Photodynamic therapy (PDT) is a form of local cancer treatment in which cell death is caused by photochemical reactions involving an exogenous photosensitizer. The photosensitizer, which is preferentially retained in malignant tissues, is photoactivated and cell death results from the generation of reactive products -most likely excited molecular (singlet) oxygen. The development of in vivo PDT dosimetry would be greatly aided by the ability to directly measure the local concentration of this product by non-invasive means. In condensed media singlet oxygen will, with some small probability, undergo a radiative transition to the ground state with emission at 1270 nm. This infrared phosphorescence may provide a means for monitoring the production of singlet oxygen in vivo. Unfortunately the background infrared fluorescence observed from tissue may be many times the expected magnitude of the 1270 nm phosphorescence, even within the bandwidth encompassing the peak. The principal aim of this project was the design of a system optimized for the in vivo detection of the singlet oxygen emission. The system makes use of the most sensitive commercially available detector and uses phase sensitive detection to discriminate against infrared fluorescence. The system's performance matched theoretical expectations for the photosensitizer Photofrin II in aqueous and methanol solutions. However, a discrepancy in the observed and theoretical values was noted for aluminum chlorosulphonated phthalocyanine suggesting a deviation from simple first order kinetics. Singlet oxygen phosphorescence was not observed during PDT of cell suspensions or mouse tumours even though considerable cell death and tumour necrosis were observed. The most likely explanation of this failure is that, due to quenching by biomolecules, the lifetime of singlet oxygen in cells or tissue is much lower than in solution so that the probability of emission is reduced accordingly. Quantitative calibration of the system yielded a lower limit of approximately 0.1 us on the singlet oxygen lifetime in tissue. This suggests that singlet oxygen is generated in a protein environment. / Thesis / Master of Science (MS)

The objective of this study was to determine the photothermal and photochemical tissue response to carbon nanotube inclusion in laser therapy using experimental and computational methods. In this study, we specifically considered varying types and concentrations (0.01-1 mg/ml) of carbon nanotubes (CNTs), e.g., multi-walled carbon nanotubes (MWNTs), single-walled carbon nanotubes (SWNTs), and single-walled carbon nanohorns (SWNHs). In order to determine the photothermal effect of CNT inclusion, the thermal conductivity and optical properties of tissue representative phantoms with CNT inclusion were measured. Thermal conductivity of tissue phantoms containing CNTs was measured using the hot wire probe method. For identical CNT concentrations, phantoms containing MWNTs had the highest thermal conductivity. Optical properties (absorption and reduced scattering coefficients) of solutions and tissue phantoms containing carbon nanotubes were measured with spectrophotometry and determined by the inverse adding doubling (IAD) method. Inclusion of CNTs in phantoms increased light absorption with minimal effect on scattering and anisotropy. Light absorption of MWNTs was found to be higher than SWNTs and SWNHs. The photochemical response to laser irradiation (wavelength 1064 nm) of CNTs was measured with spin-trap electron paramagnetic resonance (EPR) spectroscopy. Only SWNHs appeared to produce significant levels of ROS production in response to laser excitation in the presence of NADH. We detected the predominant presence of trapped hydroxyl radical (•OH) with a trace of the trapped super oxide (O2•-) radical. These free radicals are highly reactive and could be utilized to cause targeted toxicity to cancer cells. The distribution of CNTs at the cellular level, in phantoms, and in kidney tumors was measured using transmission electron microscopy (TEM) imaging. Samples were imaged following various time periods (2-48h) of incubation and CNTs were observed inside the cell cytoplasm, nucleus, vacuole, and outside cells for the above mentioned time periods. CNTs in phantoms and tumor tissue were randomly and uniformly distributed in the entire volume. Computational model geometries were developed based on CNTs distribution in cells, tissue phantoms, and kidney tumor tissue. In the computational part of this research the temperature response to laser irradiation alone or with CNT inclusion was determined using Penne's bioheat equation which was solved by finite element methods. Experimentally measured thermal conductivity and absorption and reduced scattering coefficients were used as input parameters in Penne's bioheat equation. The accuracy of the model predicted temperature distribution was determined by comparing it to experimentally measured temperature in tissue phantoms and kidney tumors following CNT inclusion and laser therapy. The model determined temperature distribution was in close correspondence with the experimentally measured temperature. Our computational model can predict the effectiveness of laser cancer therapy by predicting the transient temperature distribution. / Ph. D.

