Dissertations / Theses on the topic 'Humane Animal Treatment'

This thesis has made a significant contribution to future chlamydial research by uncovering the chlamydial pathogenic mechanisms which will potentially help in the development of targeted vaccine against the pathogen. This thesis has made important new contributions to our understanding of Chlamydia pneumoniae specific adaptations to stress responses and has provided new perspectives on the survival of this successful pathogen. This thesis has used two well established microbial stressors and has identified major differences in stress responses between human and animal Chlamydia pneumoniae isolates.

Thesis (MSc (Physiological Sciences))--University of Stellenbosch, 2009.
Breast cancer is currently the primary cause of cancer-related death in women worldwide. Conventional treatments such as radiation and chemotherapy have many deleterious and long lasting side-effects, some of which are permanent, such as infertility. As certain tumour cells can also acquire resistance to chemotherapy, the need for the development of a less severe, yet more effective, targeted anti-cancer treatment exists. Curcumin, a plant polyphenol from Curcuma longa, has long been thought to possess antitumour, antioxidant, anti-arthritic, anti-amyloid, anti-ischemic and anti-inflammatory properties. Numerous studies conducted over the past sixty years confirm this. We aimed at examining the effect of curcumin on cell viability and the different modes of cell death, namely apoptosis, necrosis and autophagy, in the MCF-12A (non-tumorigenic mammary epithelial) and MCF-7 (mammary adenocarcinoma) cell lines. Cells were incubated with different doses of curcumin to evaluate the dose response through a MTT assay. Thereafter, cells were incubated with 200 μM curcumin for 48 hrs and stained with markers and DNA stains for apoptosis (Hoechst, Caspase-3, PARP), necrosis (Propidium Iodide) and autophagy (LC3B and Beclin-1). Cells were examined via fluorescence microscopy, Western Blot- and FACS analyses. MTT results showed no significant decrease in viability in the MCF-12A cell line after curcumin treatment. However, a significant decrease in viability was observed in MCF-7 cells after treatment with 200 μM curcumin (p < 0.05). Treated MCF-7 cells also show clear LC3B expression. FACS results show a significant difference in Hoechst mean fluorescence intensity in MCF-7 cells after curcumin treatment (p < 0.05). This study provides evidence that MCF-7 cells respond to a 200 μM dose of curcumin treatment through metabolic change and induction of the autophagic pathway. The model system used in this study provides groundwork for further cell culture based studies regarding breast cancer and curcumin.

It is difficult to overestimate the social and psychological significance of human-animal interactions. Till now, studies on human-companion animal interactions primarily focussed on positive aspects and relationships, while studies on animal phobias have almost exclusively focussed on spider and snake phobia. The problem with negative human-animal relationships in general, and animal phobia in particular, is in essence a superficial understanding of the determination of physiological changes and parameters associated with its description and treatment. The main aim of this study was to provide theoretical and physiological information regarding the determination of a biochemical parameter which can be used to enhance effective diagnosis and treatment of individuals suffering from dog phobia. A trimodal approach was followed to describe anxiety and fear responses associated with dog phobia. Subjects were assigned to two groups: an experimental group consisting of females suffering from dog phobia, and a control group. The study consisted of three experimental stages: the first stage (resting stage) measured baseline values, the second stage (preintervention stage) measured values in the presence of a dog stimulus prior to the intervention program, and the third stage (postintervention stage) measured values in the presence of a dog stimulus after completion of the intervention program. Cognitive-affective aspects were initially measured by means of the Fear Survey Schedule, as well as by means of an anxiety scale and stressor schedule during the experimental stages. Motor-behavioural aspects were measured as the termination distance of the dog approach during the pre- and postintervention stages, as well as assessed by a psychologist through direct observation of non-verbal communication cues during the behavioural approach tests. The measurement of physiological aspects focussed on the determination of plasma adrenocorticotropic hormone (ACTH) levels during the experimental stages. The main results were as follow: • the experimental group scored significantly higher average scores on the animal, dog, blood/injection and total fear categories of the Fear Survey Schedule than the control group; • the intervention program was effective in treating motor-behavioural and cognitive¬affective aspects of the phobia response; • the effect of the intervention program on the plasma ACTH-Ievels was inconclusive. No significant differences were found between the experimental group's average plasma ACTH-Ievels during the experimental stages, as well as between the experimental and control groups during the resting and preintervention stages. The average plasma ACTH-Ievels of the control group was significantly lower than that of the experimental group during the postintervention stage; • total stressor schedule values suggest that subjects in the experimental group have a predisposition to be generally more anxious and fearful than subjects in the control group; • two-thirds of the dog phobia subjects reported classical conditioning as the etiological pathway; • various auditory and visual cues were found to be the focal point of perception in women suffering from dog phobia; and • group qualitatively evaluated their current fear level for dogs as substantially lower than at the onset of the project. In conclusion, the determination of plasma ACTH-Ievels as a single parameter is not adequate to support the complex interaction between overt motor-behavioural, cognitive-affective and physiological patterns during the treatment of women experiencing dog phobia.
Dissertation (MSc (Veterinary Ethology))--University of Pretoria, 1999.
Production Animal Studies
unrestricted

Increasing evidence illustrates an involvement of stress in a large variety of physical and mental illness. Together with the evolutionary development of the social behavior in humans, the traditional interpretations of the attachment theory and the social support theory underscores the importance of affection, belonging and appreciation for human well-being. Not only can an imbalanced stress system be the cause of severe pathological consequences, insufficient social contact can also hamper recovery. Frequent usage of animals in various settings steadily illustrates both physiological and psychological benefits on both the young and the old, the healthy and the ill. Through the study of neurobiological factors, with oxytocin as a central mediator of social behavior and its  impact in turn on the stress- and cortisol system, this paper examines the possibility of animals to function as social support. The potential of animals to reduce the suffering in patients with stress related psychiatric disorders, such as the highly frequent exhaustion disorder, human-animal interactions might offer a non-invasive complementary tool to current treatment methods.

Thesis (PhD)--Stellenbosch University, 2013.
ENGLISH ABSTRACT: Introduction Cisplatin has been widely used to treat solid tumours and much success has come from the use of this drug in the treatment of head and neck, ovarian, testicular, cervical and small-cell lung cancers. However, the success of cisplatin treatment is limited due to its dose-limiting toxicity and its resulting side-effects, such as nephro- and ototoxicity. The devastating side-effects induced by cisplatin treatment provided the platform for this study whereby the aim was to lower the concentration of cisplatin while maintaining its cancer-specific cytotoxic action. Equally concerning is, cisplatin resistance which is becoming increasingly common, and this radically limits the clinical efficacy and utility of the drug. Adjuvant therapy has thus become necessary in an attempt to possibly curb or lessen the extent of cisplatin resistance. Due to the large body of evidence implicating the importance of autophagy in cancer, the prospect of targeting this mechanism has generally been accepted. Various chemotherapy agents induce autophagy in cancer cells; however the effect of cisplatin on autophagic induction has not been very well explored. We thus hypothesise that the manipulation of the autophagic pathway will sensitise cancer cells to a low concentration of cisplatin treatment. Furthermore, due to the functional interaction between Bcl-2 and Beclin-1 and its role in the regulation of autophagy, ratio analysis of Beclin-1 to Bcl-2 as means of detecting the role of autophagy within the cell under homeostatic and treatment/stress conditions has been conducted. Additionally, Bcl-2 has a prominent role in the malignant cell and it’s over-expression has been found to confer resistance in a variety of cancerous cell lines. We therefore hypothesise that the silencing of Bcl-2 prior to cisplatin treatment will sensitise cervical cancer cells to apoptosis and increase the Beclin-1/Bcl-2 ratio in favour of apoptosis. Materials and Methods Three human cervical cell lines were used: a non-cancerous ectocervical epithelial cell line (Ect1/E6E7) and two cancerous cervical cell lines (HeLa and CaSki). In order to determine a concentration of cisplatin that was non-toxic to the non-cancerous Ect1/E6E7 cell line, a dose-response was performed. With the use of an autophagy inhibitor (bafilomycin A1) and an autophagy inducer (rapamycin), autophagic flux capacities were assessed in each cell line through the Western blotting technique. In order to assess whether the chosen concentration of cisplatin induced autophagy, flow cytometry with the use of a Lysotracker™ dye was utilised, as well as analysis of autophagy protein levels (LC-3 II, Beclin-1 and p62). Autophagy modulation was achieved through two methods: pharmacological modulation with use of two recognised agents, namely bafilomycin A1 and rapamycin, and biological manipulation with the use of ATG5 and mTOR mRNA silencing. The effects of different treatment regimes on cell death was assessed with the use of PARP and caspase-3 cleavage through Western blotting, caspase-3/-7 activity (Caspase-Glo®), PI inclusion, LDH release and MTT reductive capacity. Additionally the effects of these treatment regimes on cell-cycle progression were also analysed. Beclin-1 and Bcl-2 expression was determined through Western blotting and immunocytochemistry before and after treatment with cisplatin in HeLa and CaSki cells. To assess the reliance of the cervical cancer cells on Bcl-2 after cisplatin treatment, Bcl-2 knock-down was achieved through RNA interference, where after the Beclin-1/Bcl-2 ratio was assessed as well as apoptosis with the use of cleaved PARP analysis (Western blotting) and Caspase-Glo©. For the ex vivo analysis, biopsies were collected from patients undergoing routine colposcopy screenings and hysterectomies at Tygerberg Hospital, Tygerberg, Western Cape. A total of 10 normal, 29 low-grade squamous intraepithelial lesions (LSIL), 33 high-grade squamous intraepithelial lesions (HSIL) and 13 carcinoma biopsies were collected for analysis, where after the expression profiles of two autophagy markers (mTOR and LC-3 II), as well as one anti-apoptotic marker (Bcl-2) were assessed. Protein levels were analysed through Western blot and confirmed through immunohistochemistry. Results Dose-response curves revealed that 15 μM of cisplatin did not induce cell death in the normal cervical epithelial cell line (Ect1/E6E7) and was therefore utilised through-out the remainder of the study. It was additionally determined that the CaSki cells were more resistant to cisplatin treatment when compared to the HeLa and Ect1/E6E7 cells. Autophagic flux analysis revealed that, although all three cell lines were cervix derived, their autophagic flux capacities differed. It was observed that the chosen concentration of cisplatin was able to induce autophagy in all three cell lines, with the HeLa cells demonstrating a particularly pronounced response. Autophagy modulation in conjunction with cisplatin treatment revealed the following: Autophagy inhibition with bafilomycin A1 lead to significant increases in caspase-3 and PARP cleavage and LDH release in both cervical cancer cell lines. The inhibition of autophagy through silencing of ATG5 induced caspase-3 cleavage and agrees with results obtained from pharmacological inhibition of autophagy with bafilomycin A1. In addition to autophagic induction, a low concentration of cisplatin induced the up-regulation of Bcl-2, which when silenced significantly improved cisplatin-induced apoptosis in both cervical cancer cell lines. Analysis of the expression profiles of mTOR and LC-3 in normal, pre-malignant (LSIL and HSIL) and cancerous cervical tissue revealed that autophagy is significantly up-regulated in HSILs and carcinoma of the cervix. Additionally, Bcl-2 expression is significantly increased in cervical carcinoma tissue, which agrees with results from other studies. Conclusion Autophagic flux capacities between the three cell lines investigated, derived from the same organ, differ significantly. This should be taken into consideration when autophagic modulation is being used as an adjuvant treatment. With regard to chemotherapy treatment in cervical cells, a low-concentration of cisplatin significantly induces autophagy in malignant and non-malignant cervix-derived cell lines where it serves a pro-survival mechanism. Inhibition of autophagy with bafilomycin A1 and ATG5 siRNA confirmed this survival effect in both cancerous cell lines where apoptosis was significantly increased. Interestingly, rapamycin pre-treatment together with cisplatin did not induce significant levels of apoptosis in HeLa cells where autophagy induction may have provided additional protection from the cytotoxic effects of cisplatin. Therefore the inhibition of autophagy through pharmacological and biological inhibition improves the cytotoxicity of a low concentration of cisplatin and provides a promising new avenue for the future treatment of cervical cancer. Bcl-2 up-regulation in response to cisplatin treatment also serves as a protective mechanism by which cervical cancer cells survive. The extent of apoptotic cell death observed after biological inhibition of Bcl-2 reiterates the fact that this response may be exploited in order to favour the use of lower concentrations of cisplatin. Analysis of clinical specimens emphasised the value of the in vitro work: Cervical cancer biopsies had increased expression of both LC-3 II and Bcl-2, indicating autophagy induction and apoptosis inhibition, respectively. Thus two novel methods of improving cisplatin cytotoxicity have been demonstrated in the following study. Treatment regimens may administer more frequently and prolonged due to the minimal side-effects that accompanies low-dose cisplatin treatment.
AFRIKAANSE OPSOMMING: Inleiding Sisplatien word algemeen gebruik vir die behandeling van soliede gewasse. Baie sukses is reeds deur die gebruik van díe middel behaal in die behandeling van kop en nek, ovariale, terstikulêre, servikale en klein-sel kankers. Die sukses van Sisplatien-behandeling word wel ingeperk deur die dosis-beperkende toksisiteit en die gevolglike newe-effekte soos nefrotoksisiteit. Hierdie verwoestende newe-effekte wat deur sisplatien behandelings geïnduseer word, het as die platform vir hierdie studie gedien. Die doel was om die sisplatien konsentrasies te verlaag, maar terselfdertyd die kankerspesifieke sitotoksisiteit te behou. Nog ʼn punt van kommer is dat sisplatien-weerstandigheid aan die toeneem is, wat die kliniese effektiwiteit en gebruik van hierdie middel geweldig beperk. Byvoegmiddels het dus noodsaaklik geraak in die poging om die sisplatien-weerstandigheid te verhoed. As gevolg van verskeie bewyse wat die belangrikheid van outofagie in kanker impliseer, is die vooruitsig om hierdie meganisme te teiken, algemeen aanvaar. Verskeie chemoterapeutiese middels induseer outofagie in kanker selle, hoewel die effek van Sisplatien op outofagiese induksie nog nie goed ondersoek is nie. Ons hipotese is dus dat die manipulasie van die outofagiese pad die kankerselle sensitiseer tot ʼn lae konsentrasie van sisplatien. Verder, as gevolg van die funksionele interaksie tussen Bcl-2 en Beclin-1, en hul rol in die regulering van outofagie, is verhouding-analises van Beclin-1 tot Bcl-2 uitgevoer met die doel om die rol van outofagie in die sel onder homeostatiese en behandeling/stres kondisies te bepaal. Verder is Bcl-2 bekend daarvoor om ʼn prominente rol te speel in kwaadaardige selle, en die ooruitdrukking daarvan is gevind om weerstandigheid aan te help in ʼn verskeidenheid van kankeragtige sellyne. Ons hipotetiseer dus dat geenonderdrukking van Bcl-2 voor die behandeling met sisplatien die servikale kanker selle sal sensitiseer tot apoptose en ʼn verhoging in die verhouding van Beclin-1/Bcl-2 veroorsaak, wat in die guns van apoptose is. Materiale en Metodes Drie menslike servikale sellyne was gebruik: ʼn nie-kankeragtige servikale epiteel sellyn (Ect/E6E7) en twee kankeragtige servikale sellyne (HeLa en CaSki). Om ʼn konsentrasie van sisplatien te bepaal wat nie-toksies tot die nie-kankeragtige Ect1/E6E7 sellyn is, was ʼn dosisrespons uitgevoer. Met die gebruik van ʼn outofagiese inhibeerder (bafilomycin A1) en ʼn outofagiese induseerder (rapamycin), is die outofagiese-fluks kapasiteite van elke sellyn deur die Western Blotting tegniek geassesseer. Om te bepaal of die gekose konsentrasie van sisplatien outofagie induseer, is vloeisitometrie met ʼn Lysotracker™ kleurstof gebruik, sowel as analises op outofagie proteïenvlakke (LC-3 II, Beclin-1 en p62). Outofagie modulering is behaal deur twee metodes: farmakologiese modulering met twee erkende middels, naamlik bafilomycin A1 en rapamycin, en biologiese manipulasie met die gebruik van ATG5 en mTOR geenonderdrukking. Die effekte van die verskillende behandeling skedules op seldood was geassesseer deur gebruik te maak van PARP en kaspase-3 splitsing deur Western Blotting, kaspase-3/-7 aktiwiteit deur Caspase-Glo ®, PI-insluiting, LDH vrystelling en MTT reduserende kapasiteit. Verder is die effekte van hierdie behandeling skedules op selsiklus progressie ook geanaliseer. Beclin-1 en Bcl-2 uitdrukking was ook bepaal deur Western Blotting en immunohistochemie voor en na behandeling met sisplatien in HeLa en CaSki selle. Om die afhanklikheid van die servikale kankerselle op Bcl-2 na sisplatien behandelings te toets, is Bcl-2 onderdruk deur RNA-inmenging, waarna Beclin-1/Bcl-2 verhouding geassesseer is, sowel as opoptose deur die gebruik van gesplitste PARP analises (Western Blotting) en Caspase-Glo©. Vir die ex vivo analises is biopsies vanaf pasiënte wat roetine kolposkopie en histerektomies ondergaan, verkry (Tygerberg Hospitaal, Tygerberg, Westelike Provinsie). ʼn Totaal van 10 normale, 29 lae-graad plaveisel intraepiteel letsels (LSIL), 33 hoe-graad plaveisel intraepiteel letsels (HSIL) en 13 karsinoom biopsies is verkry vir analises. Die uitdrukkingsprofiel van twee outofagiese merkers (mTOR en LC-3 II), asook een merker vir apoptose (Bcl-2), was geassesseer. Proteïen vlakke was ook deur Western Blotting geanaliseer en deur immunohistochemie bevestig. Resultate Dosisrespons kurwes het getoon dat 15 μM sisplatien nie seldood in die normale sellyn (Ect1/E6E7) geïnduseer het nie, en was daarom gebruik deur die res van hierdie studie. Verder is daar ook gevind dat CaSki selle meer weerstandig tot sisplatien behandelings is wanneer vergelyk word met die HeLa en Ect1/E6E7 selle. Outofagiese-fluks analises het getoon dat, alhoewel al drie sellyne vanaf die serviks afkomstig is, daar verskille is in hul outofagiese-fluks kapasiteit. Daar is ook waargeneem dat die gekose konsentrasie van sisplatien in staat was om outofagie te induseer in al drie sellyne, met HeLa selle wat die mees merkbare respons getoon het. Modulering van outofagie in samewerking met sisplatien behandelings het die volgende onthul: inhibisie van outofagie deur bafilomycin A1 het gelei tot ʼn beduidende verhoging in kaspase-3, PARP splitsing en LDH vrylating in beide servikale kankersellyne. Geenonderdrukking van ATG5 induseer kaspase-3 splitsing en stem ooreen met resultate wat verkry is deur farmakologiese inhibisie van outofagie met bafilomycin A1. Bykomend tot outofagiese indusering, het ʼn lae konsentrasie sisplatien die opregulering van Bcl-2 geïnduseer. Wanneer Bcl-2 geenonderdrukking in hierdie scenario toegepas was, het dit ʼn beduidende verbetering in sisplatien-geïnduseerde apoptose in beide servikale kankersellyne getoon. Analises van die uitdrukkingsprofiel van mTOR en LC-3 in normale, pre-maligne (LSIL en HSIL) en kankeragtige servikale weefsel, het getoon dat outofagie beduidend opgereguleer is in HSILs en servikale karsinome. Verder is Bcl-2 uitdrukking ook gevind om beduidend verhoog te wees in servikale karsinoomweefsel, wat ooreenstem met resultate verkry in ander studies. Gevolgtrekking Outofagiese-fluks kapasiteite tussen die drie sellyne, afkomstig van dieselfde orgaan, toon beduidende verskille. Hierdie bevinding moet in ag geneem word wanneer outofagiese-modulering as ʼn bevorderingsbehandeling gebruik word. Met betrekking tot chemoterapie behandeling in servikale selle; ʼn lae konsentrasie van sisplatien veroorsaak ʼn beduidende indusering van outofagie in kwaadaardige en nie-kwaadaardige serviks-afkomstige sellyne, en dien as ʼn oorlewingsmeganisme. Inhibisie van outofagie met bafilomycin A1 en ATG5 siRNA het hierdie beskermings effek bevestig, aangesien apoptose beduidend verhoog was in beide kankersellyne. Interessant genoeg het rapamycin pre-behandeling tesame met sisplatien nie beduidende vlakke van apoptose in HeLa selle geïnduseer nie. Outofagie induksie mag dalk addisionele beskerming teen die sitotoksiese effekte van sisplatien gebied het. Daarom het die inhibisie van outofagie deur farmakologiese en biologiese inhibering die sitotoksisiteit van ʼn lae konsentrasie sisplatien bevorder, wat ʼn belowende bevinding is vir die toekomstige behandeling van servikale kanker. Bcl-2 opregulering as gevolg van sisplatien behandelings dien ook as beskermings meganisme waarby servikale kankerselle oorleef. Die mate van apoptotiese seldood wat waargeneem word na biologiese inhibering van Bcl-2, wys weer op die feit dat hierdie respons uitgebuit kan word vir die gebruik van laer konsentrasies van sisplatien. Analises van die kliniese monsters het ook die waarde van die in vitro werk versterk: Servikale kanker biopsies het verhoogde uitdrukking van beide LC-3 II en Bcl-2 getoon, wat aandui dat outofagie geïnduseer en apoptose geïnhibeer word. Daar is dus twee nuwe metodes vir die verbetering van sisplatien-toksisiteit in hierdie studie gedemonstreer. Behandeling regimes kan meer gereeld en vir langer tydperke toegepas word, aangesien die newe-effekte van lae-dosis sisplatien behandelings minimaal is.
MRC for funding

This study aims to deconstruct current conceptions about animal-assisted interventions by investigating relationships between human beings and birds of prey. Interactions between birds of prey, or “raptors,” provide novel cases from which to reexamine failed attempts to provide empirical data in support of alternative therapies. Previous research addressing the efficacy of animal-assisted interventions is simply not robust enough to be considered a feasible treatment option by medical professionals. By extension, models of self-regulation in psychology are often presented using reductionist models and oversimplified therapeutic outcomes. Taken together, raptor-human relationships help to highlight the shortcomings of each, as well as potential solutions towards developing comprehensive frameworks for measuring efficacy of multispecies interactions. This study was conducted at a small nature park in Largo, FL where a number of native raptor species are housed, cared for, and trained each day by volunteers. These volunteers made up the sample size for this study with forty participants (n = 40) between the ages of eighteen and seventy five. Drawing on both my own experiences as a raptor handler, as well as the qualitative data collected from volunteers, I employed a neuroanthropological approach to reveal underlying dynamics of the program via a two-stage research plan. Stage 1 of the study addresses the Raptor Program itself in facilitating human-animal interactions. Stage 2 addresses the mechanisms at play during firsthand encounters with birds of prey. Findings suggest that programmatic and regulatory drivers within the program must operate together, often simultaneously, for an animal-assisted intervention organization to be successful. Further, this study calls for the ongoing development of novel methodological approaches in future research to determine the efficacy of animal-assisted interventions at large.

