Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Context: There are serious deficiencies in the current tests and criteria available for the diagnosis of diabetes. A novel screening method for the earlier and more efficient detection of type 2 diabetes would be a significant clinical advance. Objective: The hexosamine biosynthetic pathway (HBP) usually acts as a fuel sensor and its activation leads to O-GlcNAcylation of target proteins in a glucose-responsive manner. O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) are responsible for O-GlcNAc addition and removal, respectively. As higher HBP flux is linked to insulin resistance/type 2 diabetes, we hypothesized that increased O-GlcNAcylation of leukocyte proteins can detect the onset of pre- and overt diabetes. Materials and methods: 74 participants from Bellville and Stellenbosch (Western Cape, South Africa) were recruited and characterized as normal, pre-diabetic or diabetic. Leukocytes (granulocytes and lymphocytes) isolated from study subjects were evaluated for O-GlcNAcylation, OGA and OGT expression by flow cytometry, immunofluorescence microscopy and Western blotting. Results: Leukocyte O-GlcNAcylation increased in both pre-diabetic and diabetic individuals, with leukocyte sub-population data showing the greatest sensitivity. OGA expression and O-GlcNAc/OGA ratios elevated in parallel with increasing glucose concentrations. OGT expression did not significantly change for any of the study subjects investigated. Conclusions: The initial and significant increases in leukocyte O-GlcNAcylation demonstrate great potential for the earlier detection of pre-diabetic and diabetic individuals. OGA expression and O-GlcNAc/OGA ratios may also have diagnostic value. Together our data show strong promise for eventual diagnostic utility and the more efficient detection of type 2 diabetes. / AFRIKAANSE OPSOMMING: Die konteks: Daar is ernstige tekortkominge in die huidige toetsing en kriteria vir die diagnose van diabetes. ʼn Nuwe metode vir die vroeë en meer effektiewe opsporing van tipe 2 diabetes sal beduidende kliniese voordeel inhou. Doelstelling: Onder normale omstandighede tree die heksosamienbiosintetiese pad (HBP) as energie sensor op, en die aktivering daarvan gee aanleiding tot O-GlcNAsetilering van proteïene in ʼn glukose-afhanglike wyse. O-GlcNAs transferase (OGT) en O-GlcNAse (OGA) is onderskeidelik verantwoordelik vir O-GlcNAs toevoeging en verwydering. Aangesien hoër HBP fluks verband hou met insulienweerstandigheid /tipe 2 diabetes, stel ons ʼn hipotese voor dat opsporing van verhoogde O-GlcNAsilasie van leukosietproteïene, die aanvang van pre-diabetes en diabetes kan voorspel. Materiale en metodes: 74 vrywillige deelnemers van Bellville en Stellenbosch (Wes Kaap Provinsie, Suid Afrika) is gewerf en gekarakteriseer as normaal, pre-diabeties of diabeties. Leukosiete (granulosiete en limfosiete), uit bloed van deelnemers geïsoleer, is vir O-GlcNAsilasie, OGA en OGT uitdrukking deur vloeisitometrie, immunofluoressensie-mikroskopie en Western blotting, ondersoek. Resultate: Leukosiet O-GlcNAsetilering is verhoog in beide pre-diabetiese en diabetiese individue, met leukosiet sub-populasie wat die mees sensitiewe data gelewer het. OGA uitdrukking en O-GlcNAs/OGA verhoudings in parallel verhoog tot ʼn toename in glukose konsentrasies. OGT uitdrukking het nie betekenisvol verander in enige van die individue wat ondersoek is nie. Gevolgtrekkings: Die vroeë en betekenisvolle toename in leukosiet O-GlcNAsetilering toon groot potensiaal vir die vroeë opsporing van pre-diabetiese en diabetiese individue. OGA uitdrukking en O-GlcNAs/OGA verhoudings het ook moontlik diagnostiese waarde. Ons data toon belowende resultate vir die gevolglike diagnostiese waarde en ʼn meer effektiewe opsporing van tipe 2 diabetes.

Thesis (MSc)--Stellenbosch University, 2012. / Background: While Selection of reverse transcriptase (RT) mutation has been reported frequently, protease (PR) mutations on antiretroviral therapy (ART) including boosted Protease inhibitor (PI) have not been reported as much in Zambia. Affordable in-house genotyping assays can been used to expand the number of patients receiving drug resistance geno-typing, which can aid in determining prevalence of RT/PI emerging mutations. Methods: A previously published drug resistance genotyping assay was modified and used to genotype RT and PR genes. 19 patients virologically failing first-line regimen and 24 failing second-line regimen were studied to determine resistance patterns. Virological failure was defined as failing to maintain <1000 copies/mL during ART. Only major and minor RT and PR mutations (IAS-USA 2010) were considered for analysis. The in-house assay was validated by comparing sequence data of 7 previously ViroSeq tested samples and 5 randomly selected samples to determine reproducibility. Results: The in-house assay efficiently amplified all 12 validation samples with the lowest sample scoring 99.4% sequence homology. The most common RT mutation was M184V (79% n=19) and (71% n=24) first and second-line respectively. No significant differences were reported in all the other RT mutations between first-line and secondline regimens. Drug resistant PI mutations (I54V, M46I and V82A all present 20.8%) were only found in the second-line regimen and were insignificant, p= 0.0562. Conclusion: The in-house assays can be used as alternatives for commercial kits to genotype HIV-1C in Zambia without compromising test quality. The insignificant PI drug resistant mutations which were found, despite virological failure in patients, could indicate a possibility of other mutations within the HIV-1 genome that could reduce PI susceptibility.

