiese analise is selle geliseer en proteïene geskei deur twee dimensionele gel elektroforese (2D-elektroforese). PD-Quest sagteware is gebruik om statisties beduidende proteïen verskille aan te dui. Die proteïene van belang is uitgesny en onderwerp aan in-gel vertering. Peptiede is geskei met behulp van hoë druk vloeistof chromatografie (HPLC) analise en aminosuurvolgordes is bepaal deur massa spektrofotometrie. Proteïene is geïdentifiseer met behulp van Mascot deur van die Swiss Prot databasis gebruik te maak. Die resultate toon dat die sperm motiliteit (onvolwasse; 26.1±1.75% totale motiele selle vs. volwasse; 60.93±3.24% totale motiele selle; p <0,001) en morfologiese parameters (onvolwasse; 64.1±2.75% normale kop morfologie vs. volwasse; 87.63±3.24% normale kop morfologie; p <0,001) tussen die twee populasies beduidend verskil. Na 2Delektroforese is 16 proteïen kolle geïdentifiseer wat beduidend verskil het, tussen die totale sperm proteoom van onvolwasse spermpopulasies en volwasse spermpopulasies. 56% van die proteïene wat beduidend verskil het, was meer uitgedruk in die onvolwasse spermpopulasie ten opsigte van die volwasse sperm populasie. Funksies is toegeskryf aan hierdie proteïene waarvan net vier proteïene naamlik Tubulin -3C/D ketting, Tubulin -2C ketting, Buite digte vesel proteïen 2 en A-Kinase anker proteïen 4 voorloper direk verband hou met sperm motiliteit en morfologie. In die tweede deel van die studie is die uitdrukking van nukluêre proteïene in menslike spermatozoa tussen onvolwasse en volwasse spermpopulasies ondersoek. Sperme was van gesonde skenkers (n=156) versamel en verder geskei van seminale plasma deur middel van ‘n PureSperm® gradiënt sentrifugasie tegniek. Vir die proteomiese analise van nukluêre proteïene is selle gefraksioneer in vier verskillende sub-sellulêre proteïen fraksies, in plaas van analise van die totale spermproteoom. Die resultate toon aan dat die sperm motiliteit (onvolwasse; 32.33±0.51% totale motiele selle vs. volwasse; 88.67±0.85% totale motiele selle; p <0,001) en morfologiese parameters (onvolwasse; 13.51±0.87% normale kop morfologie vs. volwasse; 20.89±1.20% normale kop morfologie; p <0,001) tussen die twee populasies beduidend verskil. Na 2D-elektroforese is 21 kern proteïen kolle geïdentifiseer wat betekenisvol uitgedruk was tussen onvolwasse en volwasse spermpopulasies. 95% van die nukluêre proteïene wat beduidend verskil het, was minder uitgedruk in die onvolwasse spermpopulasie ten opsigte van die volwasse spermpopulasie. Slegs een kern proteïen naamlik 78kDa Glukose gereguleerde proteïen was meer uitgedruk in die onvolwasse spermpopulasie in vergelyking met die volwasse spermpopulasie. Funksies is toegeskryf aan hierdie proteïene wat direk verband hou met sperm motiliteit, morfologie en energie metabolisme. Ten slotte, in die huidige studie is unieke tegnieke geïmplementeer om proteïen verskille tussen onvolwasse en volwasse spermpopulasies te ondersoek. Uit hierdie resultate is dit duidelik dat proteïen uitdrukking in die totale sperm proteoom en in die kern proteïen fraksie beduidend verskil en onvolledig is in die onvolwasse spermpopulasie ten opsigte van die volwasse spermpopulasie. Op grond van hierdie bevindinge word aanbeveel dat verdere studies op menslike sperme gedoen moet word ten einde die rol van individuele proteïene in sperm funksie te kan bepaal. Proteomika is ‘n ideale tegniek om die iodiopatiese oorsake van manlike infertiliteit te identifiseer, aangesien dit kan help in die identifisering van unieke reseptore (en seintransduksie paaie) wat gebruik kan word om sperm disfunksie te verbeter deur farmaseutiese behandeling.

Several factors may determine the rate. at which exogenous carbohydrate (CHO) is utilised by the human working muscles during prolonged (> 90 min moderate-intensity (63% of peak sustained power output [PPO]) exercise. These include i) the rate of gastric emptying of an ingested fluid, ii) the rate of digestion, absorption and subsequent transport of glucose into the systemic circulation, and iii) the rate of glucose uptake and oxidation by the working muscles. To test the hypothesis that the rate of gastric emptying is the primary factor limiting the rate of CHO delivery to the working muscles during exercise, uniformly labelled ¹⁴carbon (U-¹⁴C) tracer techniques were used in association with conventional gas exchange measurements and post-exercise gastric aspiration to compare the rates of gastric emptying, intestinal CHO delivery and ingested CHO oxidation from 15 g/100 ml solutions of glucose, maltose, a 22 chain-length glucose polymer, and an isocaloric 'soluble' starch preparation. Two groups of six highly-trained male cyclists or triathletes each ingested two of the test drinks which were given as a 400 ml loading bolus immediately before and then as eight 100 ml feedings at 10 min intervals during 90 min of continuous cycling at a work rate of 63% of PPO (~70% of maximal oxygen consumption [VO₂ₘₐₓ]).

Students Learn when they are actively engaged and thinking in class. The activities in this book are the primary classroom materials for teaching Anatomy and Physiology, sing the POGIL method. The result is an "I can do this" attitude, increased retention, and a feeling of ownership over the material.
https://dc.etsu.edu/etsu_books/1027/thumbnail.jpg

Ca2+-binding proteins (CaBP) alter Ca2+ signals, triggering cellular processes such as gene transcription regulation in neurons. CaBP1/CD is a calmodulin (CaM)-like Ca2+ binding protein that may regulate neuronal functions through interactions with effectors such as voltage-gated Ca2+ (Cav) channels and inositol trisphosphate receptors (InsP3Rs). To gain insight into the potential cellular functions of CaBP1/CD, we analyzed the expression and localization of CaBP1/CD variants in mouse brain. Of the three CaBP1/CD splice variants that have been characterized (CaBP1-S, CaBP1-L, and caldendrin (CD)), CD was the major variant expressed in mouse brain by western blot and quantitative polymerase chain reaction. These results reflected the expression of CaBP1/CD since they were not reproduced in mice with targeted disruption of the gene encoding CaBP1/CD (CaBP1 knock-out). By immunoperoxidase labeling, CaBP1/CD was localized in multiple cell-types including pyramidal cells in the cerebral cortex and hippocampal CA3 neurons and inhibitory neurons in the cerebellum. In the cerebellum, CaBP1/CD was not detected in Purkinje neurons but strongly colocalized with voltage-sensitive Shaker-type potassium channel, Kv1.2, in the pinceau formation formed between basket cells and the Purkinje cell axon initial segment. We conclude that CaBP1/CD is expressed in a subset of principal neurons where it may regulate Ca2+ signaling and neuronal excitability.

Exercise-induced muscle damage (EIMD) is known to be produced by novel or unaccustomed exercise, especially high force eccentric contractions. Histological myofibre disruption, force loss and muscle soreness are associated with EIMD and have implications for sporting performance. Traditional practices of assessing the extent of disruption to the myofibres is by performing needle biopsies and subsequently analysing the histology of the fibres. Recently there has been interest in investigating whether changes in force production and contractile properties of muscle following damaging exercise correlate strongly with the magnitude of disruption to the myofibres. The main aim of this study was to investigate whether changes in force production and contractile properties of muscle following damaging eccentric exercise correlated with myofibre disruption. In order to test the hypotheses set down in the study 56 mice (C57 BU/10 strain) were randomly assigned to two groups (active and passive). Each main group was then divided into 5 subgroups. Anaesthetised mice performed either 120 active (eccentric contractions) or passive (no muscle contraction) lengthening repetitions after which they were allowed to recover. The right foot was fixed to a foot plate housing a force transducer which was directly attached to the axle of a stepping motor. A stimulating electrode was surgically placed around the peroneal nerve and P. and 1/ 150 Hz ratios were determined. Animals in the active group then performed 5 bouts of 24 stimulated lengthening repetitions at 0.3 amps with a stimulation frequency of IOO Hz. The passive group's protocol was identical with the exception that no stimulation was provided. One repetition for both active and passive groups consisted of a 300 millisecond plantar flexion movement of the foot plate and a 4.7 second dorsi flexion recovery movement to the starting position. Active and passive subgroups were terminated at 3, 6, 10, 15 and 20 days following exercise, prior to which P. and 1/ 150 Hz ratio were determined. Tibialis anterior (TA) muscles were excised at this time from both exercised and contralateral limbs and prepared for later histological examination. Significant differences were evident between the two groups for Po following each bout of 24 lengthening repetitions, 10 minutes following lengthening and on days 3 and 20 of recovery. The only significant differences between the groups in 1/ 150 Hz ratio occurred 10 minutes following lengthening and at day six of recovery (p

Thesis (PhD (Biomedical Sciences. Medical Physiology))--University of Stellenbosch, 2010.
ENGLISH ABSTRACT: Psychosocial stressors have frequently been associated with an increased risk for developing The contributions of the cholinergic (Lobeline) and opioid (Naltrexone) systems in place preference behaviour were determined by employing a post-methamphetamine pharmacological treatment strategy. These two treatments failed to reverse the methamphetamine-induced place preference. However, administration of the drugs did lead to alterations in striatal dopamine and serotonin levels which may infer beneficial effects against the biochemical alterations induced by methamphetamine. We used both 2-D gel-based proteomics and isobaric tagging for relative and absolute quantitation (iTRAQ) to identify proteins in the frontal cortex, and nucleus accumbens shell and core of rats that were subjected to maternal separation, methamphetamine or both regimes. The proteins were associated with cytoskeletal modifications, altered energy metabolism, degenerative processes, interruptions in normal neurotransmission and enhanced intracellular signalling. We found that more proteins were quantitatively expressed in rats that were exposed to maternal separation followed by methamphetamine treatment than those animals subjected to the individual interventions independently. Additional proteins recruited by the combination of MS followed by MA which remained unchanged with independent treatments included malate dehydrogenase, V-type proton ATPase subunit E1, beta-synuclein, brevican core protein, eukaryotic translation initiation factor 4H, histidine triad nucleotide binding protein 1 and stress-induced phosphoprotein in the nucleus accumbens shell subregion. Additional proteins recruited in the core subregion with the combination treatment included thymosin beta-4, calretinin, Arpp-21 protein, alpha-synuclein, ubiquitin carboxylterminal hydrolase isozyme L1, cytochrome c, brain acid soluble protein 1, prosaposin and stress-induced phosphoprotein 1. Although, on a behavioural level via the use of CPP we found that MS did not exacerbate the rewarding effects of MA, the proteomic data does infer a role played by early life stress by the recruitment of additional proteins. We therefore propose that the molecular mechanisms by which early adverse events predispose animals to the addictive state may involve a complex assembly of cellular processes within the brain. depression, anxiety or substance abuse in adult life. Animal studies have also suggested that stressful experiences may result in altered behavioural responses to drugs of abuse as evidenced by enhanced cocaine self-administration and psychostimulant-induced hyperlocomotor activity. The main aim of our study was to establish whether adversity early in life would render individuals more vulnerable to later drug usage. We adopted maternal separation as our animal model of early life adversity and treated these animals with methamphetamine during the adolescent stage of their life. A conditioned place preference (CPP) paradigm was subsequently used to determine the rewarding effects of methamphetamine. To obtain an understanding of the underlying molecular mechanisms of methamphetamine-induced behaviour, we measured neurochemical changes on a neuroendocrine, neurotrophic, neurotransmitter and proteome level. Firstly, we established that methamphetamine-induced place preference behaviour lasted for at least 2 weeks after the last methamphetamine administration. Contrary to expectation, this behaviour was not affected by prior exposure to maternal separation. However, rats subjected to maternal separation caused a decrease in apomorphine-induced locomotor behaviour in methamphetamine-treated rats. Maternal separation therefore preferentially affected the behavioural repertoire of the dorsal striatum rather than that of the ventral striatum. A general down regulation of neuroendocrine activity (ACTH and corticosterone levels) was observed in animals subjected to maternal separation or methamphetamine treatment, as well as those subjected to the combination of the two interventions. Increased concentrations of plasma prolactin levels in maternally separated as well as normally reared animals subjected to methamphetamine-CPP were found which suggested a reduction in dopamine inhibition. Maternal separation resulted in increased NGF levels in the ventral hippocampus of methamphetamine treated rats. This suggested that the ventral hippocampus may particularly be vulnerable to the effects of early life stress. The increased neurotrophin concentrations may reflect a compensatory response to stress and drug exposure.
AFRIKAANSE OPSOMMING: Psigososiale stressors word gereeld geassosieer met ‘n verhoogde risiko vir die ontwikkeling van depressie, angs en dwelm misbruik in volwassenheid. Diere studies het ook al bewys dat vroeë lewensstres in die vorm van moederlike skeiding lei tot veranderde gedrag teenoor dwelm misbruik. Hierdie veranderde gedrag veroorsaak deur moederlike skeiding sluit die verhoodge kokaïne toediening en psigostimulant geinduseerde verhoging in lokomotoriese aktiwiteit in. Die hoofdoel van die studie was om vas te stel of vroeë lewensstres mense meer vatbaar laat vir latere dwelm misbruik. ‘n Moederlike skeidings diere model was gebruik om vroeë lewensstres voor te stel and het verder hierdie diere behandel met metamfetamiene gedurende adolesensie. Die gekondisioneerde plek voorkeur model was gebruik om die euforiese / verslawende effekte van metamfetamiene te bepaal. Om die onderliggende molekulêre meganismes van metamfetamien geinduseerde gedrag te verstaan het ons neurochemiese veranderinge op ‘n neuroendokriene, neurotrofiese, neurotransmissie en proteinvlak vasgestel. Eerstens het ons was gestel dat metamfetamien geinduseerde plek voorkeur vir ten minste twee weke na die laaste metafetamien toediening voortduur. In teenoorstelling met verwagting, het moederlike skeiding nie metamfetamien geinduseerde plek voorkeur beinvloed nie, maar eerder apo-morfien geinduseerde lokomotoriese aktiwiteit geaffekteer. Moederlike skeiding stres het by voorkeur die gedrags funksie van die dorsale striatum beinvloed eerder as die ventrale gedragsfunksie. ‘n Algemene afregulering van neuroendokriene aktiwiteit was waargeneem (adrenokortikotrofiene en kortikosteroon vlakke) in diere wat aan moederlike skeiding of metafetamien behandeling sowel as die kombinasie behandeling blootgestel was. Verhoogde plasma prolaktien vlakke was gevind in moederlike skeidings rotte sowel as kontrole diere wat verder blootgestel is aan metamfetamien behandeling wat ‘n inhibisie van die dopamiene sisteem toon. Moederlike skeiding het ook ‘n verhooging in neurotrofiene (NGF) in die ventrale hippokampus van metamfetamien behandelde rotte veroorsaak. Hierdie bevinding stel voor dat die ventrale hippokampus veral vatbaar is vir die effekte van vroeë lewensstres. ‘n Verhoging in neurotrofien konsentrasies mag ‘n kompenserende teenslag van die brein wees teen stres en dwelm blootstelling. Die bydrae van die cholinergiese (Lobeline) en opiaat (Naltrexone) sisteme in plek voorkeur gedrag was bepaal deur farmaseutiese behandeling te volg na metamftemien toediening. Lobeline en naltrexone was egter nie suksesvol om die metamfetamien geinduseerde plek voorkeur te wysig nie. Alhoewel die toediening van die twee behandelings het tot veranderinge in neurotransmissie (dopamiene en serotoniene) gelei wat moontlik tot voordelige effekte teen die biochemiese veranderinge van metamfetamien kan lei. Om veranderinge op proteinvlak in die frontale korteks en nukleus akkumbens middel en buitenste subareas vas te stel het ons gebruik gemaak van twee-dimensie gel elektroforese en isobariese merkers vir relatiewe en absolute kwantifisering (iTRAQ) gevolg deur massa spektrofotometrie. Geindentifiseerde proteine was geassosieer met sitoskeletale modifikasies, veranderde energie metabolisme, afbrekende prosesse, onderbrekings met normale neurotransmissie en intrasellulêre seintransduksie. Meer proteine was beduidend in die diere wat aan beide moederlike skeiding en metamfetamien behandeling blootgestel was. Addisionele proteine wat deur die kombinasie behandeling geaffekteer is in die buitenste subarea van die nukleus akkumbens sluit ‘malate dehydrogenase’, ‘V-type proton ATPase subunit E1’, ‘beta-synuclein’, ‘brevican core protein’, ‘eukaryotic translation initiation factor 4H’, ‘histidine triad nucleotide binding protein 1’ en ‘stress-induced phosphoprotein’ in. Additionele proteine geaffekteer in die middelste subarea van die nukleus akkumbens sluit ‘thymosin beta-4’, ‘calretinin’, ‘Arpp-21 protein’, ‘alpha-synuclein’, ‘ubiquitin carboxylterminal hydrolase isozyme L1’, ‘cytochrome c’, ‘brain acid soluble protein 1’, ‘prosaposin’ en ‘stress-induced phosphoprotein 1’ in. Vanuit ‘n gedrags benadering deur die gebruik van metamfetamien geinduseerde plek voorkeur het moederlike skeiding nie diere meer vatbaar gemaak vir die effekte van metamfetamien nie, maar die protein data wys wel dat vroeë lewens stres ‘n rol speel deur dat meer proteine geaffekteer word deur die kombinasie van moederlike skeiding gevolg deur later metamfetamien toediening. Ons stel voor dat die molekulêre meganismes waardeur vroeë lewensstres diere meer vatbaar maak vir die verslawende effekte van stimulante behels ‘n komplekse samestelling van sellulêre prosesse in die brein.

African runners dominate distance running both in South Africa and internationally. Therefore, the aim of this thesis was to compare selected exercise and skeletal muscle characteristics in well-trained African and Caucasian 10 km runners to determine if evidence exists of differences between these groups with respect to these physiological and biochemical characteristics. Furthermore, the relationship between exercise and skeletal muscle characteristics was investigated. Sedentary individuals from each population group were also studied to determine if differences existed in untrained skeletal muscle between groups. Maximal oxygen consumption and peak treadmill speed were measured using an incremental treadmill protocol whilst submaximal exercise characteristics were measured during a specifically designed protocol consisting of four sequential submaximal workloads relative to the peak treadmill speed of the individual. The final workload was maintained until fatigue with resistance to fatigue defined as total test time. Running economy was measured at a treadmill speed of 16.1 km/hr. Race pace characteristics were measured directly at race pace. Characteristics measured during exercise tests were oxygen uptake, minute ventilation, respiratory exchange ratio and heart rate whilst plasma lactate concentration was determined immediately after exercise. Skeletal muscle characteristics were determined by needle biopsy of the vastus lateralis muscle. Skeletal muscle enzymes citrate synthase, phosphofructokinase, 3-hydroxyacyl CoA dehydrogenase, hexokinase and carnitine palmityl transferase were assayed spectrophotometrically. Skeletal muscle buffering capacity was measured using by titration and fibre type proportions were analysed histochemically. Comparisons between groups were made with the Student's t-test for unpaired data whilst the relationships between variables were analysed using the Pearson's correlation coefficient. The first major finding was that when exercising at the same relative percentage of individual maximal treadmill velocity, African distance runners were able to exercise for longer than the Caucasians (1376±227 vs 1137±126 sec, p<0.01) with lower plasma lactate accumulation (4.8±3.2 vs 7.7±2.8 mmol/l,p<0.05). Time to fatigue was significantly related to a lower plasma lactate concentration (r=-0.63) and a lower respiratory exchange ratio (r=-0.53). The second major finding indicated that African runners were able to race 10 km at a higher percentage of their maximal oxygen uptake (93.5 vs 86.0%, p<0.005), whilst eliciting only a comparable plasma lactate concentration and respiratory exchange ratio. The third main finding was that the African runners were more economical than the Caucasian runners (p<0.05). The fourth main finding is that the African runners had a 50% greater activity of citrate synthase (p<0.005) and 3-hydroxyacyl CoA dehydrogenase (p<0.01) in the vastus lateralis than the Caucasians and this could not be explained by fibre type proportions, because the proportion of type I fibres was lower in the African runners (p<0.05). Citrate synthase activity, was related to the runners' ability to resist fatigue at high intensity relative to their individual peak treadmill velocity (r=0.70, p<0.05). A higher CS activity was related to a lower plasma lactate concentration and a lower RER. The sixth main finding of this thesis was that skeletal muscle buffering capacity of the Caucasian runners was higher than that of the African runners (p<0.05). A methodological study of buffering capacity in rats showed the buffering capacity was largely dependent upon fibre type and protein concentration, however these parameters could not explain the difference observed between the African and Caucasian runners. Furthermore, despite the differences in skeletal muscle characteristics observed between African and Caucasian runners in the current thesis, there was no evidence of these differences being inherently present in sedentary African and Caucasian individuals. In conclusion, the current series of studies do provide evidence of differences in selected exercise and skeletal muscle characteristics between African and Caucasian distance runners, with the African runners possessing exercise and skeletal muscle profiles that are considered to be more advantageous for endurance performance.