The severity and number of people affected by cancer is a worldwide problem with millions of people affected annually. The search for treatment and cures of cancer continues to be a global effort. As part of this global effort, many natural products have been tested against cancer cell lines, most from plants located in tropical regions. However, this study reports that extracts of Atriplex confertifolia, a native North American plant, has significant bioactivity against human breast cancer cell lines MCF-7, 435, and 231, and HeLa cells (cervical cancer cells). The bioactivity of A. confertifolia extracts of these cells lines was compared to an FDA-approved cancer drug and an industry-standard leukocyte control cell line. Active portions of the extracts were found primarily in the polar fractions of the plant. A dose-response curve of the extracts clearly showed significant cell death similar to the FDA-approved drug. The plant extracts did not inhibit the viability of the leukocyte cell line. Cancer cell death was followed as a function of time and concentration. Cell death appears to be a result of apoptosis.

Thesis (PhD)--Stellenbosch University, 2013.
ENGLISH ABSTRACT: Although antiretroviral treatment decreases HIV-AIDS morbidity/mortality, long-term effects include onset of insulin resistance and cardiovascular diseases. Increased oxidative stress and dysregulation of the ubiquitin-proteasome system (UPS) are implicated in protease-inhibitor (PI)-mediated cardio-metabolic pathophysiology. We hypothesized that PI treatment (Lopinavir/Ritonavir) elevates myocardial oxidative stress and concomitantly inhibits the UPS, thereby attenuating cardiac function. Lopinavir/Ritonavir was dissolved in 1% ethanol (vehicle) and injected into mini-osmotic pumps that were surgically implanted into Wistar rats for eight weeks vs. vehicle and sham controls. Subsequently, we evaluated metabolic parameters and heart function (ex vivo and in vivo methods) at baseline and following ischemia-reperfusion. PI-treated rats exhibited weight gain, increased serum LDL-cholesterol, higher tissue triglycerides (heart, liver), but no evidence of insulin resistance. It also upregulated hepatic gene expression of acetyl-CoA carboxylase β and 3-hydroxy-3-methylglutaryl-CoA-reductase, key regulators of fatty acid oxidation and cholesterol synthesis, respectively. Further, PI-treated hearts displayed impaired UPS, increased superoxide dismutase (SOD) activity and unaltered superoxide levels, and elevated peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC-1α) peptide levels. Perfusion data revealed contractile dysfunction at baseline and following ischemia-reperfusion, while post-ischemic hearts exhibited decreased ATPase specific activity vs. matched controls. Early changes initiated by PI treatment resemble the metabolic syndrome and reflect a pre-atherogenic profile. Moreover, the effects of PIs on cardiac contractile function may in part be triggered by impaired UPS activity together with strain on the mitochondrial energetic system. Our study alerts to cardio-metabolic side effects of PI treatment and raises the question of the most appropriate co-therapies for patients on chronic antiretroviral treatment.
AFRIKAANSE OPSOMMING: Alhoewel anti-retrovirale behandeling MIV-VIGS morbiditeit/mortaliteit verlaag, bestaan daar langtermyn effekte soos die aanvang van insulienweerstandigheid en kardiovaskulêre siektes. Verhoogde oksidatiewe stres en wanregulering van die ubikwitien-proteosoomsisteem (UPS) word geïmpliseer met protease-inhibeerder (PI) gemediëerde kardio-metaboliese patofisiologie. Ons hipotetiseer dat PI behandeling (Lopinavir/Ritonavir) miokardiale oksidatiewe stres verhoog, en gevolglik die UPS inhibeer waardeur dit kardiale funksie verander. Lopinavir/Ritonavir is in 1% etanol (draer) opgelos en in ‘n mini-osmotiese pomp ingespuit wat chirurgies in Wistar rottes ingeplant is vir agt weke vs. draer en valskontroles. Gevolglik het ons die metabolise parameters en hartfunksie (ex vivo en in vivo metodes) op basislyn en na afloop van ischemie-reperfusie ondersoek. PI-behandelde rotte het ‘n toename in massa getoon asook verhoogde serum LDL-cholesterol, hoër weefseltrigliseriede (hart, lewer), maar geen bewys van insulienweerstandigheid nie. Dit het ook hepatiese asetielko-ensiem A karboksilase β en 3-hidrokise-3-metielglutariel KoA reduktase geenuidrukking opwaarts gereguleer, wat sleutel reguleerders van vetsuuroksidasie en cholesterolsintese onderskeidelik is. Verder, het PI-behandelde harte ingeperkte UPS, verhoogde SOD aktiwiteit en onveranderde superoksiedvlakke vertoon, asook verhoogde peroksisoomproliferator-geaktiveerde reseptor-γ ko-aktiveerder 1-α (PGC-1α) peptiedvlakke. Perfusie data toon kontraktiele wanfunskionering gedurende basislyn en na afloop van ischemie-reperfussie, terwyl post-ischemiese harte verlaagde ATPase spesifieke aktiwiteit vs gepaarde kontrole vertoon. Vroeë veranderinge wat deur PI behandeling veroorsaak word, kom ooreen met die metabolise sindroom en reflekteer op ‘n pre-aterogeniese profiel. Bowendien kan die effekte van PIs op kardiale kontraktiele funksie deels veroorsaak word deur die ingeperkte UPS aktiwiteit tesame met die las op die mitochondriale energie sisteem. Ons studie waarsku teen kardio-metaboliese newe effekte met PI behandeling en rig die vraag; wat die mees gepaste ko-behandeling vir pasiënte op chroniese anti-retrovirale behandeling is.

Thesis (Ph. D.)--University of California, Riverside, 2009.
Includes abstract. Includes bibliographical references (leaves 201-210). Issued in print and online. Available via ProQuest Digital Dissertations.

Thesis (MSc)--Stellenbosch University, 2013.
ENGLISH ABSTRACT: Introduction: Breast cancer is a major contributor to mortality in women worldwide. Although, anthracyclines, such as doxorubicin, are among the most valuable treatments for breast cancer, their clinical use is limited due to detrimental side-effects such as cardiotoxicity. Additionally, evidence suggests that cancer cells are becoming increasingly resistant to chemotherapeutic agents. The consequence of poor vascularisation within tumours subsequently leads to a nutrient deprived microenvironment which cancer cells are known to adapt to via metabolic remodelling and increasing autophagy. Autophagy is an intracellular degradation system, which is induced as a survival mechanism in response to starvation and other environmental stressors. Recent studies have shown that starvation protects non-tumourigenic cells against chemotherapy-induced cell death. Furthermore, patients who starved prior to chemotherapy reported reduced side-effects. However, these studies investigated the effects of long-term starvation, which maybe clinically challenging. Therefore, this concept, under shorter and more tolerable periods of starvation still needs to be investigated. We hypothesis, that short-term starvation will sensitize breast cancer cells to doxorubicin-induced cell death. In order to test this hypothesis this study was approached by the following aims: (i) to establish a time point at which MCF12A breast epithelial cells are protected against starvation; (ii) to determine the effect of short-term starvation on doxorubicin induced cell death; (iii) to assess autophagy and; (iv) to assess these above mentioned aims using an in vivo model. Methods: MDAMB231 cells and MCF12A cells were starved for 0, 3, 6, 12, 24 and 48 hours using Hanks Balanced Salt Solution. Cell viability was assessed using the trypan blue, MTT and Caspase-Glo assays. MDAMB231 cells and MCF12A cells were subjected to the following conditions: (1) control; (2) 5 μM doxorubicin; (3) starvation of 3 hours and (4) a combination of starvation and doxorubicin. Following treatment an MTT assay to assess cell viability was performed. MDAMB231 cells were further examined using Live-Cell Imaging and western blot analysis. C57BL6 tumour bearing mice were treated with doxorubicin (5 mg/kg) or in combination with starvation of 24 hours. Upon termination of the protocol, tumour tissue was assessed using western blot analysis. In both in vitro and in vivo analyses cleaved-caspase 3 and cleaved-PARP were used as markers for apoptosis, LC3 and p62 as autophagic markers and p-AMPK and p-mTOR as markers of oxygen and energy sensing, respectively. Results and discussion: Three hours of starvation was chosen for in vitro experiments since no significant reduction in cell viability or increases in apoptosis occurred at this time-point in the normal MCF12A breast epithelial cells. As expected, doxorubicin induced a significant decrease in cell viability in the cancerous MDAMB231 cells. Short-term starvation in combination with doxorubicin treatment caused a further significant decrease in cell viability in MDAMB231 cells compared to the doxorubicin group alone. Interestingly, starved MCF12A cells were protected against doxorubicin-induced cytotoxicity as cell viability significantly increased. A significant decrease in autophagy was further observed with the combined treatment of doxorubicin and starvation which corresponded with a significant increase in cell death. In contrast, although the in vivo study also demonstrated a significant elevation in cell death and autophagy in response to doxorubicin treatment, the combined treatment (starvation and doxorubicin) did not have an additive effect when compared to the doxorubicin group alone. Conclusion: Our in vitro results clearly demonstrate that short-term starvation sensitizes breast cancer cells to doxorubicin-induced cell death. Additionally, decreased levels of autophagy appear to contribute to this phenomenon of sensitization. Although doxorubicin treatment resulted in increased apoptosis in vivo, 24 hours starvation in combination with doxorubicin did not sensitize the tumours to doxorubicin treatment. Thus, for future in vivo studies more time points should be considered in order to translate the beneficial effects of short-term starvation observed in our in vitro study to an animal model.
AFRIKAANSE OPSOMMING: Inleiding: Borskanker is ‘n belangrike faktor wat bydrae tot sterftes in vrouens wêreldwyd. Alhoewel antrasikliene soos doxorubicin, waardevol is vir die behandeling van borskanker, word die kliniese gebruik daarvan beperk deur newe-effekte soos kardiotoksisiteit. Verder, word daar al hoe meer bewys dat kankerselle toenemend weeerstandbiedend word teen chemoterapeutiese middels. Swak vaskularisasie van tumore lei tot ‘n mikro-omgewing met beperkte voedingstowwe waaby kankerselle kan aanpas deur middel van metaboliese hermodelering en ‘n toename in autofagie. Autofagie is ‘n intrasellulêre degraderingsisteem wat as ‘n oorlewingsmeganisme aangewend word tydens verhongering en ander omgewingstressors. Onlangse studies het getoon dat verhongering nie-tumourigeniese (normale) selle teen chemoterapie-geïnduseerde seldood beskerm. Verder is daar ook geraporteer dat pasiënte wat gevas het voor chemoterapie, verminderde newe-effekte getoon het. Hierdie studies het egter gefokus op ‘n relatief lang-termyn vas, wat klinies nogal uitdagend kan wees. Daarom moet hierdie konsep nog op korter, meer hanteerbare tye getoets word. Ons hipotese is dus dat kort-termyn vas borskankerselle kan sensitiseer tot doxorubicin-geïnduseerde seldood. Om hierdie hipotese te toets, is die volgende doelwitte gestel: (i) om ‘n tydspunt te bepaal waar MCF12A borsepiteelselle beskerm is teen verhongering; (ii) om die effek van kort-termyn verhongering op doxorubicin-geïnduseerde seldood te toets; (iii) om autofagie te karakteriseer in ons model en; (iv) om hierdie doelwitte ook in ‘n in vivo model te toets. Metodes: MDAMB231 en MCF12A selle is verhonger vir 0, 3, 6, 12, 24 and 48 ure deur van Hanks se gebalanseerde soutoplossing gebruik te maak. Sellewensvatbaarheid is bepaal deur middel van trypan blou, MTT en die Caspase-Glo tegnieke. MDAMB231 en MCF12A selle is onderwerp aan die volgende omstandighede: (1) kontrole; (2) 5 μM doxorubicin; (3) verhongering van 3 ure en (4) ‘n kombinasie van verhongering en doxorubicin. Na behandeling is die sellewensvatbaarheid deur middel van die MTT tegniek bepaal. MDAMB231 selle is verder ondersoek deur middel van “Live-Cell Imaging” en die westelike klad tegniek. C57BL6 tumor-draende muise is behandel met doxorubicin (5 mg/kg) of met ‘n kombinasie van verhongering van 24 ure en doxorubicin. Aan die einde van die protokol, is die kankerweefsel geanaliseer deur die westelike klad tegniek. In beide in vitro en in vivo analises, is gekliefde- caspase 3 en -PARP as merkers vir apoptose, LC3 and p62 as merkers vir autofagie en p-AMPK en p-mTOR as suurstof- en energie sensors respektiewelik gemeet. Resultate en bespreking: Vir die in vitro eksperimente, is ‘n tydspunt van 3 ure gekies as gevolg van die feit dat geen afname in sellewensvatbaarheid en ‘n toename in apoptose in hierdie tydsgleuf tydens verhongering in die normale MCF12A borsepiteelselle plaasgevind het nie. Soos verwag, het doxorubicin behandeling ‘n insiggewende afname in sellewensvatbaarheid in die kankeragtige MDAMB231 selle veroorsaak. Die kombinasie-terapie van verhongering en doxorubicin het ‘n verdere verhoging in seldood teweeg gebring in die MDAMB231 selle, maar het die normale MCF12A borsepiteelselle teen doxorubicin-geïnduseerde toksisiteit beskerm. Die kombinasie-behandeling is ook geassosieer met ‘n afname in autofagie. Alhoewel, die in vivo studie ook getoon het dat doxorubicin alleen insiggewende hoeveelheid seldood teweeggebring het, het die kombinasie-behandeling nie die additiewe effek, soos in die in vitro studie, teweeg gebring nie. Gevolgtrekking: Die in vitro resultate het duidelik getoon dat kort-termyn verhongering borskankerselle kan sensitiseer vir doxorubicin terapie. Verder het dit geblyk dat ‘n afname in autofagie tot die fenomeen van sensitisering bygedrae het. Alhoewel doxorubicin behandeling in vivo tot ‘n toename in apoptose in die tumor gelei het, het die kombinasie behandeling nie die kankerweefsel ten op sigte van doxorubicin gesensitiseer nie. Daar sal dus vir toekomstige in vivo studies meer tydsgleuwe van behandeling ondersoek moet word om die optimum verhongeringsperiode te vind sodat die in vitro resultate ook in vivo van toepassing kan wees.
NRF and CANSA for financial support

