Microbiome research in the past decade has revealed an astounding prevalence of bacteria in various tissues in the human body. Concurrent progress in synthetic biology has generated a converging interest in the genetic programming of bacteria to locally produce therapeutic payloads and supplant physiological niches. This dissertation presents the development of bioengineering tools that address several key challenges for the clinical translation of therapeutic bacteria. In particular, we focus on the engineering of bacteria for tumor and granuloma applications. Bacteria have been demonstrated to selectively grow within solid tumors, primarily due to the reduced immune surveillance in the necrotic and hypoxic cores. This natural tropism to tumors presents a unique opportunity to engineer bacteria as drug delivery vehicles for cancer therapy. While the recent advancement in microbial engineering has constructed ranges of therapeutic bacteria, a universal bottleneck for clinical development is the lack of tools to rapidly characterize therapeutic candidates in a complex physiological environment. To recapitulate bacterial tumor colonization in vitro, we developed a method that selectively grows bacteria within the necrotic core of tumor spheroids. This platform enabled high-throughput cocultures and predicted in vivo therapeutic outcomes, identifying potent anticancer proteins deliverable by tumor-homing Salmonella typhimurium. To ensure safety when using bacteria that produce cytotoxic payloads, we prevented bacterial spread to unintended locations by confining bacterial growth in a tumor-specific environment. We constructed hypoxia, pH, and lactate sensors and regulated bacterial growth based on sensor activation. To improve tumor specificity, we engineered gene circuits to sense hypoxia and lactate in an AND-logic gate manner. Leveraging the coculture platform, we characterized sensor activities and circuit functionalities in tumor spheroids. This engineered strain showed improved tumor specificity in an animal tumor model. Moving towards clinical applications, a key challenge is to ensure bacterial delivery to tumors without activating adverse immune responses. Approaches such as surface decoration can evade immune systems, but static modification may result in bacterial overgrowth. We developed a genetically-encoded microbial encapsulation system with a tunable, dynamic expression of capsular polysaccharides. We constructed an inducible gene circuit to regulate encapsulation, which exhibited tunable protection of the probiotic Escherichia coli Nissle 1917 (EcN) from host immune factors. By dynamically balancing low immunogenicity and protection, transient encapsulation increased the maximum tolerated dose of bacteria by approximately 10-fold when systemically injected in vivo. This strategy enhanced antitumor efficacy in multiple tumor models. Building on our work of therapeutic bacteria for cancer, we explored the use of engineered bacteria to infiltrate other pathogenic regions in the body. Specifically, we discovered that probiotic EcN colonizes granulomas, pathological features that develop at infection sites including tuberculosis. Granulomas share key similarities with solid tumors, including hypoxia and necrosis, and pose significant challenges for delivering therapeutic agents to eradicate the pathogen Mycobacterium tuberculosis within. We engineered the probiotics to locally produce antimicrobial proteins against Mycobacterium within granulomas. We developed a novel dual lysis mechanism to simultaneously enhance therapeutic protein release and limit bacterial overgrowth. To improve specificity, we constructed hypoxia-dependent bacterial growth coupled with quorum-mediated gene activation. Finally, we showed that our engineered probiotics reduced levels of Mycobacterium strains. Altogether, the presented technologies utilize a multiscale framework from circuit design to in vitro and in vivo models and advance bacteria as next-generation drug delivery vehicles capable of sensing and responding to diseases in the body.

Cancer is a generic term for a large group of diseases where cells of the body lose their normal mechanisms for growth so that they grow in an uncontrolled way. One of the most common treatments of cancer is chemotherapy that aims to kill abnormal proliferating cells; however normal cells and other organs of the patients are also adversely affected. In practice, it¿s often difficult to maintain optimum chemotherapy doses that can maximise the abnormal cell killing as well as reducing side effects. The most chemotherapy drugs used in cancer treatment are toxic agents and usually have narrow therapeutic indices, dose levels in which these drugs significantly kill the cancerous cells are close to the levels which sometime cause harmful toxic side effects. To make the chemotherapeutic treatment effective, optimum drug scheduling is required to balance between the beneficial and toxic side effects of the cancer drugs. Conventional clinical methods very often fail to find drug doses that balance between these two due to their inherent conflicting nature. In this investigation, mathematical models for cancer chemotherapy are used to predict the number of tumour cells and control the tumour growth during treatment. A feedback control method is used so as to maintain certain level of drug concentrations at the tumour sites. Multi-objective Genetic Algorithm (MOGA) is then employed to find suitable solutions where drug resistances and drug concentrations are incorporated with cancer cell killing and toxic effects as design objectives. Several constraints and specific goal values were set for different design objectives in the optimisation process and a wide range of acceptable solutions were obtained trading off among different conflicting objectives. Abstract v In order to develop a multi-objective optimal control model, this study used proportional, integral and derivative (PID) and I-PD (modified PID with Integrator used as series) controllers based on Martin¿s growth model for optimum drug concentration to treat cancer. To the best of our knowledge, this is the first PID/I-PD based optimal chemotherapy control model used to investigate the cancer treatment. It has been observed that some solutions can reduce the cancer cells up to nearly 100% with much lower side effects and drug resistance during the whole period of treatment. The proposed strategy has been extended for more drugs and more design constraints and objectives. / Libyan Ministry of Higher Education