Glycogen depletion has frequently been shown to result in a decrease in respiratory exchange ratio (RER). However, the metabolic response to glycogen depletion has generally been studied in overnight fasted subjects or in subjects who were already fatigued, or hypoglycaemic, or both, raising the question of whether the differences seen were due to general "carbohydrate deficiency" or due specifically to muscle or liver glycogen depletion. If euglycaemia and especially hyperglycaemia is maintained, the " carbohydrate deficiency" is overcome. In addition, because insulin stimulates muscle glucose uptake and not liver glucose uptake during euglycaemia (except at very high concentrations), insulin infusion would differentiate between liver and muscle glycogen depletion, since if the decrease in RER previously observed is abolished with insulin infusion while euglycaemia is maintained, this would indicate that the decrease is specifically due to muscle glycogen depletion. Thus, the aim of this study was to investigate the metabolic effect of glycogen content while an adequate amount or an excess of carbohydrate was provided in the form of an intravenous glucose infusion and when plasma insulin concentrations are raised.

Kinaesthetic Learning Activities (KLA) are techniques for enhancing the motor learning process to provide a deep understanding of fundamental skills in particular disciplines. With KLA learning takes place by carrying out a physical activity to transform empirical achievements into representative cognitive understanding. In disciplines such as medical education, frequent hands-on practice of certain motor skills plays a key role in the development of medical students' competency. Therefore it is essential that clinicians master these core skills early on in their educational journey as well as retain them for the entirety of their career. Transferring knowledge of performing dexterous motor skills, such as clinical examinations, from experts to novices demands a systematic approach to quantify relevant motor variables with the help of medical experts in order to form a reference best practice model for target skills. Additional information (augmented feedback) on certain aspects of movements could be extracted from this model and visualised via multi-modal sensory channels in order to enhance motor performance and learning processes. This thesis proposes a novel KLA methodology to significantly improve the quality of palpation training in medical students. In particular, it investigates whether it is possible to enhance the existing abdominal palpation skills acquisition process (motor performance and learning) with provision of instructional concurrent and terminal augmented feedback on applied forces by the learner's hand via an autonomous multimodal displays. This is achieved by considering the following: identifying key motor variables with help of medical experts; forming a gold standard model for target skills by collecting pre-defined motor variables with an innovative quantification technique; designing an assessment criteria by analysing the medical experts' data; and systematically evaluating the impact of instructional augmented feedback on medical students' motor performance with two distinct assessment approaches(a machine-based and a human-based). In addition, an evaluation of performance on a simpler task is carried out using a game-based training method, to compare feedback visualisation techniques, such as concurrent visual and auditory feedback as used in a serious games environment, with abstract visualisation of motor variables. A detailed between-participants study is presented to evaluate the effect of concurrent augmented feedback on participants' skills acquisition in the motor learning process. Significant improvement on medical students' motor performance was observed when augmented feedback on applied forces were visually presented (H(2) = 6:033, p < :05). Moreover, a positive correlation was reported between computer-generated scores and human-generated scores, r = :62, p (one-tailed) < :05. This indicates the potential of the computer-based assessment technique to assist the current assessment process in medical education. The same results were also achieved in a blind-folded (no-feedback) transfer test to evaluate performance and short-term retention of skills in the game-based training approach. The accuracy in the exerted target force for participants in the game-playing group, who were trained using the game approach (Mdn = 0:86), differed significantly from the participants in control group, who trained using the abstract visualisation of the exerted force value (Mdn = 1:56), U = 61, z = -2:137, p < :05, r = -0:36. Finally, the usability of both motor learning approaches were surveyed via feedback questionnaires and positive responses were achieved from users. The research presented shows that concurrent augmented feedback significantly improves the participants' motor control abilities. Furthermore, advanced visualisation techniques such as multi-modal displays increases the participants' motivation to engage in learning and to retain motor skills.

G protein-coupled receptors (GPCRs) are seven-transmembrane domain receptors that sense extracellular signal and activate intracellular signaling pathways. The serotonin 5HT2A receptor (or 2AR) is one of the GPCRs coupled to Gq proteins, activating PLC and hydrolyzing PIP2. This hydrolysis causes a diffusion of bound PIP2 away from the channel binding site resulting in G protein-gated inwardly rectifying K+ channel (GIRK) inhibition and a downstream stimulation of Ca2+ release from endoplasmic reticulum stores. Previous experiments have demonstrated that the serotonin 5HTA receptor is a target of serotonergic psychedelic drugs, such as LSD, and partially mediates the action of many atypical antipsychotic drugs. However, the portion responsible for the functional activity and response of these drugs is unknown. The purpose of this study was to functionally characterize four deconstructed analogs of risperidone, an atypical antipsychotic agent, using two assays: by application to 5HT2A receptors in Xenopus oocytes and by calcium epifluorescence imaging in a HEK293 cell line stably expressing 2AR. Our experiments revealed that two analogs, RHV-006 and RHV-008, are partial agonists by themselves and greatly antagonize the effects of serotonin. RHV-006 and RHV-008 contain the piperidine and benzisoxizole ring systems of risperidone. RHV-023 and RHV-026, on the other hand, are more efficacious agonists than RHV-006 and RHV-008 but display a non-antagonistic effect with serotonin. RHV-023 and RHV-026 contain both the piperidine and benzisoxizole ring systems in addition to part of the diazabicyclo ring, thus containing more of risperidone’s structure than RHV-006 and RHV-008.

Thesis (PhD)--Stellenbosch University, 2014.
ENGLISH ABSTRACT: Insights into the role of oxidative stress and pancreatic β-cell dysfunction in the pathogenesis of type 2 diabetes (T2D) reveals an opportunity for the development of novel therapeutics that directly protect and preserve β-cells. The protective role of dietary antioxidants, such as plant polyphenols, against oxidative stress induced diseases, including T2D, is increasingly under scrutiny. Polyphenol-rich extracts of Cyclopia spp, containing mangiferin, may provide novel therapeutics. An aqueous extract of unfermented Cyclopia maculata, containing more than 6 % mangiferin, was assessed for its protective effect in pancreatic β-cells in vitro, ex vivo and in vivo under conditions characteristic of T2D. The effect of mangiferin was also evaluated in vitro and ex vivo, with N-acetyl cysteine (NAC) as an antioxidant control. In this study, we established in vitro toxicity models in RIN-5F insulinoma cells based on conditions β-cells are exposed to in T2D; i.e. lipotoxicity, inflammation and oxidative stress conditions. To achieve this, cells were exposed to the following stressors: palmitic acid (PA), a pro-inflammatory cytokine combination and streptozotocin (STZ), respectively. Thereafter, the ability of the C. maculata extract, mangiferin and NAC to protect RIN-5F cells from the effects of these stressors was assessed by measuring β-cell viability, function and oxidative stress. Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, adenosine triphosphate and annexin-V and propidium iodide assays. Cell function was evaluated by measuring glucose stimulated insulin secretion, cell proliferation and cellular calcium. To assess oxidative stress in the RIN-5F cells, diaminofluorescein-FM and dihydroethidium fluorescence, and superoxide dismutase enzyme activity were measured. The in vitro findings were then verified in isolated pancreatic rat islets using methods and models established in the RIN-5F experiments. The protective effect of the extract, NAC and metformin was assessed in STZ induced diabetic Wistar rats, using two treatment regimes, i.e. by treating rats with established diabetes and by pretreating rats prior to induction of diabetes by STZ. Glucose metabolism, oxidative stress and pancreatic morphology were assessed by performing an oral glucose tolerance test, measuring serum insulin, triglycerides, nitrites, catalase and glutathione. Hepatic thiobarbituric acid reactive substances and nitrotyrosine were also assessed. Immunohistochemical labelling of pancreata with insulin, glucagon and MIB-5 was used for morphological assessment. The extract improved β-cell viability, function and attenuated oxidative stress, most apparently in STZ and PA induced toxicity models comparable with NAC both in vitro and in isolated islets. Mangiferin was not as effective, showing only marginal improvement in RIN-5F cell and islet function, and oxidative stress. Pretreatment of STZ induced diabetic Wistar rats with extract was as effective as, if not better than, metformin in improving glucose tolerance, hypertriglyceridaemia and pancreatic islet morphology related to improved β-cell function. This study demonstrated that the aqueous extract of unfermented C. maculata was able to protect pancreatic β-cells from STZ and PA induced toxicity in vitro and ex vivo. In vivo, pretreatment with the extract improved glucose metabolism and pancreatic islet morphology in STZ induced diabetic Wistar rats.
AFRIKAANSE OPSOMMING: Insigte oor die rol wat oksidatiewe stres en pankreas β-sel disfunksie in die patogenese van tipe 2-diabetes (T2D) speel, bied 'n geleentheid vir die ontwikkeling van nuwe terapeutiese middels wat β-selle direk daarteen beskerm. Die beskermende rol van antioksidante in die dieët soos plantaardige polifenole teen oksidatiewe stres geinduseerde siektes soos T2D, is toenemend onder die soeklig. Polifenolryk ekstrakte van Cyclopia spp wat mangiferin bevat mag nuwe terapeutiese middels lewer. ‘n Waterekstrak van ongefermenteerde Cyclopia maculata wat meer as 6% mangiferin bevat, is ondersoek vir sy beskermende effek op pankreas ß-selle in vitro, ex vivo en in vivo teen kondisies kenmerkend aan T2D. Die effek van mangiferin is ook in vitro en ex vivo geëvalueer, met N-asetielsistien (NAC) as 'n antioksidant kontrole. In hierdie studie is in vitro toksisiteitsmodelle in RIN-5F insulinoomselle gevestig. Die modelle is gebaseer op toestande waaraan β-selle blootgestel word tydens T2D; d.w.s. lipotoksisiteit, inflammasie en oksidatiewe stres. Hiervoor is die selle aan die volgende stressors blootgestel: palmitiensuur (PA), ‘n pro-inflammatoriese sitokien mengsel en streptozotosien (STZ). Vervolgens is die vermoë van die C. maculata ekstrak, mangiferin en NAC om die RIN-5Fselle teen hierdie stressors te beskerm, beoordeel deur die meting van β-sellewensvatbaarheid, funksie en oksidatiewe stres. Sellewensvatbaarheid is bepaal met 3-(4,5-dimetielthiazol-2-yl)-2,5-difenieltetrazolium bromied, adenosientrifosfaat en anneksien-V and propidium jodied toetse. Selfunksie is geëvalueer d.m.v. glukose gestimuleerde insuliensekresie, selproliferasie en sellulêre kalsium bepaling. Oksidatiewe stres in die RIN-5Fselle is geëvalueer d.m.v. diaminofluorescein-FM en dihidroethidium fluoressensie bepalings, asook meting van superoksied dismutase ensiemaktiwiteit. Die in vitro bevindings is daarna in geїsoleerde rot pankreaseilande bevestig deur die metodes en modelle wat in die RIN-5F eksperimente gebruik is. Die antidiabetiese effekte van die ekstrak, NAC en metformien in STZ-geїnduseerde diabetiese Wistar rotte is bepaal d.m.v. twee behandlingsregimes, d.w.s. die behandeling van rotte met gevestigde diabetes of deur die behandeling voor die induksie van diabetes te begin. Glukose metabolisme, oksidatiewe stres en veranderinge in die pankreasmorfologie is ondersoek d.m.v. orale glukose toleransie toetse en die bepaling van serum insulien, trigliseriedes, nitriete, katalase en glutationien. Hepatiese tiobarbituursuur reaktiewe stowwe en nitrotirosien is ook geëvalueer. Immunohistochemiese kleuring van pankreas snitte is gebruik vir morfologiese assessering van insulien, glukagon en MIB-5. Die ekstrak het mees opvallend β-sel lewensvatbaarheid en funksie verbeter, terwyl oksidatiewe stres verminder is in die STZ- en PA-geїnduseerde toksisiteitmodelle. Bogenoemde effekte van die ekstrak in vitro en in die geїsoleerde eilande was vergelykbaar met die van NAC. Mangiferin was minder effektief, met slegs ‘n marginale verbetering in die funksie van RIN-5Fselle en eilande, asook t.o.v. oksidatiewe stres. Behandeling van die Wistar rotte met die ekstrak voor induksie van diabetes met STZ was net so effektief, of selfs beter as metformien in terme van verbeterde glukosetoleransie, trigliseriedvlakke en die morfologie van pankreas eilande wat verband gehou het met β-sel funksie. Hierdie studie het getoon dat die waterekstrak van ongefermenteerde C. maculata pankreas β-selle teen veral STZ- en PA-geїnduseerde toksisiteit in vitro en ex vivo beskerm het. In vivo het behandeling met die ekstrak voor en na induksie van diabetes, glukosemetabolisme en die morfologie van pankreas eilande in STZ-geїnduseerde diabetiese Wistar rotte verbeter.

Thesis (MScMedSc)--Stellenbosch University, 2014.
ENGLISH ABSTRACT: Introduction: Nitric oxide (NO) is an important chemical messenger in the cardiovascular system. Despite considerable progress in this field, there remains an on-going need for affordable and user-friendly NO measurement techniques. Therefore, in this study we aimed to develop and characterise NO-detection techniques not previously used in our laboratory, and, in addition, characterise an ex vivo method to measure the functional effects of the endothelium and NO production in the vasculature. Methods: Three different NO-detection techniques were compared: (i) Amperometric NO sensors. Here, NO-increasing effects of known NO synthase (NOS) activators were investigated (insulin, acetylcholine and biosynthetic human insulin). Three different NO sensors were evaluated on cultured endothelial cells and aortic tissue. Putative NOincreasing effects of shear stress were also investigated; (ii) Nitrite (NO2 -) + nitrate (NO3 -) sensors. Here, I aimed to measure NO release from cultured endothelial cells; (iii) Colorimetric NO2 - measurement assay with the Griess reagent. Here, NO2 - production by endothelial cells was measured with a plate reader. In the second part of the study an organ bath - isometric tension technique was established to measure endothelium-dependent function of aortic rings. Functional differences in aortic rings isolated from diet-induced obese rats compared to lean rats were investigated. Ring contraction was induced with phenylephrine and relaxation with acetylcholine. These investigations were further supported by western blot analyses of selected critical proteins. Lastly, the effects of perivascular adipose tissue (PVAT) on contraction and relaxation were investigated in endothelium-containing or denuded aortic ring segments. Results: Although some success was achieved with the amperometric sensors regarding calibration, any experimental results obtained were difficult to repeat due to instability of the sensors. With the NO2 -/NO3 - sensor we were not able to carry out any planned experiments due to failure to properly calibrate and standardise the sensors. Success was achieved with the Griess method. All the drugs used as positive controls (DEA/NO, fenofibrate, oleanolic acid and IL-1ß) proved to be potent inducers of NO2 - release from endothelial cells. Interestingly, the isometric tension studies showed a higher % relaxation in high fat (HF) diet aortic rings compared to those from lean animals. Western blot data showed downregulation of eNOS activation and iNOS expression in obese groups, which was suggestive of endothelial dysfunction. Interestingly, proteins associated with oxidative stress (p22phox and nitrotyrosine) were downregulated in obese groups. The presence of PVAT exerted anti-contractile effects on the rings from HF rats, however in denuded aortic rings, PVAT showed a significant pro-contractile response in both lean and HF groups. PVAT also exerted anti-relaxation effects in aortic rings from both lean and HF rats. Conclusion: We managed to successfully establish two new techniques for our laboratory (Griess method and the organ bath – isometric tension method) which can complement the more established techniques in our laboratory in order to aid us in future vascular research. Finally, the isometric tension technique used in the obese rat studies generated interesting data, which further assisted in characterising the dietinduced obesity rat model in our laboratory.
AFRIKAANSE OPSOMMING: Inleiding: Stikstofoksied (NO) is ‘n belangrike chemiese boodskapper in die kardiovaskulêre sisteem. Ondanks vordering in die veld, bestaan daar ‘n aangaande behoefte aan bekostigbare en gebruikersvriendelike NO-metingstegnieke. Gevolglik het ons in hierdie studie daarna gemik om NO-metingstegnieke wat nie vantevore in ons laboratorium beskikbaar was nie, te ontwikkel en karakteriseer. Verder het ons ten doel gehad om ‘n ex vivo model te karakteriseer om die funksionele effekte van vaskulêre endoteel en NO produksie te meet. Metodes: Drie verskillende NO-metingstegnieke was ondersoek: (i) Amperometriese NO sensors. Hier het ons die verhogende effekte op NO van bekende aktiveerders van NO sintetase (NOS) ondersoek (Insulien, asetielcholien en biosintetiese menslike insulien). Drie verskillende NO-sensors was ge-evalueer in gekultuurde endoteelselle en aortaweefsel. Die vermeende NO verhogende effekte van die wrywingskragte opgewek deur laminere vloei (“shear stress”) is ook ondersoek. (ii) Nitriet (NO2 -) + nitraat (NO3 -) sensors. Hier het ons beplan om NO-vrystelling deur gekultuurde endoteelselle te meet. (iii) Kolorimetriese meting van NO2 - met die Griess reagens. Hier het ons m.b.v. ‘n mikroplaat leser die NO2 - - vrystelling deur endoteelselle gemeet. In die tweede deel van die studie het ons ‘n orgaan bad–isometriese spanningstegniek opgestel om endoteelafhanklike funksie van aortaringe te meet. Funksionele verskille in aortaringe van vetsugtige rotte is vergelyk met kontrole rotte. Ringkontraksie is met fenielefrien geïnduseer en verslapping met asetielcholien. Hierdie ondersoeke is verder ondersteun deur Western blot analises van sleutelproteïene in die aortaweefsel. Laastens het ons die effekte van perivaskulêre vetweefsel (PVAT) op kontraksie en verslapping in aortaringe met of sonder intakte endoteel ondersoek. Resultate: Alhoewel ‘n mate van sukses behaal was met die kalibrasie van die amperometriese sensors, was eksperimentele resultate moeilik om te herhaal a.g.v. sensor-onstabiliteit. Geen eksperimente kon met die NO2 -/NO3 - sensors uitgevoer word nie weens ‘n onvermoë om ordentlike kalibrasie en standardisering uit te voer. Ons het egter wel sukses behaal met die Griess-metode. Al die middels wat as positiewe kontroles gebruik was (DEA/NO, fenofibraat, oleanoliese suur and IL-1ß) het geblyk kragtige induseerders van NO2 - produksie vanaf endoteelselle te wees. Die isometriese spanningsstudies het ‘n hoer % verslapping getoon in die hoë vet (HF) dieet aortaringe in vergelyking met die kontroles. Western blot data het ‘n afregulering van eNOS en iNOS getoon in die HF diere, wat aanduidend is van endoteel disfunksie, terwyl proteïene geassosieer met oksidatiewe stress (p22phox en nitrotirosien) afgereguleer was in die HF groep. Die aanwesigheid van PVAT het ‘n anti-kontraktiele effek gehad op die ringe van die HF groep. Toe die endoteel egter verwyder was, het PVAT in beide kontrole en HF ringe ‘n beduidende pro-kontraktiele effek gehad. Verder het PVAT ook anti-verslappingseffekte op aortaringe beide kontrole en HF rotte uitgeoefen. Gevolgtrekking: Ons het daarin geslaag om twee nuwe tegnieke vir ons laboratorium suksesvol te vestig (Griess metode en die orgaanbad-isometriese spanningstegniek) wat in die toekoms die meer gevestigde tegnieke in ons laboratorium kan komplementeer. Laastens het die isometriese spanningstegniek wat in die dieetstudies gebruik is, data opgelewer wat ons verder sal help om die vetsug model in ons laboratorium te karakteriseer.