Thesis (MSc)--Stellenbosch University, 2013.
ENGLISH ABSTRACT: Introduction: Among the cancers that afflict females world-wide, neoplastic disease of breast tissue is the most frequently diagnosed form and the leading cause of cancer-related death. Conventional treatment entails the use of doxorubicin, an anticancer agent belonging to the anthracycline family of chemotherapeutic drugs. Cancer cells are becoming increasingly resistant to doxorubicin therapy. The existence of hypoxic zones, which is a common feature of solid tumours, has been shown to promote the selection of therapy resistant clones in proliferating cancer cells. By modifying cellular homeostasis, neoplastic cells are capable of tolerating the hypoxic insult and thriving within the hostile microenvironment of the tumour. This adaptation is known as ‘the hypoxic response’ and is mediated through the action of the transcriptional regulator, HIF-1. Its expression in cancer tissue has been associated with a dismal prognosis as it promotes the degree of malignancy to an advanced stage. Hypothesis & Aims: We hypothesized that the targeting of HIF-1α would circumvent the ‘protective’ hypoxic response conferred upon breast cancer and improve the cytotoxicity of doxorubicin treatment. In this study, the first aim was to identify the hypoxic conditions at which the MCF-7 breast cancer cell line manifests a doxorubicin-resistant phenotype. This was followed by examination of the molecular pathways contributing to the hypoxic resistance by elucidating the potential relationship with the hypoxic regulator HIF-1α. Once the involvement of HIF-1α was established, the next aim was to evaluate whether the attenuation of HIF-1α would terminate the resistant phenotype and sensitize the neoplastic MCF-7 cells to doxorubicin treatment. Finally, the reproducibility of the in vitro experiment and efficacy of treatments within an animal model was evaluated. 2-Methoxyestradiol is a naturally occurring metabolite originating from 17β-estradiol. It has recently been exploited as an anticancer agent due to its anti-proliferative and anti-angiogenic properties. Among its various mechanisms of action, this compound has been shown to inhibit the expression of HIF-1α. It is for this reason that this study employed 2-methoxyestradiol in the adjuvant therapeutic treatment, along with doxorubicin. Methods: The in vitro experimental model employed the use of the breast adenocarcinoma estrogen receptor (ER-positive cell line, MCF-7. These neoplastic cells were propagated under standard culture conditions until reaching ~70-80% confluency, after which treatment commenced. The treatment regime comprised a 12 hour exposure to the doxorubicin (1 μM) chemotherapeutic agent, either alone or in combination with HIF-1α inhibitors, 2-methoxyestradiol (10 μM) or siRNA duplex (400 nM), with parallel incubations under normoxic (21%) and hypoxic (~0.1%) conditions. To serve as a positive control for HIF-1α expression, cells were treated with CoCl2 (100 μM). Molecular techniques employed included the Caspase-Glo® 3/7 Assay, western blotting, and the bioreductive MTT Assay. Mitochondrial integrity was assessed by live cell imaging/fluorescent microscopy. Cellular viability was monitored at all times. The experiment was then translated into a pre-clinical in vivo model where C57BL/6 mice bearing E0771 xenografts (4 week growth) were allocated into the following treatment groups: (1) control (2) doxorubicin (5 mg.kg-1), (3) 2-methoxyestradiol (45 mg.kg-1), and (4) the combination of the two previously mentioned groups. Body weight and the rate of tumour growth were monitored throughout the experiment. Results: Treatment with CoCl2 effectively stabilized HIF-1α under normoxic conditions. 2-Methoxyestradiol was capable of attenuating HIF-1α expression under both normoxia and hypoxia as compared with siRNA transfection, which was only effective under normoxia. HIF-1α stabilization was accompanied by an increase in autophagy along with the morphological transformation of mitochondria from an elongated network to shorter disc-like forms. On the other hand, HIF-1α attenuation caused an induction in the expression of the apoptotic markers, cleaved caspase 3 and cleaved PARP, as well as the restoration of the normoxic morphology. The exposure of MCF-7 cells to 1 μM doxorubicin for 12 hours produced a differential effect in the bioreductive MTT assay between normoxic and hypoxic conditions (42.97 ± 3.095% vs. normoxic dox, p<0.01), while stimulating the apoptotic and autophagic pathways. Compared to the control, a significant expression of phospho-AMPK became evident at 21% O2, while the levels remained stable at ~0.1% O2 after doxorubicin exposure. Furthermore, chemotherapeutic treatment caused the morphology of the mitochondria to appear dot-like. Although the combination of the two drugs removed the differential effect witnessed in the MTT assay, there was no significant change when compared to doxorubicin. Levels of apoptotic cell death decreased under both oxygen conditions. While HIF-1α and autophagy decreased under normoxia, they remained elevated under hypoxia. In the in vivo component of the study, the administration of doxorubicin and 2-methoxyestradiol, alone or in combination, did not affect the rate of tumour growth or induce systematic toxicity in any of the experimental mice. When drugs were administered separately, a decrease in apoptosis along with a concomitant increase in autophagy and p-AMPK expression became noticeable while neither treatment had any significant effect on the expression of HIF-1α. Adjuvant administration, however, was capable of attenuating HIF-1α along with autophagy. Discussion: By inducing (CoCl2) and inhibiting (2-methoxyestradiol; siRNA duplex) HIF-1α, it was established that the autophagic pathway in the in vitro experimental setting of this study was dependent on the expression of HIF-1α. The bioreductive MTT assay measures the metabolic state of a cell, which is an indirect indication of cellular viability. Based on this, hypoxia was shown to confer survival to neoplastic MCF-7 cells based on the differential effect witnessed after doxorubicin treatment. Apart from the induction of apoptosis and its associated mitochondrial fragmentation, the chemotherapeutic drug increased the activation of the metabolic sensor, AMPK, which upregulated autophagy during normoxia. While this autophagic process may assist in the killing mechanism, we speculate that the autophagy upregulated under hypoxia may be responsible for the survival effect and is most likely dependent on HIF-1α. In contrast to eliciting a synergistic cytotoxic effect, the combination of doxorubicin with 2-methoxyestradiol produced an antagonistic effect on cellular viability instead. We propose that under normoxia, the combined treatment may stimulate the MCF-7 neoplastic cells to enter a state of growth arrest, or senescence, since the results indicate that the decrease in HIF-1α-dependent autophagy did not significantly affect cellular viability. Under hypoxia, despite the incorporation of the pharmacological HIF-1α inhibitor (2-methoxyestradiol), the expression levels of HIF-1α remained unaffected. We speculate that this could be the result of a potentiated stabilization of HIF-1α caused by the build-up of ROS and TCA intermediates which may be the outcome of mitochondrial dysfunction inflicted upon adjuvant therapy under hypoxia. Furthermore, it is also likely that the slight mitogenic effect observed within the MTT assay may be caused by the conversion of 2-methoxyestradiol to a chemically-reactive estrogen derivative, possibly by the action of doxorubicin, and the fact that an ER-positive cancer cell line was employed in this study. With regards to the in vivo experimental model, we speculated that the failure of the molecular changes to manipulate the growth of the tumour could have been the result of an ineffective time- and/or dose regime. Conclusion: We therefore reject our hypothesis based on the fact that an antagonistic rather than synergistic effect was witnessed when the tumorigenic MCF-7 cell line was treated with adjuvant therapy. The results warrant the need for extensive testing on the pharmacodynamics of 2-methoxyestradiol, and more informative techniques to compliment the study.
AFRIKAANSE OPSOMMING: Inleiding: Borskanker is die mees algemeen gediagnoseerde kanker asook die hoof oorsaak van kanker-verwante sterftes in vrouens wêreldwyd. Konvensionele behandeling behels die toediening van doxorubicin, ‘n anti-kankermiddel wat aan die antrasiklien-familie van chemoterapeutiese middels behoort. Kankerselle begin egter toenemend weerstandbiedend raak teen doxorubicin behandeling. Daar is al bewys dat die voorkoms van hipoksiese sones, wat ‘n algemene eienskap van soliede tumore is, die seleksie vir weerstandbiedende klone van prolifererende kankerselle, veroorsaak. Neoplastiese selle kan hierdie hipoksiese toestande weerstaan en in hierdie ongunstige mikro-omgewing floreer deur sellulêre homeostase te modifiseer. Hierdie aanpassing staan bekend as die ‘hipoksiese respons’ en word bemiddel deur die aksies van die transkripsiefaktor reguleerder, HIF-1. Die verhoogde uitdrukking van HIF-1 in kankerweefsel word oor die algemeen geassosieer met ‘n swak prognose omdat dit die maligniteit vehoog. Hipotese en Doelwitte: Die hipotese van hierdie studie behels dus die volgende: Deur HIF-1α te inhibeer, sal die ‘beskermende’ hipoksiese respons wat in borskankerselle voorkom omseil kan word en sodoende die sitotoksisiteit van doxorubicin terapie verhoog. Die eerste doelwit van hierdie studie was dus om die hipoksiese kondisies te identifiseer waar MCF-7 selle ‘n doxorubicin-weerstandbiedende fenotipe vertoon. Daarna is die molekulêre paaie wat bydrae tot hierdie hipoksiese weerstand ondersoek asook hul moontlike verwantskap met die hipoksiese reguleerder, HIF-1α. Nadat die rol van HIF-1α bevestig is, was die volgende doelwit om te bepaal of die inhibisie van HIF-1α die weerstandbiedende fenotipe sal onderdruk en neoplastiese MCF-7 selle sal sensitiseer vir doxorubicin behandeling. Laastens is die herhaalbaarheid en effektiwiteit van behandeling in die in vitro eksperimente ook in ‘n diermodel getoets. 2-Methoxyestradiol is ‘n metaboliet van 17β-estradiol wat natuurlik in die liggaam voorkom. Dit is ook onlangs as ‘n anti-kanker middel geïdentifiseer as gevolg van die anti-verdelende en anti-angiogeniese eienskappe. Een van die eienskappe van 2-methoxyestradiol is dat dit ook die uitdrukking van HIF-1α kan onderdruk. Dit is dan ook vir hierdie rede dat 2-methoxyestradiol in hierdie studie as bykomende terapie saam met doxorubicin gebruik is. Metodes: Die in vitro eksperimentele model behels die gebruik van ‘n borsadenokarsinoom, estrogeenreseptor (ER)- positiewe sellyn, MCF-7. Hierdie neoplastiese selle is onder standaard weefselkultuur omstandighede gekweek totdat konfluensie van ~70-80% bereik is, waarna behandeling begin het. Die behandelingsprosedure behels ‘n 12 uur blootstelling aan doxorubicin (1 µM) chemoterapeutiese middel alleen of in kombinasie met die HIF-1α inhibitore, 2-methoxyestradiol (10 µM) of siRNA duplex (400 nM) in normoksiese (21% O2) en hipoksiese (~0.1% O2) toestande. Die selle is ook met CoCl2 behandel wat gedien het as ‘n positiewe kontrole vir HIF-1α uitdrukking. Molekulêre tegnieke wat tydens hierdie studie gebruik is, sluit die “Caspase-Glo® 3/7” bepaling in, asook die westelike kladtegniek en die MTT bepaling. Mitochondriale integriteit is bepaal deur middel van lewende sel afbeeldings/fluoresensie mikroskopie. Sellewensvatbaarheid is ten alle tye gemonitor. Hierdie eksperment is verder ook in ‘n pre-kliniese in vivo model uitgevoer waar C57BL/6 muise met E0771 xenografte (4 weke groei) geïnduseer is en in die volgende behandelingsgroepe verdeel is: (1) kontrole; (2) doxorubicin (5 mg.kg-1); (3) 2-methoxyestradiol (45 mg.kg-1); en (4) die kombinasie van laasgenoemde twee groepe. Die liggaamsgewig en die tempo van tumorgroei is tydens die hele eksperiment gemonitor. Resultate: CoCl2 behandeling het HIF-1α effektief gestabiliseer tydens normoksiese omstandighede. 2-Methoxyestradiol het HIF-1α uitdrukking tydens normoksiese en hipoksiese toestande onderdruk wanneer dit vergelyk is met siRNA transfeksie wat slegs tydens normoksiese toestande effektief was. HIF-1α stabilisering het gepaardgegaan met ‘n toename in autofagie asook morfologiese veranderinge in die mitochondria vanaf ‘n verlengde netwerk tot korter skyfagtige vorme. Aan die ander kant het HIF-1α onderdrukking ‘n toename in die apoptotiese merkers, nl kliewing in caspase-3 and PARP veroorsaak wat gepaard gegaan het met die herstel van die tubulêre mitochondriale netwerk. Die blootstelling van die MCF-7 selle aan 1 µM doxorubicin vir 12 ure het ‘n differensiële effek in die bioreduktiewe MTT bepaling tot gevolg gehad tussen normoksiese en hipoksiese toestande (42.97 ± 3.095%, p<0.1), terwyl die apoptotiese- en autofagiese paaie in beide toestande gestimuleer is. ‘n Insiggewende toename in fosfo-AMPK uitdrukking was sigbaar tydens normoksiese toestande van 21% O2, terwyl dit onveranderd gebly het tydens hipoksiese toestande van 0.1% ~O2 na doxorubicin behandeling. Die morfologie van die mitochondria het ‘n ‘kollerige’ voorkoms tydens doxorubicin behandeling gehad. Alhoewel die behandeling van die selle met beide middels gelyktydig, die differensiële effek soos weerspieël in die MTT bepaling ophef, is daar geen insiggewende verandering wanneer met doxorubicin behandeling vergelyk word nie. Apoptotiese seldood verminder met gelyktydige behandeling van biede middels tydens normoksiese en hipoksiese toestande. HIF1-α en autofagie het afgeneem tydens normoksiese toestande, maar bly vehoog tydens hipoksie. In die in vivo model, het die toediening van doxorubicin en 2-methoxyestradiol alleen en in kombinasie nie tumorgroei geaffekteer nie en ook nie sistemiese toksisiteit in enige van die eksperimentele muise tot gevolg gehad nie. Die afsonderlike toediening van die middels het ‘n afname in apoptose in ‘n toename in autofagie en p-AMPK uitdrukking tot gevolg gehad, terwyl afsonderlike toediening van die middels nie ‘n effek op HIF-1α uitdrukking gehad het nie. Die gelyktydige toediening van biede middels het egter ‘n onderdrukking van HIF1-α teweeggebring. Bespreking: Deur HIF-1α te induseer (CoCl2) en te inhibeer (2-methoxyestradiol en siRNA) in hierdie in vitro eksperimentele omstandighede, bevestig hierdie resultate dat autofagie afhanklik is van die uitdrukking van HIF-1α. Die bioreduktiewe MTT bepaling meet die metaboliese staat van die sel wat indirek sellewensvatbaarheid bepaal. Gebasseer op hierdie bepaling is bewys dat hipoksie ‘n weerstandbiedende fenotipe veroorsaak teen doxorubicin behandeling in neoplastiese MCF-7 selle. Doxorubicin veroorsaak ‘n toename in apoptose met geassosieerde mitochondriale fragmentering asook ‘n aktivering van die metaboliese sensor, AMPK, wat autofagie stimuleer in normoksiese omstandighede. Alhoewel ‘n toename in autofagie seldood kan stimuleer, spekuleer ons dat ‘n toename in autofagie tydens hipoksie verantwoordelik kan wees vir seloorlewing wat heel moontlik ook afhanklik van HIF-1α is. In kontras met die verwagting dat die kombinasie behandeling ‘n sinergistiese sitotoksiese effek sou teweegbring, dui ons resultate dat daar ‘n antagonistiese effek op sellewensvatbaarheid was. Ons stel voor dat die gekombineerde behandeling tydens normoksiese toestande MCF-7 neoplastiese selle stimuleer om in ‘n toestand van groeistaking in te gaan aangesien die resultate daarop dui dat ‘n afname in HIF-1α afhanklike autofagie nie sellulêre lewensvatbaarheid beïnvloed het nie. Tydens hipoksie, ten spyte van die bykomdende behandeling met die HIF-1α inhibitor (2-methoxyestradiol), het die vlakke van HIF-1α onveranderd gebly. Ons spekuleer dat dat dit die gevolg kan wees van die stabilisering van HIF-1α as gevolg van ‘n toename in ROS en TCA intermediate wat die gevolg van mitochondriale wanfunksie kan wees tydens bykomende terapie onder hipoksiese toestande. Dit is ook moontlik dat die mitogeniese effek wat waargeneem is met die MTT bepaling die gevolg kan wees van die omsetting van 2-methoxyestradiol na ‘n chemiese-reaktiewe estrogeen derivaat; moontlik as gevolg van die aksie van doxorubicin en die feit dat die sellyn wat in hierdie studie gebruik is, ‘n ER-positiewe kankersellyn is. Met verwysing na die in vivo eksperimentele model, spekuleer ons dat die molekulêre veranderinge wat nie in die tumorgroei weerspieël word nie, die resultaat van oneffektiewe tyds- en dosis behandelingswyses is, of foutiewe toediening van die middel kan wees. Gevolgtrekking: Ons verwerp dus ons hipotese gebaseer op die feit dat bykomende (adjuvante) behandeling eerder ‘n antogonistiese effek as ‘n sinergistiese effek op seldood in MCF-7 selle het. Hierdie resultate regverdig die nodigheid van intensiewe toetsing op die farmakodinamika van 2-methoxyestradiol asook die gebruik van meer informatiewe tegnieke om hierdie studie te komplimenteer.
CANSA and Marie Stander

Thesis (MSc)--Stellenbosch University, 2013.
ENGLISH ABSTRACT: Contusion injuries cause significant muscle damage, activating a series of cellular events. Satellite cells (SC), the key role players in muscle regeneration, are activated to proliferate and develop into mature myoblasts, which could fuse to form new myotubes or to repair damaged fibres. Evidence suggests that anti-oxidants, such as those found in grape seed extract (GSE), enhance repair, but their effect on SCs is still unclear. This study aimed to harvest and culture primary rat myoblasts to investigate the effect of chronic in vivo GSE supplementation on SCs following a standardised crush injury. Using a modified pre-plate technique, myoblasts were harvested from rat muscle and then compared with the immortal C2C12 cell line for proliferation and differentiation competence. Several media options were compared: i) DMEM with or without L-glutamine, ii) Ham‘s F10 or iii) DMEM with L-glutamine and Ham‘s F10 combined. Primary myoblasts proliferated and differentiated at a much slower rate than C2C12 cells. The combined media was selected for further use. To investigate the effects of GSE on the recovery, rats were supplemented daily with GSE or placebo 14 days prior to a standardised mass-drop crush injury to the gastrocnemius. SCs were isolated and cultured from uninjured (NI, baseline) and from injured rats 4 hours (4h), 3 days (3d) or 14 days (14d) post-injury. Expression of myogenic proteins Pax7, M-cadherin, MyoD, CD56, desmin and CD34 was determined by flow cytometry. Myoblasts were sorted according to their CD56 and CD34 expression and three sub-sets were collected and re-cultured, namely CD56+/CD34-, CD56-/CD34+ and CD56+/CD34+. After 24 hours, sorted cells were stained for desmin expression. Pax7, M-cadherin and MyoD were present in 100% of isolated cells from all groups confirming their myogenic SC identity. For all groups, desmin was expressed only in ~80% of SCs. Lower adhesion competency in GSE supplemented groups resulted in lower yield obtained for culturing. Expression of CD56 increased significantly 3d post-injury in the placebo group. In contrast, with GSE, CD56 already increased 4h post-injury and decreased again 3d post-injury. Although CD34 expression did not differ dramatically, expression pattern resembled that of CD56. Immunocytochemistry revealed a range in morphology and desmin expression of sorted myoblasts. More myoblasts with high desmin expression were observed in the two CD56+ sub-sets (irrespective of CD34 expression), indicating that CD56 is still expressed in more mature myoblasts. Flow cytometry revealed a population of myoblasts expressing particularly high levels of desmin, primarily in the non-injured baseline GSE group. We hypothesise that this result is an indication of preparedness of myoblasts to respond earlier to injury, enabling quicker repair. This cell population with high desmin content is restored in skeletal muscle after repair (14d), only when supplemented with GSE. In conclusion, GSE attenuated adhesion competence of primary myoblasts in culture, but resulted in earlier maturation of SCs, possibly due to baseline preparedness of myoblasts in uninjured muscle for a quick response. Both reduced adhesion competence and early progression of myoblasts could enhance wound healing in skeletal muscle.
AFRIKAANSE OPSOMMING: Kneuswonde veroorsaak aansienlike skade aan skeletspier, wat ‘n reeks sellulêre prosesse in werking stel. Satellietselle, die hoofrolspelers tydens spierregenerasie, vermenigvuldig en ontwikkel tot volwasse mioblaste, wat saamsmelt om nuwe spiervesels te vorm. Antioksidante, soos die wat in druiwepit-ekstrak voorkom, bespoedig herstel, maar hul uitwerking op satellietselle is steeds onduidelik. Die doel van hierdie studie was om mioblaste uit rotspiere te isoleer en te kweek om die effek van langdurige in vivo aanvulling van druiwepit-ekstrak op satellietselle na ‘n kneusbesering te bepaal. 'n Aangepaste protokol is gebruik om primêre mioblaste te isoleer, wat daarna met C2C12 selle, ten opsigte van hul vermenigvuldigings- en differensiasievermoë vergelyk is. Verskeie groeimedia is gebruik: i) DMEM met of sonder L-glutamien, ii) Ham F10 en iii) ‘n kombinasie van DMEM, L-glutamien en Ham F10. Primêre mioblaste het stadiger vermenigvuldig en gedifferensieer as C2C12 selle. Die gekombineerde medium is vir verdere gebruik gekies. Om die uitwerking van druiwepit-ekstrak op spierherstel te ondersoek, is rotte vir 14 dae onderwerp aan daaglikse aanvullings van druiwepit-ekstrak of placebo voor ‘n gestandardiseerde kneusbesering aan die gastrocnemius. Satellietselle is geïsoleer vanuit onbeseerde spier (basiskontrole) en vanuit beseerde spier 4 ure (4h), 3 dae (3d) en 14 dae (14d) na die besering. Die uitdrukking van spierverwante proteïene Pax7, M-cadherin, MyoD, CD56, desmin en CD34 is vasgestel met 'n vloeisitometer. Mioblaste is daarna gesorteer op grond van hul CD56- en CD34-uitdrukking. Drie sub-groepe is versamel en verder gekweek, nl. CD56+/CD34-, CD56-/CD34+ en CD56+/CD34+. Na 24 uur is gesorteerde selle gekleur om desmin-uitdrukking te bepaal. Pax7, M-cadherin en MyoD is deur 100% satellietselle in alle groepe uitgedruk, wat hul spierverwante identiteit bevestig, alhoewel slegs 80% selle in alle groepe desmin uitgedruk. Druiwepit-ekstrak het die vermoë van selle om aan plate te heg onderdruk, wat gelei het tot ‘n laer opbrengs van mioblaste. Drie dae na die besering in die placebo groep het die CD56-uitdrukking beduidend toegeneem. In teenstelling hiermee het CD56-uitdrukking in die druiwepit-ekstrak groep 4 ure na die besering beduidend toegeneem en weer afgeneem na 3 dae. Hoewel daar nie sulke dramatiese verskille was tussen groepe ten opsigte van CD34-uitdrukking nie, was daar ‘n soortgelyke tendens as vir CD56-uitdrukking. Immunositochemie het ‘n verskeidenheid van morfologieë en variërende desminvlakke in gesorteerde mioblaste blootgestel. In die twee CD56+ groepe is meer mioblaste wat hoë desmin vlakke uitdruk gevind, wat aandui dat CD56 uitgedruk word deur meer volwasse mioblaste, ongeag van CD34-uitdrukking. Tydens vloeisitometrie is ‘n populasie selle wat hoë desminvlakke uitdruk, hoofsaaklik in die onbeseerde en 14d druiwepit-ekstrak groepe gevind. Dit is ‘n aanduiding dat sommige mioblaste voorbereid is om na 'n besering vinniger te reageer. Na die herstelproses word hierdie groep selle hernu in die teenwoordigheid van druiwepit-ekstrak-aanvulling. Die resultate het gevolglik daartoe gelei dat druiwepit-ekstrak die hegtingsvemoë van mioblaste verlaag, maar dat die aanvulling in vivo tot vroeër ontwikkeling van mioblaste lei, waarskynlik deur satellietselle voor te berei vir 'n vinnige respons na ‘n besering. Beide die onderdrukking van aanhegting aan kultuurplate en die vroeë ontwikkeling van mioblaste, kan die herstel van die skeletspier verbeter.
NRF and the Harry Crossley bursary for funding

