A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfillment of the requirements for the Master of Science degree. 2 November 2017. / Screening methodologies have identified specific targets that could serve as potential therapeutic markers in cancer drug design, and the Retinoblastoma binding protein 6 (RBBP6) which is predominately expressed in lung and breast cancers is one critical protein identified. This study seeks to understand the 3D structure of RBBP6 domains, with emphasis on cancer. Three of these domains have been studied in this project, i.e. the Domain With No Name (DWNN), RING Finger, and the p53-binding domain. The ubiquitin-like structure of the DWNN has implicated this domain as a ubiquitin-like modifier of other proteins such as p53, whilst the RING Finger domain has intrinsic E3 Ligase activity like MDM2 the prototypical negative regulator of p53. The DWNN and RING Finger domains have resolved solution NMR structures, whilst the p53-binding domain has none. Thus, the first initiative undertaken was to model the RBBP6 p53-binding domain using I-TASSER and eThread-Modeller web-severs. Our results demonstrated that this domain mainly constitutes of alpha-helices and loop structures. Structural quality validations of both I-TASSER and eThread-Modeller models were further assessed using Swiss-Model and ProSA (Protein structure analysis) web-servers. Analyses were focussed on specific statistical parameters (Anolea, DFire, QMEAN, ProCheck and the ProSA Z-score). Results from these analyses show that the first I-TASSER model is the best possible representation of the RBBP6 p53-binding domain depicting minimal deviation from native state. Furthermore, screening and docking studies were performed using Schrödinger-Maestro v10.7: Glide SP and drug-like molecules that would potentially serve as agonist or antagonist of RBBP6 were identified. / MT 2018

A simple method to verify the total treatment time generated by the treatment planning system (TPS) when the CONTURA MLB or the SAVI applicator are used for APBI treatments has been developed. The method compares the time generated by the TPS to a predicted time, calculated by inserting parameters obtained from the TPS in equations generated in this Thesis. The equations were generated by linearly fitting data from clinical cases that had been treated using the Contura MLB or the SAVI applicator at the Lynn Cancer Institute of the Boca Raton Regional Hospital. The parameters used were the PTV coverage, Air Kerma Strength, Balloon Volume (Contura data fit) and Evaluation PTV (SAVI data fit). As an outcome of this research, it is recommended that the plan should be reevaluated when the percent difference between the generated and the predicted times exceeds 5% for the Contura MLB, or 10% for the SAVI. / by Andreas Kyriacou. / Thesis (M.S.)--Florida Atlantic University, 2011. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2011. Mode of access: World Wide Web.

It is imperative that the cancer research community has the means with which to effectively locate, access, manage, analyse and interpret the plethora of data values being generated by novel technologies. This thesis addresses this unmet requirement by using pancreatic cancer and breast cancer as prototype malignancies to develop a generic integrative transcriptomic model. The analytical workflow was initially applied to publicly available pancreatic cancer data from multiple experimental types. The transcriptomic landscape of comparative groups was examined both in isolation and relative to each other. The main observations included (i) a clear separation of profiles based on experimental type, (ii) identification of three subgroups within normal tissue samples resected adjacent to pancreatic cancer, each showing disruptions to biofunctions previously associated with pancreatic cancer (iii) and that cell lines and xenograft models are not representative of changes occurring during pancreatic tumourigenesis. Previous studies examined transcriptomic profiles across 306 biological and experimental samples, including breast cancer. The plethora of clinical and survival data readily available for breast cancer, compared to the paucity of publicly available pancreatic cancer data, allowed for expansion of the pipeline’s infrastructure to include functionalities for cross-platform and survival analysis. Application of this enhanced pipeline to multiple cohorts of triple negative and basal-like breast cancers identified differential risk groups within these breast cancer subtypes. All of the main experimental findings of this thesis are being integrated with the Pancreatic Expression Database and the Breast Cancer Campaign Tissue Bank bioinformatics portal, which enhances the sharing capacity of this information and ensures its exposure to a wider audience.

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in partial fulfilment of the requirements for the degree of Master of Science, 2017 / Aim: The aim of this study was to quantify random and systematic setup errors in a population of head and neck cancer patients for the purposes of evaluating departmental positioning and immobilization techniques, verification and treatment protocols, as well as validating the treatment margins used. Methods and Materials: All patients had more than one phase of radiation, each consisting of different megavoltage photon field arrangements. Some phases were also treated with electron fields in addition to the photon fields. Random and systematic setup errors in all three principal directions were calculated for two groups of patients, using record and verify system couch position data. For one group (20 patients) the positioning and immobilization device system was mechanically localized to the treatment couch, and for the other group (38 patients), it was visually centered on the treatment couch. Within both groups of patients, the patient position was either verified online with portal imaging or verified offline on a conventional radiotherapy simulator. Results: For the patient group treated with the base plate visually centered on the treatment table the population random and systematic setup errors calculated for the photon fields were only indicative of setup uncertainties in the anterior-posterior direction. For the patient group treated with the base plate localized to the treatment couch, the population random and systematic setup errors were found to be within the 5 mm clinical to planning target volume expansion margin used at Livingstone Hospital. Due to treatment couch position differences from fraction to fraction, setup errors made during this study could not reliably be determined for electron field treatments Conclusions: Results indicate that the base plate should be localized to the treatment couch when calculating random and systematic setup errors for photon fields using the couch position as a surrogate for patient position. For this method to be used to calculate setup errors for electron fields, shielding should always be fastened to the same position at the endface of the applicator. Offline and online verification did not significantly influence systematic setup errors. / XL2018