Cancer is increasingly prevalent globally and influenced by factors such as obesity and smoking. Cancer itself and chemotherapies, like cisplatin, can affect our reproductive organs and increase the risk of involuntary childlessness. In the ovaries there are oocytes that are surrounded by cumulus cells (CC) and the CC provides the oocyte with nutrients like pyruvate, which is important for the oocytes ability to mature. If the oocyte does not receive sufficient amount of nutrients the ovulation may be compromised. The CC are not well studied, and few prior studies have been done specifically to observe the impact of chemotherapies on CC. Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1A) is important for the mitochondrial biogenesis in cells. The purpose of the project was to evaluate a method for RNA extraction in CC and observe the impact of cisplatin exposure on the PPARGC1A expression. CCs were aspirated from bovine ovaries and exposed to cisplatin during in vitro maturation. RNA was eluted using the QIAcube and then quality assured. The primer β2 microglobulin (B2M) was used as an endogenous control in the qPCR. The results showed that PPARGC1A was minimally expressed in bovine CC and was inadequate for use when evaluating if cisplatin changes the RNA expression in CC. However, the QIAcube proved to be a suitable method for RNA extraction from bovine CC and B2M showed to be suitable as an endogenous control in bovine CC.

Thesis (D. Tech.) -- Central University of Technology, Free State, 2009 / Superficial cancerous lesions are commonly treated through low energy X-ray or electron radiation in radiotherapy. The treatment units that produce the radiation are equipped with square, rectangular and round applicators of different sizes. These applicators attach to the treatment units and define the radiation field size applied during treatment. An applicator is chosen to fit the shape of the cancerous lesion on the patient as closely as possible. Since cancerous lesions are irregular in shape, there will always be an area of healthy tissue between the edge of the lesion and the edge of the standard field shape. This healthy tissue will be irradiated along with the lesion during treatment which is undesirable since the cancer wound heals through reparative growth of the surrounding healthy tissue after treatment. Traditional techniques that were developed to shield this healthy tissue and thus shape the radiation field to the shape of the lesion present various shortcomings. This study introduces a new thermal-spray process for producing radiation field shaping shields which overcomes most of the shortcomings encountered with the traditional field shaping techniques. Since none of the commercially available thermal-spray equipment could be used to produce field shaping shields, new thermal-spray equipment was designed and fabricated tailor made to the application. Different techniques to determine the contours of the treatment area on the patient were investigated. These included a patient contact technique using a plaster bandage impression and a non-contact technique using 3D laser scanning. From the plaster bandage impression a plaster model can be produced onto which a high density low melt material such as Wood’ s alloy can be thermally sprayed to produce a field shaping mask. A model can also be produced from the 3D laser scanning data through laser sintering (LS) in nylon polyamide powder or through computer numerical controlled (CNC) milling in a block of low density polyurethane. The thermal-spray technique was evaluated by comparing the field shaping ability of radiation shields produced through the technique to the field shaping ability of shields produced through the traditional techniques. Radiographic film was used for this purpose and the results are presented in the form of isodensity charts. The required thicknesses of thermal-sprayed field shaping masks to shield radiation of various energies were also determined. The thicknesses were determined through radiation transmission measurements of known thicknesses of sprayed sheets of Wood’ s alloy. X-ray imaging showed that there were no defects present within thermal-sprayed layers of Wood’ s alloy that may negatively affect the shielding ability of masks produced through the technique.