Dissertação de Mestrado Integrado em Medicina Veterinária
ABSTRACT - Currently the dissemination of antimicrobial resistance has become a major public health concern worldwide. The pressure they exert on the bacterial microbiota enables a preferential selection of resistant bacteria. Urinary tract infection (UTI) is a frequent clinical problem in companion animals and among the most common reasons for antimicrobial therapy. The present study aimed to evaluate the influence of antimicrobial therapy in companion animals on colonization by extended spectrum β-lactamase ESBL, cephalosporinases (AmpC) and carbapenemases – producing Enterobacterales and to verify if antimicrobial resistance is transmitted to the pet owners with whom they share a home. The study sample included 14 animals (11 dogs and 3 cats) with UTI and 25 humans belonging to 14 families. Dog and cat owners were asked to deliver fecal samples from themselves and their animals at specific times since diagnosis of infection up to two months after its resolution. Cultures of fecal samples in MacConkey medium with cefotaxime and meropenem were performed to select resistant bacteria, followed by PCR to detect β lactamase producing genes. No carbapenemase-producing Enterobacterales were found, however, in 24.2% of the fecal samples ESBL and AmpC-producing Escherichia coli or Klebsiella pneumoniae were identified. TEM-1 β-Lactamase was the most identified in animals (23.5%) and humans (28.6%). ESBL-producing genes of the CTX-M type were the most detected, in particular blaCTX-M-15. Approximately 9% of the resistance genes detected were cephalosporinases (blaCMY-2). The administration of antibiotics to pets has been reflected in an increase in antimicrobial resistance not only in themselves but also in humans. In two families, both human and animal were colonized at the same time with an ESBL/AmpC-producing bacteria. In one of this families, transmission of a bacteria, or a plasmid carrying the blaCTX-M-15 gene, may have occurred from a cat to a human, and from this to another human from the same household. Further studies are needed to ascertain the presence of interspecies transmission of AMR in this study.
RESUMO - Infecção do tracto urinário em animais de companhia: o tratamento influencia a transmissão e colonização por Enterobacterales produtoras de ESBL, AmpC e Carbapenemase para humanos? - Atualmente um dos principais problemas de saúde pública é a disseminação da resistência bacteriana aos antibióticos. A pressão por estes exercida na microbiota bacteriana possibilita uma seleção preferencial das bactérias resistentes. A infeção do trato urinário (ITU) é uma afeção frequentemente observada em clínica de pequenos animais, sendo uma das razões mais comuns para instituição de terapêutica antimicrobiana. Devido ao contacto próximo entes eles, os animais de companhia podem ter um papel muito importante na transmissão de bactérias resistentes ao Homem. O presente estudo teve como objetivo avaliar a influência da terapêutica antimicrobiana em animais de companhia, na colonização por Enterobacterales produtoras de β-Lactamase de espectro alargado (ESBL), cefalosporinases (AmpC) e carbapenemases e verificar se ocorre transmissão de resistência antimicrobiana aos tutores com quem os animais de companhia partilham casa. A amostra em estudo incluiu 14 animais (11 cães e 3 gatos) com ITU e 25 humanos pertencentes a 14 famílias. Foi pedido aos tutores dos cães e gatos que entregassem amostras fecais, suas e dos seus animais, em tempos específicos desde o diagnóstico de infeção até dois meses após a sua resolução. Foram realizadas culturas das amostras fecais em meio MacConkey com cefotaxima e com meropenem para seleção de bactérias resistentes, seguidas de PCR para deteção de genes produtores de β-lactamases. Não foram encontradas Enterobacterales produtoras de carbapenemases, no entanto, em 24,2% das amostras fecais identificaram-se Eschericia coli ou Klebsiella pneumoniae produtoras de ESBL e AmpC. A β-Lactamase TEM-1 foi a mais identificada em animais (23,5%) e humanos (28,6%). Os genes produtores de ESBLs do tipo CTX-M foram os mais detetados, em particular o blaCTX-M-15. Aproximadamente 9% dos genes de resistência detetados eram de cefalosporinases (blaCMY-2). A administração de antibióticos a animais de companhia refletiu-se num aumento de resistência antimicrobiana não só neles mesmos, como nos humanos. Em duas famílias, tanto o humano como o animal estavam na mesma altura colonizados com uma bactéria produtora de ESBL/AmpC. Num dos casos, poderá ter ocorrido transmissão de uma bactéria, ou de um plasmídeo portador do gene blaCTX-M-15, de um gato para um humano, e deste para um outro humano do mesmo agregado familiar. São no entanto necessários mais estudos para aferir sobre a transmissão de resistência antimicrobiana inter-espécies neste estudo.
N/A

Thesis (MSc)--Stellenbosch University, 2014.
ENGLISH ABSTRACT: Introduction: Mammalian circadian rhythms form an integral physiological system allowing for the synchronisation of all metabolic processes to daily light/dark cycles, thereby optimising their efficacy. Circadian disruptions have been implicated in the onset and progression of different types of cancers, including those arising in the breast. Several links between the circadian protein Per2 and DNA damage responses exist. Aberrant Per2 expression results in potent downstream effects to both cell cycle and apoptotic targets, suggestive of a tumour suppressive role for Per2. Due to the severe dose limiting side effects associated with current chemotherapeutic strategies, including the use of doxorubicin, a need for more effective adjuvant therapies to increase cancer cell susceptibility has arisen. We therefore hypothesize, that the manipulation of the circadian Per2 protein in conjunction with doxorubicin may provide a more effective chemotherapeutic strategy for the treatment of breast cancer. The aims of this project were thus to: (i) Characterize the role of Per2 in normal breast epithelial cells as well as in ER+ and ER- breast cancer cells; (ii) to determine the role of Per2 in doxorubicin-induced cell death, (iii) to determine the role of Per2 in autophagy and finally (iv) to assess whether the pharmacological inhibition of Per2 with metformin, can sensitize chemo-resistant MDA-MB-231 breast cancer cells to doxorubicin-induced cell death. Methods: An in vitro model of breast cancer was employed using the normal MCF-12A breast epithelial, estrogen receptor positive (ER+) MCF-7 and estrogen receptor negative (ER-) MDA-MB-231 breast adenocarcinoma cell lines. Circadian rhythmicity of Per2 protein expression was determined using western blotting, and Per2 cellular localization was assessed using fluorescent confocal microscopy. Per2 was then silenced by means of an endoribonuclease-prepared siRNA, and silencing efficiency was determined with the use of western blotting. The roles of Per2 in doxorubicin-induced cell death and autophagy were assessed by treating MDA-MB-231 breast cancer cells under the following conditions (1) Control, (2) 2.5 μM doxorubicin or 10 nM bafilomycin A1 (3) 30 nM esiPer2 and (4) 30 nM esiPer2 in combination with 2.5 μM doxorubicin or 10 nM bafilomycin A1. Following treatments cell viability was assessed using the MTT assay, western blotting for markers of apoptosis including p-MDM2 (Ser166), p-p53 (Ser15), cleaved caspase-3 and –PARP as well as markers of autophagy (AMPKα, mTOR and LC3). Furthermore, cell cycle analysis, G2/M transition and cell death (Hoechst 33342 and propidium iodide staining) were assessed by means of flow cytometry. The pharmacological inhibition of Per2 was achieved by treating MDA-MB-231 cells with 40 mM metformin as well as in combination with 2.5 μM doxorubicin. MTT cell viability assays, cell cycle analysis (flow cytometry) and western blotting for apoptosis (Per2, p-AMPKα (Thr172), p53, caspase-3 and PARP) were assessed. Results and discussion: A circadian pattern of Per2 protein expression was observed in the normal MCF-12A and MDA-MB-231 cancer cells with protein levels peaking at ±700% and ±500% of baseline was observed. However, no rhythmic expression was observed in the MCF-7 cancer cells. Immunostaining for Per2 showed localization OF Per2 in the cytoplasm as well as in the nucleus of both the MCF-12A and MDA-MB-231 cells. Concentration curves showed a significant reduction in cell viability following 2.5 μM doxorubicin treatment for 24 hours. Per2 protein expression was significantly reduced with both esiPer2 and metformin treatment. Silencing of Per2 in combination with doxorubicin treatment resulted in cell cycle arrest with a significant increase in apoptosis, indicating that Per2 silencing effectively sensitized the MDA-MB-231 cancer cells to the anti-carcinogenic properties of doxorubicin. Modulation of Per2 protein expression was effectively achieved with the use metformin although this decrease occurred independently of AMPKα phosphorylation. A significant increase in apoptosis was observed following treatment with metformin in combination with doxorubicin treatment. However, no changes in cell cycle regulation were observed. Per2 appears to be involved in the regulation of autophagy as a significant increase in autophagy flux was observed when Per2 was silenced. Additionally, this increase in autophagic flux resulted in a significant increase in MDA-MB-231 cancer cell death which was enhanced further when autophagy was inhibited with bafilomycin A1 subsequent to Per2 silencing. Conclusions: Per2 protein expression was shown to display a 24 hour circadian rhythm in the MCF-12A cells, and to a lesser extent in the MDA-MB-231 cells. However, the MCF-7 cells failed to show rhythmic changes in Per2 protein expression. Per2 was shown to be located predominantly in the cytoplasm, with nuclear localization observed when cytoplasmic fluorescent intensity was lower. Per2 silencing effectively sensitized the chemo-resistant MDA-MB-231 breast cancer cells to both doxorubicin-induced cell death and autophagic inhibition.
AFRIKKANSE OPSOMMING: Inleiding: Sirkadiese ritmes vorm ‘n integrale fisiologiese sisteem wat die sinkronisasie van alle metaboliese prosesse asook lig/donker siklusse se effektiwiteit optimaliseer. Onderbreking van hierdie sirkadiese ritmes word geïmpliseer in die ontstaan en bevordering van verskillende kankertipes, insluitend borskanker. Verskeie raakpunte bestaan tussen die sirkadiese proteïen, Per2, en die DNA skade-respons. Abnormale Per2 uitdrukking veroorsaak afstroom effekte op beide die selsiklus en apoptotiese teikens wat moontlik aanduidend van ‘n tumor-onderdrukkende rol vir Per2 kan wees. Daar bestaan ‘n groot nood vir meer effektiewe adjuvante terapieë om kankersel vatbaarheid vir chemoterapie te verhoog as gevolg van dosis-beperkende newe-effekte wat geassosieer word met huidige chemoterapeutiese strategieë, insluitende dié van doxorubicin. Ons hipotese is dus dat die manipulering van die sirkadiese Per2 proteïen tesame met doxorubicin ‘n meer effektiewe chemoterapeutiese strategie vir die behandeling van borskanker sal wees. Die doelwitte van hierdie projek was dus om: (i) Die rol van Per2 in normale borsepiteelselle sowel as in ER+ en ER- borsepiteel kankerselle te karakteriseer; (ii) die rol van Per2 in doxorubicin-geïnduseerde seldood te bepaal; (iii) te bepaal of Per2 ‘n rol in autofagie speel en laastens (iv) te bepaal of die farmakologiese inhibisie van Per2 met metformin chemo-weerstandbiedende MDA-MB-231 kankerselle kan sensitiseer vir doxorubicin-geïnduseerde seldood. Metodes: ‘n In vitro model vir borskanker is gebruik wat normale MCF-12A borsepiteelselle, estrogeen reseptor positiewe (ER+) MCF-7 en estrogeen reseptor negatiewe (ER-) MDA-MB-231 bors adenokarsenoomselle insluit. Sirkadiese ritmisiteit van Per2 proteïen uitdrukking is deur middel van die westelike kladtegniek bepaal en die sellulêre ligging van Per2 deur middel van fluoresensie mikroskopie. siPer2 is voorberei deur middel van endoribonuklease-siRNA en die effektiwiteit daarvan is deur middel van westelike kladtegniek getoon. Die rol van Per2 in doxorubicin-geinduseerde seldood en autofagie is bepaal deur MDA-MB-231 borskankerselle onder die volgende omstandighede te toets: (1) Kontrole, (2) 2.5 μM doxorubicin of 10 nM bafilomycin A1 (3) 30 nM esiPer2 en (4) 30 nM esiPer2 in kombinasie met 2.5 μM doxorubicin of 10 nM bafilomycin A1. Na die behandeling, is sellewensvatbaarheid bepaal deur gebruik te maak van ‘n MTT toets; westelike kladtegniek is gebruik om vir merkers van apoptose soos p-MDM2 (Ser166), p-p53 (Ser15), gekliefde caspase-3 en -PARP asook vir merkers van autofagie (AMPKα, mTOR en LC3) te toets. Die selsiklus, G2/M oorgang en seldood (Hoechst 33342 en propidium iodide kleuring) is deur middel van vloeisitometrie bepaal. Per2 is ook farmakologies geïnhibeer deur MDA-MB-231 selle met 40 mM metformin asook in kombinasie met 2.5 μM doxorubicin te behandel. Daarna is sellewensvatbaarheid (MTT) sowel as die selsiklus (vloeisitometrie) en apoptose (westelike kladtegniek vir Per2, p-AMPKα (Thr172), p53, caspase-3 and PARP) gemeet. Resultate en bespreking: ‘n Sirkadiese patroon vir Per2 proteïen uitdrukking is in die normale MCF-12A selle asook in die MDA-MB-231 kankerselle waargeneem met proteïenvlakke wat hul piek by ±700% and ±500% onderskeidelik in vergelyking met basislyn bereik het. Geen ritmiese patroon van Per2 proteïen uitdrukking is egter in die MCF-7 kankerselle waargeneem nie. Immunokleuring om Per2 ligging te bepaal het getoon dat Per2 in the sitoplasma sowel as in die nukleus in beide MCF-12A en MDA-MB-231 selle voorgekom het. Konsentrasie kurwes het aangetoon dat daar ‘n insiggewende vermindering in sellewensvatbaarheid voorgekom het na die behandeling van die selle met 2.5 μM doxorubicin vir 24 uur. Per2 proteïen uitdrukking is insiggewend verlaag met beide esiPer2 en metformin behandeling van die selle. esiPer2 aleen of in kombinasie met doxorubicin behandeling het selsiklus staking tot gevolg gehad asook ‘n beduidende toename in apoptose veroorsaak wat dus aangedui het dat esiPer2 effektief was om MDA-MB-231 kankerselle te sensitiseer vir die anti-karsinogeniese doxorubicin behandeling. Modulering van Per2 proteïen uitdrukking was effektief met metformin behandeling, alhoewel die afname onafhanklik van AMPKα fosforilasie plaasgevind het. ‘n Insiggewende toename in apoptose is waargeneem na metformin behandeling in kombinasie met doxorubicin. Geen veranderinge in die selsiklus is egter onder hierdie omstandighede waargeneem nie. Per2 blyk betrokke te wees in die regulering van autofagie aangesien ‘n insiggewende verhoging in autofagie omsetting waargeneem is na esiPer2 behandeling. Die toename in autofagie omsetting is geassosieer met ‘n insiggewende toename in seldood in MDA-MB-231 kankerselle wat verder verhoog is wanneer autofagie met bafilomycin A1 (autofagie inhibitor) in kombinasie met esiPer2 behandel is. Gevolgtrekkings: Per2 proteïen uitdrukking het ‘n 24 uur sirkadiese ritme in die MCF-12A normale selle, en tot ‘n mindere mate in die MDA-MB-231 selle getoon. Die MCF-7 selle het egter geen ritmiese patroon van Per2 proteïen uitdrukking getoon nie. Per2 kom hoofsaaklik in die sitoplasma voor en het slegs in die nukleus voorgekom wanneer die sitoplasmiese fluoresensie intensiteit laer was. esiPer2 was dus effektief om die chemo-weerstandbiedende MDA-MB-231 borskankerselle te sensitiseer vir doxorubicin-geïnduseerde seldood.
National Research Foundation

Thesis (MSc)--University of Stellenbosch, 2007.
ENGLISH ABSTRACT: Tumour necrosis factor alpha (TNFα) is a pleiotropic cytokine and has a wide variety of dose dependent cellular effects ranging from cell growth and differentiation, to inducing apoptosis. It has long been implicated in muscle and non-muscle inflammatory disorders, such as muscle wasting in chronic disease states, and rheumatoid arthritis. However, a physiological role for TNFα in muscle regeneration has been proposed as elevated levels of the cytokine are present when muscle regeneration processes are initiated: TNFα is secreted by infiltrating inflammatory cells, and by injured muscle fibres. Adult skeletal muscle contains a population of resident stem cell-like cells called satellite cells, which become activated, proliferate and differentiate following muscle injury to bring about repair of damaged muscle. Much research on the effects of TNFα on satellite cell differentiation has been conducted in recent years. It is however difficult to get a complete characterisation of the cytokine’s action as all models used slightly differ. We aimed therefore at providing comprehensive assessment of the effects of increasing doses of chronically supplemented TNFα on differentiating C2C12 cells. Cells were allowed to differentiate with or without TNFα supplementation for 7 days. Differentiation was induced at day 0. The effect on differentiation was assessed at days 1, 3, 5, and 7 by western blot analysis, and supplementary immunohistochemical analysis at days 1, 4, and 7 of markers of differentiation - muscle regulatory factors: MyoD and myogenin, markers of the cell cycle p21, PCNA, and the integral signalling molecule, p38MAPK. TNFα supplementation at day 1 tended to positively regulate early markers of differentiation. With continued supplementation however, markers of differentiation decreased dose dependently in treated cultures as the initial effect appeared to be reversed: A trend towards a dose dependent decrease in MyoD, myogenin and p21 protein existed in treated cultures at days 3, 5, and 7. These findings were significant at day 5 (p21, p<0.05), and day 7 (myogenin, p<0.05). A significant dose dependent decrease in p38 phosphorylation was evident at day 3 (p<0.05), while phospho-p38 was dose dependently increased at day 7 (p<0.05). Taken together, these data show that TNFα supplementation for 24 hours following the induction of differentiation in vitro, tends to increase levels of early markers of differentiation, and with continued TNFα supplementation decrease markers of differentiation in a dose dependent fashion. This study provides a comprehensive characterisation of the dose and time dependent effects of TNFα on satellite cell differentiaton in vitro. The model system used in the current study, allows us to make conclusions on more chronic disease states.
AFRIKAANSE OPSOMMING: Tumor nekrose faktor alfa (TNFα) is ‘n pleiotropiese sitokien wat ‘n wye verskeidenheid, dosis afhanklike, sellulêre effekte te weeg bring. Hierdie sellulêre effekte sluit sel groei en differensiasie tot sel dood in. TNFα is by beide spier en niespier inflammatoriese stoornisse soos spier tering in kroniese siektetoestande, en rumatiese artritis betrek. ‘n Fisiologiese rol vir TNFα is egter voorgestel aangesien verhoogde vlakke van die sitokien tydens inisiasie van spier herstel meganismes teenwoordig is: TNFα word deur infiltrerende inflammatoriese selle, asook deur beseerde spier vesels afgeskei. Volwasse skeletspier bevat ‘n populasie stamselagtige selle, sogenoemde satelliet selle. Laasgenoemde word geaktiveer, prolifereer en differensieër volgende spierbesering, om sodoende herstel van beskadigde spier te weeg te bring. Baie navorsing op die effekte van TNFα op satelliet sel differensiasie is onlangs uitgevoer. Dit is egter aansienlik moeilik om volgens hierdie navorsing‘n algehele beeld van TNFα se aksies te vorm aangesien alle modelle wat gebruik word verskil. Ons doel was daarom om ‘n omvangryke assessering van toenemende konsentrasies kronies gesupplementeerde TNFα op differensieërende C2C12 selle op ‘n enkele model uit te voer. Selle was vir 7 dae met of sonder TNFα supplementasie gedifferentieër. Differensiasie was by Dag 0 geïnduseer. TNFα se effek op differensiasie is op dae 1, 3, 5, en 7 deur middel van western blot analise geassesseer. Aanvullende immunohistochemiese bepalings op dae 1, 4, en 7 is verder deurgevoer. Merkers vir differensiasie het die spier regulatoriese faktore MyoD en miogenien, sel siklus merkers p21 en PCNA, asook die integrale sein transduksie molekule p38MAPK ingesluit. TNFα supplementasie by dag 1 het geneig om vroeë merkers van differensiasie positief te reguleer. Met voortdurende supplementasie is die vroeë positiewe effekte (op ‘n dosis afhanklike manier) egter omgekeer: ‘n neiging teenoor (‘n dosis afhanklike) vermindering in MyoD, miogenien en p21 proteïen het in behandelde kulture op dae 3, 5, en 7 bestaan. Hierdie bevindinge was beduidend by dag 5 (p21, p<0.05), en dag 7 (miogenien, p<0.05). A beduidende dosis afhanklike afname in p38 fosforilasie was duidelik by dag 3 (p<0.05), terwyl fosfo-p38 by dag 7 verhoog het met verhoogde konsentrasie TNFα (p<0.05). Bogenoemde saamgevat, dui aan dat TNFα supplementasie 24h volgende die induksie van differensiasie in vitro, verhoogde vlakke van vroeë differnsiasie merkers te weeg bring. Met voortdurende TNFα supplementasie, word differensiasie merkers egter met toenemende dosis verminder. Hierdie studie voorsien ‘n omvattende karakterisering van die dosis- en tyd afhanklike effekte van TNFα op satelliet sel differesiasie in vitro. Die model sisteem in hierdie studie gebruik, maak afleidings oor meer kroniese siektetoestande moontlik.