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, 2014. / ENGLISH ABSTRACT: The aim of this study was to investigate the powerful muscle regulator, myostatin, and its regulators in response to an acute bout of plyometric training. The participants were recruited and screened by characterization by means of isometric force production tests, baseline blood creatine kinase levels and VO2 max results. The selected individuals (n=15) were subjected to a baseline muscle biopsy for comparative purposes. The study made use of plyometric jumping, as source of eccentric exercise, to serve as an exercise intervention after which muscle biopsies (4 hours post and 24 hours post) and blood draw (4 hours post, 24 hours post and 48 hours post) samples were taken. Maximal voluntary isometric contractions of the knee extensors were also measured immediately after the exercise protocol and after 1 week recovery. Creatine kinase (CK) analysis on the serum samples was used to conclude muscle damage. The muscle biopsy samples were used for protein quantification (Western blot) and gene expression assessment (semi-quantitative and real-time PCR). The results showed decreased force production immediately after eccentric exercise (p < 0.05), while returning back to baseline values at 1 week post exercise and CK results showed a significant increases at 4 hours (p<0.05), 24 hours (p<0.001) and 48 hours (p<0.01) after exercise. There were no significant differences in myostatin precursor protein (43 kDa), phosphorylated Smad2,3, Smad7 or activin receptor IIb in response to eccentric exercise. However, the follistatin protein was increased at both 4 hours and 24 hours after exercise (p<0.01). RNA analysis of the extracellular matrix (ECM) protein, decorin, revealed the existence of the splice variants A1 and A2 in human skeletal muscle. The RT-PCR analysis (n=4) of these variants showed no significant difference when comparing pre- to post-exercise. The decorin core protein was also investigated by means of antibody probing and results revealed the need for ABC chondroitinase enzyme treatment before immunoblotting of human skeletal muscle samples. The results concerning knee extensor force reduction and circulating creatine kinase showed the effectiveness of plyometric jumping in producing skeletal muscle damage in the lower limbs of unfit individuals, unaccustomed to eccentric exercise. In conclusion, myostatin, and its associated signalling cascade, are not activated in early muscle regeneration, but follistatin is increased during this phase possibly aiding and initiating the muscle repair process. Future studies: Variants of decorin are expressed in human skeletal muscle, increasing the complexity that should be taken into account in studies concerning the regulation of decorin in a human model. Investigation into myostatin protein at different post-translational levels needs more clarification. Published methods and materials used in different laboratories are not consistent and investigators should attempt to standardise protocols in order to compare results between studies more effectively. Of importance, these results show that the myostatin at protein level report different results compared to mRNA analysis and that more investigation into myostatin regulatory factors, with special reference to follistatin and decorin, is needed in future human models. / AFRIKAANSE OPSOMMING: Die doel van hierdie studie was om die kragtige spiere reguleerder, miostatin, en sy reguleerders in reaksie op 'n akute aanval van pliometriese spronge te ondersoek. Die deelnemers is gewerf en gekeur deur karakterisering deur middel van isometriese krag produksie toetse, basislyn bloed kreatien kinase vlakke en VO2maks resultate. Die geselekteerde individue (N = 15) is onderhewig aan 'n basislyn spierbiopsie vir vergelykende doeleindes. Die studie het gebruik gemaak van pliometriese spronge (essentriese spier aksie) as die oefening intervensie waarna spierbiopsie (4 uur na en 24 uur na) en bloed (4 uur na, 24 uur na en 48 uur na) monsters geneem is. Isometriese kontraksies van die knieverlengers is ook gemeet onmiddellik na die oefening protokol en na 1 week se herstel. Kreatine kinase (KK) ontleding van die serum monsters is gebruik om spierskade aftelei. Die spierbiopsie monsters was gebruik vir proteïen kwantifisering (Western klad) en die assessering van geen uitdrukking (semi-kwantitatiewe en real-time PCR). Die resultate het gewys dat krag produksie afgeneem het onmiddellik na essentriese oefening (p <0.05), terwyl dit terugkeer na die oorspronklike waardes 1 week na oefening en KK resultate toon 'n beduidende toename by 4 uur (p <0,05), 24 uur (p <0,001) en 48 uur (p <0,01) na oefening. Daar was geen betekenisvolle verskille in Miostatien voorloper proteïen (43 kDa), gefosforileerde Smad2,3, Smad7 of Activin reseptoor IIb in reaksie op essentriese oefening. Dit is egter die follistatien proteïen wat verhoog by beide 4 uur en 24 uur na oefening (p <0,01). RNS ontleding van die ekstrasellulêre matriks (ESM) proteïen, decorin, het die bestaan van die splitsing variante A1 en A2 in menslike skeletspier, aan die lig gebring. Die RT-PCR analise (n = 4) van hierdie variante het geen betekenisvolle verskille getoon wanneer voor met na-oefening vergelyk is. Die decorin kern proteïen is ook ondersoek deur middel van teenliggaam afhanklike metodes en resultate het die behoefte aan ABC chondroitinase ensiem behandeling voor immunokladding van menslike skeletspier monsters gesteun. Die resultate aangaande knieverlenger krag vermindering en sirkuleerende kreatien kinase het die doeltreffendheid van pliometriese spronge in die vervaardiging van skeletspier skade in die onderste ledemate van individue ongewoond aan essentriese oefening verseker. Ten slotte, Miostatien, en sy verwante sein kaskade, is nie geaktiveer vroeg in spier herstelling, maar follistatien is tydens hierdie fase verhoog en help moontlik met die aanvang van die spier herstel. Toekomstige studies: variante van decorin word uitgedruk in menslike skeletspier, wat die kompleksiteit aangaande decorin verhoog en dit is iets wat in ag geneem moet word in studies wat handel oor die regulering van decorin in mens modelle. Ondersoek na miostatien proteïen op verskillende na-translasie vlakke moet meer duidelikheid verkry. Gepubliseer metodes en materiaal wat gebruik word in verskillende laboratoriums is nie konsekwent en ondersoekbeamptes moet probeer om protokolle te standaardiseer sodat resultate van studies meer effektief kan vergelyk word. Van belang is, die resultate wys dat miostatien op proteïen vlak verskillende resultate vertoon in vergelyking met boodskapper-RNS ontleding en dat meer ondersoek na miostatien regulerende faktore, met spesiale verwysing na follistatien en decorin, nodig is in toekomstige menslike modelle.

Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Introduction The discovery of Doxorubicin in the 1960s has drastically improved the survival rates of cancer patients, however, its success is limited by dose-dependent cardiotoxicity. While much of the literature has focused on acute cardiotoxicity which is minor and generally reversible, chronic cardiotoxicity poses a serious threat to cancer survivors since it can lead to dilative cardiomyopathy, congestive heart failure and even death. The mechanisms that contribute to cardiotoxicity are still a matter of controversy, however, oxidative stress-induced myocardial damage and apoptosis are thought to be the major role players. Reperfusion injury, also characterized by oxidative stress and apoptosis, occurs as a result of restoring blood flow to an ischemic heart. Fortunately, pre- and post-conditioning are techniques employed to minimize this damage and are thought to do so by activating the reperfusion injury salvage kinase (RISK) and survivor activating factor enhancement (SAFE) pathways. The RISK pathway involves the pro-survival kinases, Erk1/2 and Akt, while the SAFE pathway, triggered by TNF-α, involves Jak2 and STAT3. Since both reperfusion injury and Doxorubicin-induced cardiotoxicity share similar characteristics, this study aimed to determine whether the RISK and SAFE pathways are activated in response to long-term Doxorubicin treatment. Furthermore, this study aimed to determine whether TNF-α is produced during treatment, since its role in Doxorubicin-induced cardiotoxicity is still relatively unknown. Methods H9c2 cardiomyocytes and differentiated C2C12 myotubes were treated daily with increasing concentrations of Doxorubicin for a total of 120 hours. Cell viability, apoptosis and necrosis were assessed using the MTT, Caspase-Glo® 3/7 and lactate dehydrogenase assays respectively. TNF-α production was measured using Quantikine® ELISA kits and various assays were used to assess oxidative stress, anti-oxidant capacity and anti-oxidant status. The protein expression of the RISK and SAFE pathways were analysed by western blotting using both phospho-specific and total antibodies. Results and Discussion Treatment with Doxorubicin caused a time- and dose-dependent decrease in cell viability in both cell lines and this was accompanied by an increase in apoptosis. In the H9c2 cardiomyocytes, treatment with 0.2 μM Doxorubicin yielded significant levels of TNF-α after 120 hours and we can speculate that these low levels partially protected the cells from the toxic effects of Doxorubicin by activating the SAFE pathway, since both Jak2 and STAT3 were phosphorylated at this concentration. Treatment with 1 μM Doxorubicin caused a larger and biphasic pattern of TNF-α release, which may have then contributed to the decrease in cell viability, since the SAFE pathway was not activated at this concentration. Akt was phosphorylated during the first 72 hours of treatment with the low dose of Doxorubicin, but chronic treatment prevented this phosphorylation. While Erk1/2 was not phosphorylated at all at the low dose of Doxorubicin, neither Akt nor Erk1/2 was phosphorylated at the high dose and their inhibition may contribute to the cardiotoxic effects of Doxorubicin. In the C2C12 myotubes, a significant amount of TNF-α was produced after 120 hours of treatment with the low dose of Doxorubicin. Treatment with the high dose of Doxorubicin induced significant TNF-α production at every time point. While STAT3 was phosphorylated at the serine residue after treatment with the low dose of Doxorubicin, treatment with the high dose induced phosphorylation at the tyrosine residue in a time-dependent manner. p-Jak2 expression was significantly down-regulated at both concentrations of Doxorubicin, suggesting that STAT3 proteins can by-pass activation by Jak2. The Erk1/2 leg of the RISK pathway was also not activated for the majority of the treatment period, however, p-Akt expression was increased at the low concentration of Doxorubicin relative to total Akt expression. Conclusion These observations indicate that treatment with Doxorubicin causes a severe, dose-dependent loss in viability which is likely to mediated by high concentrations of TNF-α (induced by high concentrations of Doxorubicin) and down-regulation of protective signaling pathways. TNF-α may confer partial protection at low concentrations by activating the SAFE pathway. However, activation of the SAFE pathway could not provide sufficient protection from Doxorubicin, most probably because the RISK pathway was not simultaneously activated. Our results also clearly highlight the differences between acute and chronic treatment since a single high dose of Doxorubicin produced vastly different responses to cumulative treatment with a low dose. Before one can extrapolate these results into the clinical setting, further research is required to provide a better understanding of the RISK and SAFE pathways and whether stimulation thereof will provide a protective effect. In addition, although our study has shown that TNF-α is produced in response to Doxorubicin treatment, its true role, whether beneficial