Thesis (PhD)--Stellenbosch University, 2013.
ENGLISH ABSTRACT: Introduction: Coronary heart disease (CHD) is the leading cause of death worldwide with 3.8 million men and 3.4 million women dying globally each year. Although existing myocardial reperfusion strategies such as thrombolysis and percutaneous coronary intervention (PCI), if applied in a timely manner, limit myocardial infarct size, the mortality and morbidity remains significantly high. Ischaemic preconditioning (IPC) may offer the potential to attenuate myocardial ischaemia/reperfusion injury through cardioprotective signaling pathways which is recruited at the time of myocardial reperfusion, thereby improving clinical outcomes in patients with coronary artery disease. Ischaemic preconditioning is a phenomenon whereby short intermittent episodes of coronary occlusion followed by reperfusion protect the myocardium against a subsequent period of sustained ischaemia. This protection is reflected in the limitation of infarct size and improved functional recovery of the ischaemic heart during reperfusion. Despite intensive research efforts, the promise of an effective cardioprotective strategy using the endogenous protective mechanisms of the heart which underlies IPC, has not yet been materialized. Although progress has been made in terms of signaling mechanisms in the preconditioned heart, the identification of the myocardial reperfusion phase as the critical “window” for cardioprotection, requires the elucidation of the signal transduction pathways during the reperfusion phase after IPC. In view of the above, the aims of the present study were to investigate: i. the involvement of the RISK pathway and p38 MAP kinase pathway in IPC during early and late reperfusion ii. the involvement of heat shock protein-27 (HSP-27), heat shock protein-70 (HSP-70), GSK-3β, CAMKII, AMPK and the transcription factor CREB in the context of IPC during early reperfusion iii. the involvement of autophagy and apoptosis during early and late reperfusion after IPC iv. the correlation of the protein kinases with the hemodynamic parameters of the heart v. the mechanism of IPC by means of two-dimensional (2D) proteomics Methods: The isolated perfused working rat heart model was used with functional recovery as end-point. Hearts were preconditioned (IPC) for 3x5 min global ischaemia, alternated with 5 min reperfusion. Hearts were subjected to 25 min sustained global ischaemia, followed by 5, 10, 15 or 30 min reperfusion when hearts were snap-frozen for western blotting analysis. Alternatively, hearts were reperfused for 30 min to record hemodynamic parameters and measure functional recovery. Non-preconditioned (Non-IPC) hearts were stabilized for 30 min and subjected to 25 min sustained global ischaemia followed by 5, 10, 15 or 30 min reperfusion when hearts were snap-frozen. Alternatively Non-IPC hearts were reperfused for 30 min to serve as control for the 30 min reperfused IPC group. Activation of the protein kinases was determined by western blotting analysis. For the proteomic study mitochondrial and cytosolic proteins were isolated from heart tissue and separated in the first dimension by isoelectric focusing, followed by separation in the second dimension by two dimensional gel electrophoresis. The PD Quest software programme was used to identify significantly expressed protein spots. Protein spots of interest were excised and subjected to in-gel digestion and the resulting peptides were analysed by mass spectrometry. Proteins were identified by Mascot and the Swiss Prot database. Results: Western blotting analysis demonstrated that the RISK pathway and p38 MAPK are activated very early in reperfusion, but the activation is not sustained during the reperfusion period. Autophagy is also upregulated during this early reperfusion phase; it is attenuated in the middle reperfusion phase and increase for a second peak of upregulation in the late reperfusion phase. In addition, we identified CAMKII as a novel marker of functional recovery in IPC after reperfusion. The proteomic analysis identified twenty differentially expressed mitochondrial and thirty six differentially expressed cytosolic proteins between Non-IPC and IPC hearts. Functions ascribed to the majority of these individual proteins were directly related to cardiac metabolism. Conclusion: Activation of the majority of the protein kinases investigated in the present study is associated with the hemodynamic parameters of the heart instead of functional recovery. Results indicated that the variable signaling patterns could be attributed to differences in heart rate and the effect thereof (ejection fraction, minimum and maximum rate of contraction), as a result of sympathetic stimulation due to psychological stress in the animals before slaughtering. Proteomics results demonstrated that IPC hearts which failed after ischaemia /reperfusion are metabolically compromised and “worse off” compared to non-IPC hearts.
AFRIKAANSE OPSOMMING: Inleiding: Koronêre hartsiekte is die vernaamste oorsaak van sterftes wêreldwyd met 3.8 miljoen mans en 3.4 miljoen vrouens wat jaarliks sterf. Alhoewel bestaande miokardiale herperfusie strategieë soos trombolise en perkutane koronêre intervensie (PKI), wanneer betyds toegepas, miokardiale infarktgrootte beperk, bly mortaliteit en morbiditeit steeds hoog. Isgemiese prekondisionering (IPK) beskik oor die potensiaal om miokariale isgemie/herperfusie skade te verminder deur beskermende seinoordragpaaie tydens miokardiale herperfusie te aktiveer en sodoende die pasiënte wat aan koronêre arterie siekte ly, se prognose te verbeter. Isgemiese prekondisionering verwys na die verskynsel waartydens kort episodes van isgemie opgevolg deur herperfusie, die miokardium teen ‘n daaropvolgende langdurige isgemiese insident beskerm. Hierdie beskerming word gereflekteer in die beperking van infarktgrootte en verbeterde funksionele herstel van die isgemiese hart tydens herperfusie. Ten spyte van intensiewe navorsingspogings is die presiese meganisme van endogene beskerming tydens IPK nog nie ten volle ontrafel nie. Die identifisering van die miokardiale herperfusie fase se kritiese “vensterperiode” van beskerming, noodsaak ‘n volledige analise van die seinoordragpaaie wat geaktiveer word tydens die herperfusie fase na IPK. In die lig van bogenoemde, was die doel van die huidige studie om die volgende te ondersoek: i. die betrokkenheid van die RISK seinoordragpad en p38 MAP kinase tydens vroeë en laat herperfusie na IPK ii. die betrokkenheid van “heat shock protein-27” (HSP-27), “heat shock protein- 70” (HSP-70), GSK -3β, CAMKII, AMPK en die transkripsie faktor, CREB, in die konteks van IPK tydens vroeë herperfusie iii. die betrokkenheid van outofagie en apoptose tydens vroeë en laat herperfusie na IPK iv. die korrelasie van die proteïenkinases met die hemodinamiese parameters van die hart v. die meganisme van IPK deur middel van twee dimensionele proteomika Metodes: Die geïsoleerde werkende rothart model, met funksionele herstel as eindpunt, is gebruik. Harte is geprekondisioneer (IPK) met 3x5 min globale isgemie, afgewissel met 5 min herperfusie. Daarna is harte blootgestel aan 25 min volgehoue globale isgemie, gevolg deur 5, 10, 15 of 30 min herperfusie, waartydens harte gevriesklamp is. Alternatiewelik, is harte blootgestel aan 30 min herperfusie ten einde funksionele herstel te meet en hemodinamiese parameters te registreer. Nie-geprekondisioneerde (Non-IPK) harte is gestabiliseer vir 30 min, waarna dit onderwerp is aan 25 min volgehoue globale isgemie, gevolg deur 5, 10, 15 of 30 min herperfusie, waartydens harte gevriesklamp is vir westelike klad analise. Alternatiewelik, is Non-IPK harte onderwerp aan 30 min herperfusie om te dien as kontrole vir die 30 min IPK groep. Aktivering van die proteïenkinases is bepaal deur westelike klad analise. Vir die proteomiese studie, is onderskeidelik mitokondriale en sitosoliese proteïene geïsoleer en geskei in die eerste dimensie met behulp van isoelektriese fokusering, gevolg deur skeiding in die tweede dimensie met behulp van twee dimensionele gel elektroforese. Die PDQuest sagteware program is gebruik om proteïenkolle te identifiseer wat statisties beduidende verskille toon. Proteïenkolle van belang is uitgesny en onderwerp aan in-gel tripsinering en die peptiede wat sodoende verkry is, is deur middel van massa spektrometrie geanaliseer. Proteïene is geïdentifiseer deur Mascot en die Swiss Prot databasis. Resultate: Westelike klad analise het aangetoon dat die RISK pad en p38 MAPK geaktiveer is tydens vroeë herperfusie, maar die aktivering word nie volgehou tydens die hele herperfusie periode nie. Outofagie word gestimuleer tydens die vroeë herperfusie fase; dit word onderdruk in die middel herperfusie fase en bereik ‘n tweede piek van stimulering in die laat herperfusie fase. Die proteomiese analise het onderskeidelik twintig differensieel gereguleerde mitokondriale proteïene en ses en dertig differensieel gereguleerde sitosoliese proteïene geïdentifiseer tussen Non-IPK en IPK. Die grootste persentasie van hierdie proteïene is direk betrokke by miokardiale energie metabolisme. CAMKII is geidentifiseer as ‘n unieke merker van funksionele herstel in IPK tydens reperfusie. Gevolgtrekking: Aktivering van die meeste van die proteïenkinases wat ondersoek is in die huidige studie, is geassosieer met die hemodinamiese parameters van die hart, in plaas van funksionele herstel. Die resultate het aangetoon dat die varierende patrone van kinase aktivering toegeskryf kan word word aan verskille in harttempo en die effek daarvan (ejeksie fraksie, minimum en maksimum tempo van kontraksie), as gevolg van simpatiese stimulasie toegeskryf aan sielkundige stres in die diere voor slagting. Proteomiese analise het getoon dat IPK harte wat faal na isgemie/reperfusie metabolies gekompromiseer is en “slegter daaraan toe” is, in vergelyking met Non-IPK harte.

It has been well established that both carbohydrate-loading before and carbohydrate ingestion during exercise can enhance endurance performance by supplying carbohydrate for oxidation. However, the precise mechanism(s) underlying the proposed ergogenic effects of these procedures remain to be established. The studies in this thesis were therefore designed to examine the effects of carbohydrate-loading and carbohydrate ingestion on fuel substrate kinetics.

Thesis (PhD)--Stellenbosch University, 2012.
ENGLISH ABSTRACT: Background: Myocardial oxidative fuel supply is increased in obese conditions. How this metabolic environment and altered cardiometabolic phenotype associated with prediabetic obesity impacts on cardiac function and tolerance to ischaemia/reperfusion injury remains uncertain. While obese individuals are likely to be treated with PPARα agonists, controversy exists as to how activation of the PPARα receptor influences cardiovascular function and post-ischaemic recovery. Aims: To determine in a model of hyperphagia-induced obesity 1) whether protracted obesity is associated with left ventricular (LV) mechanical dysfunction; 2) the responsiveness of these hearts to insulin stimulation; 3) whether insulin can afford cardioprotection against ischaemia/reperfusion damage; and 4) how obesity and chronic PPARα agonist (K-111) treatment influences myocardial function, substrate metabolism, mitochondrial function and post-ischaemic outcomes. Methods: Male Wistar rats were fed standard rat chow or a high caloric diet. 1) In vivo LV mechanical function was assessed echocardiographically in 32 week fed animals. Ex vivo LV function was measured in the presence of glucose, insulin and/or fatty acid (FA); 2) Ex vivo myocardial insulin sensitivity was assessed by measuring insulin stimulated glycolytic flux in 16 week fed rats. Insulin was also administered prior to and during regional ischaemia to determine its effect on post-ischaemic function and infarct size; 3) K-111 was added to the drinking water during the last 10 weeks of feeding (feeding period of 18 weeks); a) Ventricular mitochondrial function was determined polarographically in the presence of either glutamate or palmitoyl-L-carnitine as substrates; b) Myocardial carbohydrate and lipid metabolism, and in a separate series of perfusions, myocardial infarct size were determined in the presence of physiological or high insulin (30 or 50μIU/ml) and FA (0.7 or 1.5mM) concentrations. Results: 1) Obese animals maintained normal in vivo LV mechanical function. Glucose perfused hearts from obese animals had depressed aortic outputs compared to the control group (32.58±1.2 vs. 46.17±0.91 ml/min; p<0.001) which was abolished by the presence of FA; 2) Hearts from obese animals had reduced insulin stimulated glycolytic flux rates (1.54±0.42 vs. 2.16±0.57 μmol/g ww/min, p<0.01). Although insulin reduced infarct size in the obese group (20.94±1.60 vs. 41.67±2.09 %, p<0.001), its cardioprotective effect was attenuated in the presence of FA; 3) By simulating the in vivo metabolic environment of control and obese animals in ex vivo perfusions, elevated insulin and FA levels associated with obesity increased infarct sizes in the obese group compared to the control group (47.44±3.13 vs. 37.17±2.63 %, p<0.05); 4) While chronic K-111 treatment reversed systemic metabolic abnormalities associated with obesity, neither obesity nor the drug influenced myocardial and mitochondrial function or postischaemic outcomes. K-111 was able to reduce palmitate oxidation in the obese group. Conclusion: Elevated levels of circulating FFA may be important in maintaining normal LV mechanical function in the obese condition. While obesity had no impact on myocardial mitochondrial function and post-ischaemic outcomes during comparable perfusion conditions, the specific metabolic environment associated with obesity may augment post-ischaemic injury. K-111 is effective in reducing obesity related metabolic abnormalities, but has no effects on myocardial function, mitochondrial function or ischaemic tolerance.
AFRIKAANSE OPSOMMING: Agtergrond: Miokardiale oksidatiewe substraat voorsiening is verhoog in vetsug. Hoe hierdie metaboliese omgewing en veranderde miokardiale metaboliese fenotipe in prediabetiese vetsug miokardiale funksie en iskemie/herperfusie skade beïnvloed, is onseker. Alhoewel vetsugtige individue met PPARα agoniste behandel kan word, is die resultate verkry van hierdie reseptor aktivering op miokardiale funksie en iskemiese skade teenstrydig. Doelwitte: Om te bepaal of 1) verlengde vetsug linker ventrikulêre (LV) funksie beïnvloed; 2) hierdie harte sensitief vir insulien stimulasie is; 3) insulien die hart teen iskemie/herperfusie beskadiging beskerm; en of 4) vetsug en chroniese K-111 behandeling miokardiale funksie, substraat metabolisme, mitochondriale funksie en post-iskemiese herstel in vetsugtige, insulienweerstandige rotte beïnvloed. Metodes: Manlike Wistar rotte is met gewone rotkos, of ʼn hoé kalorie dieet gevoer. 1) In vivo LV funksie in 32 week gevoerde rotte is met behulp van eggokardiografie bepaal. Ex vivo LV funksie is met of sonder insulien en/of vetsure in die perfusaat bepaal; 2) Die ex vivo insuliensensitiwiteit is in 16 weke gevoerde rotte bepaal deur miokardiale glikolise te meet. Insulien is ook voor en tydens streeksiskemie toegedien, ten einde sy effek op miokardiale beskerming te bepaal; 3) K-111 is in die drink water van rotte toegedien vir die laaste 10 weke van hul dieet (voedingsperiode van 18 weke); a) Ventrikulêre mitochondriale funksie is polarografies bepaal in die aanwesigheid van glutamaat of palmitiel-L-karnitien; b) Miokardiale koolhidraat- en lipied metabolisme, en in ʼn aparte groep rotte, infarktgrootte, is bepaal in die teenwoordigheid van fisiologiese of hoë insulien- (30 of 50μIU/ml) en vetsuurvlakke (0.7 of 1.5mM). Resultate: 1) Vetsugtige rotte het normale in vivo LV funksie gehandhaaf. Glukose geperfuseerde harte van vet rotte se LV funksie was laer as die van kontroles (Aorta omset: 32.58±1.2 vs. 46.17±0.91 ml/min; p<0.001), maar dit het verbeter in teenwoordigheid van vetsure; 2) Harte van vetsugtige rotte het verlaagde insuliengestimuleerde glikolise getoon (1.54±0.42 vs. 2.16±0.57 μmol/g ww/min, p<0.01). Alhoewel insulien infarktgrootte in die vetsugtige groep verlaag het (20.94±1.60 vs. 41.67±2.09 %, p<0.001), is sy beskermende effekte in die teenwoordigheid van vetsure verlaag; 3) deur die in vivo metaboliese omgewing van kontrole en vetsugtige rotte in die perfusaat van die harte ex vivo te simuleer, is dit aangetoon dat die verhoogde vlakke van insulien en vetsure, geassosieer met vetsugtigheid, infarktgroottes in die vetsugtige groep teenoor die kontrole groep verhoog het (47.44±3.13 vs 37.17±2.63 %, p<0.05); 4) Hoewel chroniese gebruik van K-111 die metaboliese abnormaliteite gepaardgaande met vetsug normaliseer het, het beide vetsug en die middel geen invloed op miokardiale of mitochondriale funksie of vatbaarheid vir iskemiese beskadiging gehad nie. K-111 het miokardiale palmitaatoksidasie in die vetsugtige behandelde groep verlaag. Gevolgtrekking: Verhoogde bloed vetsuurvlakke in vetsug mag n belangrike rol in die handhawing van sistoliese funksie speel. Dit blyk dat die spesifieke in vivo omgewing geassosieer met vetsug wel tot verhoogte vatbaarheid vir iskemie/herperfusie skade mag lei. K-111 is effektief om die sistemiese metaboliese abnormaliteite gepaard met vetsugtigheid te verbeter, maar het geen effek op miokardiale funksie, mitochondriale funksie of vatbaarheid vir iskemie gehad nie.

Acute myocardial infarction (AMI) is the leading cause of death worldwide. Currently, the best method of treating cardiac ischemia is early reperfusion which, itself, induces myocardial damage. The mTOR complex is a key regulator of cardioprotection against cell stressors. We hypothesized that reperfusion therapy with Rapamycin, a potent mTOR inhibitor, would reduce infarct size in adult mouse hearts. Rapamycin was administered at the onset of reperfusion following 30 min in situ LAD ligation. After 24 hours of reperfusion, myocardial infarct size and apoptosis were significantly reduced in rapamycin-treated mice compared to control. Rapamycin inhibited pro-apoptotic protein Bax and phosphorylation of ribosomal protein S6 (target of mTORC1), while it induced phosphorylation of AKT (target of mTORC2). Rapamycin also induced phosphorylation of ERK, while significantly reduced phosphorylation of p38. Thus, our study shows that reperfusion therapy with Rapamycin provides cardioprotection through induction of the phosphorylation of Akt and ERK.

The measurement and analysis of the human diaphragm electromyogram (EMGdi), as obtained with an esophageal electrode, requires objective control of the disturbances and filtering effects which can influence the signal. One issue of importance is that an increase in the muscle-to-electrode distance (MEdist) acts as a low-pass filter, filtering out the high frequency components of the EMG power spectrum (the MEdist filter). Due to the numerous factors which can influence the EMGdi, control of signal quality is also of utmost importance. The aims of this study were: (1) to evaluate the effect of the MEdist filter on EMGdi, as measured with a multiple array esophageal electrode, (2) to take advantage of the MEdist filter in order to locate the position of the diaphragm with respect to the electrodes, and (3) to evaluate the influence of changes in chest wall configuration on EMGdi center frequency (CF) values, while controlling for both signal quality and the MEdist filter.
Five normal male subjects performed static contractions of the diaphragm at seven predetermined chest wall configurations. The EMGdi was measured with an array of eight steel rings mounted on a catheter, forming seven sequential pairs of electrodes, with an interelectrode distance of 10 mm. EMGdi signal quality was evaluated by computer algorithms. The pair of electrodes whose EMGdi signals (and power spectrums) were the least influenced by the MEdist filter was assumed to be closest to the diaphragm.
The results of the study indicated (1) EMGdi power spectrums and their associated CF values were strongly affected by the position of the diaphragm with respect to the multiple array esophageal electrode. CF decreased by approximately 1 Hz per mm displacement away from the diaphragm. (2) By controlling for the MEdist filter, there was no relationship found between changes in chest wall configuration and CF values. These data demonstrate that changes in chest wall configuration, and hence diaphragm length, do not influence the CF values of the EMGdi, if the distance between the electrodes and the diaphragm and signal quality are controlled for.

From our everyday life, we know that our hearts beat with a rhythm which is not perfectly periodic. Even an isolated spontaneously beating cardiac cell, devoid of neural, hormonal, and intracardiac regulatory input, does not beat perfectly regularly. I investigate the hypothesis that the beat-to-beat fluctuations in transmembrane potential of spontaneously beating cardiac cells are due to stochastic gating of the ionic channels in the cell membrane.
Recordings of transmembrane potential from small clusters of spontaneously beating 7-day-old embryonic chick ventricular cells were analyzed to characterize the voltage waveform and the regularity of beating. I constructed a deterministic Hodgkin-Huxley-type ionic model which reproduces spontaneous activity in our experimental recordings, as well as the experimental results of applying various ion channel blockers (D-600, almokalant, and Ba2+). The model consists of six currents: a calcium current (ICa), three potassium currents (IKs, I Kr, IK1), a background current ( Ib), and a seal-leak current (I seal).
The deterministic Hodgkin-Huxley-type model was then reformulated into a stochastic single-channel model. The single-channel model reproduces the irregularity of beating seen experimentally: e.g. the coefficient of variation of interbeat interval was 4.4% vs. 3.9% in the clusters. In the model, IKs is the current giving the major contributions to fluctuations in interbeat interval.
Phase resetting of the spontaneous activity of cardiac pacemaker cells by a brief stimulus pulse was simulated in Hodgkin-Huxley-type models and single-channel models of slow-upstroke (central) and fast-upstroke (peripheral) rabbit sinoatrial node cells. In the Hodgkin-Huxley-type models the phase-resetting response is continuous, but can be extremely delicate in the fast-upstroke model, in that a tiny difference in the stimulus timing can change the stimulus response from a delayed action potential to an advanced one. Therefore, the noise in the fast-upstroke single-channel model can cause a stimulus with fixed amplitude and fixed timing to have widely different effects: sometimes it will induce an action potential but in other cases it will delay an action potential, as seen previously in experiments on cardiac preparations.