Thesis (PhD)--Stellenbosch University, 2012.
ENGLISH ABSTRACT: Introduction – Due to spatial separation from the native vascular bed, solid tumours develop regions with limited access to nutrients essential for growth and survival. The promotion of a process known as macroautophagy may facilitate in the maintenance of intracellular amino acid levels, through breakdown of cytoplasmic proteins, so that they remain available for macromolecular biosynthesis and ATP production. Several studies point to the potential ability of some cancers to temporarily increase autophagy and thereby prolong cell survival during metabolic stress. The validity of these claims is assessed when a commonly used breast cancer cell line and an epithelial breast cell line are starved of amino acids in this study. Furthermore, we go on to hypothesize that acute amino acid deprivation during treatment will result in an elevated sensitivity of MDAMB231 cells to doxorubicin toxicity but limit its cytotoxic side-effects in MCF12A cells. Methods and study design- Human breast cancer cells (MDAMB231) and breast epithelial cells (MCF12A) cultured in complete growth medium were compared to those incubated in medium containing no amino acids. Steady state autophagy levels were monitored using classical protein markers of autophagy (LC3-II and beclin-1) and the acidic compartmentalization in cells (Lysotracker™ red dye) in conjunction with autophagy inhibition (bafilomycin A1 and ATG5 siRNA). Cell viability was monitored using several techniques, including caspase 3/7 activity. ATP levels were assessed using a bioluminescent assay, while mass spectrometry based proteomics was used to quantify cellular amino acid levels. Similar techniques were used to monitor autophagy during doxorubicin treatment, while cellular doxorubicin localization was monitored using immunofluorescence microscopy. Finally, a completely novel GFP-LC3 mouse tumour model was designed to assess autophagy and caspase activity within tumours in vivo, during protein limitation and doxorubicin treatment. Results - Amino acid deprivation resulted in a transient increase in autophagy at approximately 6 hours of amino acid starvation in MDAMB231 cells. The amino acid content was preserved within these cells in an autophagy-dependent manner, a phenomenon that correlated with the maintenance of ATP levels. Inhibition of autophagy during these conditions resulted in decreased amino acid and ATP levels and increased signs of cell death. MCF12A cells displayed a greater tolerance to amino acid starvation during 24 hours of amino acid starvation. Evidence indicated that autophagy was important for the maintenance of amino acid and ATP levels in these cells and helped prevent starvation-induced cell death. Furthermore, data showed that concomitant amino acid withdrawal resulted in decreased cellular acidity in MDAMB231 cells, and increased acidity in MCF12A cells, during doxorubicin treatment. These changes correlated with evidence of increased cell death in MDAMB231 cells, but a relative protection in MCF12A cells. A novel model was used to apply these techniques in vivo, and although mice fed on a low protein diet during high dose doxorubicin treatment had increased mean survival and smaller tumour sizes, evidence suggested that autophagy is protecting a population of cells within these tumours. Conclusions - This novel approach to tumour sensitization could have several implications in the context of cancer therapy, and given the delicate relationship that autophagy has with the cancer microenvironment, efforts to determine the mechanisms involved in autophagy and sensitization could lead to new and innovative treatment opportunities for cancer management.
AFRIKAANSE OPSOMMING: Inleiding – As gevolg van hul skeiding van die oorpronklike vaskulêre netwerk, ontwikkel soliede gewasse areas met beperkte toegang tot noodsaaklike voedingstowwe. Die bevordering van 'n proses wat as makro-autofagie bekend staan, kan die handhawing van intrasellulêre aminosuur vlakke fasiliteer. Voorafgenoemde proses word waarskynlik deur die afbreek van sitoplasmiese proteïene teweegebring om sodoende vir makro-molekulêre biosintese en ATP produksie beskikbaar te kan wees. Verskeie studies dui daarop dat sommige kankersoorte die vermoë het om autofagie tydelik te verhoog, en daarby sel oorlewing gedurende metaboliese stress te verleng. Die geldigheid van hierdie eise word evalueer wanneer 'n algemeen beskikbare borskanker sellyn, en 'n borsepiteelsellyn in hierdie studie van aminosure verhonger word. Verder, veronderstel ons dat akute aminosuur ontneming gedurende behandeling 'n verhoogde sensitiwiteit van MDAMB231 selle tot doxorubicin toksisiteit tot gevolg sal hê, maar terselfdetyd die middel se sitotoksiese newe-effekte in MCF12A selle sal beperk. Metodes en studie ontwerp – Menslike borskanker- (MDAMB231) en bors epiteel selle (MCF12A) wat in volledige groeimedium gekweek is, is vergelyk met selle wat in aminosuur vrye medium gekweek is. Basislyn autofagie-vlakke is gemonitor deur die gebruik van klassieke autofagie proteïen merkers (LC3-II en beclin-1) en die asidiese kompartementalisering in selle (Lysotracker™ rooi kleurstof) saam met autofagie inhibisie (bafilomycin A1 and ATG5 siRNA). Sellewensvatbaarheid is deur die gebruik van verskeie tegnieke, insluitend caspase 3/7 aktiwiteit, gemonitor. ATP-vlakke is deur die gebruik van 'n bioluminiserende tegniek gemeet, terwyl massa-spektrometrie-gebaseerde “proteomics” gebruik is om sel aminosuur vlakke te kwantifiseer. Soortgelyke tegnieke is gebruik om autofagie gedurende doxorubicin behandeling waar te neem, terwyl sellulêre doxorubicin lokalisasie deur die gebruik van immunofluoresensie mikroskopie gemonitor is. Ten slotte, is 'n unieke GFP-LC3 muismodel in hierdie studie ontwikkel. Hierdie model is gebruik om autofagie en caspase aktiwiteit in gewasse in vivo te bestudeer tydens proteïen beperking en doxorubicin behandeling. Resultate – Aminosuur ontneming het tot 'n tydelike verhoging in autofagie na ongeveer 6 ure van aminosuur verhongering in MDAMB231 selle gelei. Die aminosuur inhoud van hierdie selle het op 'n autofagie-afhanklike manier behoue gebly. Hierdie verskynsel het met die handhawing van ATP-vlakke gekorreleer. Autofagie inhibisie gedurende hierdie kondisies het 'n verlaging in aminosuur en ATP-vlakke teweeggebring, sowel as vermeerderde tekens van seldood tot gevolg gehad. MCF12A selle het 'n groter toleransie tot aminosuur verhongering tydens die 24 uur aminosuur verhongeringsperiode getoon. Getuienis het aangedui dat autofagie belangrik vir die handhawing van aminosuur en ATP-vlakke in hierdie selle was, en gehelp het om verhongerings-geïnduseerde seldood te voorkom. Verder het data gewys dat aminosuur ontrekking tot verminderde sellulêre asiditeit in MDAMB231 selle, en verhoogde asiditeit in MCF12A selle gedurende doxorubicin behandeling gelei het. Hierdie veranderinge stem ooreen met getuienis van toenemende seldood in MDAMB231 selle, maar 'n relatiewe beskerming in MCF12A selle. 'n Unieke model was gebruik om hierdie tegnieke in vivo toe te pas. Alhoewel verhoogde oorlewing en kleiner gewasse in muise op 'n lae proteïen dieet gedurende hoë dosis doxorubicin behandeling opgemerk is, het bewyse voorgestel dat autofagie 'n populasie selle binne die gewasse beskerm. Gevolgtrekkings – Hierdie unieke benadering tot tumor sensitisering kan verskeie implikasies in die konteks van kanker behandeling hê. Gegewe die delikate verhouding van autofagie met die kanker mikro-omgewing, kan pogings om die meganismes betrokke in autofagie en sensitisering te bepaal, tot nuwe en innoverende behandelings vir kanker lei.

Includes bibliographical references (leaves 196-220) Studies the growth, body composition, feed intake of grazing animals, the use of anabolic agents, fatty acid composition of ruminants fats; and, the effect of saturated fat on human health.

Thesis (MSc)--University of Stellenbosch, 2007.
ENGLISH ABSTRACT: Introduction Cardiovascular disease is one of the leading causes of death in the world. Formation of harmful reactive oxygen species (ROS) is associated with several pathological conditions, and contributes to ischaemia/reperfusion injury. Antioxidants can be added to the diet in an attempt to decrease the prevalence of cardiovascular disease by decreasing the harmful effects of ischaemia/reperfusion injury. Red Palm Oil (RPO) consists of saturated, monounsaturated and polyunsaturated fatty acids and is rich in antioxidants such as -carotene, tocopherols and tocotrienols. It has previously been shown that RPO-supplementation improved reperfusion mechanical function. In these studies it was found that RPO might exert its beneficial effects during reperfusion through increased PKB/Akt pathway activity, which may lead to inhibition of apoptosis and improved mechanical function. Aims The aims of this study were: 1) to determine whether RPO-supplementation protected against ischaemia/reperfusion injury in the isolated perfused rat heart, 2) to confirm RPO-supplementation’s effect on the PKB/Akt pathway activity and, 3) to elucidate the regulators in the PKB/Akt pathway that RPOsupplementation influenced. Methods Male Wistar rats were divided into 4 groups, 2 control groups and 2 experimental groups. The 2 control groups were fed a standard rat chow (SRC) for 4 weeks. The two experimental groups received SRC and RPOsupplementation for 4 weeks. Hearts were excised and transferred to a Langendorff perfusion apparatus and perfused with Krebs-Henseleit buffer. Mechanical functional recovery was measured after 25 min of total global noflow ischaemia. The following parameters were also measured during various time points in the protocol: left ventricular develop pressure, heart rate, coronary flow, rate pressure product. Hearts were also freeze-clamped for biochemical analysis at 10 min during reperfusion. The biochemical analysis was aimed at determining PKB/Akt involvement. In a second protocol, hearts were subjected to the same perfusion protocol, but wortmannin was also added to the perfusion fluid, in order to inhibit PI3- kinase. Results Hearts from the RPO-supplemented rats showed an improved RPP recovery (92.26 ± 5.89 % vs 63.86 ± 7.74 %) after 10 min of reperfusion. This finding corroborated the findings of previous studies. Hearts of the RPOsupplemented rats perfused with wortmannin, showed increased RPP recoveries at several time points. Biochemical results showed that wortmannin did indeed inhibit PI3-K phosphorylation in the RPO-supplemented group, as was expected. The RPO-supplemented group that was perfused with wortmannin had an increased PKB/Akt (Ser473) phosphoyrylation, when compared to the wortmannin control group. It was also found that the combination of RPO and wortmannin had prosurvival effects. Discussion This study showed that RPO-supplementation offered protection against ischaemia/reperfusion injury in the Langendorff-perfusion apparatus at 10 min into reperfusion. Thereafter the significance of the protection was lost. This protection has been confirmed in several previous studies and several mechanisms have been proposed for this protection. Since no conclusive evidence exists on the precise mechanism of protection, our investigation focused on the regulators of the pro-survival PKB/Akt pathway. An improved functional recovery was also seen in the RPO-supplemented group that was perfused with wortmannin. This was an unexpected finding, because Wortmannin is a known PI3-kinase inhibitor (as was confirmed by our biochemical data). PI3-kinase phosphorylation leads to PKB/Akt phosphorylation and therefore, activation of a pro-survival pathway. It would be expected that wortmannin would inhibit PKB/Akt and thus decrease the survival of the cells. The RPO-supplementation thus reversed wortmannin’s detrimental effect to such an extent that the functional recovery was far better than RPO-supplementation alone. In the RPO + wortmannin group, PKB/Akt (Ser473) phosphorylation was increased, contrary to previous findings. This is an indication that RPO may have the ability to override wortmannin’s inhibitory effect on PI3-kinase, or that PKB/Akt (Ser473) may be phosphorylated independently of PI3-kinase.
AFRIKAANSE OPSOMMING: Inleiding Kardiovaskulêre siektes is een van die hoof oorsake van sterftes in die wêreld. Die vorming van skadelike reaktiewe suurstof spesies word geassosieer met verskeie patologiese kondisies en dra ook by tot isgemie/reperfusie skade. ‘n Moontlike manier om die voorkoms van isgemie/herperfusie skade asook kardiovaskulêre siektes te voorkom, is om antioksidante by die dieet te voeg. Rooi Palm Olie (RPO) bevat versadigde, mono-onversadigde en polionversadigde vetsure. RPO bevat ook ‘n oorvloed van antioksidante soos β- karoteen en tokoferole en tokotriënole. Dit is bewys in vorige studies dat RPO-aanvulling verbeter funksionele herstel. Hierdie voordelige effekte mag dalk wees agv verhoogde PKB/Akt pad aktiwiteit. Die PKB/Akt pad word geassosieer met die inhibisie van apoptose en verhoogde meganiese funksie. Doelwitte Die doelwitte van hierdie studie was om te bepaal of 1) RPO-aanvulling beskermende effekte teen isgemie/herperfusie skade in die geisoleerde rotharte het, 2) Bevestig of RPO-aanvulling wel die PKB/Akt pad beïnvloed 3). om die effekte wat RPO-aanvulling het op die reguleerders van die PKB/Akt pad te onthul. Metodes Manlike Wistar rotte is in 4 groepe verdeel. 2 Groepe kontrole rotte is ‘n standaard rotkosmengsel gevoer vir 4 weke. Die 2 eksperimentele groepe het ook ‘n standaard rotkosmengsel gekry plus ‘n RPO-aanvulling vir 4 weke. Harte is uitgesny en op ‘n Langendorff perfusie sisteem gemonteer en met Krebs-Henseleit buffer geperfuseer. Meganiese funksie herstel is gemeet na 25 min totale globale geen-vloei isgemie. Linker ventrikulêre ontwikkelde druk, harttempo, koronêre vloei en tempo druk produk is gemeet by verskillende tydpunte. Sommige harte is na 10 min herperfusie vir biochemiese analiese gevriesklamp. Die biochemiese analisiese was beoog om die PKB/Akt pad betrokkenheid te bepaal. ‘n Tweede stel harte is aan dieselfde perfusie protokol blootgestel, maar wortmannin (PI3-kinase inhibitor) is ook bygevoeg by die perfusie vloeistof. Resultate Die groep wat met RPO aangevul is, het na 10 min herperfusie, ‘n verbeterde tempo druk produk herstel getoon (92.26 ± 5.89 % vs 63.86 ± 7.74. Hierdie bevinding is ook met ander studies bevestig. ‘n Interessante bevinding was dat die groep wat met RPO aangevul is en met wortmannin geperfuseer is, ‘n verbeterde meganiese funksionele herstel getoon het. Biochemiese resultate het getoon dat wortmannin wel PI3-K fosforilering geinhibeer het. Die harte van die rotte in die groep wat aangevul is met RPO en daarna met wortmannin geperfuseer is, het ‘n toename in PKB/Akt (Ser473) fosforilering getoon, relatief tot die wortmannin geperfuseerde harte van die rotte in die kontrole groep. Hierdie groep (RPO-aanvulling en wortmannin perfusie) het beskermende effekte getoon. Bespreking Hierdie studie het getoon dat RPO-aanvulling beskerming gebied het teen isgemie/herperfusie skade in die Langendorff geperfuseerde rothart na 10 min herperfusie. Daarna is die beduidenheid van die beskerming verloor. Hierdie bevindings ondersteun die resultate van vorige studies. Verskeie moontlike meganismes is voorgestel vir die beskerming, maar die presiese meganisme is nog nie duidelik nie. In hierdie studie is daar gekyk na die reguleerders van die PKB/Akt pad. Geen vorige studies het al gefokus op RPO-aanvulling en sy effek op die reguleerders van die PKB/Akt pad nie. ‘n Onverwagte bevinding is dat harte van die rotte in die RPO + wortmannin groep ‘n verbeterde funksionele herstel getoon het. Wortmannin is ‘n PI3- kinase inhibitor. PI3-K fosforilering lei tot PKB/Akt fosforilering, wat tot sel beskerming lei. Dus, aangesien wortmannin PI3-K inhibeer, sou dit verwag word dat wortmannin sel beskerming sal verminder. Die RPO het egter die wortmannin se nadelige effekte tot so ‘n mate oorskrei dat die funksionele herstel baie beter was as die RPO-aanvulling alleen. Die verhoogde PKB/Akt (Ser473) fosforilering, wat gesien is in die RPO + wortmannin groep kan toegeskryf word aan RPO se vermoë om wortmannin se nadelige effekte te oorskrei. ‘n Moontlike verduideliking vir hierdie bevinding mag wees dat rooi palm olie PKB/Akt (Ser473) op ‘n PI3-K onafhanklike manier fosforileer.