or detrimental, remains to be determined. / AFRIKAANSE OPSOMMING: Inleiding Die ontdekking van Doksorubisien (DOKS) in die 1960’s het die oorlewingsyfer van kankerpasiënte drasties verhoog, maar DOKS-gebruik gaan egter ook gepaard met dosis-afhanklike kardiotoksisiteit. Terwyl die literatuur grootliks fokus op akute kardiotoksisiteit, wat minimaal en algemeen omkeerbaar is, hou kroniese kardiotoksisiteit ‘n ernistige bedreiging vir kankeroorlewendes in, aangesien dit kan lei tot dilatiewe kardiomiopatie, kongestiewe hartversaking, en selfs dood. Die spesfikieke meganismes wat bydrae tot kardiotoksisiteit is tans steeds onbekend, maar oksidatiewe stres-geinduseerde miokardiale skade en apoptose word beskou as hoof bydraende faktore. Reperfussie skade, ook gekaraktiseer deur die teenwoordigheid van oksidatiewe stres en apoptose, kom voor as gevolg van die herstel van bloedtoevoer na ‘n isgemiese hart. Om die skade te minimaliseer word voor- en nakondisionerings tegnieke geïmplimenteer wat die RSHK (Reperfussie Skade Herwinnings Kinase) en OAFV (Oorlewerings Aktiverings Faktor Versterkings)-weë aktiveer. Die RSHK weg maak gebruik van pro-oorlewings kinases Erk1/2 en Akt, terwyl die TNF-α geaktiveerde OAFV weg Jak2 en STAT3 betrek. Aangesien beide reperfussie skade en DOKS-geinduseerde kardiotoksisiteit soortgelyke eienskappe deel, is die doel van hierdie studie om vas te stel of die RSHK en OAFV-weë geaktiveer word in langtermyn DOKS behandeling. Boonop is nog ‘n doel van hierdie studie om vas te stel of TNF-α geproduseer word tydens behandeling, aangesien die rol daarvan in DOKS-geinduseerde kardiotoksisiteit steeds onbekend is. Metodes H9c2 kardiomiosiet en gedifferensieerde C2C12 miobuise was daagliks behandel met toenemende konsentrasies van Dox vir 120 ure. Die effekte van DOKS op sel lewensvatbaarheid, apoptose en nekrose is onderskeidelik ondersoek deur middel van die MTT, Caspase-Glo® 3/7 en LDH toetse. TNF-α produksie is bepaal deur van die Quantikine® toets gebruik te maak, en verskeie metodes is gebuik om die oksidatiewe stres, anti-oksidantkapasiteit en anti-oksidantstatus te bepaal. Die proteïenuitdrukking van die RSHK (Erk1/2 en Akt) en OAFV (Jak2 en STAT3) weë was ontleed deur middel van westerse afklattingstegniek deur van beide fosfospesifieke en totale teenliggaampies gebruik te maak. Resultate en Bespreking Behandeling met DOKS het ‘n tyd en dosis-afhanklike afname in sel lewensvatbaarheid in beide sellyne veroorsaak, wat gepaard gegaan het met ‘n toename in apoptose. In die H9c2 kardiomiosiete, het ‘n lae DOKS dosisbehandeling (0.2 μM) betekenisvolle vlakke van TNF-α na 120 uur opgelewer en ons kan spekuleer dat hierdie lae vlakke gedeeltelik die selle van die toksiese effekte van DOKS deur die aktivering van die OAFV weg beskerm het omrede beide Jak2 en STAT3 by hierdie konsentrasie gefosforileer is. Die hoë DOKS dosis (1 μM) het ‘n groter en bifasiese patroon van TNF-α vrystelling vertoon, wat kon bydra tot die DOKS-geinduseerde afname in sel lewensvatbaarheid. Akt is gedurende die eerste 72 uur van behandeling gefosforileer met die lae DOKS dosis, maar kroniese behandeling het hierdie fosforilering verhoed. Terwyl Erk1/2 glad nie gefosforileer is by die lae DOKS dosis nie, is nie Akt of Erk1/2 by die hoë dosis gefosforileer nie, en kan hierdie inhibering bydrae tot die kardiotoksiese effekte van DOKS. In die C2C12 miobuise, is ‘n betekenisvolle hoeveelheid TNF-α na 120 uur van behandeling geproduseer by die lae DOKS dosis. Behandeling met die hoë DOKS dosis het betekenisvolle TNF-α produksie geinduseer by elke tydspunt. Terwyl STAT3 gefosforileer is by die serienresidu na behandeling met die lae DOKS dosis, het behandeling met die hoë dosis fosforilering by die tirosienresidu op ’n tydsafhanklike wyse plaasgevind. p-Jak2 uitdrukking was betekenisvol verminder by beide DOKS konsentrasies, wat aanduidend is dat die STAT3 proteïene nie geaktiveer hoef te word deur Jak2 nie. Die Erk1/2 been van die RSHK weg is ook nie geaktiveer gedurende die oorhoofse behandelingstydperk nie, alhoewel, p-Akt wel uitgedruk is by die lae konsentrasie van DOKS relatief tot die totale Akt uitdrukking. Gevolgtrekkings Die resultate van hierdie studie toon dat DOKS-behandeling tot ‘n dosis-afhanklike verlies in sel lewensvatbaarheid lei. Hierdie effek word waarskynlik bemiddel deur die teenwoordigheid van hoë konsentrasies TNF-α, en ook die afregulering van die beskermende seinweë. TNF-α kan moontlik gedeeltelike beskerming bied by lae konsentrasies deur aktivering van die OAFV weg. Die aktivering van die OAFV weg kon egter nie voldoende beskerming teen DOKS bied nie; moontlik as gevolg van die afwesigheid van die gelyktydige RSHK weg aktivering. Ons resultate vertoon die verskille tussen die akute en kronies behandeling aangesien ‘n enkele hoë-dosis van DOKS, in vergelyking met ‘n kumulatiewe lae-dosis, grootliks verskillende resultate opgelewer het. Voordat hierdie resultate klinies verder ondersoek kan word is verdere navorsing nodig om TNF-α en die RSHK en OAFV-weë beter te verstaan, en om vas te stel of stimulering van hierdie seinoordragpaaie ‘n beskermende effek teweeg sal bring.

Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Introduction Normal cerebral cortex development depends on extensive neuronal migration during embryogenesis, permitting the formation of accurate synaptic circuits and a highly ordered laminar neocortex. The motility of a migrating neuron is achieved by a dynamic microtubule cytoskeleton that alternates between states of stabilization/lengthening and destabilization/shortening. This dynamic instability of the microtubule cytoskeleton is controlled by numerous microtubule-stabilizing and -destabilising proteins that bind directly to microtubules. Autophagy (“self-eating”), a major bulk intracellular degradation system, involves the fusion of autophagosomes with lysosomes, followed by proteolysis and recycling of cellular constituents. Like neuronal migration, autophagy is a microtubule-dependent process. The dynamic microtubule network serves as a track for autophagosomes to be transported to the lysosomes. WDR47 is a protein that is expressed in the brain during development, but of which the function is largely unknown. Novel interactions have recently been identified between Reelin and WDR47 and between the microtubule-destabilising protein superior cervical ganglion 10 (SCG10) and WDR47. These findings suggest that WDR47 may be regulating microtubule-dependent processes such as neuronal migration and autophagy. We hypothesize that WDR47 may play a role in regulating neuronal migration and/or autophagy, and that this regulation may be mediated by a tubulin stability-regulating role of WDR47. Aims and Methods Our aims are to assess the cellular localization of WDR47 in GT1-7 cells and to determine whether WDR47 is able to influence neuronal migration, filopodia extension, surface adhesion, ultra-structure, autophagy, tubulin stability, and tau or SCG10 protein levels. GT1-7 neuronal cells were cultured under normal conditions and transfected with WDR47 siRNA for 24 hours, followed by western blot verification of the knock-down. A 36 hour neuronal in vitro cell migration assay was performed and images of the wound were captured every 6 hours; the migration distances and the wound areas for the different time points were measured and analysed. A 24 hour migration assay was performed, capturing images every hour, and the direction of migration was determined. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were performed to analyse neuronal surface morphology and ultra-structure. Western blot analysis of SCG10, acetylated α-tubulin, Tau, LC3, and Sequestosome 1/p62 (SQTM1) protein levels was performed. Super-resolution structured Illumination microscopy (SR-SIM) three-dimensional (3-D) imaging of WDR47-YFP transfected cells, confocal microscopy of LC3 and acetylated tubulin, co-localization analysis of WDR47 and acetylated tubulin, and fluorescence recovery after photo-bleaching (FRAP) analysis were performed. Results WDR47 siRNA treatment significantly reduced the average migration distance and the migration velocity, resulted in fewer filopodia-like extensions as well as perturbed surface adhesion of migrating neurons, and lead to an increased presence of endoplasmic reticulum (ER) structures as well as an expanded nuclear envelope. LC3-II protein levels were significantly lower with WDR47 siRNA treatment, but were significantly increased with WDR47 siRNA treatment in conjunction with Bafilomycin A1 treatment, indicating increased autophagic flux. SCG10 protein levels were significantly decreased with WDR47 siRNA treatment. SR-SIM and confocal microscopy of WDR47 siRNA treated cells revealed a robust presence of highly convoluted acetylated tubulin in the perinuclear region as well as decreased LC3 fluorescence signal. Confocal microscopy revealed co-localization of WDR47 with acetylated tubulin. - Discussion and Conclusion: The results suggest that WDR47 is involved in regulating neuronal migration, neuronal surface adhesion and filopodia formation, microtubule dynamics, and likely also autophagic flux. Taken together, we propose that WDR47 is regulating microtubule dynamics by facilitating assembly of microtubule-regulating proteins such as SCG10, thereby affecting microtubule-dependent processes such as neuronal migration and autophagy. / AFRIKAANSE OPSOMMING: Inleiding Normale serebrale korteks ontwikkeling is hoogs afhanklik van neuronale migrasie tydens embriogenese, en is belanrik vir die vorming van akkurate sinaptiese netwerke en 'n hoogs geordende laminêre neokorteks. Die vermoё van 'n neuron om te migreer berus op 'n hoogs dinamiese mikrotubulien sitoskelet wat verleng/stabiliseer of verkort/destabiliseer soos tubulien-eenhede begevoeg of verwyder word. Hierdie dinamiese onstabiliteit van die mikrotubulien sitoskelet word beheer deur verskeie mikrotubulien-stabiliserende en - destabiliserende proteïene wat direk bind aan mikrotubuliene. Autofagie ("self-eet"), 'n grootmaat intrasellulêre degradasie stelsel, behels die fussie van autofagosome met