The motivation of this research is to propose innovative nonlinear and optimal control design algorithms, which can be used in real life. The algorithms need to be computationally efficient, should deal with control constraints and should operate under state feedback. To show the efficacy of algorithms, automatic therapy for different cancer problems is chosen to be the field of application. In this thesis, first an advanced control design technique called ’optimal dynamic in-version’ has been successfully experimented with control constraints. The proposed approach has subsequently been shown to be quite effective in proposing automatic drug delivery schemes with simultaneous application of chemo and immunotherapy drugs for complete elimination of cancer cells in melanoma (a skin cancer) as well as glioma (a brain cancer). As per the current practice, the amount of drug dosages are generally given based on some apriori statistical study with a very small sample size, which in reality may either also lead to drug toxicity (due to excessive drug) or may become ineffective (due to insufficient drug) for a particular patient. Subject to the fidelity of the mathematical model (which has been taken from published literature), it has been shown in this thesis that nonlinear control theory can be used for computation of drug dosages, which can then be used in a feedback strategy, thereby customizing the drug for the patient’s condition, to cure the disease successfully. Next, attention has been shifted to impulsive control of systems. Such impulsive con-trol systems appear in many other applications such as control of swings, control of spacecrafts and rockets using reaction control system, radiotherapy in cancer treatment and so on. Two impulsive control design philosophies are proposed in this thesis. In one approach, recently proposed model predictive static programming (MPSP) has been extended for impulsive control systems and has been named as impulsive-MPSP (I-MPSP). In other approach, another recent development, namely the Pseudospectral method has been utilized to consider both the magnitude of the control impulses as well as the time instants at which they are applied as the decision variables. It can be noted, that to the best of the knowledge of the author, the time instants of control application, being considered as decision variables is being proposed for the first time in the nonlinear and optimal control framework. Both I-MPSP and Pseudospectral methods are computationally quite efficient and hence can be used for feedback control (I-MPSP happens to be computationally more efficient than the Pseudospectral method). Applicability of the proposed extensions have been shown by solving various benchmark problems such as (i) a scalar linear problem, (ii) Van der Pol’s oscillator problem and (iii) an inverted pendulum problem. Finally the applicability of the proposed I-MPSP strategy has been shown by solving challenging problems such as radiotherapy treatment of head and neck and adenocarcimona cancers. Radio-therapy model is considered with oxygen effect, in which radiosensitivity parameters are considered in different forms. Head and neck cancer is considered with constant radiosensitivity parameters and adenocarcinoma is considered with constant, linear, quadratic and saturation model of radiosensitivity parameters. Note that toxicity constraints on normal tissue, which are nonlinear control constraints, are also successfully incorporated in this control design.

Thesis (D. Tech. (Biomedical Technology)) -- Central University of Technology, Free State, 2014 / Medicinal plants are part of indigenous people‟s cultural heritage, thus since ancient times treatment of various diseases using medicinal plants has been part of human culture. The value of medicinal plants to mankind has been very well proven. It is estimated that 70% to 80% of people worldwide rely mainly on traditional health care systems, especially on herbal medicines (Stanley and Luz, 2003). In many societies the medicinal properties of plants were discovered mostly through trial and error, but use was also influenced by the belief systems of the people involved and often became entangled with religious and mythical practices (Mathias et al., 1996). Besides that, medicinal plants are proving to be rich resources of constituents that can be used in drug development and synthesis. Medicinal plants have been a source of a wide variety of biologically active compounds for many centuries and have been used extensively as crude material or as pure compounds for treating various disease conditions. Between 1% and 10% of plants out of an estimated 250 000 to 500 000 species of plants on earth are used by humans (Boris, 1996). 