Les effets des acides retinoiques tout-trans (tra) et 13-cis (cra) sont etudies in vivo dans le modele experimental du gliome c6 de rat. Le cra et le tra associes a des corticoides (methylprednisolone 1 mg/kg/jour) afin de reduire les reactions oedemateuses occasionnees par la necrose tumorale, provoquent des modifications morphologiques importantes avec des criteres de redifferenciation, et induisent des survies prolongees, jusqu'a 30% avec 20 mg/kg/jour de cra. Des effets secondaires se manifestent sous forme d'hemorragies intratumorales, associes a une activation locale de la fibrinolyse, due a la secretion d'activateur tissulaire du plasminogene (t-pa) par les cellules tumorales. Le recepteur nucleaire beta a l'acide retinoique, majoritairement exprime dans le systeme nerveux central, fait l'objet d'une etude moleculaire dans les tumeurs cerebrales humaines. On observe une absence d'expression des transcripts et de la proteine dans les gliomes dont la frequence augmente avec le grade de malignite, en l'absence d'alterations majeures du gene en southern. Dans la perspective d'une application therapeutique chez l'homme, ces resultats devraient permettre un meilleur ciblage des patients susceptibles d'etre sensibles a cette therapie, du fait de la correlation entre la presence du recepteur et la sensibilite a l'acide retinoique. L'etude des relations entre differenciation et expression de t-pa doit permettre l'evaluation d'effets secondaires non decrits jusqu'a present

Thesis (MSc)--University of Stellenbosch, 2007.
ENGLISH ABSTRACT: Human studies on the response of muscle to contusion injury are limited, probably due to the large variability in injury severity and the non-specificity of clinical symptoms reported. To circumvent this problem, several experimental animal models have been designed to study muscle damage and regeneration after contusion injuries. However, the majority of techniques currently used to induce contusion injury are very invasive and therefore not optimal. Furthermore, published studies regarding clinical treatment of such injuries are limited. The main aims of this study were therefore: a) to establish and characterise an in vivo model of non-invasive contusion injury, and b) to assess the effect of pre-injury chronic administration of the antioxidant supplement Oxiprovin™ - a natural grape seed extract (GSE) - on skeletal muscle recovery after experimentallyinduced injury. Two groups of male Wistar rats were subjected to 14 days of oral administration of isovolaemic placebo (sterile isotonic saline) or GSE (20 mg/kg/day) prior to induced contusion. Contusion injury was induced with the mass-drop technique, and recovery parameters assessed for up to 14 days post-injury. Placebotreated rats on average exhibited a 56 % higher creatine kinase (CK) activity when compared to the GSE-treated rats when area under the curve (AUC) was calculated for 14 days post-injury (p < 0.001). In the placebo group, plasma oxygen radical absorbance capacity (ORAC) was unchanged over time, but muscle ORAC was significantly increased by day 7 post-injury (p < 0.001). In the GSE group, a significant decrease in both plasma (p < 0.01) and muscle ORAC (p < 0.001) was evident 4 hr after injury, followed by a significant increase by day 3 (p < 0.05 and p < 0.001 respectively). CD34+ satellite cell (SC) numbers (quiescent and activated) peaked earlier in GSE-treated rats when compared to placebo-treated rats (4 hours vs. day 7 post-injury). Total satellite cell number (CD56+) also peaked earlier in GSE-treated rats than in placebo-treated rats (4 hours vs. 3 days post-injury), while M-cadherin+ SC numbers (quiescent, activated or proliferating) in both treatment groups were significantly increased 4 hours post-injury (p < 0.001), but more so in the placebo group. In GSE-treated rats when compared to placebo-treated rats, newly generated muscle fibres (displaying central nuclei and MHCf +) both appeared (day 3 vs. day 7 post-injury) and peaked in number (day 3 vs. day 7 post-injury; increase from baseline p < 0.001 for both) earlier. The results of this study demonstrate that we have successfully established an in vivo model for non-invasive contusion injury in rats. Furthermore, we have shown that Oxiprovin™: a) increased the ability to scavenge reactive species generated after injury and b) resulted in the activation of satellite cells and formation of newly generated muscle fibres at an earlier time point, thus accelerating the recovery of skeletal muscle after a standardised contusion injury.
AFRIKAANSE OPSOMMING: Eksperimente aangaande die reaksie van spier op kneusings in mense is beperk, waarskynlik as gevolg van die groot verskeidenheid simptome wat mag voorkom en die verskille in die ernstigheid van beserings. Ten einde hierdie problem te oorbrug, is verskeie eksperimentele diermodelle opgestel om kneusings en die herstel van spier daarna te ondersoek. Die tegnieke wat grootendeels vandag gebruik word om kneusings te veroorsaak, maak inbraak op die spier deur die spier te ontbloot voor besering, en is dus nie ideaal nie. Daar is ook nie baie bewyse aangaande die mees geskikte manier om so ‘n besering klinies te behandel nie. Die doel van hierdie studie was dus om: a) ‘n in vivo model van kneusings op te stel en te omskryf, en b) die effek van chroniese toediening van die antioksidant Oxiprovin™ - ‘n natuurlike druifsaad ekstrak (DSE) – op die herstel van skeletspier na ‘n kneusing te ondersoek. Twee groepe manlike Wistar rotte is onderwerp aan mondelikse toediening van isovolemiese plasebo (steriele isotoniese soutoplossing) of DSE (20 mg/kg/dag) vir ‘n tydperk van 14 dae voor kneusing. Kneusing is geïnduseer met die “massdrop” tegniek, en parameters van herstel is ondersoek tot en met 14 dae na besering. Plasebo-behandelde rotte het gemiddeld 56 % hoër kreatien kinase (KK) aktiwiteit in vergelyking met DSE-behandelde rotte (p < 0.001), toe die oppervlak onder die kurwe (OOK) tot en met 14 dae na besering bereken is. Geen verskil oor tyd is in die plasebo groep opgemerk toe plasma suurstof radikaal absorpsie kapasiteit (SRAK) bepaal is nie, maar ‘n betekenisvolle toename in spier SRAK teen dag 7 (p < 0.001) is waargeneem. ‘n Betekenisvolle afname in beide plasma (p < 0.01) en spier (p < 0.001) SRAK van die DSE is teen 4 hr waargeneem, gevolg deur ‘n betekenisvolle toename teen dag 3 na besering (p < 0.05 en p < 0.001 onderskeidelik). Die aantal CD34+ satelliet selle (SS – rustend en geaktiveerd) het beduidend vroeër in die DSE groep gestyg in vergelyking met die plasebo groep (4 uur vs. 7 dae na besering). Die totale aantal SS (CD56+) het ook vroeër in die DSE-behandelde rotte as die plasebobehandelde rotte gestyg (4 uur vs. 3 dae na besering), terwyl die aantal Mcadherin+ SS (rustend, geaktiveerd of prolifererend) betenisvol gestyg het in beide groepe teen 4 uur (p < 0.001) na besering, maar hoër in die plasebo groep was. Die aantal nuutgevormde spiervesels (met sentraal geleë nukleï en MHCf +) het beide vroeër verskyn en gepiek in die DSE-behandelde rotte in vergelyking met die plasebo-behandelde rotte (dag 3 vs. dag 7 na besering). Die resultate van hierdie studie dui aan dat ons instaat was om ‘n in vivo model van nie-indringende kneusing in rotte op te stel. Verder, het ons ook bewys dat Oxiprovin™ toediening die vermoë verleen het om: a) reaktiewe spesies wat na beserings gevorm word, meer doeltreffend te verwyder en b) satelliet selle vroeër te aktiveer en die vorming van nuwe skeletspiervesels te vervroeg, om sodoende die herstel van skeletspier na ‘n gestandardiseerde kneusing vinniger te bewerkstellig.

Over the past two decades movement of cattle towards the north of Uganda has enabled the Trypanosoma brucei rhodesiense focus in south-eastern Uganda to spread into previously unaffected districts. This thesis brings together important epidemiological data regarding the impact of mass cattle drug treatment on the point prevalence of several different species of trypanosome in a newly endemic area of human sleeping sickness. Crucially the findings illustrate mass drug treatment is effective in reducing the prevalence of T. b. rhodesiense in cattle, thus minimising the reservoir potential of these animals in the epidemiology of human disease. During 2006 a control programme was launched to halt the northward spread of this zoonotic parasite. This programme, entitled ‘Stamping Out Sleeping Sickness’ (SOS) proposed to reduce the prevalence of Human African Trypanosomiasis (HAT) in the newly affected districts by reducing the prevalence of this parasite in the main animal reservoir of infection – domestic cattle. Cattle were mass treated using trypanocides to clear infections. Previous work demonstrated the prevalence of T. brucei s. l. and T. b. rhodesiense in cattle was higher in the districts of Dokolo and Kaberamaido than in the other SOS intervention districts (Selby 2011). To determine whether animals in these areas were also exposed to pathogenic cattle trypanosomes samples were screened for the presence of T. vivax and T. congolense savannah using PCR. Chapter three of this thesis determined the prevalence of these trypanosomes in cattle in these districts. Before treatment had taken place the prevalence of T. vivax was 2% (4/200, 95% CI 3.57 – 0.12%) in Dokolo and 7.3% (21/310, 95% CI 10.17 - 4.24 %) in Kaberamaido. The prevalence of T. congolense savannah at baseline was 3.5% (7/200, 95% CI 7.08–1.42 %) in Dokolo and 9.1% (21/230, 95% CI13.6–5.7 %) in Kaberamaido. Monitoring was conducted three, nine and 18 months post treatment and both pathogens were detected at all time points. The impact the treatment had on point prevalence varied by trypanosome species and between the two districts. Several clusters of villages in Dokolo and Kaberamaido continued to report cases of HAT after the initial SOS intervention due in part to their proximity to livestock markets (Batchelor et al., 2009). In 2008 re-treatment of these ‘high risk’ areas was undertaken. Monitoring was performed before and six months after treatment. Cattle blood samples were collected at 20 village sites from ten ‘case-positive villages’ (from which human sleeping sickness cases had been reported six months prior to June 2007) and from ten ‘case-negative villages’ (no reported human sleeping sickness cases six months prior to June 2007). These samples were screened for all of the aforementioned trypanosomes using species specific PCR protocols. Chapter five details the results of this screening, and assessed whether re-treatment in Dokolo and Kaberamaido was effective in reducing the prevalence of trypanosomiasis. The re-treatment had a dramatic effect, significantly reducing the point prevalence of overall trypanosomiasis in the 20 villages screened from 38.1% (95% CI = 40.5 – 35.79%) at baseline to 26.9% (95% CI 28.96 – 24.97, p < 0.0001) at six months. Looking at each species separately, point prevalence of three out of four detected species of trypanosome fell significantly, including T. b. rhodesiense, which was reduced to 25% of its baseline prevalence. Finally the two SOS treatment cycles were compared both statistically and spatially with emphasis on trends at village level and the occurrence of mixed infections.

Thesis (MSc)--University of Stellenbosch, 2006.
ENGLISH ABSTRACT: Introduction: The ability of different polyunsaturated fatty acids (PUFAs), especially n-3 PUFAs, to prevent the development of cancer has been under intense investigation the past three decades. Numerous studies have shown that these fatty acids can kill cancer cells in vitro as well as in vivo whilst normal cells remain unaffected. Unfortunately, the cellular and molecular mechanisms responsible for this phenomenon are still poorly understood. This study investigated the signalling pathways modulated by docosahexaenoic acid (DHA) in an adenocarcinoma cell line, in order to shed some light on these unknown mechanisms. Materials & Methods: NCM460 (normal colon epithelial) and CaCo2 (colon adenocarcinoma) cells were cultured and treated with low doses of palmitic acid (PMA), oleic acid (OA), arachidonic acid (AA), and DHA. The effects of these fatty acids on the proliferation of the cells were measured with the MTT assay. The composition of membrane phospholipids of CaCo2 cells was determined after 48h supplementation with different fatty acids by gas chromatography. Also, CaCo2 cells were treated with DHA (10 μM) only and proteins were harvested at fixed time points ranging from 2 minutes to 48 hours. The protein inhibitors wortmannin (PI3 kinase inhibitor), PD 98059 (MEK inhibitor) and SB 203580 (p38 inhibitor) and also RNA interference (RNAi) of the p38 MAPK protein were used to investigate cross-talk between signalling pathways. ERK, p38 MAP kinase, Akt, and p53 were then analysed by Western blotting using phospho-specific and total antibodies. The cleavage of the apoptotic proteins, caspase-3 and PARP were also analysed. Results and discussion: MTT assays revealed that none of the fatty acids were toxic to normal cells. In addition, DHA was shown to be most effective to kill CaCo2 cells whilst protecting NCM460 cells and a subsequent dose response experiment revealed that lower concentrations are most suitable for this purpose. DHA was also shown to be readily incorporated into phospholipids, along with AA. This is associated with increased membrane fluidity, which could affect the localisation, and downstream effects, of various signalling proteins within the membrane. Western blot analysis revealed a rapid increase in activity in most proteins under investigation, especially ERK and Akt (Ser473). Long-term DHA supplementation suppressed the full activation of Akt. This down regulation of survival signalling could lead to cell death in CaCo2 cells. In addition, it was shown that after 48h, DHA induced the cleavage of caspase-3 and PARP, which is indicative of apoptosis. RNAi experiments suggested a possible role for p38 MAPK in the phosphorylation of p53 at Ser15, a site which is associated with DNA damage. Conclusion: DHA exerts its effects by means of cellular signal transduction pathways, particularly by suppression of the important survival-related kinase, Akt. This could have implications for future therapeutic interventions in cancer patients, as fatty acids are safe to use and do not interfere with the functionality of normal tissue.
AFRIKAANSE OPSOMMING: Inleiding: Die vermoë van verskillende poli-onversadigde vetsure (POVSe), veral n-3 POVSe, om die ontstaan van kanker te voorkom, is intens nagevors die afgelope drie dekades. Menigte studies het aangevoer dat hierdie vetsure kankerselle in vitro asook in vivo kan doodmaak, terwyl normale selle nie daardeur beïnvloed word nie. Ongelukkig word die sellulêre and molekulêre meganismes onderliggend tot hierdie verskynsel nie goed begryp nie. Hierdie studie het verskeie seintransduksie-paaie wat deur dokosaheksaenoësuur (DHS) in ‘n adenokarsinoom sellyn gemoduleer word, ondersoek. Materiale & Metodes: NCM460 (normale kolonepiteel) en CaCo2 (kolon adenokarsinoom) selle is onderhou in ‘n selkultuur-laboratorium en behandel met lae dosisse palmitiensuur (PMS), oleïensuur (OS), aragidoonsuur (AS), en DHS. Die invloed van hierdie vetsure op die proliferasie van die selle is d.m.v. die MTT toets bepaal. The samestelling van membraan-fosfolipiede van CaCo2 selle is na 48h behandeling met die verskillende vetsure bepaal deur middel van gaschromatografie. Die CaCo2 selle is ook met DHA (10 μM) alleenlik behandel en teen vaste tydpunte wat wissel van 2 minute tot 48h, waarna proteïene geëkstraeer is. Die proteïen-inhibitore wortmannin (PI3 kinase inhibitor), PD 98059 (MEK inhibitor), en SB 203580 (p38 inhibitor) asook RNAinterferensie (RNAi) teen die p38 MAPK proteïen is ingespan om oorvleueling tussen seintransduksie–weë te ondersoek. ERK, p38 MAPK, Akt, en p53 is geanaliseer deur middel van die Western–klad metode met fosfo–spesifieke en totale antiliggame. Die kliewing van die apoptotiese proteïene caspase-3 en PARP is ook bepaal. Resultate en bespreking: MTT toetse het ontul dat geen vetsure toksies was vir die normale selle nie. Daar is ook gevind dat DHS die mees effektiewe vetsuur was om CaCo2 selle te dood, terwyl NCM460 selle beskerm word. Gevolglik het ‘n dosis-respons eksperiment getoon dat laer konsentrasies die beste geskik is vir hierdie doel. Daar is ook gevind dat DHA maklik in fosfolipiede geïnkorporeer word, tesame met AS. Dit word geassosieer met verhoogde membraan-vloeibaarheid, wat die ligging, en ook stroom-af werking, van verskeie seintransduksie proteïene in die membraan, kan beïnvloed. Westernklad analises het ‘n vinnige verhoging in die aktiwiteite van die meeste proteïene onder die soeklig, getoon, veral ERK en Akt (Ser473). Langdurige DHS behandeling het die maksimale aktiwiteit van Akt onderdruk. Hierdie afname van oorlewing-gerigte seine kan lei tot seldood in CaCo2 selle. Daar is boonop geving dat DHS die kliewing van caspase-3 en PARP geïnduseer het na 48, wat dui op apoptose. Uit die RNAi eksperiment kon daar ook ‘n moontlike rol vir p38 MAPK in die fosforilering van p53 by Ser15, wat geassosieer word met DNS-skade, getoon word. Gevolgtrekking: DHS beoefen sy effekte deur middel van seintransduksie paaie, veral deur die oorlewing-geassosieerde kinase, Akt, te onderdruk. Dit kan implikasies hê vir toekomende terapeutiese ingrypings in kankerpasiënte, aangesien vetsure veilig is om te gebruik en nie skadelik is vir normale weefsel nie.

ABSTRACT External otitis and its treatment. Is a group III steroid without antibiotics sufficient therapy? – Experimental and clinical studies Per Emgård, Department of Otorhinolaryngology, University of Umeå and Ystad Hospital, Umeå and Ystad, Sweden External otitis is one of the most common ear, nose and throat (ENT) diagnoses in out-patient clinics. The clinical course of external otitis includes itching, pain, redness, swelling and effusion of the external auditory canal (EAC) with normal tympanic membrane status. The inflammatory condition is often associated with infection by bacteria, e.g. Pseudomonas aeruginosa, or skin bacteria such as Staphylococcus species. Fungi are present only in a low percentage of cases and if present Candida albicans infection is the most frequent in northern countries such as Sweden and the UK. Topical therapy is recommended in most countries and dominates the therapy in most studies. Topical drugs used are usually a combination of antibiotics and a steroid. However, external otitis is treated with surprisingly many strategies – eleven different ones in Sweden, for example, and 18 in the UK. The aims of the present studies were to – -establish an animal model, infected and uninfected, suitable for testing various treatment strategies of external otitis; and -perform a clinical study in patients to elucidate whether a group III steroid alone is as efficient for treatment of external otitis as is the commonly used topical drug containing a combination of a steroid and antibiotics. The animal model was established through mechanical irritation of the external ear canal skin of Sprague-Dawley rats. An evaluation scale for characterization of the clinical status of the ear canal was introduced, recording redness, swelling and occurrence of effusion in a standardized way. Specimens of the ear canal skin were analysed by histological techniques. A topical solution of 0.05% bethametasone dipropionate (BD) was compared with a 1% hydrocortisone solution with antibiotics oxytetracycline and polymyxin B added (HCPB), administered in the external otitis model infected or non-infected with bacteria (P. aeruginosa) and a fungus (C. albicans). The same drugs were tested in a randomized parallel-group multi-centre study in 51 patients. The clinical status of the external otitis patients was evaluated on a similar scale as used in the animal model. Early normalization of the ear canal skin status and frequency of relapses during the 6-month follow-up period were used as end-points of the study. The studies showed the following: -An animal model for external otitis, infected or uninfected, could be established. -A new scale for evaluation of the external ear canal status with regard to redness, swelling and occurrence of effusion was introduced for the animal model as well as for the investigations in patients. -Treatment with a group III steroid topical solution without antibiotics was superior to treatment with a group I steroid with antibiotics added in achieving resolution of external otitis. -The effectiveness of the topical drugs in the clinical studies in external otitis patients was similar to that in animal external otitis models. We conclude that a group III steroid solution cures external otitis more effectively than does a solution containing a group I steroid combined with antibiotics, whether infected by bacteria or by fungi. No difference was evident regarding adverse effects. Furthermore, costs favour a solution without any antibiotic components. In view of these observations a group III steroid solution is preferred for remedy of external otitis in the clinical situation. Key words: external otitis, external auditory canal (EAC), animal model, treatment, betamethasone, hydrocortisone, antibiotics, human study, Pseudomonas aeruginosa, Candida albicans.