lisosome, gevolg deur proteolitiese afbraak van sellulêre organelle en proteine. Soos neuronale migrasie is autofagie 'n mikrotubulien-afhanklike proses. Die dinamiese mikrotubulien netwerk dien as 'n spoor vir die vervoer van autofagosome na lisosome. WDR47 is 'n proteïen wat voorkom in die brein tydens ontwikkeling, maar waarvan die funksie grootliks onbekend is. Interaksies was onlangs geïdentifiseer tussen beide Reelin en WDR47 en die mikrotubulien-destabiliserende proteïen SCG10 en WDR47. Hierdie bevindinge dui daarop aan dat WDR47 n rol speel in die regulering van tubulienstabiliteit en sodoende mikrotubulien-afhanklike prosesse. Ons veronderstel dat WDR47 'n rol kan speel in die regulering van neuronale migrasie en/of autofagie en dat hierdie regulasie moontlik afhanklik is van 'n tubulien-stabiliteit-regulerende rol van WDR47. - Doelwitte en Metodes: Ons doelwitte is om die sellulêre lokalisering van WDR47 in GT1-7 neurone te evallueer en om te bepaal of WDR47 n effek het op neuronale migrasie, oppervlak adhesie en filopodia formasie, ultra-struktuur, autofagie, tubulien-netwerke en -stabiliteit, en Tau of SCG10 proteïenvlakke. GT1-7 neuronale selle is gekweek onder normale omstandighede en vir 24 uur getransfekteer met WDR47 siRNA, gevolg deur verifikasie met Western-blot analise. 'n 36 uur neuronale in vitro sel migrasie toets is uitgevoer en fotos van die wond is elke 6 uur geneem. Die migrasie afstande en die wondareas vir die verskillende tydpunte is gemeet en ontleed. 'N 24-uur-migrasie toets is uitgevoer, 'n foto van die wond is elke uur geneem, en die rigting van migrasie is bepaal. Skandering elektronmikroskopie (SEM) en transmissieelektronmikroskopie (TEM) is uitgevoer om neuronale oppervlakmorfologie en ultrastruktuur te observeer. Western blot analise van SCG10, geasetieleerde α-tubulien, Tau, LC3 en Sequestosome 1/p62 (SQTM1) proteïenvlakke is uitgevoer. Super-resolusie gestruktureerde verligting mikroskopie (SR-SIM) driedimensionele (3-D) beelding van WDR47-YFP getransfekteerde selle, konfokale mikroskopie vir visualisering van LC3 en tubulien, co-lokalisering analise van beide WDR47 en LC3 en WDR47 en tubulien, asook fluorescentie hersteling na foto-bleek (FRAP) analise is uitgevoer. Resultate Die gemiddelde migrasie-afstand en die migrasiesnelheid (μm/min) het beduidend afgeneem met WDR47 siRNA behandeling. SEM analise van WD47 siRNA-behandelde neurone het minder filopodia en veranderde oppervlak adhesie vertoon, en TEM analise het 'n verhoogde teenwoordigheid van endoplasmiese retikulum (ER) strukture, en 'n uitgebreide kernmembraan vertoon. LC3-II proteïenvlakke was beduidend laer met slegs WDR47 siRNA behandeling, maar beduidend hoёr met WDR47 siRNA behandeling in samewerking met Bafilomycin A1 behandeling. Hierdie resultate dui aan op toeneemende autofagie met WDR47 siRNA behandeling. Verder, beduidend laer vlakke van SCG10 proteïenvlakke is waargeneem met WDR47 siRNA behandeling. SR-SIM en konfokale mikroskopie van WDR47 siRNA behandelde selle het 'n robuuste teenwoordigheid van hoogs buigende geasetieleerdetubulien in die area rondom die nukleus, 'n afgeneemde LC3 Bespreking en Gevolgtrekking Die resultate dui daarop aan dat WDR47 betrokke is by die regulering van neuronale migrasie, filopodia vormasie, oppervlak adhesie, mikrotubuliendinamika, en waarskynlik ook autofagie. Ons stel voor dat WDR47 mikrotubuliendinamika afekteer deur die regulering van proteïene soos SCG10, en sodoende mikrotubulienafhanklike prosesse soos neuronale migrasie en autofagie fasiliteer.

Assignment (MPhil)--University of Stellenbosch, 2003. / ENGLISH ABSTRACT: The mechanisms of blood flow (BF) control to skeletal muscle during dynamic exercise are still not clearly understood. The paraplegic subject (P) has reduced sympathetic innervation to the lower limbs. The current study was designed to focus on the contribution of neural control, specifically the sympathetic nervous system (SNS), as part of the central vascular mechanism to skeletal muscle BF during dynamic exercise. Aims: We studied BF parameters in P vs. able-bodied subjects (AB) to determine whether the paraplegic can serve as a model for assessing the contribution of the SNS to changes in active vs. inactive muscle BF during exercise. Further questions addressed include: the influence of level of fitness on resting and exercise BF, how lesion level affects BF control in the paraplegic, the 'muscle pump' theory and its hypothesized role in exercise hyperemia and whether blood pooling occurs in the legs of paraplegics. Method: Noninvasive duplex Doppler studies of the large conduit arteries (brachial and common femoral) were performed on 10 elite paraplegic athletes (EP), 10 sedentary paraplegics (SP) en 10 sedentary able-bodied subjects (AB). The paraplegic groups were further subdivided by lesion level with T6 being the critical level. Tests were carried out at rest and after 2 bouts of arm ergometer exercise: a maximal incremental test and 3 minutes at 