Thesis (PhD (Biomedical Sciences. Medical Physiology))--Stellenbosch University, 2008.
The overall objective of this thesis is to provide additional data to assist clinicians and experimental neurologists alike in the quest for better understanding, more accurately diagnosing and more successfully treating patients suffering from Parkinson’s disease (PD). The general theme of the thesis is the interaction between certain environmental stimuli, including the exposure to adverse events during early central nervous system (CNS) development and the manifestation of elements of neurodegeneration, whether by means of neurochemical changes or expressed as a dysfunctional voluntary motor system. The first chapter provides a general introduction to the research theme of the thesis. This includes, in particular, a discussion on current understanding concerning the etiology and clinical profile of PD, the relative contribution made by genetic factors compared to environmental ones, and current treatment strategies for treating the disease. Mention is also made of the failure of these therapeutic applications for reversing or protecting against the disease, due to the side-effects associated with them. The material covered in chapter 1 provides the basis for the more complete discussion concerning these various aspects, contained in the chapters to follow. The overall aim was also to characterise the effects of commonly used toxin-induced animal models of PD, and the extent of vulnerability that the CNS displays towards them. The destruction of dopaminergic neurons following the administration of 6-OHDA at targeted points along the nigrostriatal tract is used extensively to model PD pathology in rats and is an established animal model of the disease. However, mature or even aged animals are mainly used in these studies, while the effects that the toxin might have on the developing CNS remain unclear. The study reported in chapter 4 aimed to elucidate some of 6-OHDA’s actions on the young adolescent (35 days-old) CNS by comparing the motor and biochemical effects of a unilateral infusion of the toxin into two anatomically distinct basal ganglia loci: The medial forebrain bundle (MFB) and the striatum. Animals were randomly assigned to receive either a direct delivery of 6-OHDA (12μg/4μl) into the MFB or an indirect injection, into the striatum. Although both lesion types were used, the MFB model is considered a more accurate portrayal of end-stage PD, while the striatum-model better reflects the long-term progressive pathology of the disease. The different lesions’ effects on motor function were determined by observing animal’s asymmetrical forelimb use to correct for weigh shifting during the vertical exploration of a cylindrical enclosure. Following the final behavioral assessment, the concentration of dopamine (DA) and DA metabolites remaining in the post-mortem brains were determined using 4 HPLC electrochemistry (HPLC-EC) and the levels compared between the two groups. The HPLC-EC results revealed a compensatory effect for DA production and DA turnover on the lesioned hemisphere side of the toxin-infused animal group. Thus, following 6-OHDA treatment, there appears to be extensive adaptive mechanisms in place within the remaining dopaminergic terminals that may be sufficient for maintaining relatively high extracellular and synaptic concentrations of DA. However, since substantial changes in motor-function were observed, it is suggested that the capacity of the remaining dopaminergic neurons to respond to increased functional demands may be limited. In addition, the behavioral results indicate that the distinct indices relating to different functional deficits depend on the lesioning of anatomically distinct structures along the nigrostrial tract. It has long been known that far fewer women are diagnosed with PD than men are. This seeming protection offered to females against degenerative disease of the CNS may relate to estrogen, although the hormone’s mechanism of action on the dopaminergic system is poorly defined. With an estimated 10-15 million women using oral contraceptives (OCs) in the United States alone, the aim of chapter 2 was to examine the evidence for a possible relationship between PD and the female reproductive hormone estrogen. A review of the current literature available on the topic was performed by consulting Medline, and by performing a search of the case-reports contained within the World Health Organization’s (WHO) International Drug Monitoring database, for possible PD-related symptoms that may arise from estrogen replacement therapy (ERT). The results, whilst conflicting, seem to suggest that estrogen protects women from obtaining the disease, or at least some features of it. Intensive research efforts are called for, with sufficient power to establish the relationship between ERT and the onset and development of parkinsonism. Chapter 3 reports on the results obtained from an experiment that subjected young Sprague-Dawley rats, 35 days of age, to a lower and a higher dose of 6-OHDA delivered to the MFB. Control rats received equivalent saline infusions. At 14 days post-surgery, the rats were evaluated for forelimb akinesia. For the higher dose of 6- OHDA the female rats were less impaired than males in making adjustment steps in response to a weight shift and in the vibrissae-evoked forelimb placing test. In addition, Tyrosine hydroxylase (TH) immunoreactivity was significantly higher for the female rats. Early gender differences in cell survival factors and/or other promoters of neuroplasticity may have contributed to the beneficial outcome seen in the females. For example, nerve growth factor (NGF) was found to be higher in the female rats following administration of the DA neurotoxin. It is unclear whether gonadal steroids are involved, and, if so, whether female hormones are protective or whether male hormones are prodegenerative. Determining the mechanisms for the improved outcome seen in the young female rats may lead to potential treatment strategies against PD. 5 Many studies have shown that early life stress may lead to impaired brain development, and may be a risk factor for developing psychiatric diseases, including clinical depression. However, few studies have investigated the impact that early stress may have on the onset and development of neurodegenerative disorders such as PD. The study reported on in chapter 5 conjointly subjected rat pups to a maternal separation (MS) paradigm that is a well characterised model of adverse early life events, and a unilateral, intrastriatal injection of 6- OHDA. The combined effects of these models on motor deficits and brain protein levels were investigated. Specifically, the animals were assessed for behavioral changes at 28 days postlesion with a battery of tests that are sensitive to the degree of DA loss sustained. The results show that animals that had been subjected to MS display poorer performance in the vibrissae and single-limb akinesia test compared to non-MS control animals (that had also been subjected to the toxin exposure). In addition, there was a significant increase in the loss of TH staining in MS rats compared to non-MS ones. The results from this study therefore suggest that exposure to adverse experiences during the early stages of life may contribute towards making dopaminergic neurons more susceptible to subsequent insults to the CNS occurring during mature stages of life. Therefore, taken together, early exposure to stress may predispose an individual towards the onset and development of neurodegenerative disease, which especially becomes a threat during the later stages of adult life. Moreover, within the framework of these characteristics, the capacity of a widely-used pharmacological agent (statins) was tested for possible future therapeutic application in PD (chapter 7). Although the precise cause of sporadic PD remains an enigma, evidence suggests that it may associate with defective activity of complex I of the mitochondrial electron transport chain. Mitochondrial DNA transmit and express this defect in host cells, resulting in increased oxygen free radical production, depressed antioxidant enzyme activities, and greater susceptibility to apoptotic cell death. Simvastatin is a member of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) group of drugs that are widely used for lowering cholesterol levels in patients who display elevated concentrations of low-density lipoprotein cholesterol. The study aimed to investigate the effects that statin-treatment have on motor-function and at the mitochondrial-protein level, using rotenone, a mitochondrial complex I inhibitor, as a rat-model of PD. Adult male Sprague-Dawley rats were treated either with simvastatin (6mg/day for 14 days) or with a placebo. Two different tests to assess motor function were used: The apomorphine-rotation test, and the vibrissae-elicited forelimb placement test. Following the drug administration protocol, the nigrostriatal tract was unilaterally lesioned with either rotenone (3 μg/4 μl) or, for the controls, were sham-operated by infusing the vehicle (DMSO:PEG) only. Five days later the rats were killed and a highly purified concentration of isolated mitochondria was prepared from the substantia nigra (SN) sections. 2- 6 Dimensional electrophoresis (2-DE) with subsequent identification of the spots using electronspray ionization quadruple time-of-flight mass spectrometrical (ESI-Q-TOF MS) was performed and the results BLAST-searched using bio-informatics tools for naming the identified peptides. The motor test results indicate that while unilateral rotenone causes behavioral asymmetries, treatment with simvastatin improved motor function relative to the rotenoneinduced ones. Mass Spectroscopy identified 23 mitochondrial proteins that differ significantly in protein expression (p < 0.05) following simvastatin treatment. The altered proteins were broadly classified according to their cellular function into 6 categories, with the majority involved in energy metabolism. This study effectively illustrated how neuroproteomics, with its sophisticated techniques and non-biased ability to quantify proteins, provides a methodology with which to study the changes in neurons associated with neurodegeneration. As an emerging tool for establishing disease-associated protein profiles, it also generates a greater understanding as to how these proteins interact and undergo post-translational modifications. Furthermore, due to the advances made in bioInformatics, insight is created concerning their functional characteristics. Chapter 4 summarises the most prominent proteomics techniques and discuss major advances made in the fast-growing field of neuroproteomics in PD. Ultimately, it is hoped that the application of this technology will lead towards a presymptomatic diagnosis of PD, and the identification of risk factors and new therapeutic targets at which pharmacological intervention can be aimed. The final chapter (chapter 8) provides a retrospective look at the academic work that had been performed for the purpose of this thesis, recaps on the main findings, and also highlights certain aspects of the project and provides relevant suggestions for future research. Lastly, the appendix provides a detailed overview of the methods followed for the experiments described in this thesis. It provides not only a comprehensive description of the techniques that had been followed, but provides information concerning the care taken with the animals (i.e. post-surgery) in order to control for the potential influence of experimental variables on the results.

Aim The purpose of this study was to examine high versus low resistance training loads performed to muscular failure and its effect on muscular strength, power and strength endurance. Method 11 men and 3 women (age 26,4 ±4,4 years, weight 79,9 ±10,7 kg, height 179,4 ±76 cm) were recruited to train for 2 days/week for 8-weeks in the leg press and leg extension. One leg was randomly allocated to a high load (HL) program performing 3-5 reps and the other leg was allocated to the low load (LL) program, performing 20-25 reps. All sets were executed to muscular fatigue. The participants were measured for 1RM strength, strength endurance and muscular power before and after the study. Results HL and LL leg significantly improved strength gains in the LP exercise by 20,3%, respectively 21%, P< 0,001, but no difference was noted between legs P= 0,876. HL displayed significant increases in the LE exercise by 10,3%, P< 0,05, while no significant improvement occurred for the LL leg, -2,7%, P> 0,05. Strength remained insignificantly similar between protocols P> 0,05. The mean power results indicated no significant improvements within protocols, HL P= 0,309, LL P= 0,112. There was also no significant difference between the two protocols after the intervention P= 0,646. As for muscular strength endurance, the LL performed more repetitions which was significantly greater than for the HL leg 26,5 reps, respectively 23,9 reps, P= 0,045. Conclusion This study concludes that similar strength gains can be accomplished when training with heavier or lighter loads as long as all resistance training is performed to muscular failure. It was also determined that performing lower loads to failure is superior for local strength endurance. Finally, traditional resistance training has no benefit for augmenting muscular power whether training with higher or lighter loads to exhaustion

Kursen Projektarbete 

Thesis (MScMedSc)--Stellenbosch University, 2012.
ENGLISH ABSTRACT: Introduction: The worldwide escalation in the incidence of obesity and its strong association with insulin resistance, type 2 diabetes and the cardiovascular complications that accompany these disease states have elicited interest in the underlying mechanisms of these pathologies. Preliminary data generated in our laboratory showed that obesity is associated with abnormalities in the insulin signalling pathway. Specifically, we found a down-regulation of protein kinase B (PKB/Akt), which is known to mediate the metabolic effects of insulin. One of the downstream targets of PKB/Akt is glycogen synthase kinase-3 (GSK-3), which is inhibited by this phosphorylation. Detrimental effects of unopposed activity of GSK-3 have recently been described. This may play a pivotal role in some of the adverse consequences of insulin resistance in the heart. Hypothesis: Chronic inhibition of GSK-3 will induce myocardial hypertrophy or exacerbate the development of existing hypertrophy in a pre-diabetic model of diet induced obesity and insulin resistance. Objectives: (1) Assess the extent of the development of myocardial hypertrophy in a rat model of diet induced obesity (DIO) and insulin resistance. (2) Assess the effect of inhibition of GSK-3 protein on the development of myocardial hypertrophy. Methods: Two groups of age-matched male Wistar rats were used. Control animals received standard rat chow, while obese animals received a high caloric diet for 20 weeks. After 12 weeks, half of the animals in both groups received GSK-3 inhibitor treatment (CHIR118637, 30mg/kg/day, Novartis). At the end of 20 weeks, three series of experiments were conducted. (i) The animals were subjected to echocardiography to determine in vivo myocardial function, and biometric, metabolic and biochemical parameters were evaluated. (ii) The ability of the cardiomyocytes to accumulate deoxy-glucose after stimulation with insulin was determined, and (iii) the localization of key proteins was monitored using fluorescence microscopy and cell size was determined using light microscopy and flow activated cell sorter analysis. Results and discussion: The high caloric diet increased body weight (p<0.005) and intraperitoneal fat mass (p<0.01) when compared to controls. Complications associated with obesity, such as impaired glucose tolerance (p<0.05), hyperinsulinemia (p<0.0005) and an increased HOMA-IR index (p<0.01) were observed. Additionally, cardiomyocytes from the DIO animals had a significantly impaired response to insulin, specifically when 10nM (p<0.05) and 100nM (p<0.05) of insulin were used as stimulus. We also found a dysregulation in PKB/Akt, indicated by a down-regulation of phosphorylated PKB/Akt (p<0.01). The diet promoted the development of myocardial hypertrophy, since the ventricular weight (p<0.05) and ventricular weight to tibia length ratio were increased (p<0.01). Echocardiography experiments showed an increase in end diastolic diameter in the DIO animals (p<0.05). Additionally, there was an increase in the cardiomyocyte cell width in the DIO rats (p<0.0001) and a tendency for peri-nuclear localization of NFATc3. GSK-3 inhibition promoted the development of insulin resistance in control animals, as indicated by an increase in the body weight (p<0.05), serum insulin levels (p<0.01) and HOMA-IR index (p<0.01). In the DIO animals, the GSK-3 inhibitor treatment improved insulin resistance, as a decrease in serum insulin concentration (p<0.05) was observed. The cardiomyocytes from the treated DIO animals also showed an increase in glucose uptake (p<0.05) when stimulated with 100nM of insulin. The GSK-3 inhibitor promoted the development of myocardial hypertrophy in the control animals, indicated by an increase in ventricular weight (p<0.05) and cardiomyocyte cell width (p<0.0001), but did not exacerbate hypertrophy in the DIO animals. Conclusion: Both the high caloric diet and the GSK-3 inhibitor promoted the development of insulin resistance and myocardial hypertrophy in the rats. In the DIO animals the GSK-3 inhibitor treatment ameliorated insulin resistance and did not promote the further development of myocardial hypertrophy.
AFRIKAANSE OPSOMMING: Inleiding: Die huidige styging in vetsugtigheid en die sterk assosiasie daarvan met insulien weerstandigheid, tipe 2 diabetes en kardiovaskulêre komplikasies soos hipertrofie, het ‘n belangstelling in die onderliggende meganismes van hierdie siektetoestande ontlok. Voorlopige data uit ons laboratorium het getoon dat vetsug geassosieerd is met abnormaliteite in die insulien seintransduksie-pad soos byvoorbeeld ‘n afregulering van miokardiale proteïen kinase B (PKB/Akt), wat bekend is om die metaboliese effekte van insulien te medieer. Een van die proteïene wat deur PKB/Akt gefosforileer en daardeur geïnhibeer word, is glikogeen sintase kinase-3 (GSK-3). Negatiewe effekte van onge-opponeerde aktiwiteit van GSK-3 is beskryf en dit mag ‘n sleutelrol speel in sommige van die nadelige gevolge van insulien weerstandigheid in die hart. Hipotese: Chroniese onderdrukking van GSK-3 sal miokardiale hipertrofie ontlok of die bestaande hipertrofie in ‘n pre-diabetiese model van dieet-geïnduseerde vetsug en insulien weerstandigheid vererger. Doelstellings: (1) Om die omvang van die ontwikkeling van miokardiale hipertrofie in ‘n rotmodel van dieet-geïnduseerde vetsug te ondersoek en (2) om die effek van inhibisie van GSK-3 op die ontwikkeling van hipertrofie te ondersoek. Metodes: Ouderdomsgepaarde manlike Wistarrotte is in hierdie studie gebruik. Die diere is vir ‘n periode van 20 weke aan verskillende diëte onderwerp, naamlik standaard kommersiële rotkos vir die kontrole diere en ‘n hoë kalorie dieet vir die eksperimenteel vet diere (DIO). Helfte van elke groep diere is vir 8 weke met ‘n GSK-3 inhibitor behandel (CHIR118637, 30mg/kg/day, Novartis). Na die 20 weke is 3 eksperimentele reekse uitgevoer: (i) Die diere is eggokardiografies ondersoek om in vivo miokardiale funksie te bepaal en biometriese, metaboliese en biochemiese parameters is evalueer. (ii) Die vermoë van kardiomiosiete om de-oksiglukose na insulien stimulasie te akkumuleer, is bepaal, en (iii) die lokalisering van sleutelproteïene is met behulp van fluoressensie mikroskopie en die selgrootte met behulp van ligmikroskopie bepaal. Resultate en bespreking: Die hoë kalorie dieet het gepaard gegaan met ‘n beduidende toename in liggaamsgewig (p<0.005) en intraperitoneale vetmassa (p<0.01) in vergelyking met diere op die kontrole dieet. Newe-effekte geassosieerd met vetsug nl. onderdrukte glucose toleransie (p<0.05), hiperinsulinemie (p<0.0005) en ‘n verhoogde HOMA-IR index (p<0.01) is ook waargeneem. Daar was ook ‘n beduidend ingekorte respons van glukose opname deur kardiomiosiete van die vet diere na stimulasie met 10nM (p<0.05) en 100nM (p<0.05) insulien. Disregulering van PKB/Akt is gevind in die vorm van ‘n afregulering van die fosforilering van die proteïen (p<0.01). Die dieet het ook gelei tot die ontwikkeling van miokardiale hipertrofie aangesien die ventrikulêre gewig (p<0.05) asook die verhouding van die ventrikulêre gewig teenoor tibia lengte beduidend toegeneem het (p<0.01). Eggokardiografie het ‘n toename in ventrikulêre end-diastoliese dimensie in die DIO diere aangetoon (p<0.05). Tesame hiermee het die breedte van kardiomiosiete van die DIO diere toegeneem (p<0.0001) en daar was ook ‘n peri-nukluêre lokalisering van NFATc3. Behandeling van kontrole diere met ‘n GSK-3 inhibitor het insulienweerstandigheid ontlok soos afgelei uit ‘n verhoging in liggaamsgewig (p<0.05), serum insulien-vlakke (p<0.01) en die HOMA-IR waarde (p<0.01). In teenstelling het behandeling van die DIO diere met die GSK-3 inhibitor tot ‘n verbetering van insulienweerstandigheid gelei aangesien ‘n verlaging in serum insulien konsentrasies gevind is (p<0.05). Kardiomiosiete vanaf die behandelde DIO diere het ook ‘n verhoogde insulien-gestimuleerde glukose opname met 100nM insulien getoon (p<0.05). Behandeling met die GSK-3 inhibitor het die ontwikkeling van miokardiale hipertrofie in die kontrole diere teweeggebring, soos aangetoon deur ‘n toename in die ventrikulêre gewig (p<0.05) en ‘n groter selwydte in kardiomiosiete terwyl dit geen invloed op die bestaande hipertrofie van die vet diere gehad het nie. Gevolgtrekking: Die huidige studie het getoon dat die betrokke dieet asook behandeling met ‘n GSK-3 inhibitor insulienweerstandigheid sowel as die ontwikkelling van miokardiale hipertrofie in rotte ontlok. In die DIO diere het die behandeling met die GSK-3 inhibitor bloedglukose en insulien-vlakke verlaag en het nie hipertrofie vererger nie.