2 Plants used for medicinal purposes contribute significantly to the development of major medical drugs that are used today. Most common medicines have compounds extracted from plants as their primary active ingredients and many have provided blueprints for synthetic or partially synthesized drugs (Simpson and Ogorzaly, 2001). There has been a major resurgence of interest in traditionally used medicinal plants, with a number of international and local initiatives actively exploring the botanical resources of southern Africa with the intention to screen indigenous plants for pharmacologically active compounds (Gurib-Fakim et al., 2010; Rybicki et al., 2012). South Africa is considered a “hot spot” for biodiversity and more than 22 000 plant species occur within its boundaries. This represents 10% of the world‟s species, although the land surface of South Africa is less than 1% of the earth‟s surface (Coetzee et al., 1999). Plants have also been used by man for various purposes, among others as arrow and dart poisons for hunting, poisons for murder, hallucinogens used for ritualistic purposes, stimulants for endurance and hunger suppression, as well as medicine (Duke et al., 2008; Cragg and Newman, 2005). A derivative of the polyhydroxy diterpenoid ingenol isolated from the sap of Euphorbia peplus (known as “petty spurge” in England or “radium weed” in Australia), which is a potential chemotherapeutic agent for skin cancer, is currently under clinical development by Peplin Biotech for the topical treatment of certain skin cancers (Kedei et al., 2004; Ogbourne et al., 2004). Combretastatin A-4 phosphate, 3 a stilbene derivative from the South African bush willow, Combretum caffrum, acts as an anti-angiogenic agent causing vascular shutdowns in tumors (Newman et al., 2005; Holwell et al., 2002). Further reliance on plants for drug development is demonstrated by the use of galantamine hydrobromide, an alkaloid obtained from the plant Galanthus nivalis used traditionally in Turkey and Bulgaria for the treatment of Alzheimer‟s disease (Howes et al., 2003; Heinrich and Teoh, 2004). The plant chemicals used for the above-mentioned purposes are secondary metabolites, which are derived biosynthetically from plant primary metabolites (e.g. carbohydrates, amino acids and lipids). Secondary metabolites are organic compounds that are exclusively produced by plants and that are not directly involved in the normal growth, development and reproduction of a plant (Firn and Jones, 2003). Yet, they have many functions that are important for the plant‟s long-term health and appearance. Plants, being stationary, have to cope with a number of challenges, including engineering their own pollination and seed dispersal, local variation in the supply of the simple nutrients that they require to synthesize their food and the coexistence of herbivores and pathogens in their immediate environment. Plants have therefore evolved secondary biochemical pathways that allow them to synthesize a spectrum of organic molecules, often in response to specific environmental stimuli, such as herbivore-induced damage, pathogen attacks, or nutrient deprivation (Reymond et al., 2000; Hermsmeier et al., 2001). 4 The biosynthesis of secondary metabolites is derived from the fundamental processes of photosynthesis, glycolysis and the Krebs cycle to afford biosynthetic intermediates which, ultimately, result in the formation of secondary metabolites also known as natural products (Dewick, 2002). It is hypothesized that secondary metabolism utilizes amino acids and the acetate and shikimate pathways to produce “shunt metabolites” (intermediates) that have adopted an alternate biosynthetic route, leading to the biosynthesis of secondary metabolites (Sarker et al., 2006). Modifications in the biosynthetic pathways that produce secondary metabolites are probably due to natural causes (e.g. viruses or environmental changes) or unnatural causes (e.g. chemical or radiation processes) in an effort to adapt or provide longevity for the plant (Sarker et al., 2006). Plants‟ secondary metabolites can be classified into several groups according to their chemical classes, such alkaloids, terpenoids and phenolics (Harbone, 1984; Wink, 2003).

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.

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 (MBA)--Stellenbosch University, 2008. / ENGLISH ABSTRACT: The cost of medical care, in particular the cost of cancer care, has seen significant increases globally in the last few years. These cost increases in part are a result of tremendous advancements in new health technologies to diagnose, treat and care for cancer sufferers. The development of these highly specialised treatment modalities is not expected to slow down in the next few years, as potentially new treatments are already in the pipeline. On the other hand, cancer is becoming more prevalent. affecting more people worldwide. The condition remains life threatening, causing patients to become dependent and desperately hopeful of their requested treatments. Managed care, which includes the processes of rationing, has been implemented by medical aid schemes in the private health care industry in an effort to curtail the escalating costs of health care. Currently medical aids in the country are under immense pressure to comply with financially demanding legislation as well as to increase their membership risk by keeping contributions low and subsequently improve access to private health care in the country. Notwithstanding the fact that rationing might be justified from an economic perspective, the implications of transposing free market principles into an almost sacred health care environment challenges current morals and ethics in this arena. The price consciousness in cancer care is almost creating a scenario where clinical reasons are becoming subservient to fiscal reasons or, put differently, it is placing a price tag on human lives. In its