75% of maximal. Diameter, mean velocity, pulsatile index and blood flow were measured/calculated. Results: Resting heart rate was significantly higher in the paraplegic groups (EP = 80 bpm ± 10, SP = 83 bpm ± 12) vs. the AB group (69 bpm ± 7), p < 0.05. Resting diameter in the common femoral artery (CFA) was similar in EP (5.93 mm ± 1.54) and SP (6.52 mm ± 0.95), but significantly lower than in AB (7.87 mm ± 1.38), p < 0.05. Similar resting pulsatile index (PI) in the CFA were contrary to that previously reported, casting doubt on venous blood pooling theories. Post-exercise values need to be interpreted with caution in view of the large resting differences in CFA diameter. Percentage change values are therefore more appropriate. These differences were not statistically significant, but may suggest interesting trends. Large variability existed for most resting and post-exercise values. Conclusion: The paraplegic subject is an ideal model for the study of the influence of the SNS on blood supply to exercising skeletal muscle. The difference in CFA diameter at rest in the paraplegic vs. the AB group confirms previous results and is probably due to structural/non-physiological changes. Our observation that the BA and CFA diameters in EP and SP subjects do not differ significantly at rest, suggests that training does not have a spillover vasomotor effect on lower limb conduit arteries in paraplegia. Similar BF and PI values post-exercise in the SP and AB groups challenge the muscle pump theory. The SNS has an important role in the control of skeletal muscle blood flow - both at rest (vascular tone) and during exercise (redistribution). Suggestions for future research are made. / AFRIKAANSE OPSOMMING: Die meganismes betrokke by die beheer van bloedvloei (BV) gedurende dinamiese oefening is nog onduidelik. Die parapleeg (P) het verminderde simpatiese innervasie na die onderste ledemate. Die huidige studie fokus op die bydrae van die simpatiese senuwee sisteem (SSS), as deel van die sentrale vaskulêre meganisme, tot skeletale spier BV tydens dinamiese oefening. Doelstellings: Ons het BV parameters in P vs. nie-gestremde proefpersone (kontrole) bestudeer om vas te stelof die parapleeg as model gebruik kan word om die bydrae van die SSS tot veranderings in die BV in aktiewe- en onaktiewe spiere gedurende oefening, te ondersoek. Verdere aspekte wat ondersoek is, sluit in: die invloed van tiksheidvlak ten opsigte van rustende en oefenings BV, of die verlammingsvlak by die parapleeg BV kontrole beïnvloed, die 'spierpomp-teorie' en sy hipotetiese rol in oefeninghiperremie, asook die vraag of bloedsaamstorting in die bene van parapleë plaasvind. Metode: Nie-indringende duplex Doppler studies van die groot geleidingsarteries (bragiaal [BA] en gemene femoral [CFA]) is by 10 elite paraplegiese atlete (EP), 10 sedentêre parapleë (SP) en 10 sedentêre nie-gestremde proefpersone (AB) uitgevoer. Die paraplegiese proefpersone is verder onderverdeel deur die vlak van T6 as kritiese verlammingsvlak te gebruik. Toetse is tydens rus en na 2 arm-ergometer oefeningsessies uitgevoer: een maksimale inkrementeie toets en een van 75% van maksimum intensiteit. Deursnit, gemiddelde vloeispoed, pulsatiewe indeks en bloedvloei is gemeet en/of bereken. Resultate: Rustende hartspoed was beduidend hoër in die paraplegiese groepe (EP = 80 slaelminuut ± 10 en SP = 83 slm ± 12) vs. die AB groep (69 slm ± 7), p < 0.05. Rustende deursnit in die gemene femorale arterie (CFA) was dieselfde in EP (5.93 mm ± 1.54) en SP (6.52 mm ± 0.95), maar beduidend laer as in AB (7.87 mm ± 1.38), p < 0.05. Die feit dat rustende pulsatiewe indeks (PI) in die CFA dieselfde in albei groepe was, laat twyfelontstaan oor die veneuse bloedopdammings teorieë soos weergegee in die literatuur. Na-oefeningswaardes moet omsigtig evalueer word met inagneming van die groot rustende verskille in CFA deursnit. Persentasieverskilwaardes is dus meer toepaslik. Hierdie veskille was nie statisties beduidend nie, maar suggereer interessante tendense. Groot variasie het voorgekom vir beide rustende en na-oefenings waardes. Gevolgtrekking: Die parapleeg is 'n ideale model vir studies om die invloed van die SSS op bloedvloei aan aktiewe skeletale spier te bestudeer. Die verskil in rustende CFA deursnit in die parapleeg vs. die AB groep bevestig vorige resultate en is waarskynlik te wyte aan strukturele, nie-funksionele veranderinge. Ons bevindinge dat die BA en CFA deursneë nie beduidend verskil in die SP en EP groep gedurende rus nie, dui daarop dat gereëlde oefening nie 'n oorloop vasomotor effek op die onderste ledemate in die parapleeg het nie. Die feit dat daar geen verskil aangetoon kon word tussen BV en PI waardes na-oefening in die SP en AB groepe, betwis die spierpomp teorie. Die studie toon dat die SSS 'n belangrike rol in die beheer van skeletale spier bloedvloei speel - beide met rus (vaskulêre tonus) en gedurende oefening (herdistribusie). Voorstelle vir toekomstige navorsing word gemaak.