Thesis (PhD (Physiology (Human and animal))-- University of Stellenbosch, 2007.
Definition: Stem cells are unspecialised cells with the capacity for long-term self-renewal and the ability to differentiate into multiple cell-lineages. The potential for the application of stem cells in clinical settings has had a profound effect on the future of regenerative medicine. However, to be of greater therapeutic use, selection of the most appropriate cell type, as well as optimisation of stem cell incorporation into the damaged tissue is required. In adult skeletal muscle, satellite cells are the primary stem cell population which mediate postnatal muscle growth. Following injury or in diseased conditions, these cells are activated and recruited for new muscle formation. In contrast, the potential of resident adult stem cell incorporation into the myocardium has been challenged and the response of cardiac tissue, especially to ischaemic injury, is scar formation. Following muscle damage, various growth factors and cytokines are released in the afflicted area which influences the recruitment and incorporation of stem cells into the injured tissue. Transforming Growth Factor-β (TGF-β) is a member of the TGF-β-superfamily of cytokines and has at least three isoforms, TGF-β1, -β2, and -β3, which play essential roles in the regulation of cell growth and regeneration following activation and stimulation of receptor-signalling pathways. By improving the understanding of how TGF-β affects these processes, it is possible to gain insight into how the intercellular environment can be manipulated to improve stem cell-mediated repair following muscle injury. Therefore, the main aims of this thesis were to determine the effect of the three TGF-β isoforms on proliferation, differentiation, migration and fusion of muscle progenitor cells (skeletal and cardiac) and relate this to possible improved mechanisms for muscle repair. The effect of short- and long-term treatment with all three TGF-β isoforms were investigated on muscle progenitor cell proliferation and differentiation using the C2C12 skeletal muscle satellite and P19 multipotent embryonal carcinoma cell-lineages as in vitro model systems. Cells were treated with 5 ng/mℓ TGF-β isoforms unless where stated otherwise. In C2C12 cells, proliferating cell nuclear antigen (PCNA) expression and localisation were analysed, and together with total nuclear counts, used to assess the effect of TGF-β on myoblast proliferation (Chapter 5). The myogenic regulatory factors MyoD and myogenin, and structural protein myosin heavy chain (MHC) were used as protein markers to assess early and terminal differentiation, respectively. To establish possible mechanisms by which TGF-β isoforms regulate differentiation, further analysis included determination of MyoD localisation and the rate of MyoD degradation in C2C12 cells. To assess the effect of TGF-β isoforms on P19 cell differentiation, protein expression levels of connexin-43 and MHC were analysed, together with the determination of embryoid body numbers in differentiating P19 cells (Chapter 6). Furthermore, assays were developed to analyse the effect of TGF-β isoforms on both C2C12 and P19 cell migration (Chapter 7), as well as fusion of C2C12 cells (Chapter 8). Whereas all three isoforms of TGF-β significantly increased proliferation of C2C12 cells, differentiation results, however, indicated that especially following long-term incubation, TGF-β isoforms delayed both early and terminal differentiation of C2C12 cells into myotubes. Similarly, myocyte migration and fusion were also negatively regulated following TGF-β treatment. In the P19 cell-lineage, results demonstrated that isoform-specific treatment with TGF-β1 could potentially enhance differentiation. Further research is however required in this area, especially since migration was greatly reduced in these cells. Taken together, results demonstrated variable effects following TGF-β treatment depending on the cell type and the duration of TGF-β application. Circulating and/or treatment concentrations of this growth factor could therefore be manipulated depending on the area of injury to improve regenerative processes. Alternatively, when selecting appropriate stem or progenitor cells for therapeutic application, the effect of the immediate environment and subsequent interaction between the two should be taken into consideration for optimal beneficial results.

Assignment (MPhil)--University of Stellenbosch, 2006.
ENGLISH ABSTRACT: The aim of this study was to investigate practical methods of monitoring AIDS related wasting and lipodystrophy in a resource-poor clinical setting with HIV infected women as the population group of interest. Measurement of body composition changes using anthropometry is both cost- and time-efficient. Various different skinfolds were taken and two different equations (the equations of Pollock et al. (1975) and Durnin and Womersley (1974) for calculating body fat were used to determine the most promising method or methods of monitoring body composition changes in a clinical setting. Detailed anthropometric measurements were performed, as well as selected measurements for haematological parameters and quality of life (QoL) for a group of 8 participants on antiretroviral medication (ART group) and 6 participants who were not on treatment (TN group). New variables namely, intra-abdominal indicator (IAI) and a percent of ideal body mass to percent of ideal arm circumference ratio (%IBW:%IAC) were investigated as possible indicators of lipodystrophy. Although measurements were taken at various timepoints, three specific time-points were chosen for data-analysis for the ART group and two time points for the TN group. These three time-points were, baseline (on the day of recruitment for TN participants and within one month before the initiation of treatment for ART participants), short-term (2 to 12 weeks after treatment initiation or the baseline measurement or for the ART and the TN participants) and long-term (within one and a half year of treatment initiation for the ART group). ART and TN participants did not differ for many variables at baseline. The major differences between ART and TN were in measured and derived variables of the arm, especially percent of ideal arm circumference (%IAC) and upper arm fat area (UAFA), which were significantly lower in the ART group. CD4+ and QoL improved significantly for the ART participants from baseline to long-term. This was not associated with changes in muscle mass, but rather some fat mass variables. Participants on antiretroviral medication exhibited changes relating to abdominal obesity. It was concluded that antiretroviral therapy contributed greatly to the QoL of the participants and it probably aided in the recovery from wasting for at least one participant in this study. Measures of the arm can be used in a rural clinical setting to effectively monitor patients with regard to AIDS related wasting. The new variables IAI and %IBW:%IAC could be helpful in the monitoring of lipodystrophy and should be investigated in future research.
AFRIKAANSE OPSOMMING: Die doelwit van hierdie studie is om praktiese metodes te ondersoek om VIGS-verwante uittering en lipodistrofie te meet in ‘n plattelandse kliniese omgewing (waar hulpbronne dikwels beperk is) met MIV ge-infekteerde vroue as populasiegroep. Die gebruik van antropometrie om veranderinge in liggaamssamestelling te meet is beide koste- en tydeffektief. Verskeie velvoumetings is geneem en twee verskillende vergelykings (die vergelykings van Pollock et al. (1975) en Durnin en Womersley (1974)) is gebruik om liggaamsvetinhoud te bereken, met die doel om ‘n belowende metode te vind om veranderinge in liggaamssamestelling te meet in ‘n kliniese omgewing. Verskeie antropometriese metings is geneem, sowel as uitgesoekte hematologiese en lewenskwaliteitmetings (QoL) vir ‘n groep van agt deelnemers wat antiretrovirale medikasie ontvang het (ART groep) en ses deelnemers wat nie hierdie behandeling ontvang het nie (TN groep). Nuwe veranderlikes (binnebuikindikator (IAI) en die verhouding van persentasie van ideale liggaamsmassa tot persentasie van ideale armomtrek (%IBW:%IAC)) is ondersoek as moontlike aanwysers van lipodistrofie. Drie spesifieke tydpunte vir die ART groep en twee tydpunte vir die TN groep is gekies uit die verskeie tydpunte waarby metings geneem is, nl. basislyn (gedefinieer as die dag wat TN deelnemers in die studie opgeneem is en 0 tot 4 weke voor die begin van behandeling vir die ART deelnemers), korttermyn (2 tot 12 weke nadat behandeling begin is of na die basislyn meting) en lang-termyn (binne een en ‘n half jaar nadat behandeling begin is vir die ART groep). By die basislyn tydpunt het min van die ART en TN deelnemers se gemete veranderlikes verskil. Die ART en TN groepe het hoofsaaklik verskil ten opsigte van veranderlikes wat betrekking het op die arm, veral persentasie van ideale armomtrek (%IAC) en bo-arm vetarea (UAFA). Hierdie twee veranderlikes was beduidend laer in die ART groep as in die TN groep. CD4+ seltelling en lewenskwaliteit tellings het beduidend verbeter vir die ART deelnemers van die basislyn tot die lang-termyn tydpunt. Hierdie veranderinge is nie samehangend met veranderinge in spiermassa nie, maar eerder met sommige vetmassa veranderlikes. Deelnemers wat antiretrovirale medikasie ontvang het, het veranderinge getoon wat gedui het op ‘n verhoogde neerlegging van vet in die buikarea. Ten slotte is bevind dat antiretrovirale medikasie bygedra het tot die verbeterde lewenskwaliteit van die deelnemers en dat dit waarskynlik ook die omkeer van uittering van ten minste een deelnemer aangehelp het. Daar is ook bevind dat armverwante metinge gebruik kan word in die plattelandse kliniese omgewing om pasiënte suksesvol te monitor ten opsigte van VIGSverwante uittering. Die nuwe veranderlikes, IAI en %IBW:%IAC kan moontlik gebruik word om lipodistrofie-verwante veranderings te meet en die gebruik van hierdie veranderlikes behoort ondersoek te word in verdere navorsing.

Des stratégies de thérapie génique sont en cours d'étude pour le traitement de maladies humaines. Ce type de thérapie s'adresse en premier lieu aux anomalies héréditaires récessives causées par la déficience en un gène unique dont le traitement nécessite l'addition du gène normal, mais pourrait être utilise également pour le traitement de cancers, de leucémies ou de maladies infectieuses. Le recours aux modèles animaux est indispensable pour : 1) juger directement de l'efficacité du transfert de gènes in vivo; 2) explorer les effets biologiques et potentiellement thérapeutiques de molécules exprimées à des niveaux physiologiques ou non physiologiques; 3) améliorer la méthodologie qui sera finalement appliquée à l'homme. Nos travaux s'inscrivent précisément dans le cadre du développement et de l'amélioration de modèles animaux murins. Étant donné les propriétés antivirales de l'interféron (IFN), nous développons une approche de thérapie génique basée sur l'expression constitutive de l'IFN dans les cellules hématopoïétiques et dirigée contre les maladies virales fatales à évolution lente et chronique comme le sida. D'autre part, nous nous intéressons à une maladie génétique héréditaire causée par la déficience en un gène unique codant pour l'adénosine déaminase (ADA); le dysfonctionnement de ce gène entraine une immunodéficience combinée sévère (SCID) chez l'homme. Aussi, la seconde partie de nos travaux concerne l'amélioration de la technologie relative à l'expression du gène codant pour l'ADA humaine dans les cellules hématopoïétiques in vivo, dans un modèle murin. L’approche commune à ces deux projets repose sur l'expression des ADNc (ADN complémentaires) des gènes codant pour l'IFN-beta murin et l'ADA humaine dans les cellules hématopoïétiques par l'intermédiaire de vecteurs rétroviraux.

Thesis (MEdPsych)--Stellenbosch University, 2014.
ENGLISH ABSTRACT: High-risk behaviour among adolescents places heavy burdens on the public health, social welfare and criminal justice systems of many countries. Today, Equine-Assisted Learning (EAL) is increasingly used as an adjunct and experiential intervention programme to support the learning and personal development of adolescents at risk. It combines counselling and educational programmes with interactive activities involving horses (Thomas, 2009). The aim of this investigation was to conceptualize the experiences of these at-risk adolescents in residential care (AIRC), since gaps exist in both international and national research pertaining to studies which focus on exploring adolescents' experiences of taking part in EAL programmes (Holder, 2011). An interactive, qualitative and multiple case study design was employed in this study. The research was conducted at a Western Cape residential care facility with five adolescents between the ages of 12 and 18, after they had completed a three-week EAL programme. The programme comprised five sessions of one hour each. Data was collected through a projective technique (collage), semi-structured individual interviews, and a focus group interview. The research findings suggested that the at-risk adolescents experienced this Equine-Assisted Learning programme as positive and that it contributed to strengthening their relationships, enhancing attachment, self-esteem, empathy, communication, social competence, and a sense of mastery, as well as opening up future possibilities for them. As a relatively new field of study in South Africa, Equine-Assisted Learning can make a significant contribution to fostering the learning and development of adolescents in residential care.
AFRIKAANSE OPSOMMING: Die hoë-risiko gedrag van adolessente plaas 'n swaar las op lande se openbare gesondheid, maatskaplike welsyn- en kriminele regstelsels en sodoende word 'Equine Assisted Learning' (EAL) toenemend gebruik as 'n aanvullende en ervarings-intervensieprogram om die leer en persoonlike ontwikkeling van adolessente in nood te ondersteun. In EAL, word berading en opvoedkundige programme met interaktiewe aktiwiteite wat perde betrek, gekombineer (Thomas, 2009). Die doel van die studie was om hoë-risiko adolessente in residensiële sorg se ervarings van 'n EAL intervensieprogram te konseptualiseer, aangesien daar op beide internasionale en nasionale vlak 'n gebrek aan navorsing is wat fokus op die verkenning van adolessente se ervaring van deelname aan EAL programme (Holder, 2011). 'n Interaktiewe, kwalitatiewe en veelvuldige gevallestudie ontwerp is in hierdie studie ontplooi. Die navorsing is uitgevoer in 'n Wes-Kaapse residensiële sorg fasiliteit met die hulp van vyf adolessente tussen die ouderdomme van 12 en 18, nadat hulle drie weke lank aan 'n EAL program deelgeneem het. Die program het bestaan uit vyf sessies wat elk een uur geduur het. Data is ingesamel deur gebruik te maak van projektiewe tegniek (collage), semi-gestruktureerde individuele onderhoude asook 'n fokusgroep-onderhoud. Navorsingbevindings het voorgestel dat adolessente wat risiko beloop, die EAL program as positief ervaar. Dit blyk ook dat dit bygedra het tot die bevordering van sekere aspekte soos verhoudings, hegting, selfbeeld, empatie, kommunikasie, sosiale bevoegdheid, 'n gevoel van bemeestering en die daarstel van toekomstige moontlikhede. 'Equine Assisted Learning' is 'n relatiewe nuwe studieveld in die Suid-Afrikaanse konteks, en kan daarom betekenisvol bydra tot die bevordering van leer en ontwikkeling van adolessente in residensiële sorg.

Thesis (MEd(Psych))-- Stellenbosch University, 2013.
ENGLISH ABSTRACT: The background experiences of adolescents removed from their families and placed in residential care are often characterised by trauma, abuse and neglect, resulting in significant psychological and developmental implications. However, as a result of their negative past experiences, many of these individuals view other people, including helping professionals, with mistrust or apprehension, influencing their openness to and thus the effectiveness of traditional psychotherapeutic interventions. Equine Assisted Psychotherapy (EAP) is explored within this study as an alternative non-invasive approach which aims to meet the emotional and developmental needs of vulnerable and difficult-to-reach adolescents. Innate characteristics of horses seem to encourage individuals to engage fully in the psychotherapeutic process making them well-suited to the role of co-therapist within the therapeutic team. This study aimed to explore the experiences of adolescents living within residential care who had participated in an EAP program for approximately eight months, and the affects their participation had within their lives. A qualitative study founded within the interpretive paradigm was used to gain insight into the research questions. Participants were selected using purposive and convenient sampling techniques. Following this, data was obtained through semi-structured interviews, the researcher’s reflective journal, and a focus group discussion. Inductive data segmentation and reassembling techniques were utilised to analyse the data. The findings were integrated and presented as a case study. The research findings indicated that the participants experienced various learning and development opportunities through their participation in EAP. They were able to establish connections which led to improved psychological, social, and physical well-being. The implicit integration of the Circle of Courage principles of mastery, belonging, generosity and independence within the EAP process was also explored and documented for the first time within this text. The findings were related to resilience theory in order to depict EAP as a psychotherapeutic approach which serves to foster resilience for vulnerable adolescents, namely those placed within residential care in this study.
AFRIKAANSE OPSOMMING: Die agtergrond ervaringe van adolessente wat verwyder is uit hul ouerhuise en in residensiële sorg geplaas is, word dikwels gekenmerk deur trauma, mishandeling en verwaarlosing, met beduidende gepaardgaande sielkundige - en ontwikkelingsimplikasies. Voortvloeiend uit hul negatiewe ervaringe van die verlede, is dit moeilik vir sommige van hierdie individue om ander mense, en dit sluit professionele mense in die hulp-professies in, te vertrou en sonder bedugtheid te benader. Dit beïnvloed hul openlikheid en dus die effektiwiteit van tradisionele psigoterapeutiese intervensies. Perd-ondersteunde psigoterapie (Equine Assisted Psychotherapy, EAP) word ondersoek in hierdie studie as ‘n alternatiewe, nie-indringende benadering, waarvan die doel is om die emosionele - en ontwikkelingsbehoeftes van weerlose en moeilik-bereikbare adolessente mee aan te spreek. Dit wil voorkom asof ingebore eienskappe van perde individue aanmoedig om ten volle deel te neem aan die psigoterapeutiese proses, en dit maak die perde baie geskik om as hulp-terapeute op te tree binne die terapeutiese span. Hierdie studie beoog om die ervaringe van adolessente in residensiële sorg, en wat vir agt maande deelgeneem het aan ‘n EAP program, te ondersoek, sowel as die effek wat hierdie deelname op hul lewens gehad het. ‘n Kwalitatiewe studie gebaseer op die interpretatiewe paradigma is gebruik om insig te verkry in die navorsingsvrae. Deelnemers is geselekteer deur doelgerigte - en gerieflikheidstegnieke te gebruik. Hierna is data verkry deur semi-gestruktureerde onderhoude, die navorser se reflektiewe joernaal en ‘n fokusgroep onderhoud. Induktiewe data segmentasie en hersamestellingstegnieke is gebruik om die data te analiseer. Die bevindinge is geïntegreer en aangebied as ‘n gevallestudie. Die bevindige van die studie dui aan dat die deelnemers verskeie leer- en ontwikkelingsgeleenthede ervaar het deur hul deelname aan EAP. Hulle was in staat om konneksies te maak wat bygedra het tot sielkundige, sosiale en fisiese welsyn. The research findings indicated that the participants experienced various learning and development opportunities through their participation in EAP. Die implisiete integrasie van die beginsels van die ‘Circle of Courage’, naamlik bemeestering, behoort aan, ruimhartigheid en onafhanklikheid, is binne die EAP proses ondersoek en vir die eerste keer in hierdie teks gedokumenteer. Die bevindinge is vergelyk met veerkragtigheid teorie om EAP as ‘n psigoterapeutiese benadering te beskryf wat veerkragtigheid aanmoedig by weerlose adolessente, naamlik dié wat in residensiële sorg geplaas is en aan hierdie studie deelgeneem het.

Robot assisted therapy is becoming increasingly popular. Research has proven it can be of benefit to persons dealing with a variety of disorders, such as Autism Spectrum Disorder (ASD), Attention Deficit Hyperactivity Disorder (ADHD), and it can also provide a source of emotional support e.g. to persons living in seniors’ residences. The advancement in technology and a decrease in cost of products related to consumer electronics, computing and communication has enabled the development of more advanced social robots at a lower cost. This brings us closer to developing such tools at a price that makes them affordable to lower income individuals and families. Currently, in several cases, intensive treatment for patients with certain disorders (to the level of becoming effective) is practically not possible through the public health system due to resource limitations and a large existing backlog. Pursuing treatment through the private sector is expensive and unattainable for those with a lower income, placing them at a disadvantage. Design and effective integration of technology, such as using social robots in treatment, reduces the cost considerably, potentially making it financially accessible to lower income individuals and families in need. The Objective of the research reported in this manuscript is to design and implement a social robot that meets the low-cost criteria, while also containing the required functions to support children with ASD. The design considered contains knowledge acquired in the past through research involving the use of various types of technology for the treatment of mental and/or emotional disabilities.

Negative discourses may be triggered when the public watch social media and perceive threats to horse welfare, challenging a sport’s social licence to operate. This mixed-method study investigated sport participants’ attitudes towards welfare, and alignment with the Five Domains Animal Welfare Assessment Model in order to inform communication frameworks.

碩士
國防醫學院
海底醫學研究所
96
Hyperbaric Oxygen Therapy (HBOT) is a medical treatment using oxygen administered at greater than normal atmospheric pressure. Hyperbaric oxygen (HBO2) has been applied to treat different clinical diseases, such as decompression sickness, carbon monoxide poisoning, air embolism, chronic wounds, chronic refractory osteomyelitis, and so on. Our previous study was to treat Jurkat cell line and HCI-H929 cell line at 3.6 ATA HBO2 ,3.6 ATA HBA and incubator, respectively , for 6 hours. The results demonstrated significant apoptosis in HBO2 group by increased oxidative stress.After HBO2 treatment，the phosporylation of p38 MAPK was increased and phosporylation of ERK1/2 was decreased. Furthermore, caspase-3 was activated leading to the cleavage of PARP and apoptosis. Therefore, the study was to investigate the effect of HBO2 on human-like leukemia animal model. First, we injected 1~2.5x105 human leukemia cells into NOD/SCID mice through mice tail vein, and established human-like leukemia condition. One week after injection,the animals in HBO2 group were expored to 2.5ATA HBO2 for 2 hours everyday till death. The results showed that three weeks after injection, human CD 45 percentage in peripheral blood was decreased in HBO2 group . We sacrificed the mice and analyzed the mice’s spleenocytes.Compared with control group, HBO2 group demonstrated significant low spleen weight and spleen lymphoblast number. The oxidative stress and annexinV was higher in HBO2 group than those in control group . Loss of mitochondria membrance potential(△Ψm) was noted in HBO2 group. After HBO2treatment, Bcl-2 protein expression decrease. In summary, our results demenstrated that HBO2 treatment could increase intracellular oxidative stress leading to apoptosis. Therefore, HBO2 treatment decreased lymphoblasts in mouse spleen. Decreased expression of Bcl-2 protein might be one of the mechanisms involved in this process.