EDD (E3 isolated by differential display), located at chromosome 8q22.3, is the human homologue of the Drosophila melanogaster tumour suppressor gene 'hyperplastic discs'. Edd null mice and hyd mutants display embryonic lethality. EDD is also a multifunctional HECT family E3 ubiquitin protein-ligase, with reported roles in both progesterone action and the DNA damage response. To investigate the possible involvement of EDD in human cancer, several cancer types were analysed for allelic gain or loss (allelic imbalance, AI) at the EDD locus. AI of the EDD locus was most frequent in the serous subtype of ovarian cancer (16/22, 73%) and common in other cancers, including breast cancer (31%). AI is likely to represent amplification of the EDD gene locus rather than loss of heterozygosity, as quantitative RT-PCR and immunohistochemistry showed that EDD mRNA and protein are frequently overexpressed in breast and ovarian cancers. These data imply a potential role for EDD in cancer progression. However, depletion of EDD from cells in culture by RNA interference had very little effect on proliferation and cell survival. To identify EDD-regulated pathways, transcript analysis was performed on EDD-depleted cells. The results suggested that EDD modulates cell-cell communication and the actin cytoskeleton. Consistent with transcript analysis, depletion of EDD from two normal breast cell lines (HMEC-184 and MCF-10A) resulted in altered cell morphology, with decreased cell-cell contacts. This was concurrent with altered beta-catenin (an integral component of adherens junctions) at cell-cell contacts, which was also observed in the developing blood vessels of Edd null mice. Interestingly, total cellular beta-catenin levels were not affected. Furthermore, EDD depletion resulted in a decrease in expression of the cytoskeletal regulators twinfilin and R-RAS, with a simultaneous decrease in MAPK (ERK1 and ERK2) activity. Consistent with disruption of adherens junctions, EDD-depleted mammary acini lost tissue coordination and polarity. These data provide a significant advance in our knowledge of EDD, both in its role in regulating the organisation of cells into higher structures and its potential role in the development of cancer. This has relevance to an understanding of embryonic development and the role of tissue homeostasis in cancer progression.

Doctor of Health Science / It is well documented that for prostate cancer patients undergoing radiation therapy there is a correlation between target volume displacement and changes in bladder and rectal volumes. However, these studies have used a methodology that has captured only a subset of all treatment positions. This research used daily Computer Tomography (CT) imaging to comprehensively assess organ volumes, organ motion and their effect on dose, something that has never been performed previously, thus adding considerably to the understanding of the topic. Daily CT images were obtained using a Siemens Primus Linear Accelerator equipped with an in-room Somatom CT unit in the accelerator suite, marketed as ‘Primatom’, to accurately position the patient prior to treatment delivery. The internal structures of interest were contoured on the planning workstation by the investigator. The daily volume and location of the organs were derived from the computer to assess and analyse internal organ motion. The planned dose distribution was then imported onto the treatment CT datasets and used to compare the planned dose to i) the actual isocentre, where the isocentre was actually placed for that fraction, ii) the uncorrected isocentre, by un-doing any on-line corrections performed by the treatment staff prior to treatment delivery, and iii) the future isocentre, by placing the isocentre relative to internal organ motion on a daily basis. The results of this study did not confirm a statistically significant decrease in rectum volumes over time (hypothesis 1), however large fluctuations in bladder volume were confirmed (hypothesis 2). Internal organ motion for the rectum and bladder was demonstrated to be related to organ filling. Ideal planning volumes for these organs have been reported to minimise systematic and random uncertainty in the treatment volumes. An observed decrease in prostate volume over time, a systematic uncertainty in the location of the prostate at the time of the planning CT scan and a significant relationship between prostate centre of volume and rectum and bladder volumes has resulted in a recommendation that patients should be re-scanned during treatment to ensure appropriate clinical target volume coverage. A significant relationship between rectal and bladder volumes and the dose delivered to these organs was found (hypothesis 3). The dose delivered to the planning target volume was not related to the rectal or bladder volumes, although it was related to the motion of these organs. Despite these results only minimal effects on the dose delivered to any of the three isocentres occurred, indicating that the planned dose was accurately delivered using the methodology presented here (hypothesis 4). However the results do indicate that the patient preparation instructions need to be improved if margins are to be reduced in the future. It is unrealistic to assume that Image Guided Radiation Therapy will ever become routine practice due to infrastructure costs and time limitations. This research will inform radiation therapy centres of the variables associated with prostate cancer treatment on a daily basis, something that has never before been realistically achievable. As a result centres will be able to devise protocols to improve treatment outcomes.