Thesis (MScMedSc (Biomedical Sciences. Medical Physiology))--University of Stellenbosch, 2010.
ENGLISH ABSTRACT: Introduction: Obesity and its associated complications, such as the metabolic syndrome, hypertension and cardiovascular disease, are escalating worldwide. In recognition of this, untested remedies advertised as anti-diabetic agents are flooding the market. Many of these products have limited efficacy, limited tolerability and significant side-effects. One remedy, claiming to have anti-diabetic properties, is DiaviteTM. DiaviteTM, a herbal product, consisting solely of the dried and ground pods of the Prosopis glandulosa tree, which is currently marketed as a food supplement with blood glucose and blood pressure stabilizing properties, as well as having the ability to enhance glucose utilization. It is already freely available from agents as well as sold over the counter at pharmacies. The producers of DiaviteTM are now seeking registration for their product from the Medicines Control Council (MCC) and, therefore, require solid scientific evidence of its effects. Aims: The aims of our study were, on request of the producing company, to determine the efficacy of DiaviteTM (P. glandulosa) as an anti-diabetic agent and possible mechanisms of action of this plant product. Methology: We utilized rat models of streptozotocin (STZ)-induced type 1 diabetes and diet-induced obese (DIO) insulin resistance. Male Wistar rats were rendered (a) type 1 diabetic after a once-off intra-peritoneal injection of STZ at a dose of 40 mg/kg and (b) insulin resistant after being on a high caloric diet (DIO) for 16 weeks. Half the animals of the type 1 diabetes model as well as the insulin resistant model were placed on DiaviteTM treatment (25 mg/kg/day) for a period of 4 – 8 weeks, depending on the model. The STZ-induced type 1 diabetic rats were sacrificed and the pancreata harvested for histological analysis. Animals on the DIO diet were sacrificed and (i) intra-peritoneal fat weight determined (ii) isolated hearts subjected to ischaemia/reperfusion to determine infarct size and protein expression profiles and (iii) cardiomyocytes prepared to determine insulin sensitivity. At the time of sacrifice blood was collected for blood glucose and serum insulin level determination, for both models. In addition, a standard toxicology study was performed in Vervet monkeys over a 3 month period. Results: In our type 1 diabetic model (blood glucose > 10 mmol/L) with a β-cell reserve, DiaviteTM treatment lead to increased serum insulin levels (p < 0.001) in both control and STZ groups as well as increased small β-cell (0 - 2500 μm2) formation (p < 0.001) in the pancreas of the STZ animals. Hearts from DiaviteTM treated control and DIO insulin resistant animals presented with smaller infarct sizes (p < 0.05) after ischaemia/reperfusion compared to their controls. DiaviteTM treatment lead to the increase of basal (p < 0.01) and insulin-stimulated (p < 0.05) glucose uptake in cardiomyocytes prepared from DIO insulin resistant animals. DiaviteTM treatment also led to significantly suppressed PTEN expression and activity (p < 0.01) in the DIO insulin resistant animals. In addition, DiaviteTM treatment had (i) no obvious detrimental effects in our rat models and (ii) no toxicity over a 3 month period in vervet monkeys. Conclusion: Our present study has shown that DiaviteTM treatment lowers fasting blood glucose levels, stimulates insulin secretion and leads to the formation of β-cells. In addition, oral consumption of DiaviteTM elicits cardioprotection against an ischaemic incident. DiaviteTM treatment improves insulin sensitivity of cardiomyocytes. Furthermore, it has been established that DiaviteTM treatment has no obvious detrimental effects in either of our rat models and no short-term toxic effects over a 3 month period in Vervet monkeys (data not shown). We thus conclude that in our models, DiaviteTM proved safe and it seems as if DiaviteTM, after short-term use, is beneficial as a dietary supplement.
AFRIKAANSE OPSOMMING: Inleiding: Vetsug, en die gepaardgaande komplikasies, soos die metaboliese sindroom, hipertensie en kardiovaskulêre siektes, neem wêreldwyd toe. Daar is tans verskeie middels op die mark wat as anti-diabetiese middels geadverteer word. Baie van hierdie geadverteerde produkte het beperkte effektiwiteit en het verskeie newe-effekte. Een so ‘n middel, is DiaviteTM. DiaviteTM is 'n plantproduk, wat slegs uit die gedroogte en fyngemaakte peule van die P. glandulosa boom bestaan. Hierdie produk word tans bemark as 'n voedselaanvulling met beide bloedglukose en bloeddruk stabiliserende eienskappe, asook die vermoë om glukose gebruik te verbeter. DiaviteTM is reeds vrylik beskikbaar van agente sowel as verkrygbaar by verskeie apteke. Die produsente van DiaviteTM wil aansoek doen om registrasie vir hul produk by die Medisynebeheerraad (MCC) en hulle vereis daarom wetenskaplike bewyse van die gevolge van die gebruik van hierdie produk. Doel: Die doel van ons studie was om op versoek van die produksie maatskappy, die doeltreffendheid van DiaviteTM (P. glandulosa) as 'n anti-diabetiese behandeling te evalueer, sowel as die moontlike meganismes van werking van hierdie plantproduk. Metodes: Ons het gebruik gemaak van rot modelle van (i) streptozotocin (STZ)-geïnduseerde tipe 1 diabetes en (ii) dieet-geïnduseerde vetsugtig (DIO) insulienweerstandigheid. Manlike Wistar rotte was as (a) tipe 1 diabeties geklassifiseer na 'n eenmalige, intra-peritoneale inspuiting van STZ teen 'n dosis van 40 mg/kg en as (b) insulienweerstandig geklassifiseer, nadat hulle op 'n hoë kalorie dieet (DIO) vir 16 weke was. Die helfte van beide die tipe 1 diabetes en die insulienweerstandige groep diere was met DiaviteTM behandel (25 mg/kg/dag) vir 'n tydperk van 4 - 8 weke, afhangende van die model. Die STZ-geïnduseerde tipe 1 diabetes rotte is geslag en die pankreata geoes vir histologiese analise. Diere op die DIO dieet is geslag en (i) die intra-peritoneale vet gewig bepaal, (ii) die geïsoleerde harte blootgestel aan isgemie/herperfusie om die infarkt groottes vas te stel, sowel as die proteïenuitdrukkingsprofiele te bepaal en (iii) kardiomiosiete was berei om die insulien sensitiwiteit te bepaal. Ten tyde van die slagting is bloedmonsters geneem vir bloedglukose en serum insulien vlak bepaling, vir beide modelle. Additioneel, is 'n standaard toksologie studie met Vervet apies oor 'n 3 maande tydperk uitgevoer. Resultate: In die model van tipe 1 diabetes (bloed glukose > 10 mmol/L), met 'n β-sel reserwe, is gevind dat DiaviteTM behandeling tot verhoogde serum insulien vlakke (p < 0.001) in beide kontrole en STZ groepe lei. DiaviteTM behandeling lei ook tot ‘n hoër vlak van klein β-sel (0 - 2500 μm2) vorming (p < 0.001) in die pankreas van die STZ diere. Die harte van die DiaviteTM behandele kontrole en DIO groep het kleiner infarkt groottes (p < 0.05) getoon na isgemie/herperfusie in vergelyking met hul kontrole groepe. DiaviteTM behandeling het ook gelei tot verhoogde basal (p < 0. 01) en insulin-gestimuleerde (p < 0. 05) glukose opname in kardiomiosiete wat berei was van DIO insulinweerstandige diere. DiaviteTM behandeling het PTEN uitdrukking en aktiwiteit aansienlik onderdruk (p < 0.01) in die DIO insulienweerstandige groep diere. Daar is dus gevind dat DiaviteTM behandeling (i) geen duidelike nadelige invloed in ons rot-modelle en (ii) geen toksisiteit oor 'n 3 maande tydperk in Vervet apies getoon nie. Gevolgtrekking: Ons huidige studie toon dus dat DiaviteTM behandeling vastende bloedglukosevlakke verlaag, insulien sekresie stimuleer en die proses van β-sell vorming bevorder. Additioneel, is gewys dat wanneer DiaviteTM mondelings gebruik word, dit die hart beskerm teen isgemiese insidente. Ons het ook getoon dat DiaviteTM behandeling insuliensensitiwiteit van kardiomiosiete verhoog. Verder is daar vasgestel dat DiaviteTM behandeling geen ooglopende nadelige gevolge in beide ons rot-modelle getoon het nie en daar geen korttermyn-toksiese effekte oor 'n 3 maande tydperk in Vervet apies (data nie getoon) is nie. Ons kan dus aflei dat Diavite TM in ons modelle veilig is en na kort termyn gebruik, voordelig is as 'n dieetaanvulling.

Thesis (MScMedSc (Biomedical Sciences. Medical Physiology))--University of Stellenbosch, 2010.
ENGLISH ABSTRACT: Introduction: The discovery of the endothelium as a regulator of vascular tone, and the subsequent discovery of nitric oxide (NO) as the major endothelium-derived relaxing factor (EDRF), has opened up vast possibilities in the continued efforts to prevent and manage cardiovascular disease. Endothelial dysfunction (ED) is defined as reduced NO bioavailability and hence the reduced ability of the endothelium to maintain vascular homeostasis. ED represents the first, reversible step in the initiation of atherosclerotic disease and is thus regarded as a strong predictive tool of ischaemic heart disease (IHD). ED and its underlying mechanisms have been largely under-investigated in myocardial capillary-derived endothelial cells (cardiac microvascular endothelial cells, CMECs), and this study aimed to address this gap in the literature. Oleanolic acid (OA) is a bioactive triterpenoid derived from leaf extracts of African medicinal plants such as Syzigium cordatum (Water berry tree), and has been reported to elicit vasodilatory, hypoglycaemic and hypolipidaemic properties. However its effects particularly on CMECs and its putative role in reversing ED remain unclear, and this study aimed to investigate such effects. Aims: The aims of this study were to: (1) Establish an in vitro model of ED in cultured myocardial capillary-derived CMECs by developing protocols for the induction of ED. (2) Asses ED induction by measurement of the following biomarkers: (i) intracellular NO production, (ii) superoxide (O2-) production, (iii) nitrotyrosine expression and (iv) NADPH oxidase expression. (3) Investigate underlying cellular mechanisms of our ED model by measuring and comparing eNOS and PKB/Akt expression and activation in control and dysfunctional CMECs. (4) Investigate the effects of OA derived from leaf extracts obtained from Syzigium cordatum (Hochst.) [Myrtaceace], in both control and dysfunctional CMECs. Methods: (1) To induce ED, hyperglycaemia and inflammation were simulated by incubation with 25 mM glucose (24 hours) and 1 ng/ml TNF-á (24 hours) or 5 ng/ml TNF-á (6 and 24 hours) respectively. Reduced intracellular NO production was used as the main indicator of ED. NO production and cell viability were quantified by FACS analysis of the fluorescent probes, DAF-2/DA and propidium iodide (PI) / Annexin V respectively. Cellular mechanisms were investigated by measurement of O2- levels via FACS analysis of DHE fluorescence, and measurement of total and activated PKB / Akt and eNOS, p22-phox, nitrotyrosine expression via Western blotting. (2) Effects of OA on CMECs were investigated by pre-treatment with 30 or 40 ìM OA for 5 and 20 min followed by NO production and cell viability measurements. To investigate the effects of OA on ED, CMECs were pre-treated with 40 ìM OA 1 hour prior ED induction followed by NO, cell viability, and eNOS expression / activation measurements. Results: (1) 25 mM glucose (24hours), 1 ng/ml TNF-á (24 hours) and 5 ng/ml TNF-á (6 hours) failed to induce ED as verified by an increase in NO production in the treated cells. A model of ED was successfully achieved by incubating CMECs with 5 ng/ml TNF-á (24 hours), as verified by a significant decrease in NO production. Investigations into cellular mechanisms underlying our TNF-á-induced ED model, showed that activated eNOS and PKB / Akt levels were reduced. Furthermore, O2- levels remained unchanged, however p22-phox (NADPH) expression was significantly increased suggesting oxidative stress. Nitrotyrosine levels (an oxidative / nitrosative stress marker and indirect measure of eNOS uncoupling) remained at control levels. (2) Investigations into the effects of OA on CMECs showed that 30 ìM OA increased NO production after 5 and 20 min of incubation whereas 40 ìM increased NO production after 20 min only. Pre-treatment with 40 ìM OA significantly reversed ED by restoring NO production back to control levels. Data from cellular mechanism investigations showed that 40 ìM OA significantly increased eNOS activation in both normal and dysfunctional CMECs. Cellular viability was not negatively affected by any of the above interventions. Discussion and Conclusions: Based on our findings, reduced activation of the PKB / Akt-eNOS pathway appears to be the primary mechanistic pathway of the TNF-á-induced model of ED. Though O2- levels remained at control levels, the significant increase in p22-phox is indicative of increased expression of the O2- producing enzyme, NADPH oxidase, thus suggesting oxidative stress. However, based on our nitrotyrosine expression data, there was no strong evidence of eNOS uncoupling in our ED model. OA significantly stimulated NO production in our model of CMECs. Furthermore, our findings showed that OA is able to reverse ED. The NO production stimulatory effects of OA in our cells appear to be achieved via the increased activation of eNOS. We have, for the first time as far as we are aware, developed a TNF-á-induced model of ED in myocardial capillary-derived endothelial cells. It appears that reduced activation of the PKB/Akt-eNOS pathway is the primary mechanism leading to decreased NO production in this model. However, we did find some evidence of elevated oxidative stress, which led us to believe that eNOS uncoupling cannot be excluded as a mechanism of ED in our model. In this study, we report for the first time convincing evidence that OA has powerful NO-increasing properties in myocardial capillary-derived CMECs. Our study also show novel data, which suggest that OA is able to reverse ED in this model. Follow-up investigations could shed more light on the exact mechanisms underlying OA.s effects in this model.
AFRIKAANSE OPSOMMING: Inleiding: Die ontdekking dat endoteel 'n reguleerder van vaskulêre tonus is, en die gevolglike ontdekking dat stikstofoksied (NO) die belangrikste endoteel-afgeleide verslappingsfaktor (EDRF) is, het verskeie moontlikhede in aangaande pogings om kardiovaskulêre siektes te voorkom en hanteer, ontsluit. Endoteel-disfunksie (ED), word gedefineer as verlaagde NO biobeskikbaarheid en dus 'n ingekorte vermoë van die endoteel om vaskulêre homeostase te handhaaf. ED verteenwoordig die eerste, omkeerbare stap in die ontstaan van aterosklerotiese siekte en word dus beskou as 'n sterk instrument waarmee isgemiese hartsiekte voorspel kan word. Studies oor ED en sy onderliggende meganismes, veral in miokardiale kapillêre-afgeleide endoteelselle (kardiale mikrovaskulêre endoteelselle, CMECs), word redelik afgeskeep in die literatuur, en hierdie studie het dit ten doel gehad om die gaping in die literatuur aan te spreek. Oleanoliese suur (OA) is 'n bio-aktiewe triterpenoïede wat gevind word in blaar ekstrakte van inheemse medisinale plante soos bv. Syzigium cordatum (Waterbessie boom). OA het bewese vasodilatoriese, hipoglukemiese en hipolipidemiese eienskappe. OA se effekte op CMECs, en sy moontlike rol in die omkering van ED, is egter onbekend, en hierdie studie het dit ten doel gehad om sulke effekte te ondersoek. Doelwitte: Die doelwitte van hierdie studie was: (1) Die vestiging van 'n in vitro model van ED in gekultuurde CMECs afkomstig van miokardiale kapillêre deur protokolle vir die induksie van ED te ontwikkel. (2) Die evaluering van ED induksie deur die volgende bio-merkers te meet: (i) intrasellulêre NO produksie, (ii) superoksied (O2-) produksie, (iii) nitrotirosien uitdrukking en (iv) NADPH oksidase uitdrukking. (3) Die ondersoek na onderliggende sellulere meganismes van ED in ons model deur die meting en vergelyking van eNOS and PKB/Akt uitdrukking en aktivering in kontrole en disfunksionele CMECs. (4) Ondersoek na die effekte van OA afkomstig van blaar ekstrakte verkry van Syzigium cordatum (Hochst.) [Myrtaceace], in beide kontrole en disfunksionele CMECs. Metodes: (1) Daar was gepoog om ED te induseer deur hiperglukemie en inflammasie te simuleer met onderskeidelik 25 mM glukose (24 uur) en 1 ng/ml TNF-a (24 uur) of 5 ng/ml (6 en 24 uur) inkubasie. Verlaagde intrasellulere NO produksie was ingespan as die hoof indikator van ED. NO produksie en sellewensvatbaarheid was gekwantifiseer deur vloeisitometriese analises (FACS) van die fluoresserende agense, DAF-2/DA en propidium jodied (PI) / Annexin V onderskeidelik. Sellulere meganismes was ondersoek deur O2- vlakke via FACS analise van DHE fluoressensie te meet, asook die meting van totale en geaktiveerde PKB / Akt en eNOS, p22-phox, nitrotirosien uitdrukking via Western blot tegnieke. (2) Effekte van OA op CMECs was ondersoek deur vooraf-behandeling met 30 of 40 µM OA vir 5 en 20 min gevolg deur NO produksie en sellewensvatbaarheid metings. Resultate: (1) 25 mM glukose (24 uur), 1 ng/ml TNF-a (24 uur) and 5 ng/ml TNF-ƒaa (6 uur) kon nie daarin slaag om ED te induseer nie, soos blyk uit die verhoogde NO produksie waargeneem in die behandelde selle. 'n Model van ED was suksesvol verkry deur CMECs met 5 ng/ml TNF-a (24 uur) te inkubeer, soos waargeneem deur verlaagde NO produksie. Ondersoek na sellulere meganismes onderliggend tot ons TNF-a-geinduseerde ED model, het getoon dat geaktiveerde eNOS en PKB / Akt vlakke verlaag was. Verder is gevind dat O2- vlakke onveranderd gebly het hoewel p22-phox (NADPH) uitdrukking betekenisvol toegeneem het, wat 'n aanduiding van oksidatiewe skade is. Nitrotirosien vlakke (.n oksidatiewe / nitrosatiewe stres merker en indirekte maatstaf van eNOS ontkoppeling) het onveranderd rondom kontrole vlakke gebly. (2) Ondersoek na die effekte van OA op CMECs het getoon dat 30 µM OA tot verhoogde NO produksie na 5 en 20 min inkubasie gelei het, terwyl 40 µM slegs na 20 min NO-verhogende effekte gehad het. Vooraf behandeling met 40 µM OA het ED betekenisvol omgekeer deur NO terug na kontrole vlakke te laat herstel. Ondersoek na sellulere meganismes het getoon dat 40 µM OA eNOS aktivering betekenisvol verhoog het in beide normale en disfunksionele CMECs. Sellulere lewensvatbaarheid was nie negatief geaffekteer deur enige van bogeneemde ingrepe nie. Bespreking en afleidings: Gebaseer op ons bevindinge, blyk verlaagde aktivering van die PKB/Akt-eNOS pad die primere meganistiese pad in ons TNF-a-geïnduseerde model van ED te wees. Alhoewel O2- vlakke rondom kontrole vlakke gebly het, was die betekenisvolle toename in p22-phox .n aanduiding van verhoogde uitdrukking van die O2- produserende ensiem, NADPH oksidase, wat dus suggererend van oksidatiewe stres was. Aan die ander kant was daar nie sterk bewyse van eNOS ontkoppeling in ons ED model nie, gebaseer op die nitrotirosien uitdrukking data. OA het duidelik NO produksie in ons model van CMECs gestimuleer. Verder wys ons resultate dat OA in staat is om ED om te keer. Die NO produksie-stimulerende effekte van OA in ons selle blyk die gevolg te wees van verhoogde aktivering van die PKB / Akt-eNOS pad. Ons het hier vir die eerste keer, sover ons bewus is, 'n TNF-a-geinduseerde model van ED in CMECs afkomstig van miokardiale kapillere gevestig. Dit blyk dat verlaagde aktivering van die PKB/Akt-eNOS pad die primere meganisme was waardeur verlaagde NO produksie in ons model veroorsaak was. Ons het egter wel bewyse van verhoogde oksidatiewe stress gevind, wat ons laat glo dat eNOS ontkoppeling nie heeltemal as .n meganisme van ED in ons model uitgesluit kan word nie. In hierdie studie toon ons vir die eerste maal oortuigende bewyse dat OA kragtige NO-verhogende eienskappe in miokardiale kapillere-afgeleide CMECs het. Ons studie bring ook nuwe data na vore, wat suggereer dat OA in staat is om ED in hierdie model om te keer. Opvolgstudies sal meer lig kan werp op die onderliggende meganismes van OA in hierdie model.

Exercise tolerance, measured as peak oxygen consumption (VO₂ peak), is very low in patients with end-stage renal failure undergoing maintenance haemodialysis. Due to their associated anaemia and low peak heart rates during maximal exercise it has been argued that the reduced blood oxygen carrying capacity and central cardiovascular limitations are primarily responsible for the poor exercise tolerance of these patients. However, others suggest that peripheral (skeletal muscle) limitations including impaired substrate utilization, muscle weakness caused by peripheral neuropathy and myopathy, malnutrition and general physical deconditioning are responsible for the poor exercise tolerance. The present thesis was therefore designed to study whether central cardiovascular function or anaemia or muscle weakness causes patients with end-stage renal failure to terminate exercise at workrates well below those achieved by healthy controls.