true glory, the rationale of rationing is to challenge the individual patient needs against that of the bigger medical aid society. The distributive justice principles of rationing are creating immense conflict between the virtue-based, principle-based and contemporary ethics, which are currently governing medical practice in the country. As a result rationing creates serious vexing funding decisions with long-ranging effects. Its against this background that the study further consider the implications of managed care and rationing as it creates serious questions about the fairness, decision-making power and authority of managed care organizations. The implication of this is that the treating physician seems to have lost all autonomy and control in trying to treat and care for his cancer patient. Hence the perception that managed care does not act in the best interest of the vulnerable and desperate cancer suffering patient. As a result of th is view of managed care it becomes important to ensure the fairness and or legitimacy of managed care and rationing decisions. Therefore, the final section of the study considers the fair and just rationing of medical care as well as setting limits that are morally and ethically acceptable, in a cancer related setting. The studies of Daniels and Sabin are utilized extensively in particular the suggested criteria required by managed care organisations to ensure their rationing decisions are fair and legitimate. The implications of this and the assurances to cancer sufferers in a medical scheme is that the decisions to fund new health technologies are based on a process that is transparent and collaborative and that cost consideration of treatment has merit if it is made within the confines of this process. / AFRIKAANSE OPSOMMING: Die koste van mediese sorg, en spesifiek die koste van kankersorg, het in die afgelope paar jaar wereldwyd aansienlik toegeneem. Hierdie toename in koste is gedeeltelik die resultaat van geweldige vooruitgang in nuwe gesondheidstegnologiee om kankerlyers te diagnoseer, te behandel en vir hulle te sorgo Daar word nie verwag dat die ontwikkeling van hierdie hoogs gespesialiseerde behandelingsmodaliteite oor die volgende paar jaar sal afneem nie, aangesien nuwe behandelings steeds geregistreer word. Aan die ander kant is die voorkomssyfer van kanker besig om toe te neem, en be"invloed dit mense oor die hele wereld. Die toestand is steeds lewensbedreigend, en veroorsaak dat pasiente afhanklik van en desperaat vol hoop is vir die nodige behandeling. Bestuurde sorg, wat die proses van rantsoenering insluit, is deur mediesefondsskemas in die privaat gesondheidsorgbedryf ge"lmplementeer in 'n poging om die stygende koste van mediese sorg te verminder. Mediese fondse in die land is tans onder geweldige druk om aan finansieel veeleisende wetgewing te voldoen en om hulle lidmaatskaprisiko te verhoog deur bydraes laag te hou en gevolglik toegang tot privaat gesondheidsorg in die land te verbeter. Ondanks die feit dat rantsoenering moontlik vanuit 'n ekonomiese perspektief geregverdig kan word, daag die implikasies van die omsetting van vryemarkbeginsels in 'n amper heilige gesondheidsorgomgewing huidige morele waardes en etiek in hierdie veld uit. Die prysbewustheid in kankersorg skep amper 'n scenario waar kliniese redes ondergeskik aan fiskale redes gestel word of, om dit anders te stel, dit plaas 'n prys op mense se lewens. In sy volle glorie is die rasionaal van rantsoenering om die individuele pasient se behoeftes teenoor die van die groter mediesefondssamelewing te stel. Die beginsels van verdelende regverdigheid van rantsoenering skep enorme konflik tussen die deug..gebaseerde, beginselgebaseerde en kontemporere etiek wat tans die mediese praktyk in die land beheer. Gevolglik skep rantsoenering ernstige, moeilike befondsingsbesluite met effekte oor die lang termyn. Oit is teen hierdie agtergrond dat die studie die verdere implikasies van bestuurde sorg en rantsoenering moet oorweeg, aangesien dit ernstige vrae rondom die billikheid , besluitneming en outoriteit van bestuurde sorg maatskappye lig. By implikasie beteken dit dat die geneesheer wat die pasient behandel, feitlik aile beheer verloor het om die pasient vir aile praktiese doeleindes optimaal te behandel. Oaarom die persepsie dat bestuurde sorg nie in die beste belang van die kwesbare en desperaat kanker pasiente is nie. As gevolg van die persepsie van bestuurde sorg, raak dit meer belangrik om die bilikheid en regverdigheid van gesondheid sorg besluite te verseker. Met dit in ag genome, oorweeg die finale deel van die studie die bilikheid en regverdigheid van mediese rantsoenering so-ook die set van perke wat eties en moreel aanvaarbaar is, in 'n kanker verwante agtergrond. Die werke van Daniels en Sabin word in aansienlike detail hersien in besonder hul voorgestelde kriteria wat vereis word deur bestuurde sorg organisasies om te verseker hul besluite ten opsigte van rantsoenering is redelik en regverdig. Die implikasies hiervan en die versekering tot kanker Iyers in 'n mediese skema is dat die besluite om nuwe gesondheidstegnologiee te befonds, is gebasseer op In deursigtige en samehorende proses en dat aile koste oorwegings vir behandeling meriete het, indien dit is gemaak is binne die raamwerk van hierdie proses.

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.