Thesis (Ph. D.)--State University of New York at Binghamton, Department of Psychology, 2006. / Includes bibliographical references.

Curcumin is the active component of tumeric, and this polyphenolic compound has been extensively investigated as an anticancer drug that modulates multiple pathways and genes. We demonstrated that curcumin inhibited 253JB-V and KU7 bladder cancer cell growth, and this was accompanied by induction of apoptosis and decreased expression of the proapoptotic protein survivin and the angiogenic proteins vascular endothelial growth factor (VEGF) and VEGF receptor 1 (VEGFR1). Since expression of survivin, VEGF and VEGFR1 are dependent on specificity protein (Sp) transcription factors, we also investigated the effects of curcumin on downregulation of Sp protein expression as an underlying mechanism for the apoptotic and antiangiogenic activity of this compound. Curcumin decreases expression of Sp1, Sp3 and Sp4 in blader cancer cells indicating that the cancer chemotherapeutic activity of curcumin is due, in part, to decreased expression of Sp transcription factors and Sp-dependent genes. Betulinic acid (BA) and curcumin are phytochemical anticancer agents, and we hypothesized that both compounds decrease EGFR expression in bladder cancer through downregulation of specificity protein (Sp) transcription factors. BA and curcumin decreased expression of EGFR, Sp1, Sp3, Sp4 and Sp-dependent proteins in 253JB-V and KU7 cells; EGFR was also decreased in cells transfected with a cocktail (iSp) containing small inhibitory RNAs for Sp1, Sp3 and Sp4 showing that EGFR is an Sp-regulated gene. Methyl 2-cyano-3,11-dioxo-18?-olean-1,12- dien-30-oate (CDODA-Me) is a synthetic triterpenoid derived from glycyrrhetinic acid which inhibits proliferation of KU7 and 253JB-V bladder cancer cells. CDODA-Me also decreased expression of specificity protein-1 (Sp1), Sp3 and Sp4 transcription factors. Similar results were observed for a structurally-related triterpenoid, methyl 2-cyano-3,12-dioxooleana-1,9-dien-28-oate (CDDO-Me), which is currently in clinical trials for treatment of leukemia. Celastrol, a naturally occurring triterpenoid acid from an ivy-like vine exhibits anticancer activity against bladder cancer cells. Celastrol decreased cell proliferation, induced apoptosis and decreased expression of specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4 and several Sp-dependent genes like Fibroblast growth factor receptor 3 (FGFR3). In vivo studies using KU7 cells as xenografts showed that celastrol represents novel class of anticancer drugs that acts, in part, through targeting downregulation of Sp transcription factors.

Second only to lung cancer, breast is the leading type of cancer among women in the US. Despite all the medical advances over the past few decades, toxicity and increased resistance to standard drug therapy still remains a significant problem. The heterogeneic nature of all cancers has led to a shift in treatment approaches, in that more research is being carried out with combination treatments in the hope that a multidirectional targeting of cancer will be far more effective than the current single treatment options. Our goal was to gain a better understanding of the molecular mechanisms of a nonhydrolyzable ether analog of RRR-[alpha]-tocopherol, 2, 5, 7, 8-tetramethyl-2R-(4R, 8R, 12-trimethyltridecyl)chroman-6-yloxyacetic acid (abbreviated [alpha] -TEA), and to investigate its efficacy when used in combination with known chemotherapeutics 9-nitro-camptothecin (9NC), and Paclitaxel (Taxol). The data presented here looks encouraging as it shows a clinically relevant delivery method using [alpha]-TEA and 9NC has the unique ability to reduce primary tumor burden as well as macro and micrometastatic lung and lymph node lesions in an aggressive syngeneic mouse mammary model, while displaying no obvious toxic side effects. The effect of combination treatments on tumor volume appears in part to be moderated by an increase in tumor cell apoptosis and a decrease in tumor cellular proliferation. Next, the intricate molecular mechanism of how [alpha]-TEA alone and in combination with 9NC is able to induce apoptosis in 66cl-4-GFP murine mammary cancer was investigated. The data suggest that the signaling pathway that ultimately leads to apoptosis is caspase dependent, is able to upregulate pro-death players while at the same time downregulate pro-survival proteins such as c-Flip and survivin. Finally, we investigated the efficacy of [alpha]-TEA used in an allograft mouse model following treatment with Taxol. Combination treatments were able to significantly reduce primary tumor burden, decrease lung and lymph node micrometastases, tumor cell proliferation, tumor blood vessel density as well as increase tumor cell apoptosis. Based on the results presented, we propose that [alpha]-TEA when used alone and in combination is an effective, non-toxic option for cancer treatment which warrants further investigation. / text

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