La simulation médicale interactive sur ordinateur est une technologie révolutionnaire pour améliorer l'efficacité de nombreuses interventions médicales tout en réduisant le niveau de risque pour les patients. Bien que visant essentiellement l'apprentissage, ces simulations pourraient être utilisées, dans un futur proche, pour la planification d'interventions complexes ou même pour assister le praticien / clinicien dans la salle d'opération. Ce manuscrit présente une revue détaillée du domaine multi-disciplinaire de la simulation médicale, et illustre nos différentes contributions dans ce domaine. Après une vue d'ensemble, au Chapitre I, de nombreuses applications en simulation médicale, le Chapitre II décrit nos contributions sur les modèles, depuis la modélisation anatomique (afin de créer des représentations réalistes, et potentiellement adaptées au patient, de l'anatomie humaine) jusqu'à la modélisation biomécanique (pour déterminer les caractéristiques des tissus mous et définir des modèles mathématiques décrivant leur comportement). Les problématiques liées à la modélisation de matériel médical (instruments flexibles ou systèmes d'imagerie) ou encore la modélisation physiologique (pour le calcul d'écoulement sanguin par exemple) sont également abordées. Le Chapitre III s'attache à la modélisation des interactions entre instruments et tissus mous, qui occupent une part très importante dans toute intervention médicale. Les différentes techniques à mettre en oeuvre pour modéliser de telles interactions (détection de collision, modélisation des contacts et rendu haptique) sont décrites dans ce chapitre. Au Chapitre IV sont présentées plusieurs contributions liées à la validation, que ce soit pour comparer des modèles déformables ou pour l'évaluation de systèmes d'apprentissage. Le Chapitre V est dédié à la description de divers prototypes de simulateurs développés au cours de ces travaux de recherche, et le Chapitre VI présente nos récents travaux visant au développement d'une plate-forme Open Source dédiée à la simulation médicale. Cette plate-forme, appelée SOFA, est le fruit d'un travail collaboratif international à travers lequel nous espérons fédérer de nombreuses équipes de recherche. Finalement, le Chapitre VII résume nos différentes contributions et présente un ensemble de perspectives et de défis, en particulier dans les domaine de la simulation et de la planification sur des données spécifiques à des patients.

Thesis (MScMedSc (Biomedical Sciences. Medical Physiology)--Stellenbosch University, 2008.
For many centuries, scientists have engaged in a theoretical debate concerning the etiology of mood disorders, with very few ancient scholars speculating about the importance of genetic factors and affective temperaments as factors in the etiology of depression. Mood, emotion and cognition have been shown to be modulated by the serotonergic midbrain raphe system; implicated in the pathogenesis of psychiatric disorders like those of the affective spectrum. Evidence from neuroscience, genetics, and clinical investigation demonstrate that depression is a disorder of the brain. Brain imaging research is revealing that in depression, neural circuits responsible for moods, thinking, sleep, appetite, and behavior fail to function properly, and that the regulation of critical neurotransmitters is impaired. Genetics research, including studies of twins, indicates that genes play a role in depression. Vulnerability to depression appears to result from the influence of multiple genes acting together with environmental factors. Other research has shown that stressful life events, particularly in the form of loss such as the death of a close family member, may trigger major depression in susceptible individuals. Depression and anxiety have often been successfully treated by means of selective serotonin reuptake inhibitors. However, selective serotonin reuptake inhibitors do not solve all the problems inherent to the treatment of depression, for approximately 30 % of depressed patients do not respond to treatment and 20 % experience relapses whilst on treatment. Of consideration is the fact that the majority of drugs today are based on proteins, with 50 % of therapeutics on the market targeting cell membrane proteins. Up to this day the precise pathophysiology of mood disorders remains obscure, as does the neurobiology of normal mood regulation. Accordingly, there is a need for methods to identify the structural and/or signaling components which lead to changes in the brain, particularly the hippocampus, of subjects having mood disorders such as bipolar depressive disorder, chronic major depressive disorder and the like. Similarly, there is a need for the early detection, screening and diagnosis of individuals at risk for a mood disorder. As the serotonin tranpsorter is the primary target for therapeutic intervention in the treatment of numerous psychiatric disorders and considering the fact that at the structural level this protein’s function as transporter in membranes remains incompletely understood, investigating its function in psychiatric disorders are of importance . The objective of this study was to determine the role of the serotonin transporter in wild type and serotonin knockout rats, with regards to the hippocampus. Rat hippocampi were fractionated into cytosolic and membrane components, which were run and further separated in two dimensions. Firstly separation occurred by isoelectrical focusing (pI), follwed by gel iii electrophoresis (molecular weight). Gels were compared to see whether protein spots have changed between animals that have been differentially bred. Differentially expressed protein spots, as determined by PD Quest software, were excised, digested and analyzed by means of mass spectrometry. Our results indicated that metabolic, structural and cell signaling proteins were differentially expressed in both the ventral and dorsal hippocampus of the serotonin knockout rat. Futhermore, cellular stress proteins were found to be only differentially expressed in the ventral hippocampus. The majority of proteins identified in both hippocampal areas as well as both fractions, were assigned to energy metabolism. The cytosolic protein profile mirrored the pattern of the membrane protein profile. In conclusion, this proteomic study identified various protein groups that interacted with one another, thus establishing compensation for disrupted serotonin homeostasis.

Uromodulin (UMOD) is a protein made exclusively in the thick ascending limb. Clinical studies have demonstrated that rare missense mutations in UMOD result in autosomal dominant tubulointerstitial kidney diseases manifest by tubulointerstitial fibrosis (TIF), tubular cysts and a rapid progression to renal failure. In addition, several genome wide association studies reported that common single nucleotide polymorphisms in the UMOD gene are associated with an increased risk of chronic kidney disease (CKD) and hypertension. Interestingly, Dahl salt-sensitive (SS) rats exhibit many of the same pathologies observed in these clinical populations with alterations in UMOD. The goal of this study was to assess the qualitative and quantitative aspects of UMOD via western blotting, and the extent of SS hypertension and proteinuria in Dahl SS vs. a consomic rat strain in which chromosome 1 of the salt-resistant Brown-Norway (BN) rat, harboring the UMOD gene, has been introgressed into the Dahl SS background (SS.BN1). We hypothesized that differences in UMOD would be apparent in SS vs. SS.BN1 rats maintained on a low salt-diet and that the extent of SS hypertension and proteinuria would be attenuated in SS.BN1 vs SS rats. Western blot of urinary UMOD was performed in 16 week old SS (n=5), SS.BN1 (n=7) and BN (n=6) rats maintained on a low salt (LS) diet. BP (radiotelemetry) and proteinuria were assessed during LS feeding and during three weeks of high salt (HS) feeding in a different group of 8-10 week old SS (n=9) and SS.BN1 (n=8) rats. For western blotting, urine was normalized based on the protein concentration, and the density of the 85 kDa UMOD band in SS and SS.BN1 samples were normalized to the average density observed in BN rats. The UMOD band was 4.5 fold higher (p In summary, these data demonstrate striking qualitative and quantitative differences in UMOD between SS and SS.BN1 rats. The pattern of UMOD expression in SS rats is consistent with that observed in some patient populations of UMOD associated kidney disease. Finally, the evidence that SS.BN1 rats, harboring the UMOD gene from BN rats, exhibit significant protection against SS hypertension and proteinuria is consistent with the notion that an alteration in UMOD function may, in part, be responsible for such pathologies in SS rats.

Medial tibial stress syndrome (MTSS) is a stress and overuse injury that presents as pain on the medial aspect of the lower two-thirds of the tibia. It is most often caused by repetitive actions on hard surfaces such as running, marching, and dancing. Individuals most affected by MTSS are runners, members of the military, dancers, and athletes that play soccer, volleyball and basketball. While MTSS has a relatively standard presentation of pain on the medial aspect of the tibia, it can occasionally be mistaken for other injuries such as stress fractures or compartment syndrome. If a diagnosis is unsure, methods such as x-ray, bone-scan, and MRI can be utilized to better obtain the correct diagnosis. A variety of treatments exist for MTSS including, ice, massage, muscle strengthening, and rest. A combination of these various techniques is most often what is employed. In this study, the effectiveness of a set of resistance ankle exercises in combination with ice and massage was tested and compared to that of ice and massage alone. The hypothesis was that athletes receiving the exercises as part of their treatment, in addition to the icing and massaging, would have a greater decrease in pain from MTSS than athletes just receiving ice and massage as treatment. The exercises would strengthen the muscles of the lower leg that, when weak, can contribute to the development of MTSS. Results indicated that the exercises yielded a more significant decrease in pain from MTSS than ice and massage alone.

Inflammation or nerve injury sensitizes several populations of nociceptive neurons in the dorsal horn of the spinal cord, including those that express the neuropeptide Y (NPY) Y1 receptor (Y1R). Our overall hypothesis is that after tissue or nerve injury, these Y1R-expressing neurons enter a state of latent sensitization (LS) that contributes to vulnerability to the development of chronic pain; furthermore, LS is under the tonic inhibitory control of endogenous Y1R signaling. First, we evaluated the intracellular signaling pathways that become activated in Y1R-expressing neurons and participate in LS. To do this, we established behavioral models of inflammatory or neuropathic pain, allowed pain hypersensitivity to resolve, and then during this period of pain remission we administered the Y1R receptor antagonist, BIBO3304, by intrathecal injection. As observed previously with mu-opioid receptor antagonists/inverse agonists, we found that BIBO3304 reinstated pain hypersensitivity via an N-methyl-D-aspartate receptor (NMDAR)- and adenylyl cyclase type 1 (AC1)-dependent mechanism. Our subsequent behavioral pharmacological experiments then established two signaling pathways downstream of AC1 that maintain LS. The first pathway involves protein kinase A (PKA) and transient receptor potential cation channel A1 (TRPA1) and channel V1 (TRPV1). The second pathway involves exchange proteins activated by cAMP (Epac 1 and Epac 2). We next found that nerve injury decreases the co-expression of Y1R with markers of excitatory interneurons, suggesting that Y1R-expressing neurons acquire a pain-enhancing phenotype after peripheral nerve injury. In a separate set of experiments that utilized Y1R-receptor internalization as an index of NPY release, we found that nerve injury increased stimulus-evoked NPY release. We conclude that injury induces pain-facilitatory mechanisms of LS in the dorsal horn involving PKA→TRPA1 and PKA→TRPV1 at the central terminals of primary afferent neurons. Whether Epac mechanisms are located on these same presynaptic terminals and/or at Y1R-expressing excitatory interneurons remain to be determined. We also conclude that injury-induced LS is masked by a compensatory up-regulation of spinal NPY release that tonically inhibits pain. These results present a novel mechanism of injury-induced LS and endogenous control of the transition from acute to chronic pain by the NPY-Y1R system. Our work sheds light on novel targets for the treatment of chronic pain.

The proximal Interphalangeal (PIP) joint is vital for hand function. It is frequently affected by contractures as a result of injury or disease. Dynamic external fixators are one of the useful treatment methods of these contractures, enabling the ability to exercise adjacent joints during healing. The aim of this study was a development of the Compass hinge device to present a new design device for the digit PIP joint. A failed Compass hinge external fixator has been analysed. The device consists of polymer parts manufactured from polyetherimide (PEI). Finite element analysis (FEA) was used to investigate the principal stresses in the device under different loading conditions. Scanning electron microscopy (SEM) was used to investigate the fracture surfaces. The FEA showed that the maximum principal stress was greater than the fatigue strength of PEI. The SEM fractographs confirm that failure was by brittle fatigue. A new finger fixator named the PIP joint protractor hinge device comprises 17 parts which are assembled together. The proposed device materials consist of Poly-ether -ether-ketone (PEEK) and stainless steel 316LS. The design was subjected to FEA and a working model was manufactured and subjected to cyclic mechanical testing. The FEA showed that the maximum stress was 242.9 MPa and this was less than the yield strength and the fatigue endurance limits for the selected materials. Mechanical testing showed that testing reached run-out of 170,000 cycles with no cracks or damage visible in the device parts.

Amyloid beta (ABeta) and tau protein are both implicated in memory impairment in early Alzheimer’s disease, but whether and how they interact to cause synaptic dysfunction are unknown. Consequently, I firstly investigated whether tau protein is required for the robust phenomenon of ABeta-induced impairment of hippocampal long-term potentiation (LTP), a widely accepted cellular model of memory. I demonstrate that the absence of tau prevents the ABeta-induced impairment of LTP; moreover, a specific inhibitor of the tau kinase glycogen synthase kinase 3 blocks both an ABeta-induced increase in tau phosphorylation and the ABeta-induced LTP impairment. Thus, tau protein, likely in its phosphorylated form, is required for ABeta to impair LTP. Secondly, I investigated the underlying mechanisms for this ABeta-induced impairment and find that ABeta changes the balance between the two major types of glutamate receptors involved in plasticity processes, with a specific effect on GluN2B subunit-containing NMDA receptors. Since the distribution of these receptors is asymmetric between the left and right mouse hippocampus, I accessed these different types of synapses optogenetically and found that only the GluN2B-rich synapses receiving left CA3 input show ABeta-induced changes in the balance of glutamate receptors, suggesting an asymmetry in synaptic vulnerability to ABeta. Moreover, there was a left-right difference in tetanus-induced LTP and therefore, thirdly, I investigated whether mice have a hemispheric dissociation in memory processing using acute optogenetic silencing of left or right CA3 during hippocampus-dependent memory tasks. Unilateral silencing of either the left or the right CA3 caused a deficit in short-term memory, but only left CA3 silencing impaired performance on a spatial long-term memory task. Together, these results suggest that memory may be routed via distinct left-right pathways within the mouse hippocampus, and that neural pathways subserving distinct functions may also be differentially vulnerable to pathological changes at the synaptic level.

This study was designed as an investigation of the role of changes in muscle force and changes in muscle length on the EMG for the Tibialis Anterior (TA).
Using surface electrodes we examined the EMG for 4 contraction levels at 5 ankle positions over 60$ sp circ$ of ankle rotation. The change in median frequency with muscle length identified a significant shift in the power spectrum to lower frequencies with increasing muscle length.
To further investigate our results we performed three other experiments: First, using X-rays to identify the relative change in distance between two intramuscular wire electrodes we found the change in TA muscle length for this study to be 15% over the 60$ sp circ$ of ankle rotation. Second, to test for synergist contamination we used fine wire electrodes in the Extensor Digitorum Longus and the Peroneus. We found no evidence to support significant contamination. Third, we examined the role of smaller electrodes with a smaller interelectrode distance on our findings. The EMG showed drastic changes with even a slight shift in electrode position most likely due to the large number of innervation zones.
Therefore, the results indicate a shift in the power spectrum with a change in muscle length. In addition, surface EMG results are heavily dependent on the innervation zones and on the electrode geometry, all of which are important considerations in developing the EMG as an accurate diagnostic tool.

Airway hyperresponsivness (AHR) is one of most prominent pathophysiological features of asthma. Increasing evidence suggests that vagal bronchopulmonary afferents may be involved in the development of AHR. However, the underlying mechanisms are not clear. Therefore, the purpose of this dissertation was to investigate the effect of chronic airway inflammation induced by allergen sensitization on vagal bronchopulmonary afferents. The study was carried out in an animal model of allergic asthma. Brown-Norway rats were sensitized by intraperitoneal Ovalbumin (Ova) and exposed to aerosolized Ova 3 times/week for three weeks. Control rats received the vehicle. In vivo single-fiber recording technique was applied in this study. Our results showed that chronic Ova exposure caused an elevated baseline activity of pulmonary Cfibers, and a distinctly higher sensitivity of these afferents to chemical stimulants and lung inflation. After an acute Ova inhalation challenge, the increase in baseline activity and the excitability of pulmonary C-fibers were further augmented in sensitized rats, but not in control rats. In addition, sensitivity of pulmonary myelinated afferents to capsaicin was significantly elevated after chronic airway inflammation was induced by allergen. Furthermore, immunohistochemsitry data showed that, in nodose ganglia the proportion of transient receptor potential vanilloids type 1 channels (TRPV1)-expressing bronchopulmonary neurons was significantly higher in sensitized rats than in controls. This increase of TRPV1 expression was found mainly in neurofilament-positive neurons (myelinated neurons), but this effect was absent in jugular ganglia. In conclusion, allergen-induced airway inflammation caused a pronounced sensitizing effect on vagal pulmonary non-myelinated (C-fiber) afferents and elevated the sensitivity of vagal pulmonary myelinated afferents to capsaicin. The latter was accompanied by the upregulation of TRPV1 expression in these myelinated neurons.

Low density lipoprotein receptor (LDLR) is an apolipoprotein E (apoE) receptor and may play a role in Alzheimer’s disease (AD) development. A single nucleotide polymorphism (SNP), rs688, that has been identified to modulate the splicing efficiency of LDLR exon 12 and is associated with higher cholesterol and AD in some case-control populations. The exon 12 deleted mRNA is predicted to produce a soluble form of LDLR that fails to mediate apoE uptake. To gain additional insights, in this study, I seek to understand the regulation of LDLR splicing efficiency. To identify functional cis-elements within LDLR exon 12, I mutated several conserved putative exonic splicing enhancers (ESEs) to neutralize their affinity to serine/arginine-rich (SR) proteins. Transfection of wild type (WT) or mutant LDLR minigenes in HepG2 cells was performed, and splicing efficiency evaluated by quantitative RT-PCR. The results showed that two functional ESEs within exon 12, near rs688, are critical to LDLR splicing. To identify splicing factors that modulate exon 12 splicing, I co-transfected an LDLR minigene and vectors encoding different SR proteins and heterogeneous nuclear ribonucleoproteins (hnRNPs). After quantifying the splicing efficiency, I found that SRp20 and SRp38 increased exon 11- skipping. Moreover, ectopic expression of SRp38-2 and hnRNP G increased exon 11&12-skipping. Interestingly, the actions of hnRNP G did not require its RNA recognition motif (RRM). To further investigate the role of theses splicing factors on LDLR splicing, I quantified the expression level of these splicing factors as well as LDLR splicing efficiency in human brain and liver. I found that SRp38 mRNA expression is associated with LDLR splicing efficiency. In conclusion, this study discovered that rs688 is located close to the two functional ESEs within LDLR exon 12, and revealed a role of SRp38 in LDLR splicing efficiency.

Extracellular components can be internalized by either receptor-mediated or fluid-phase endocytosis. Receptor-mediated endocytosis involves the internalization of receptor-ligand complexes into coated vesicles of about 0.1 μm in diameter. The average diameter of primary pinocytic vesicles has been calculated to be 0.24 - 0.28 μm. The discrepancy in size between coated vesicles and the average pinosome diameter can be explained if, in addition to coated vesicles, another endocytic process involving vesicles larger than 0.28 μm in diameter takes place. These two vesicle types could together produce an average diameter of 0.24 μm. This hypothesis suggests that coated vesicles cannot fully account for fluid-phase uptake. Hypertonic conditions can selectively inhibit receptor-mediated endocytosis, leaving fluid-phase uptake unaffected, again suggesting that an alternative to coated pit-mediated uptake exists. In this study we determined the volume-weighted average diameter of primary pinocytic vesicles under hypertonic conditions (0.52 osm) where receptor-mediated uptake of transferrin was selectively inhibited by 42%. Fluid-phase uptake of FITC-dextran was unaffected by 0.52 osm medium. The internalization rate of ³H-galactose-labelled plasma membrane was reduced from 2.6 %/min to 1.5 %/min. The decrease in the rate of membrane internalization, without a reduction in the rate of fluid uptake at hypertonicity, implied a reduced surface to volume ratio of the pinocytic vesicles formed under these conditions. This suggested an increase in the average diameter of primary pinocytic vesicles. Membrane internalization rates were calculated on the assumption that all labelled cell-surface constituents were internalized to the same relative extent, as has been shown previously for isotonic conditions. This assumption was also shown to hold true under isotonic conditions. The reduced rate of membrane internalization under hypertonic conditions was shown not to be due to the exclusion of any labelled protein species from internalized vesicles. The larger average vesicle size determined under conditions of selective reduction of coated vesicle formation (i.e. hypertonicity), demonstrates the existence of a population of larger pinosomes involved in a possible alternative mechanism to coated-pit-mediated endocytosis.

Dysregulation of dopaminergic homeostasis has been established as the primary source of numerous neurological disorders including Parkinson’s and drug addiction. A tonic increase of dopamine (DA) in the nucleus accumbens is required for associating everyday events and behaviors with rewards. Yet many addictive exogenous compounds such as amphetamine (AMPH) and cocaine (COC) produce a much greater augmentation of synaptic DA levels that are linked to euphoria and a shift in behavior towards drug seeking. The protein responsible for maintaining extracellular levels of DA is the dopamine transporter (DAT). It is primarily located in the perisynaptic area at terminals of pre-synaptic neurons where its main function is to sequester DA from the extracellular space and to transport it back into the cell, a process that is electrogenic. AMPH and COC directly interact with DAT and alter its ionic currents. Not much is known about the effect of psychostimulant-induced DAT currents on neuronal excitability and neurotransmitter release. We use synthetic chemistry, molecular biology, and biophysics in heterologous expression systems to decipher the actions of drugs of abuse on DAT. Furthermore we demonstrate drug-induced DAT currents can activate Ca2+ channels associated with dopaminergic excitability. Lastly, we focused on investigating drug effects on excitability in a human midbrain dopaminergic cell line. Understanding how psychostimulants interact with DAT to produce the dysfunctional states of the transporter may facilitate the development of unique therapeutic strategies to treat psychostimulant dependence.

