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.

Metastasis accounts for more than 90% of cancer-related mortality, and thus, there is a compelling need for innovative therapeutic breakthroughs. TP53 mutations are detected in up to 80% of esophageal squamous cell carcinomas (ESCCs), the major subtype of esophageal cancer and one of the most lethal cancers worldwide, as well as in other SCCs. These mutations in turn correlate with poor patient prognosis and high metastatic rates. To elucidate novel mutant p53-dependent mechanisms in promoting ESCC metastasis, we generated a mouse model combining genetic and carcinogenic approaches: We treated the L2-Cre (esophageal specific promoter); LSL-Trp53R172H/-, Trp53-/- or Trp53+/+; Rosa26LSL-YFP mice with a carcinogen 4-NQO, and isolated primary and metastatic tumor cells that vary in their p53 statuses. We have shown that ESCC cells with Trp53R172H exhibit greater metastatic capabilities compared to the tumor cells harboring Trp53-/-, indicating gain-of-function (GOF) activity. Through comprehensive RNA-seq and cytokine array analyses, we identified that Colony-stimulating factor-1 (Csf-1) is significantly upregulated in a p53-R172H-dependent manner in metastatic lung lesions of ESCC. p53-R172H binds to the promoter region of Csf-1 locus in metastatic ESCC cells. Our findings demonstrate that p53-R172H-dependent Csf-1 signaling through its cognate receptor Csf-1r enhances tumor cell invasion and lung metastasis by utilizing complementary genetic and pharmacological approaches. This mechanism is mediated in part through Stat3 phosphorylation and epithelial-to-mesenchymal transition (EMT). These findings are further supported through in vivo targeting of Csf-1r. In addition, high levels of CSF-1 also correlate with mutant p53 in ESCC Tissue Microarrays (TMAs) and The Cancer Genome Atlas (TCGA) datasets. Our CUT&RUN-seq analysis on ESCC tumor cells revealed that both the Csf-1 locus and EMT-associated genes are enriched with histone 3 lysine 27 acetylation (H3K27ac). This enrichment creates a permissive environment for the interaction between Brd4 and p53-R172H, thereby regulating Csf-1 transcription. Notably, Brd4 interacts specifically with p53-R172H. Inhibiting Brd4 not only decreases tumor invasion and lung metastasis, but also reduces circulating Csf-1 levels in blood serum in vivo. Overall, our results establish a novel p53-R172H-dependent Brd4-Csf-1 signaling axis that facilitates lung metastasis in ESCC and underscores the GOF properties of p53-R172H. Our discoveries identify therapeutic vulnerabilities in metastatic ESCC, which can be applicable to other SCCs with similar transcriptomic and epigenetic profiles. These insights pave the way for developing therapeutic strategies for this difficult-to-treat disease.

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.

Tumors employ a variety of genetic resistance mechanisms to evade immune responses and immunotherapies such as PD-1 blockade. The pleiotropic cytokine interferon-gamma (IFNγ) is a potent immune effector and critical for patient response to PD-1 blockade, yet conventional systemic delivery is hindered by severe dose limiting toxicities. As such, the effects of exogenously introduced IFNγ either as monotherapy or in combination with PD-1 blockade in the context of different tumor genetic background remain poorly understood. Synthetic biology allows programming of microbes for tumor-specific delivery of therapeutic candidates that are otherwise not possible using conventional administration strategies. Herein, we engineered a strain of probiotic bacteria that home to tumors and locally release IFNγ. We validated the efficacy of our therapeutic strain, either as monotherapy or in combination with PD-1 blockade, in multiple murine tumor models. Within this dissertation, we demonstrate that a single intratumoral injection of these IFNγ-producing bacteria is sufficient to drive systemic tumor antigen–specific antitumor immunity, without observable toxicity. Although cancer cells employ various resistance mechanisms to evade immune responses, bacteria-derived IFNγ additionally overcomes primary resistance to PD-1 blockade via activation of cytotoxic CD4⁺Foxp3⁻ and CD8⁺ T cells. Moreover, by activating NK cells, bacteria-derived IFNγ also overcome acquired resistance mechanisms to PD-1 blockade, specifically loss of function mutations in IFNγ signaling and antigen presentation pathways. Collectively, this dissertation highlights the promise of combining IFNγ-producing bacteria with PD-1 blockade as a therapeutic strategy for overcoming immunotherapy-resistant, locally advanced, and metastatic disease.

Altered RAS signaling pathways drive uncontrolled cell proliferation, migration, and survival, ultimately leading to tumor development and progression Effective inhibition of RAS signaling holds the potential to benefit roughly a quarter of human cancer patients whose tumors are driven by RAS mutations. In pancreatic ductal adenocarcinoma (PDAC), the third leading cause of cancer related mortality in the US, over 90% of patient cases are driven by activating mutations in KRAS. Here we assessed two divergent approaches to target aberrant RAS signaling for PDAC treatment.In Part I we investigated the therapeutic potential of the RAS(ON) multi-selective inhibitor RMC-7977, a highly potent compound blocking the active GTP-bound forms of KRAS, HRAS, and NRAS, showing affinity for both mutant and wild type (WT) variants. We evaluated RMC-7977 in a comprehensive range of preclinical PDAC models and, following direct RAS inhibition, we observed broad and pronounced anti-tumor activity, at exposures that were well-tolerated in vivo. Pharmacological analyses revealed divergent responses to RMC-7977 in tumor versus normal tissues. Treated tumors exhibited waves of apoptosis along with sustained proliferative arrest whereas normal tissues underwent only transient decreases in proliferation, with no evidence of apoptosis. In the autochthonous KPC model, RMC-7977 treatment resulted in a profound extension of survival followed by on-treatment relapse. Analysis of relapsed tumors identified Myc copy number gain as a prevalent candidate resistance mechanism, which could be overcome by combinatorial TEAD inhibition in vitro. Together, these data established a strong preclinical rationale for the use of broad-spectrum RAS-GTP inhibition in the setting of PDAC and identified a promising candidate combination therapeutic regimen to overcome monotherapy resistance. In Part II, we investigated the therapeutic potential of combined MEK and autophagy pathways inhibition in preclinical models of PDAC. Consistent with previously published work, MEK inhibition increased the dependency of PDAC cells on autophagy, and the combination of Trametinib (a MEK inhibitor) and Hydroxychloroquine (an autophagy inhibitor) demonstrated synergistic anti-proliferative effects in murine PDAC cell lines. We added to this body of work by evaluating the combination in the clinically predictive genetically engineered mouse models (GEMMs). Trametinib/Hydroxychloroquine treatment led to significant tumor regressions, MAPK signaling inhibition, and reduced cell proliferation in the KPC mice, as well as improved survival in the KPF/FC model. Single-cell RNA sequencing analysis of treated KPC tumors revealed both treatment sensitive and resistant malignant cell populations, with the sensitive cells showing upregulated MAPK and autophagy pathway activities. Analysis of fibroblast population additionally revealed that inflammatory CAFs (iCAFs) are diminished in response to treatment, likely due to disrupted paracrine signaling mediated by IL1A secretion from malignant cells. Together, these data provided a preclinical rationale for the use of the Trametinib and Hydroxychloroquine combination in PDAC treatment. However, recent updates from ongoing clinical trials have reported limited clinical activity of this combination. The datasets generated here may help identify key discrepancies between preclinical models and clinical samples that contribute to the lack of translation, and guide the development of improved therapeutic combinations.

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.