Dissertation (PhD)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: Stress related to chronic exercise affects both the immune and endocrine systems, but there are still many issues that are poorly understood, particularly effects of stress on the functional capacity of immune cells. This thesis probed some of these issues using physiological models of physical and psychological stress. Both exercise training stress and chronic psychological stress in human subjects were shown to result in an up-regulation of spontaneous reactivity of white blood cells in vitro, using two different assays, namely a) a peripheral blood mononuclear cell (PBMC) culture assay measuring immune cell responsiveness and b) a relatively new flow cytometry technique for assessing activation status of cells by their expression of the surface marker CD69, in a lymphocyte subpopulation-specific manner. An up-regulation of immune cell activation in the absence of an additional stressor was associated with a decreased capacity to mount a response to a subsequent mitogen stimulus in vitro after chronic psychological stress and acute, extreme exercise stress. Another novel finding was that cortisol high-responders to chronic psychological stress exhibited a higher spontaneous reactivity of both CD4+ and CD8+ lymphocytes when compared to cortisol low-responders. This result indicates that chronic exposure to cortisol may decrease its usual inhibitory effect on spontaneous T lymphocyte responsiveness. After optimisation of an animal model of mild, psychological stress, we demonstrated (using an IL-6 antibody) that IL-6 is necessary for a full-blown cortisol response to chronic, intermittent mild stress. Results also suggest that IL-6 plays a role in regulation of its own secretion by PBMCs in response to a stressor, by maintaining the production of IL-1β in the face of stress. Basal serum corticosterone concentration was shown to be the main determinant of the magnitude of mitogen-stimulated PBMC secretion of IL-6 in vitro in the stress-free controls. However, after blocking of IL-6 in vivo, IL-1β was identified as a major regulator of IL-6 secretion by mitogen-stimulated PBMCs in vitro, independently of the presence or absence of stress. The implications of these novel findings are that proinflammatory cytokines are sensitively regulated during mild stress.Mean serum cortisol concentration at rest was not a useful tool to assess chronic exercise stress after training intervention. However, classification of athletes at baseline into two groups according to their resting serum cortisol concentration illustrated two distinct patterns for the responses of both cortisol and the cortisol:testosterone ratio to chronic stress. These studies on the effects of chronic stress on parameters of the endocrine stress-axis and the immune system led to the following main conclusions: a) chronic exposure to cortisol results in a decreased inhibition of spontaneous immune cell activity at rest, b) this increased spontaneous activation of immune cells at rest in the absence of a stressor, is associated with a suppression of immune capacity to respond to a subsequent challenge, c) the latter finding is not evident under stress-free conditions where cortisol promoted immune cell IL-6 secretion, and d) IL- 1β and IL-6 are involved in the regulation of each others’ secretion. / AFRIKAANSE OPSOMMING: Chroniese oefening-verwante stres beïnvloed beide the immuun- en endokriene sisteme, maar daar is nog baie aspekte wat swak begryp word, veral m.b.t. die effekte van stres op die funksionele kapasiteit van immuunselle. Hierdie tesis het sommige van dié vraagpunte ondersoek deur gebruik te maak van fisiologiese en psigologiese stres. Beide oefening program-verwante stres en chroniese psigologiese stres in proefpersone het ‘n op-regulering van spontane witbloedselreaktiwiteit in vitro tot gevolg gehad, wat d.m.v twee verskillende metodes aangetoon is, naamlik a) ‘n perifere bloed mononukluêre selkultuur (PBMS-kultuur) bepaling van immuunsel reaktiwiteit en b) ‘n relatief nuwe vloeisitometriese tegniek vir die assessering van aktiveringsstatus van selle, deur hul uitdrukking van die oppervlakmerker CD69, op ‘n limfosiet subpopulasie-spesifieke wyse. ‘n Opregulering van immuunselaktiwiteit in die afwesigheid van ‘n addisionele stressor is geassosieer met ‘n verlaagde kapsiteit om te reageer op ‘n latere mitogeniese prikkel in vitro, na chroniese psigologiese stres en akute, erge oefeningstres. Nog ‘n nuwe bevinding was dat kortisol hoog-respondeerders, in reaksie op chroniese psigologiese stres, ‘n hoër spontane reaktiwiteit van beide CD4+- and CD8+-limfosiete toon in vergelyking met kortisol laagresopndeerders. Hierdie bevinding toon aan dat chroniese blootstelling aan kortisol die inhiberende effek daarvan op spontane reaktiwiteit van T-limfosiete verminder. Na optimalisering van ‘n rotmodel van gematigde, psigologiese stres, het ons gedemonstreer (deur gebruik te maak van ‘n IL-6 teenliggaam) dat IL-6 nodig is vir ‘n volledige kortisolreaksie op chroniese, onderbroke, gematigde stres. Die resultate dui daarop dat IL-6 ‘n rol in die regulering van sy eie sekresie deur PBMSe in reaksie tot ‘n stressor speel, deur die handhawing van produksie van IL-1β in die teenwoordigheid van stres. Basale serum kortisolkonsentrasie is as die belangrikste beslissende faktor in die omvang van mitogeengestimuleerde PBMS sekresie van IL-6 in vitro in die stresvrye kontroles aangedui. Na blokkering van IL-6 in vivo, is IL-1β egter as ‘n belangrike reguleerder van IL-6 sekresie deur mitogeen-gestimuleerde PBMSe in vitro geïdentifiseer, onafhanklik van die teenwoordigheid of afwesigheid van stres. Die implikasie van hierdie nuwe bevindinge is dat proinflammatoriese sitokiene tydens gematigde stres sensitief gereguleer word.Die gemiddelde serum kortisolkonsentrasie in ‘n rustende toestand was nie ‘n gepaste instrument om chroniese oefeningstres na ‘n oefenprogram-ingreep te assesseer nie. Na basislyn klassifikasie van atlete in twee groepe volgens hul rustende serum kortisolkonsentrasie, is twee afsonderlike patrone vir die reaksie van beide kortisol en die kortisol:testosteroon verhouding egter aangetoon. Hierdie studies rakende die effekte van chroniese stres op parameters van die endokriene stres-as en die immuunsisteem het tot die volgende vernaamste gevolgtrekkings gelei: a) chroniese blootstelling aan kortisol het ‘n verlaagde inhibisie van spontane immuunselaktiwiteit tydens rustende toestande tot gevolg, b) hierdie verhoogde spontane aktivering van immuunselle tydens ‘n rustende toestand word geassosieer met ‘n onderdrukking van immuunkapasiteit om te reageer op ‘n daaropvolgende prikkel, c) laasgenoemde bevinding is nie sigbaar tydens stresvrye toestande, wanneer kortisol IL-6 sekresie bevorder, nie en d) IL- 1β en IL-6 is betrokke by die regulering van mekaar se sekresie.

Digitized from microfiche to pdf. / Thesis (PhD(Agric) (Human and Animal Physiology))--University of Stellenbosch, 1998. / Please refer to full text.