Acute kidney injury (AKI) is a major health burden associated with a 50% mortality rate. Of particular concern, the incidence of AKI has increased dramatically over the last decade. Yet, there is a paucity of available treatments to prevent AKI or to reduce the high rate of AKI-associated mortality. A common cause of AKI, especially in hospital settings, is prolonged decreases in renal blood flow (i.e., renal ischemia). Recent studies have demonstrated that activating the cholinergic anti-inflammatory pathway via vagal stimulation can mitigate AKI severity in rodent models of renal ischemia-reperfusion (IR) injury. While vagal stimulation is not a practical approach to prevent AKI in patients due its invasive nature and numerous side effects, recent studies have identified non-neuronal cholinergic cells within the kidney that could be targeted to reduce the severity of AKI. The overarching goal of this project is to examine the potential role of the renal cholinergic system in modulating the severity of and recovery from AKI in transgenic mice expressing green fluorescent protein (GFP) under control of the choline acetyl-transferase (ChAT) promoter, a protein involved in the synthesis of acetylcholine. The objectives of this study were to develop a clinically relevant model of renal IR-induced AKI in mice by identifying the duration of ischemia required for manifestation of the effects of AKI and to determine whether differences in susceptibility to AKI exists between male and female mice. Initially, male mice underwent 20 (n=3), 22 (n=3), or 25 (n=4) minutes of bilateral renal IR under isoflurane anesthesia with body temperature controlled at 37°C. Ischemia was achieved by careful placement of vascular clamps on the renal artery and vein supplying each kidney. The severity of AKI was determined by measuring serum creatinine (SCr) at 3 days post-AKI. Compared to SCr of mice that were 3 days post-sham AKI (SCr = 0.47 mg/dl, n=2), SCr of male mice from all three ischemia time categories was substantially elevated (SCr > 3 mg/dl, n=10). However, mortality associated with 22 and 25 minutes IR was striking (>90%) making studies of long-term AKI effects difficult. In contrast, 20 minutes IR resulted in AKI manifest by elevated SCr (3.43±0.7 mg/dl, n=3), widespread acute tubular necrosis and a clinically relevant mortality rate of 50%. Next, male (n=10) and female (n=5) mice were subjected to 20 minutes of IR. The mortality rate in male mice (n=10) was 50% (n=10) through 7 days post-AKI; however, all female mice survived. Additional studies showed that female mice had lower SCr 3 days post-AKI (0.63±0.1 mg/dl, n=2) with very modest levels of acute tubular necrosis as compared to the higher SCr (1.92±0.1 mg/dl, n=2) and extensive acute tubular necrosis observed in male mice. The differences observed in AKI severity and mortality rates suggest that female mice are protected against AKI as compared to male mice and future studies will explore the potential role of the renal cholinergic system in contributing to these sex differences in AKI.

Thesis (MScMedSc)--Stellenbosch University, 2014.
ENGLISH ABSTRACT: Introduction: Primary culture of isolated adult rat cardiomyocytes (ARCMs) is an important model for cardiovascular research, but successful maintenance of these cells in culture for their use in experiments remains challenging (Xu et al, 2009; Louch et al, 2011). Most studies are done on acutely isolated cardiomyocytes immediately after isolation, which is due to low survival of these cells in culture. Obstacles in culture are due to the type of medium and attachment factors (tissue culture adhesives) used to culture and grow these cells. Although we previously identified an optimum medium and adhesive for culture, an adhesive that permits cells to remain attached to the culture surface until after an ischemia/reperfusion insult was elusive. Aims: We therefore aimed to identify the best attachment factor and concentration that will allow adult rat cardiomyocytes to remain attached to the culture surfaces after ischemia/reperfusion experiments. Methods: Cardiomyocytes were isolated from adult Wistar rat hearts and cultured overnight on different concentrations (25 -200 μg/ml) of collagen 1, collagen 4, extracellular matrix (ECM), laminin/entactin (L/E) and laminin. Following overnight cultures, experiments were done in PBS and in PBS versus MMXCB to compare ARCM attachment and viability. Cardiomyocytes cultured on ECM, L/E and L (25−200μg/ml) were subjected to 1 hour of simulated ischemia using MMXCB that contained 3mM SDT and 10mM 2DG, followed by 15 minutes reperfusion. Cell viability was determined by staining cells with JC-1 and images of cells in a field view of 1.17μm/mm2 were captured using fluorescence microscopy. The cells were analysed according to morphology and fluorescence intensity. Results: Total and rod-shaped ARCMs attachment was improved when MMXCB was used as an experimental buffer instead of PBS. Regardless of the buffer used, morphological viability was poor on substrates of Col 1 and Col 4. In contrast to collagens, ARCMs attached efficiently and morphological viability was high on substrates of ECM, L/E and L in MMXCB, but this was greatly reduced in PBS. Mitochondrial viability was high in MMXCB compared to PBS on Col 1 and Col 4 at 75−175μg/ml and on ECM, L/E and L at all concentrations, except at 50 and 150μg/ml ECM, 175μg/ml L/E and 25μg/ml L. When cardiomyocytes cultured on ECM, L/E and L were subjected to simulated ischemia, total ARCMs, rod-shaped and R/G fluorescence (mitochondrial viability) was reduced at all concentrations compared to the control group. Hypercontracted cells were higher in the ischemic treated cells compared to the controls on ECM at 75−150μg/ml and 200μg/ml, L/E at 50,100μg/ml and 175μg/ml and on L at 125μg/ml. Total numbers of ARCMs attached on ECM, L/E and L in the ischemic group consisted of similar numbers of non-viable hypercontracted and viable rod-shaped cells. Conclusion: Cardiomyocytes should be cultured on ECM or L/E or L at concentrations from 25−200μg/ml in MMXCB. PBS is harmful to cultured ARCMs and should thus not be used as an experimental buffer. Ischemia/reperfusion can be simulated on ARCMs cultured on ECM, L/E or L from 25−200μg/ml, provided that a modified culture buffer is used as experimental buffer.
AFRIKAANSE OPSOMMING: Inleiding: Primêre selkulture van geïsoleerde volwasse rot kardiomiosiete (VRKMe) is ‘n belangrike model vir kardiovaskulêre navorsing, maar om hierdie selle suksesvol in kultuur te onderhou is ‘n groot uitdaging (Xu et al, 2009; Louch et al, 2011). Die meeste navorsingstudies maak gebruik van akuut geïsoleerde kardiomiosiete onmiddelik na isolasie omdat oorlewing van hierdie selle in kultuur baie laag is. Die struikelblokke in kultuur is as gevolg van die tipe medium en weefselkultuurgom wat gebruik word. Ons het voorheen 'n optimale medium en weefselkultuurgom geïdentifiseer vir VRKM kultuur oorlewing, maar die weefselkultuurgom was nie effektief genoeg om die selle aan die kultuuroppervlak te laat bly vaskleef, tot na die einde van 'n isgemie/herperfusie eksperiment nie. Doel: Die doel was dus om die beste weefselkultuurgom en konsentrasie te identifiseer, wat sal toelaat dat VRKMe verbonde bly aan die kultuuroppervlaktes tot na die einde van isgemie/herperfusie eksperimente. Metodes: Kardiomiosiete was geïsoleer vanaf volwasse Wistar rotharte en oornag in kultuur op verskillende konsentrasies (25 -200 μg/ml) van kollageen 1, kollageen 4, ekstrasellulêre matriks (ESM), laminin/entactin (L/E) en laminin onderhou. Die volgende dag was die VRKMe vir eksperimentasie in PBS en in PBS teenoor MMXCB gebruik, om selbehoud en oorlewing te vergelyk. Kardiomiosiete op ESM, L/E en L (25−200μg/ml) was aan 1 uur van gesimuleerde isgemie blootgestel, in MMXCB wat 3mM SDT en 10mM 2DG bevat het, gevolg deur 15 minute herperfusie. Sel oorlewing was bepaal deur selle te kleur met JC-1 en daarna was fluoressensiebeelde van die selle in ‘n veldgebied van 1.17μm/mm2 geneem. Die selle was volgens selmorfologie en fluoressensie intensiteit ontleed. Resultate: Met die gebruik van MMXCB as eksperimentele buffer in plaas van PBS, het die aantal totale en staafvormige VRKMe verbinding verbeter. Morfologiese onderhoud was sleg op kollageen 1 en 4, ongeag van watter buffer gebruik was. In kontras met die kollagene was die VRKM verbinding en morfologiese onderhoud op ESM, L/E en L in MMXCB effektief verbeter, maar in PBS aansienlik verminder. Mitochondriale lewensvatbaarheid in MMXCB teenoor PBS op kollageen 1 en 4 by 75−175μg/ml, sowel as op ECM, L/E en L by alle konsentrasies, was hoog, behalwe by 50 en 150μg/ml ESM, 175μg/ml L/E en 25μg/ml L. Isgemie blootstelling van kardiomiosiete gekultuur op alle konsentrasies van ESM, L/E en L, het ‘n afname in die totale, staafvormige en R/G fluoressensie (mitochondriale lewensvatbaarheid) teweeggebring. Meer hiperkontrakteerde kardiomiosiete was in die isgemie behandelde groepe as in die kontrole groepe teenwoordig, spesifiek op ESM by 75−150μg/ml en 200μg/ml, op L/E by 50,100μg/ml en 175μg/ml asook op L by 125μg/ml. In die isgemie groepe het die totale aantal VRKMe op ESM, L/E en L meestal uit ‘n gelyke hoeveelheid hiperkontrakteerde en staafvormige selle bestaan. Gevolgtrekking: Kardiomiosiete moet op ESM of L/E of L by konsentrasises van 25−200μg/ml in MMXCB gekultuur word. PBS is nadelig vir VRKMe in kultuur en moet dus nie gebruik word as eksperimentele buffer nie. Isgemie/herperfusie eksperimente kan gesimuleer word op VRKMe wat op 25−200μg/ml ESM, L/E of L gekultuur is, mits ‘n gemodifiseerde kultuur buffer gebruik word as eksperimentele buffer.

By undertaking a number of different experimental approaches at the genetic, cellular/ molecular and integrative physiology levels, I investigated functional variation in the Hypoxia-Inducible Factor (HIF) transcription pathway in humans. My studies focused on Tibetan natives. Tibetan highlanders are adapted to life in a hypoxic environment and exhibit distinct physiological traits at high altitude. Recent studies identified positive selection at two genetic loci, EPAS1 (HIF2α) and EGLN1 (PHD2), in Tibetan highlanders and demonstrated an association of EGLN1/EPAS1 genotype with haemoglobin concentration. Both are genes of the HIF pathway, which coordinates an organism’s response to hypoxia. Patients living at sea level with genetic diseases of the HIF pathway have characteristic phenotypes at both the integrative physiology and cellular levels. I investigated whether Tibetans living at sea level also possess distinct phenotypic characteristics, and whether these may be related to underlying variation within the HIF pathway. I compared Tibetans living at sea level with Han Chinese, their most closely-related major ethnic group, and found that Tibetans possess a significantly different integrative physiology phenotype. Tibetans had a lower haemoglobin concentration and haematocrit, a higher pulmonary ventilation relative to metabolism, and blunted pulmonary vascular responses to both acute (minutes) and sustained (8 hours) hypoxia. Regarding genotype- phenotype relationships within the Tibetans, I found a significant correlation between both EPAS1 and EGLN1 genotype and the induction of erythropoietin by systemic hypoxia. At an intermediate cellular level, the relative expression and the hypoxic induction of HIF- regulated genes were significantly lower in peripheral blood lymphocytes from Tibetans compared with Han Chinese. I also investigated whether the genetic variation in EPAS1 selected for in Tibetans may be functional at the molecular level by affecting transcription of EPAS1 in cells and whether certain coding variants in EGLN1 found in Tibetans affect protein (PHD2) activity in cells and in vitro. A small supplementary study was undertaken in patients with idiopathic erythrocytosis, who have elevated or inappropriately normal erythropoietin levels, to investigate if they have genetic alterations in the HIF system.

Thesis (PhD (Biomedical Sciences. Medical Physiology))--University of Stellenbosch, 2009.
ENGLISH ABSTRACT: In recent years there has been an increase in obesity and diabetes mellitus (DM). These conditions have for a long time been associated with infertility. Obesity is characterized by high levels of circulating leptin and cytokines as well as insulin resistance. Type I DM is associated with low or no insulin whereas, Type II DM is characterised by insulin resistance. As the prevalence of obesity and DM continues to rise, it is likely that the incidence of infertility associated with these pathological conditions will likewise increase. The effects of insulin and leptin on male reproductive function have been reported on the endocrine and spermatogenesis level, but their effects on cellular level of human ejaculated spermatozoa are yet to be elucidated. This study presents data on the role of insulin and leptin on human ejaculated spermatozoa and their interaction with cytokines and nitric oxide. In the first part of the study, we established the suitable concentrations of glucose, insulin and leptin that could be administered to human spermatozoa in vitro. Glucose concentration of 5.6 mM was chosen as the suitable concentration to be administered to human spermatozoa because it has previously been reported in the literature; furthermore, it is within the range of the physiological glucose levels found in the blood of fasting humans. Insulin and leptin concentrations of 10 μIU and 10 nmol were chosen respectively because they gave much improved sperm function and this was within the range of insulin and leptin levels previously measured in human ejaculated spermatozoa. This was followed by investigating the signalling pathway of insulin and its beneficial effects on human spermatozoa function. Endogenous insulin secretion from human ejaculated spermatozoa was blocked by nifedipine and its receptor tyrosine phosphorylation effects were inhibited by erbstatin while phosphatidylinositol 3-kinase (PI3K) phosphorylation activity was inhibited by wortmannin. Exogenous insulin administration significantly increased human sperm motility parameters as well as the sperm ability to acrosome react. The inhibition of endogenous insulin release from spermatozoa as well as the inhibition of the insulin receptor substrate (IRS) tyrosine phosphorylation significantly decreased motility parameters and the ability of spermatozoa to acrosome react. The study also investigated the effects of insulin and leptin on human sperm motility, viability, acrosome reaction and nitric oxide (NO) production. Both insulin and leptin significantly increased sperm motility parameters, acrosome reaction and NO production. The NO production induced by insulin and leptin was via PI3K signalling as evidenced by a reduction in NO levels when PI3K activity was inhibited by wortmannin. To investigate whether insulin and leptin could improve motility parameters of asthernozoospermic and teratozoospermic spermatozoa, the spermatozoa were separated into two fractions by means of a double density gradient technique. The gradient system was able to separate spermatozoa into high morphologically abnormal and less motile spermatozoa similar to that of asthernozoospermic and teratozoospermic patients as well as a more motile fraction. Insulin and leptin significantly increased the motility parameters of spermatozoa from the immature and less motile fraction. The fourth part of the study was aimed at investigating the effects of the cytokines, tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6), on human sperm motility, viability, acrosome reaction and NO production. The study shows that TNF-α and IL-6 significantly reduced motility parameters and acrosome reaction in a dose4 and time-dependent manner. These cytokines were also shown to significantly increase NO production from human spermatozoa. The decreased motility parameters induced by these cytokines could be attributed to their ability to induce excessive NO production. It is not yet clear how they inhibit spermatozoa to undergo the acrosome reaction. The fifth part of the study was to investigate the expression and localization of glucose transporter 8 (GLUT8) in human spermatozoa. This study shows that GLUT8 is constitutively expressed and located in the midpiece region of the human spermatozoa. The study also showed that stimulating spermatozoa with insulin led to an increase in GLUT8 expression as well as translocation to the acrosomal region. In the last part of the study we wanted to investigate why the increase in NO generation by spermatozoa due to insulin and leptin stimulation is accompanied with increased sperm function whereas NO increased due to TNF-α and IL-6 stimulation is accompanied with decreased sperm function. We observed that TNF-α and IL-6 not only increased NO production but also ROS production. This study speculates that the decrease in sperm motility and acrosome reaction when TNF-α and IL-6 were administered was due to the concomitant high increase in NO and ROS they induced. In conclusion, this study has established in vitro beneficial effects of insulin and leptin in normozoospermic and asthernozoospermic human sperm function. These hormones influence sperm function via the PI3K signalling pathway in two ways. Firstly, by increasing GLUT8 expression and translocation thereby possibly increasing glucose uptake and metabolism and secondly, by increasing NO production. The study has also established that TNF-α and IL-6 have detrimental effects on human spermatozoa in a dose and time dependent manner. These effects are mediated via their ability to stimulate both NO and ROS production in human spermatozoa. This study reports that GLUT8 is expressed in the midpiece region of human spermatozoa and that insulin stimulation upgrades its expression and leads to its translocation to the acrosomal region.
AFRIKAANSE OPSOMMING: Oor die afgelope jare was daar `n toename in obesiteit en diabetes mellitus (DM). Hierdie toestande word reeds vir ’n geruime tyd geassosieer met onvrugbaarheid. Obesiteit word gekenmerk deur verhoogde sirkulerende vlakke van leptiene en sitokiene sowel as insulien weerstandigheid. Tipe I DM word geassosieer met lae of geen insulien terwyl Tipe II DM gekenmerk word deur insulien weerstandigheid. Soos wat die voorkoms van obesiteit en DM toeneem, is dit waarskynlik dat die insidensie van onvrugbaarheid wat met hierdie patologiese toestande geassosieer word, gevolglik ook sal toeneem. Die effek van insulien en leptien op die manlike voortplantingsfunksie is alreeds aangetoon op endokriene en spermatogenese vlak, maar hul effekte op sellulêre vlak van menslike geëjakuleerde spermatosoë is nog onduidelik. Die studie vertoon data oor die rol van insulien en leptien op die menslike geëjakuleerde spermatosoë en hul interaksie met sitokiene en stikstofoksied (NO). In die eerste gedeelte van die studie, het ons ’n toepaslike konsentrasie van insulien en leptien bepaal wat aan menslike spermatosoë in vitro toegedien kan word. Glukose konsentrasies van 5,6 mM is bepaal as die gepaste konsentrasie om aan menslike spermatosoë toe te dien, omdat dit beter resultate tot gevolg het; verder is dit vergelykbaar met fisiologiese glukose vlakke in die bloed van `n vastende persoon. Insulien en leptien konsentrasies is op 10 μIU en 10 nm onderskeidelik vasgestel, aangesien dit tot beter resultate gelei het, en omdat dit vergelykbaar was met insulien en leptien vlakke wat reeds voorheen in menslike geëjakuleerde spermatosoë gemeet is. Dit was gevolg deur `n ondersoek na die insulien seintransduksie pad en sy voordelige effekte op menslike spermatosoë funksie. Endogene insulien afskeiding deur menslike geëjakuleerde spermatosoë was deur nifedipien geïnhibeer en sy reseptor tirosien fosforilasie effekte was deur erbstatin geïnhibeer terwyl fosfatidielinositol 3-kinase (PI3K) fosforilasie deur wortmannin geïnhibeer is. Eksogene insulien toediening het menslike sperm-motiliteit parameters betekenisvol laat toeneem asook die vermoë van sperme om die akrosoomreaksie te ondergaan. Die inhibisie van endogene insulien afskeiding deur spermatosoë sowel as die inhibisie van die insulien reseptor substraat (IRS) tirosien fosforilasie het die motiliteit parameters en die akrosoomreaksievermoë van spermatosoë verlaag. Die studie het ook die effekte van insulien en leptien op menslike sperm-motiliteit, -lewensvatbaarheid, -akrosoomreaksie en -NO produksie nagevors. Beide insulien en leptien het sperm-motiliteit parameters, -akrosoomreaksie en -NO produksie betekenisvol verhoog. NO produksie is deur insulien en leptien via PI3K seintransduksie geïnduseer, soos bewys deur die verlaging waargeneem in NO vlakke toe PI3K aktiwiteit deur wortmannin geïnhibeer was. Om vas te stel of insulien en leptien die motiliteit parameters van asthenozoospermiese en teratozoospermiese spermatosoë kon verbeter, het ons spermatosoë in twee fraksies met ’n dubbel digtheid gradiënt geskei. Die gradiënt sisteem was daartoe instaat om die spermatosoë in ’n onvolwasse, (morfologies abnormaal en minder motiel - soortgelyk aan dié van asthenozoospermiese en teratozoospermiese pasiënte), sowel as ’n volwasse meer motiele fraksie te skei. Insulien en leptien het die motiliteit parameters van spermatosoë van die onvolwasse en minder motiele fraksie verhoog. Die vierde gedeelte van die studie was daarop gemik om die effekte van die sitokiene tumor nekrose faktor alfa (TNF-α) en interleukin-6 (IL-6) op menslike sperm-motiliteit, -lewensvatbaarheid, -akrosoomreaksie en -NO produksie, te ondersoek. Die studie het getoon dat TNF-α en IL-6 motiliteit parameters en akrosoomreaksie in ’n tyd- en dosis-afhanklike wyse betekenisvol verlaag het. Hierdie sitokiene was ook in staat om NO produksie in menslike spermatosoë te verhoog. Die verlaging in motiliteit parameters wat deur hierdie sitokiene geïnduseer is, kan toegeskryf word aan hul vermoë om die produksie van oormatige hoeveelhede NO te stimuleer. Dit is nog nie duidelik hoe hulle die akrosoomreaksie in spermatosoë kan inhibeer nie. Die vyfde gedeelte van die studie het dit ten doel gehad om die uitdrukking en lokalisering van die glukose transporter 8 (GLUT8) in menslike spermatosoë te ondersoek. Hierdie studie kon aantoon dat GLUT8 konstitutief uitgedruk is en in die middelstuk van die menslike spermatosoë voorkom. Die studie bewys ook dat stimulering van die spermatosoë met insulien tot `n toename in GLUT8 uitdrukking sowel as translokasie na die akrosomale area, lei. In die finale gedeelte van die studie wou ons ondersoek waarom die toename in NO produksie in spermatosoë (as gevolg van insulien en leptien stimulasie) deur `n toename in spermfunksie gekenmerk word, terwyl die toename in NO produksie (as gevolg van TNF-α en IL-6 stimulasie) deur ’n afname in spermfunksie gekenmerk word. Ons het waargeneem dat TNF-α en IL-6 nie alleen NO produksie nie, maar ook reaktiewe suurstof spesies (ROS) produksie verhoog het. Ons vermoed dat die afname in sperm motiliteit en akrosoomreaksie met TNF-α en IL-6 toediening, die gevolg van die gelyktydige verhoging in NO en ROS was. In gevolgtrekking kan ons sê dat hierdie studie die voordelige in vitro effekte van insulien en leptien op asthenozoospermiese en teratozoospermiese menslike spermfunksie aangetoon het. Hierdie hormone beïnvloed spermfunksie via die PI3K seintransduksie pad op twee maniere. Eerstens, deur `n toename in GLUT8 uitdrukking en translokasie, met die gevolg dat glukose opname en metabolisme moontlik verhoog is, en tweedens, deur die toename in NO produksie. Die studie het ook vasgestel dat TNF-α en IL-6 nadelige effekte op menslike spermatosoë in `n dosis- en tyd-afhanklike wyse het. Hierdie effekte vind plaas a.g.v. hul vermoë om beide NO en ROS produksie in menslike spermatosoë te induseer. Die studie toon aan dat GLUT8 uitdrukking in die middelstuk van die menslike spermatosoon voorkom en dat insulien stimulasie GLUT8 uitdrukking opreguleer en tot translokasie na die akrosomale area lei.