Malaria disease poses a serious global health burden as recent reports have indicated that nearly half of the world’s population is at risk (WHO 2008). The World Health Organization (WHO) Expert Consultative Team has reported that 90% of all malaria deaths occur in Sub Saharan Africa. (WHO, 2008). Despite the numerous global efforts to control and manage the disease, through various ways, use of chemotherapeutic agents continues to be the major intervention strategy in the control of malaria. The WHO recommended use of Artermisinin combination therapy (ACT) has been hampered by an imbalance between demand and supply in the poor socioeconomically challenged rural populations of Sub Saharan Africa, the epicenter of malaria infection. Chloroquine (CHQ), therefore, continues to be used in most malaria endemic areas in developing countries despite development of P. falciparum resistance to the drug (WHO, 2006). Oral administration is the major delivery route for CHQ. However, CHQ is a bitter drug, with an inconvenient dosing schedule leading to incomplete courses of therapy by most malaria patients. Oral CHQ administration is also associated with adverse effects in various organ systems resulting from deposition of CHQ in these organs to elicit impairment of glucose homeostasis, renal and cardiovascular function. Alternative methods of CHQ administration such as transdermal delivery have, therefore, been suggested, in an effort not only to avoid the bitter taste, but also to modify the dosing schedule, which may improve patient comfort and compliance. Transdermal delivery of CHQ via an amidated matrix patch, which is envisaged to ensure a slow, controlled and sustained release of therapeutic concentrations of CHQ, may circumvent the previously reported adverse effects of oral CHQ. It is against this background that the current study compared the effects of transdermal CHQ patch and oral chloroquine in the management of malaria as assessed by the ability to clear parasites of P. berghei infected rats. The other aims were to investigate and distinguish between the patho physiological effects of malaria and CHQ treatments on blood glucose and plasma insulin concentration, renal and cardiovascular function in male Sprague-Dawley rats. To investigate and distinguish between the pathophysiological effects malaria infection and CHQ treatments on blood glucose homeostasis, renal and cardiovascular function markers, separate groups of non infected and P. berghei infected male Sprague Dawley rats (90g-150g) were used. The animals were treated twice daily with oral CHQ (60mg/kg) and a once off transdermal delivery of CHQ via topical application of pectin CHQ matrix patch (53mg/kg) in a 21 day study divided into pre treatment, (days 0-7) treatment (days 8-12) and post treatment (days 13-21) periods. The animals were housed individually in metabolic cages for the duration of the study. Treatment was for 5 consecutive days. Measurements of body weight, food and water intake, mean arterial pressure (MAP), blood glucose concentration, % parasitaemia, haematocrit, and 24 hour urine volume, Na+, K+, urea and creatinine outputs were done every day during the treatment period, and every third day during the pre and post treatment periods. Separate groups of non fasted conscious animals (n=6) were sacrificed on days 0, 7, 8, 9, 10, 12, 14 and 21, at 24 hours after the last treatment for oral CHQ administration and after a once off patch application on the first day of treatment. The plasma obtained was assayed for plasma insulin, lipid profile parameters and plasma Na+, K+, urea and creatinine. The harvested liver and gastrocnemius muscle were used for determination of glycogen concentration. The current study has demonstrated the sustained controlled release of CHQ from the pectin matrix patch, demonstrating the therapeutic ability to clear P. berghei malaria parasites from systemic circulation. Malaria infection and oral CHQ treatment exhibited blood glucose lowering effects which were circumvented by topical application of the pectin CHQ matrix patch. Oral CHQ elevated hepatic glycogen concentration through mechanisms that are still to be elucidated. Topical application of CHQ via pectin matrix patch did not alter hepatic and gastrocnemius muscle glycogen concentrations. Malaria infection and oral CHQ delivery reduced food intake, water intake and % body weight changes of the animals as well as inducing natriuresis, reduced urine output and increased urinary creatinine outputs. Malaria infection was also shown to elicit hyperkalaemia and kaliuresis in experimental animals. Hypotensive effects of malaria infection and oral CHQ delivery were also demonstrated in the current study. Malaria infection and oral CHQ delivery elevated plasma total cholesterol and LDL-c as well as reduction in the cardio protective particle, plasma HDL-c, concentrations. Topically delivered CHQ via pectin CHQ matrix patch did not evoke any such alterations, suggestive of its ability to circumvent the observed adverse effects of oral CHQ delivery due to sustained, controlled release of therapeutic concentrations of CHQ from the transdermal formulation. To the best of our knowledge, the results of the present study provides the first evidence of the release of therapeautic CHQ concentrations from pectin CHQ matrix patch that cleared the malaria parasites from systemic circulation as well as demonstrating the ability of the transdermal formulation to circumvent the adverse effects of oral CHQ delivery in glucose homeostasis, renal and cardiovascular function markers. This is clinically relevant as it provides a feasible and novel alternative method of CHQ delivery that could play a major role in the effective management of malaria.
Thesis (Ph.D.)-University of KwaZulu-Natal, Westville, 2011.

This dissertation of limited scope, part of a Course-work Master’s in Ancient Languages and Cultures, consists of five chapters which deal with issues relating to the perception and literary treatment of non-human animals in African and Classical traditional stories involving animal characters. The focus of the research was placed upon arguing that: human characteristics were attributed to animal creatures in the myths/traditional stories from both cultures; both cultures made attempts to explain how certain animals became domesticated and how others remained wild; mythical thinking is not a preserve of one culture, it is rather part of human nature; mythical monsters are present in both cultures and that they have always to be destroyed by man, though not easily; myths served several functions for both cultures, ranging from educational entertainment to socializing purposes, to making attempts to explain ancient man’s environment and its happenings. The study was undertaken in the hope of enabling certain recommendations to be formulated, on the basis of the findings, to effect a better and more informed strategy for teaching Classical Mythology and Classics, in general, in the Mawian/African context.
Classics & World Languages
M. A. (Specialisation in Ancient languages and culture)

Studies the growth, body composition, feed intake of grazing animals, the use of anabolic agents, fatty acid composition of ruminants fats; and, the effect of saturated fat on human health.
Thesis (M.Ag.Sc.) -- University of Adelaide, Dept. of Animal Sciences, 1989

Esta investigação visa compreender a relação entre variáveis alusivas à relação humano- animal de companhia e o bem-estar subjetivo humano. Para isso, o estudo seguiu uma metodologia quantitativa, no qual participaram 660 indivíduos que foram avaliados quanto à satisfação com as relações que têm com os seus animais de companhia, quanto ao seu bem-estar subjetivo e quanto à confiança que têm nos seus animais de companhia. Resultados mostram que os indivíduos sentem mais bem-estar quando estão satisfeitas com a relação com o seu animal de companhia, e uma das variáveis que explica isto é a confiança que sentem nos seus animais. Tal descoberta proporciona pistas acerca do papel de variáveis psicológicas como a confiança no animal de companhia, com repercussões ao nível do bem-estar do dono e possivelmente com implicações práticas por exemplo, nas intenções de abandono. Há ainda bastante caminho para desbravar neste campo de investigação, tendo em conta que a relação humano-animal de companhia aparenta ser uma fonte de ligações únicas e improváveis.
This investigation aims to understand the link between human-pet relationship parameters and human subjective well-being. In this regard, the study followed a quantitative method, in which 660 individuals participate and were evaluated in satisfaction with relationship with pets, subjective well-being and trust in pets. Results showed that individuals who are more satisfied with the relationship feel higher levels of well-being, and one of the explanatory mechanisms is the trust in pets. This achievements gives clues about the role of psychological variables such as trust in pets, with consequences for the owner well-being and possibly with practical implications, for instance, in relinquishment intentions. There still is a lot of work to do in this field, as the human-pet relationship seems to be a source of unique and unlikely connections.

Fung, Suk Chun.
Thesis (Ed.D.)--Chinese University of Hong Kong, 2011.
Includes bibliographical references (leaves 328-337).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstract also in Chinese.

碩士
臺北醫學大學
牙醫學系碩博士班
100
Background: Biopsies have contributed to our knowledge of the causes and remedies of human diseases in the biological sciences. However, few studies have directly compared dental treatments in humans and animals, especially in the field of periodontal regeneration. Besides, exploring treatment effects of guided tissue regeneration in periodontal infrabony lesions from histology and re-entry aspects by systematic review and meta-analysis are still lack. Objective: 　　The main objective of this systematic review was to compare the effects of treating periodontal infrabony lesions using guided tissue regeneration (GTR) alone or GTR + bone grafts of animal and human studies, as well as explore the GTR efficacy of animals and humans respectively from histological and re-entry aspects. Material and methods: Publications in English were searched using PubMed database combined with hand searching from January 1, 1960 to May 1, 2010. The search included any type of barrier membrane with or without grafting materials for the treatment of periodontal infrabony lesions. All studies with histological or re-entry methodology outcome parameters evaluating bone filling and/or new cementum formation ratio from defect depths were adopted. Follow-up periods of at least 4 weeks in animals and 6 months in humans were required for inclusion. Meta-analysis was used to evaluate bone fill ratio in animals and humans respectively, and new cementum formation ratio in animals. Only descriptive analysis was performed in human histological data. Results: Of 576 retrieved studies, 21(9 animal and 12 human studies) were selected for the review. The meta-analysis showed that the weighted average bone fill ratio was 52% (95% confidence interval, [CI]: 18%, 85%) in animals and 58% (CI: 29%, 86%) in humans from GTR + bone graft groups, which was not statistically significant (p = 0.790). Similar results were found in GTR groups where the weighted average bone fill ratio was 54% (CI: 37%, 72%) and 59% (CI: 42%, 77%) in animals and humans, respectively (p = 0.692). Bone fill ratios of human re-entry studies and new cementum formation ratios of animal studies showed GTR and GTR+ bone grafts had comparable outcomes, but both were superior to the control group. Most studies demonstrated periodontal regeneration following GTR or GTR + bone graft approach. Conclusion: Our analysis indicated (1) bone fill ratios in animals were comparable to those in humans in infrabony defects treated using GTR alone or with additional bone grafts. (2) regardless of bone fill ratio in human re-entry studies or new cementum formation ratio in animals, comparable outcomes of GTR and GTR + bone grafts were showed, but both were superior to control group.

Dados sobre a realidade portuguesa indicam que os níveis de saúde mental dos jovens têm vindo a diminuir. Internacionalmente, há estudos que demonstram o claro benefício da interação com os animais para o bem-estar, inclusivamente em públicos jovens. Em contexto nacional, não temos conhecimento de projetos desenhados especificamente para promover o bem-estar dos jovens, com uma abordagem de psicologia positiva, e em simultâneo desenvolver uma empatia para com os animais. Esta área da interação humano-animal está em franco crescimento, mas ainda existem lacunas e várias possibilidades no trabalho que pode ser feito. Foi realizado um diagnóstico de necessidades, com uma amostra de 105 jovens, entre os 12 e os 19 anos, o bem-estar psicológico, o mindfulness, a empatia para com os animais, a gratidão e a interação com animais foram avaliados. Os resultados obtidos são consistentes com a literatura, confirmando a importância da gratidão no bem-estar psicológico e empatia para com os animais; assim como a relevância da interação direta com animais na empatia para com estes, e de forma marginal no bem-estar psicológico. Todavia, e em oposição à literatura, o mindfulness que não encontrou suporte estatístico, pelo que estes resultados devem ser analisados com cautela. Posteriormente, foi apresentada uma proposta de um projeto denominado “CÃOPARSA - Empatizar & Bem-Estar”, que visa promover o bem-estar psicológico e aumentar a empatia para com os animais, de um grupo de jovens; numa abordagem de psicologia positiva. No final, as principais conclusões, limitações e direções futuras nesta área são discutidas.
Data on the Portuguese reality suggests that the mental health levels of the Portuguese youth have been decreasing. At the international level, some studies demonstrate the clear benefit of animal interaction for well-being, including in young audiences. Nationally, we are not aware of projects specifically designed to promote young people's well-being, with a positive psychology approach, and at the same time develop empathy towards animals. This area of human-animal interaction is growing rapidly, but there are still gaps in the work that can be done, so it is pertinent to conduct a needs assessment study. A needs assessment study was carried out with a sample of 105 young people, aged 12-19 years, where levels of psychological well-being, mindfulness, empathy towards animals, gratitude and interaction with animals were evaluated. The results obtained are consistent with the literature confirming the importance of gratitude in psychological well-being and empathy towards animals; as well as the relevance of direct interaction with animals on empathy towards animals, and marginally on psychological well-being. However, contrary to the literature in this domain, the relationship with mindfulness did not find statistical support, so these results should be analysed with caution. Subsequently, a proposal was presented for a project called “CÃOPARSA - Empathize & Well-being”, which aims to promote psychological well-being and increase empathy towards animals in a group of young people; based on a positive psychology approach. The main conclusions, limitations and future directions of this work are discussed given its contributions to this field of research.

This dissertation employs a case study research design and reviews literature from the field of animal assisted therapy (AAT) with an emphasis on equine facilitated psychotherapy (EFP), as a possible therapeutic alternative for treating inappropriate aggressive behaviour in a young boy. The research suggests that Horse Whispering can indeed be regarded as a viable therapeutic technique when intent on breaking the aggressive behaviour cycle.
Educational Studies
M.Ed.

The utilization of in vitro tests with a tiered testing strategy for detection of mild ocular irritants can reduce the use of animals for testing, provide mechanistic data on toxic effects, and reduce the uncertainty associated with dose selection for clinical trials. The first section of this thesis describes how in vitro methods can be used to improve the prediction of the toxicity of chemicals and ophthalmic products. The proper utilization of in vitro methods can accurately predict toxic threshold levels and reduce animal use in product development. Sections two, three and four describe the development of new sensitive in vitro methods for predicting ocular toxicity. Maintaining the barrier function of the cornea is critical for the prevention of the penetration of infections microorganisms and irritating chemicals into the eye. Chapter 2 describes the development of a method for assessing the effects of chemicals on tight junctions using a human corneal epithelial and canine kidney epithelial cell line. In Chapter 3 a method that uses a primary organ culture for assessing single instillation and multiple instillation toxic effects is described. The ScanTox system was shown to be an ideal system to monitor the toxic effects over time as multiple readings can be taken of treated bovine lenses using the nondestructive method of assessing for the lens optical quality. Confirmations of toxic effects were made with the utilization of the viability dye alamarBlue. Chapter 4 describes the development of sensitive in vitro assays for detecting ocular toxicity by measuring the effects of chemicals on the mitochondrial integrity of bovine cornea, bovine lens epithelium and corneal epithelial cells, using fluorescent dyes. The goal of this research was to develop an in vitro test battery that can be used to accurately predict the ocular toxicity of new chemicals and ophthalmic formulations. By comparing the toxicity seen in vivo animals and humans with the toxicity response in these new in vitro methods, it was demonstrated that these in vitro methods can be utilized in a tiered testing strategy in the development of new chemicals and ophthalmic formulations.

Service provision in veterinary oncology in Ontario was examined using a mixed methods approach. First, an interview-based qualitative study explored the service expectations of oncology clients at a tertiary referral centre. Next, a survey-based quantitative study established an understanding of oncology service in primary care practice and investigated the treatment recommendations of practitioners for dogs diagnosed with cancer. The first study, which involved 30 individual and dyadic interviews, identified “uncertainty” (attributable to the unpredictable nature of cancer and its treatment) as an overarching psychological feature of clients’ experience. Consequently, “the communication of information” (both content and process) was the foremost service expectation. For clients, it enabled confidence in the service, the ability to make informed patient care decisions, and preparedness for the potential outcomes of those decisions; it also contributed to creating a humanistic environment, which enhanced client resiliency. Findings suggest that services can support client efforts to manage uncertainty through strategic design and delivery of service, and incorporate intentional communication strategies to support clients’ psychological fortitude in managing the cancer journey. The second study, a vignette-based survey of primary care practitioners across Ontario (N=1071) which investigated veterinarian decision-making in relation to oncology care, determined that 56% of practitioners recommended referral as their first choice of intervention, while 28% recommended palliative care, 13% in-clinic treatment, and 3% euthanasia. Recommendations were associated with patient, client and veterinarian factors. Specifically, referral and treatment were recommended for younger dogs, healthier dogs, and dogs with lymphoma versus osteosarcoma; for strongly bonded clients, and financially secure clients; and by veterinarians who graduated from a North American college, had experience with treating cancer, felt confident in the referral centre, and believed treatment was worthwhile, with variation in relation to practitioner gender and the type of medicine practiced. The human-animal bond appeared to be the primary factor associated with practitioners’ advocacy for quality of medical care for patients. Through a blend of qualitative and quantitative methodologies, this thesis contributes to the evidence upon which best practices may be built so as to enhance the quality of patient and client care in veterinary oncology.
Ontario Veterinary College Pet Trust Fund 049406 and 049854

AQ4N (banoxantrone) is a prodrug that, under hypoxic conditions, is enzymatically converted to a cytotoxic DNA-binding agent, AQ4. Incorporation of AQ4N into conventional chemoradiation protocols therefore targets both oxygenated and hypoxic regions of tumors, and potentially will increase the effectiveness of therapy. This current pharmacodynamic and efficacy study was designed to quantify tumor exposure to AQ4 following treatment with AQ4N, and to relate exposure to outcome of treatment. A single dose of 60 mg/kg AQ4N enhanced the response of RT112 (bladder) and Calu-6 (lung) xenografts to treatment with cisplatin and radiation therapy. AQ4N was also given to separate cohorts of tumor-bearing mice 24 hours before tumor excision for subsequent analysis of metabolite levels. AQ4 was detected by high performance liquid chromatography/mass spectrometry in all treated samples of RT112 and Calu-6 tumors at mean concentrations of 0.23 and 1.07 microg/g, respectively. These concentrations are comparable with those shown to be cytotoxic in vitro. AQ4-related nuclear fluorescence was observed in all treated tumors by confocal microscopy, which correlated with the high performance liquid chromatography/mass spectrometry data. The presence of the hypoxic marker Glut-1 was shown by immunohistochemistry in both Calu-6 tumors and RT112 tumors, and colocalization of AQ4 fluorescence and Glut-1 staining strongly suggested that AQ4N was activated in these putatively hypoxic areas. This is the first demonstration that AQ4N will increase the efficacy of chemoradiotherapy in preclinical models; the intratumoral levels of AQ4 found in this study are comparable with tumor AQ4 levels found in a recent phase I clinical study, which suggests that these levels could be potentially therapeutic.

Text in English
Equine-Assisted Psychotherapy (EAP) is a form of Animal Assisted Therapy (AAT) used to treat individuals’ psychological problems. EAP is an interactive process in which a licensed mental health practitioner, a credentialed equine professional and suitable equines work together to address psychotherapy goals. Since the 1990s, research on EAP has grown rapidly in the United States of America (USA) and Europe, however research in the South African context is lacking. This descriptive and exploratory research study explored and described the experiences of licensed mental health practitioners and/or credentialed equine specialists who have included EAP within their practise by focusing on the role that equines play within the psychotherapeutic process. The knowledge of this study was derived from a Constructivistic epistemology. Constructivism argues that humans attain and build knowledge and meaning from their experiences. Participants were selected based on purposeful criterion sampling. Only licensed mental health practitioners or credentialed equine specialists who included EAP within their practise, registered at the Equine-Assisted Psychotherapy Institute of South Africa (EAPISA) or the Equine-Assisted Growth and Learning Association (EAGALA) were considered for participation. Semi-structured, face-to-face interviews were conducted with six participants. A post-interview follow-up email was used to gain additional feedback from each participant. Finally, data was analysed using thematic analysis. As EAP is a fairly new modality of therapy within South Africa, the contribution of this study is to add to the growing body of professional EAP literature. Thematic analysis identified seven main themes: shifting dynamics in the therapeutic relationship, setting the scene for storytelling, the equine as an intermediary and therapeutic tool, what the equine sees is what you get, instantaneous but lasting results, variations and similarities in approach and activities, the emotive motives of EAP practitioners. These themes are discussed in relation to the research findings. Findings of this study showed substantial agreement in what the equine brings to the psychotherapeutic session: unique equine attributes, opportunities for metaphorical learning, and relational aspects. The participants demonstrated a consensus in the belief that EAP can be beneficial to a large spectrum of psychotherapeutic populations in South Africa. A recommendation for future research is for larger, international studies that explore the viewpoints of psychotherapists who practice equine therapies in other countries in order to expand the knowledge base and address the competency and standards discussion in the EAP field.
Psychology
M.A. (Psychology)