Thesis (PhD)--University of Stellenbosch, 2011.
ENGLISH ABSTRACT: The Mechanism of -adrenergic preconditioning ( -PC) Ischaemic preconditioning (IPC), a potent endogenous protective intervention against myocardial ischaemia, is induced by exposure of the heart to repetitive short episodes of ischaemia and reperfusion. The protective effects of this phenomenon have been demonstrated to be mediated by release of autocoids such as adenosine, opioids and bradykinin. Release of endogenous catecholamines and activation of the beta-adrenergic receptors (b-AR) have also been shown to be involved in ischaemic preconditioning. However, the exact mechanism whereby activation of the - adrenergic signal transduction pathway leads to cardioprotection, is still unknown. In view of the above, the aims of the present study were to evaluate: (i) the respective roles of the 1-, 2- and 3-AR receptors as well as the contribution of Gi protein and PKA to -adrenergic preconditioning, (ii) the role of the prosurvival kinases, PKB/Akt and ERK 44/p42 MAPKinase in -drenergic preconditioning, (iii) whether b-AR stimulation protect via ischaemia and the formation of adenosine; the respective roles of the A1-, A2-, A3-adenosine receptors as well as the involvement of the PI3-K/PKB/Akt and ERKp44/p42 signal transduction pathways, in the cardioprotective phenomemon of -adrenergic preconditioning and (iv) the contribution of the mitochondrial KATP channels (mKATP), reactive oxygen species and NO to the mechanism of -AR-induced cardioprotection. Methods: Isolated perfused rat hearts were subjected to 35 min regional ischaemia (RI) and reperfusion. Infarct size (IS) was determined using tetrazolium staining (TTC) and data were analyzed with ANOVA. Hearts were preconditioned with 5 min isoproterenol 0.1 μM ( 1/ 2-AR agonist), or formoterol 1 nM ( 2-AR agonist) or BRL 37344 1 μM ( 3-AR agonist) followed by 5 min reperfusion. The roles of the 1-, 2- and 3-ARs as well as NO were explored by using the selective antagonists CGP-20712A (300 nM), ICI -18551 (50 nM), SR59230A (100 nM) and NOS inhibitors L-NAME (50 μM) or LNNA (50 μM) respectively. Involvement of ROS and the mK+ ATP channels was studied by administration of N-acetyl cysteine (NAC, 300 μM) and the mitK+ ATP iv channel blocker 5-HD (100 μM) during the triggering phase. The role of PKA and PI3-K/Akt was investigated by the administration of the blockers Rp-8-CPT-cAMPs (16 μM) and wortmannin (100 nM) respectively, prior to RI or at the onset of reperfusion. Pertussis toxin (PTX), 30 μg kg-1 was administered i.p., 48 h prior to experimentation. The role of adenosine and the adenosine A1, A3, A2A and A2B receptors was studied by using adenosine deaminase and the selective antagonists DPCPX (1 μM), MRS 1191(1 μM), ZM241385 (1 μM) and MRS1754 (1 μM). Activation of PKB/Akt and ERKp44/p42 was determined by Western blot. Results: Infarct sizes of hearts preconditioned with isoproterenol of formoterol were significantly smaller compared to those of non-preconditioned hearts. This was associated with an improvement in postischaemic mechanical performance. However the 3-AR agonist BRL37344 could not reduce infarct size. The 1- and 2-AR blockers CGP-20712A and ICI-118551 completely abolished the isoproterenol-induced reduction in infarct size and improvement in mechanical recovery, while the 3-AR blocker was without effect. Both Rp-8-CPT-cAMPs and wortmannin significantly increased infarct size when administered before 1/ 2-AR preconditioning or at the onset of reperfusion while it reduced mechanical recovery during reperfusion. PTX pretreatment had no significant effect on the reduction in infarct size induced by 1/ 2-AR or 2-AR preconditioning, however it reduced mechanical recovery in the latter. The NOS inhibitors had no effect on the reduction in infarct size induced by 1/ 2-AR preconditioning, but depressed mechanical function during reperfusion. The significant reduction in infarct size by 1/ 2-PC, was associated with activation of ERKp44/p42 and PKB/Akt during the triggering phase, as well as during reperfusion. DPCPX (A1-AdoR antagonist) had no effect on the 1/ 2-PC-induced reduced infarct size or ERK p44/p42 and PKB activation. A2A-AdoR, but not A2b-AdoR, blockade during the trigger phase abolished the reduction in infarct size of 1/ 2-PC. Both antagonists significantly reduced ERK and PKB activation in the trigger phase. In addition, when applied at the onset of reperfusion they significantly reduced ERK p44 / v p42 MAPK and PKB/Akt activation to an even greater extent. MRS-1191 (A3-AdoR antagonist) blocked 1/ 2-PC when applied prior to index ischaemia or when added during early reperfusion, significantly inhibiting both ERK p44 and PKB activation. Cardioprotection of 1/ 2-PC was abolished by inhibition of ROS generation with NAC in the triggering phase as well as at the start of reperfusion. However, the mitoK+ ATP channel blocker 5- HD was without effect. Conclusions: Protection afforded by an acute transient stimulation of the -ARs, depends on the activation of both 1-AR and 2-ARs but not the 3-AR. PKA as well as PI3-K activation prior to sustained ischemia and at the onset of reperfusion were essential for cardioprotection. With functional recovery as endpoint, it appears that NO is involved in 1/ 2-AR preconditioning, while the Gi protein may play a role in 2-AR preconditioning. The production of endogenous adenosine induced by transient b1/b2 stimulation of the isolated rat heart is involved in b−AR preconditioning. Cardioprotection was shown not to be dependent on the A1AdoR while activation of the A3-AdoR occurs during both the triggering and mediation phases. Both the adenosine A2A and, to a lesser extent, the adenosine A2B receptors participate in the triggering phase of b1/b2-PC. Generation of ROS during the triggering and reperfusion phases is involved in eliciting protection, but no role for the mKATP channels could be demonstrated. Finally, activation of the RISK pathway (PKB/Akt and ERKp44/p42) during the triggering phase is a prerequisite for protection. In addition, cardioprotection by b-AR is characterized by activation of the RISK pathway during reperfusion.
AFRIKAANSE OPSOMMING: Iskemiese prekondisionering (IPC) is ‘n kragtige endogene beskerming teen miokardiale iskemie, wat deur blootstelling van die hart aan kort opeenvolgende episodes van iskemie en herperfusie, ontlok word. Hierdie beskerming word medieer deur vrystelling van outakoïede soos adenosine, opioïede en bradikinien. Vrystelling van endogene katekolamiene en aktivering van die betaadrenerge reseptore (b-AR) is bewys om ook by hierdie proses betrokke te wees. Die presiese meganismes waardeur aktivering van die -adrenerge seintransduksiepad tot miokardiale beskerming lei, is nog onbekend. In die lig van bogenoemde, was die doel van die huidige studie om die volgende te evalueer: (i) die onderskeie rolle van die b1-, b2- en b3-AR sowel as die bydrae van die Gi proteïen en PKA in b- adrenerge prekondisionering, (ii) of b-AR stimulasie beskerming ontlok via iskemie en vorming van adenosien, die onderskeie rolle van die A1-, A2-, A3-adenosien reseptore (AdoRs) sowel as die PI3- K/PKB/Akt en ERKp44/p42 seintransduksie paaie, (iv) die mitochondriale KATP (mKATP) kanale, vry suurstof radikale en NO in b−AR prekondisionering. Metodes: Geïsoleerde, geperfuseerde rotharte is aan 35 minute streeksiskemie en herperfusie onderwerp. Infarktgrootte (IS) is deur die tetrazolium (TTC)-kleuringsmetode bepaal. Data is met behulp van ANOVA analiseer. Harte is geprekondisioneer vir 5 min met isoproterenol 0.1 μM ( 1/ 2-AR agonist), of formoterol 1 nM ( 2-AR agonist) of BRL 37344 1 μM ( 3-AR agonist), gevolg deur 5 min herperfusie, voor streeksiskemie. Die belang van die 1-, 2- en 3-ARs sowel as NO is ondersoek, deur onderskeidelik gebruik te maak van selektiewe antagoniste nl CGP- 20712A (300 nM), ICI -18551 (50 nM), SR59230A (100 nM) en NOS inhibitore L-NAME (50μM) of LNNA (50μM). Die rol van die mK+ ATP kanale en ROS is bepaal deur die toediening van die mK+ ATP kanaal blokker 5-HD (100 μM) en die vrye-radikaal opruimer, N-asetiel cysteine (NAC, 300 μM). Die belang van PKA en PI3-K/Akt is bepaal deur toediening van die PKA blokker Rp-8- CPT-cAMPs (16μM) en wortmannin (100nM) respektiewelik. Pertussis toxin (PTX), 30 μg kg-1 is i.p toegedien, 48 uur voor eksperimentasie. vii Die rol van adenosien en die adenosien A1, A2A, A2B en A3 reseptore is bestudeer, deur gebruik te maak van adenosien deaminase en die selektiewe antagoniste DPCPX (1 μM), MRS 1191(1 μM), ZM241385 (1 μM) and MRS1754 (1 μM),repektiewelik. Die middels is deurgaans toegedien tydens die prekondisioneringsprotokol (“snellerfase”) of tydens vroeë herperfusie. Aktivering van PKB/Akt en ERK p44/p42 is deur Western blot analise bepaal. Resultate: Infarktgrootte van harte wat geprekondisioneer is met of isoproterenol ( 1/ 2-PC) of formoterol ( 2-PC), was beduidend kleiner as díe van ongeprekondisioneerde harte. Dit is geassosieer met ‘n toename in postiskemiese meganiese herstel. Die 3-AR agonis BRL37344 ( 3- PC) het egter geen effek op infarktgrootte gehad nie. Die selektiewe 1- en 2-AR blokkers CGP- 20712A en ICI-118551 het die afname in infarktgrootte heeltemal opgehef, asook die verbetering in meganiese herstel tydens herperfusie terwyl die 3-AR blokker geen effek getoon het nie. Beide Rp- 8-CPT-cAMPs en wortmannin het infarktgrootte beduidend vergroot en meganiese herstel beduidend verlaag, wanneer dit net voor 1/ 2-prekondisionering of tydens die begin van herperfusie toegedien is. PTX voorafbehandeling het geen beduidende effek op die vermindering van infarktgrootte (geïnduseer deur 1/ 2-PC of 2-PC) gehad nie. Meganiese herstel is egter verminder in die geval van 2-PC. Die NOS inhibitore het geen effek op die vermindering in infarktgrootte geïnduseer deur b1/b2 gehad nie, maar het ook meganiese herstel onderdruk. Die beduidende afname in infarktgrootte deur b1/b2 prekondisionering is gekenmerk deur aktivering van ERKp42/p44 en PKB/Akt tydens die snellerfase. Soortgelyke aktivering van hierdie kinases is ook tydens herperfusie van b-AR geprekondisioneerde harte waargeneem. DPCPX (A1-AdoR antagonis) het geen effek op die infarkt-verminderde effek van 1/ 2- prekondisionering of op ERK p44/p42 en PKB aktivering gehad nie. A2A-AdoR, maar nie A2b – AdoR, blokkade tydens die snellerfase, het die effek van b-AR prekondisionering op infarktgroottee opgehef. Beide antagoniste het die aktivering van ERKp42/p44 en PKB/Akt tydens die snellerfase onderdruk. Wanneer toegedien tydens herperfusie, het dit die aktivering van hierdie kinases tot ‘n groter mate onderdruk. MRS-1191 (A3-AdoR antagonis) het infarktgrootte beduidend verhoog en 1/ 2-prekondisionering geblokkeer, beide wanneer dit voor indeks-iskemie toegedien is of tydens vroeë herperfusie, tesame met inhibisie van PKB en ERK p44/p44 aktivering. viii Die kardiobeskerming van 1/ 2-prekondisionering is opgehef deur middel van opruiming van vry suurstof radikale deur NAC in die snellerfase sowel as aan die begin van herperfusie. Die mK+ ATP kanaal blokker 5-HD het geen effek op b-AR prekondisionering gehad nie. Gevolgtrekking: Kardiobeskerming teweeggebring deur ‘n kort periode van stimulasie van die - ARs, is afhanklik van die aktivering van beide 1-AR en 2-ARs, maar nie 3-AR nie. PKA sowel as PI3-K aktivering, net voor volgehoue iskemie en tydens vroeë herperfusie, is aangedui om noodsaaklik vir 1/ 2-AR prekondisionering te wees. Waar funksionele herstel as eindpunt gebruik is, blyk dit dat NO wel betrokke is by 1/ 2-AR prekondisionering, terwyl die Gi protein ‘n rol mag speel in 2-AR prekondisionering. Vorming van endogene adenosien tydens b-adrenerge stimulasie is betrokke by b-AR prekondisionering. Hierdie beskerming is nie van die A1-AdoR afhanklik nie, maar aktivering van die A3-AdoR is nodig tydens beide die sneller en herperfusie fases. Beide die A2A-AdoR, en tot ‘n mindere mate die A2B–AdoR, is ook betrokke by die snellerfase. Vorming van vry suurstof radikale is nodig vir b-AR prekondisionering, nterwyl die mKATP kanale nie betrokke is nie. Ten slotte, aktivering van die RISK seintransduksiepad (ERKp42/p44 en PKB/Akt) tydens die snellerfase is ‘n voorvereiste vir die ontlokking van beskerming. Daarbenewens word b-AR prekondisionering gekarakteriseer deur aktivering van hierdie pad tydens herperfusie.
South African Medical Research Council
University of Stellenbosch

Thesis (MScMedSc)-- Stellenbosch University, 2013.
ENGLISH ABSTRACT: Preconceptual sex selection is an ethically justifiable process whereby X- and Y-chromosome bearing spermatozoa are isolated prior to fertilization of the oocyte in order to generate either a male or a female offspring. Although various separation techniques are available, none can guarantee 100% accuracy. There are various physiological differences between X- and Y-chromosome bearing spermatozoa which can be used to separate these two populations of sperm. For the purpose of this study, X- and Y-chromosome bearing spermatozoa were separated based on (1) their respective abilities to remain viable when subjected to adverse environments, including extreme pH values, increased temperatures and various hydrogen peroxide (H2O2) concentrations; (2) the ability of Y-chromosome bearing spermatozoa to swim faster and/or more progressively than X-chromosome bearing spermatozoa; and (3) the X-chromosome bearing spermatozoa’s increased size and weight when compared to the Y-chromosome bearing spermatozoa. The efficacy of live and dead cell separation through (i) Magnetic Antibody Cell Separation (MACS) and (ii) a modified swim-up technique was also assessed and compared. Changes in the sex-chromosome ratio of samples were established by double-label fluorescent in situ hybridization (FISH) before and after processing. Sperm motility (CASA) and viability (eosin/nigrosin) was assessed before and after each intervention. Ethical clearance for this study was granted by the Health Research Ethics Committee 1 (Ethics #: S13/04/068). The results indicated successful enrichment of X-chromosome bearing spermatozoa upon incubation in acidic media, increased temperatures, and H2O2. In contrast, Y-chromosome bearing spermatozoa were successfully enriched through a direct swim-up method as well as discontinuous gradient centrifugation. In conclusion, this study demonstrated the potential role for physiological differences between X- and Y-chromosome bearing spermatozoa in the development of preconceptual gender selection through sperm sorting.
AFRIKAANSE OPSOMMING: Prekonsepsie geslagselektering is 'n eties regverdigbare proses waardeur X- en Y- chromosoom draende spermatosoë geïsoleer word voordat bevrugting van die oösiet plaasvind, om óf 'n manlike óf 'n vroulike nageslag te genereer. Alhoewel verskeie skeidingstegnieke beskikbaar is, kan geeneen 100% akkuraatheid waarborg nie. Daar bestaan verskeie fisiologiese verskille tussen X- en Y- chromosoom draende spermatosoë wat skeiding van hierdie twee groepe spermatosoë moontlik kan maak. Vir die doel van hierdie studie is skeidingsmetodes vir die X- en Y- chromosoom draede spermatosoë gebaseer op (1) hul onderskeie vermoëns om lewensvatbaar te bly tydens blootstelling aan ‘n ongunstige milieu, insluitend ekstreme pH waardes, verhoogde temperature en verskeie waterstofperoksied (H2O2) konsentrasies; (2) die vermoë van die Y-chromosoom draende spermatosoon om vinniger en/of meer progressief as X-chromosoom draende spermatosoë te swem; en (3 ) die X-chromosoom draende spermatosoon se verhoogde grootte en gewig in vergelyking met die Y- chromosoom draende spermatosoon. Die effektiwiteit van die (i) Magnetiese Anti-liggaam Sel Skeidingstegniek (MACS) en (ii) 'n aangepaste weergawe van die op-swem tegniek om lewendige en dooie selle te skei is ook bepaal en vergelyk. Veranderinge in die geslagschromosoom verhouding van die monsters is bepaal deur dubbel-etiket fluoresensie in situ hibridisering (FISH) voor en na verwerking. Spermmotiliteit (CASA) en lewensvatbaarheid (eosien/nigrosin) is bepaal voor en na elke intervensie. Etiese goedkeuring vir hierdie studie is verleen deur die Gesondheids-Navorsingsetiekkomitee 1 (Etiese # : S13/04/068). Die resultate dui suksesvolle verryking van X-chromosoom draende spermatosoë deur inkubasie in suur media, verhoogde temperature, en H2O2. Y-chromosoom draende spermatosoë is verryk deur middel van 'n direkte op-swem metode sowel as diskontinue gradiënt sentrifugering . Ten slotte, hierdie studie toon die potensiële rol vir fisiologiese verskille tussen X- en Y- chromosoom draende spermatosoë in die ontwikkeling van prekonsepsie geslagselektering metodes deur skeiding van X- en Y-chromosoom draende sperme.