With the increasing number of breast cancer survivors, there is a need to enhance health education to help survivors make informed decisions about maximizing their quality of life. YouTube is one of the most popular video applications that can be used for public health education. Nonetheless, there is little research on the content of health-related information that is uploaded to YouTube relevant to breast cancer survivors. This study was intended to help fill that gap in knowledge by describing the sources, formats, and content conveyed in the most widely viewed YouTube videos on breast cancer. YouTube was searched with a cleared browsing history using the key search term “breast cancer.” The resulting videos were sorted by view count. Videos were then screened for inclusion and exclusion criteria, yielding a sample of 100 videos with the most views. Video title, link, number of views, and date of upload were coded along with content included in each video. The inter- and intra-rater reliability was acceptable (Kappa’s = .79 and .97, respectively). The sample of 100 videos was collectively viewed 135,311,626 times, suggesting that the subject of breast cancer is a popular topic on YouTube. Nearly half of the sample videos (n = 45) were uploaded by television news/media agencies. Combined/multiple formats were the most popular format (n = 61), followed by still images/text (n = 48). General information on cancer was found to be the most common (n = 71), followed by screening for breast cancer occurrence/ recurrence (n = 62), and cancer treatments/breast cancer treatments (n = 45). Several of the content categories were rarely covered in the most-watched videos—for example, cancer rehabilitation recommendations, returning to work after cancer treatment, and financial burden/management of cancer. Thus, while topics such as breast cancer screening are widely covered, topics for breast cancer survivors regarding maximizing their quality of life are less widely covered. Few videos (n = 3) contained misinformation, but these videos were viewed millions of times, emphasizing the need for ongoing monitoring to identify and remove misinformation. The findings of this study indicated that YouTube videos on breast cancer gained over 135 million views. YouTube can be a great media channel for public health education. Nonetheless, there is significant need for more high-quality YouTube videos to be created to help breast cancer survivors navigate their cancer journey.

Chromatin regulation is a major aspect of cancer development, progression, and treatment. Several small molecule inhibitors of chromatin regulators are currently used for treatment of certain hematological malignancies. However, there is still opportunity for many more patients to benefit from therapeutic approaches that target chromatin regulation, especially in the context of solid tumors. A critical unmet need is the identification of robust biomarkers that can guide the application of epigenetic inhibitors in a precise and personalized manner. In my dissertation, I aim to address this important knowledge gap by studying how perturbation of chromatin can target metabolic and lineage abnormalities in solid tumors for therapeutic benefit. To do this, I have focused on genetic and pharmacological perturbations of chromatin pathways in two cancer models: (1) lung adenocarcinoma (LUAD) with NRF2 activation and (2) neuroendocrine esophageal carcinoma (NEC). In the study on NRF2-active LUAD, we found that histone deacetylase (HDAC) inhibitors can be repurposed to reprogram the epigenomic and metabolic landscape, which leads to specific and potent anti-tumor effects in the context of NRF2 activation. Specifically, we employed a chromatin-focused genetic screen to identify dependencies on chromatin regulators. The screen revealed an NRF2-specific dependency on class I histone deacetylases. Experiments in mouse and human LUAD cell lines in vitro and in vivo indicated an NRF2-specific sensitivity to the class I HDAC inhibitor Romidepsin. Mechanistically, profiling of histone acetylation and gene expression upon Romidepsin treatment revealed a relative loss of histone H4 acetylation at promoters which was associated with reduced gene expression. Many downregulated genes were more essential for the survival of NRF2 hyperactive cancer cells, including genes involved in glutamine and serine metabolism, c-Myc and several of its targets involved in purine and pyrimidine synthesis. These transcriptional changes had corresponding effects on altering the metabolic pathways that NRF2-active cells selectively require for survival. In the study on neuroendocrine esophageal carcinoma (NEC), we identified a crucial role for epigenetic regulation of lineage fate through transcriptional control of the key epidermal transcription factor p63. This project originated from data from my collaborators that indicates a role for p63 in the suppression of basal-to-neuroendocrine identity transition in the developing esophagus. Consistently, I found that p63 is silenced in NEC through a non-genetic mechanism. Reintroducing p63 isoforms in a human NEC cell line showed that ΔNp63α was sufficient to restore squamous marker expression. An epigenetic drug screen assessing p63 gene expression and subsequent validation experiments revealed that inhibition of EZH2, a histone methyltransferase, induced expression of ΔNp63α and genes related to the squamous identity. Analysis of the chromatin state in the TP63 locus showed that EZH2 inhibition led to a loss histone H3 methylation and a gain of histone H3 acetylation and its reader BRD4. These results support the hypothesis that the squamous identity can be reactivated epigenetically in NEC through de-repression of ΔNp63α as a potential therapeutic strategy. Together, these studies contribute to our understanding of the transcriptional response to chromatin perturbation and show that this can be leveraged to modulate cell metabolism and identity, as well as to achieve therapeutic benefit in new contexts of cancer.

Rapid developments in the field of CAR T cells offer important new opportunities while at the same time increasing numbers of patients pose major challenges. This review is summarizing on the one hand the state of the art in CAR T cell trials with a unique perspective on the role that Europe is playing. On the other hand, an overview of reproducible processing techniques is presented, from manual or semi-automated up to fully automated manufacturing of clinical-grade CAR T cells. Besides regulatory requirements, an outlook is given in the direction of digitally controlled automated manufacturing in order to lower cost and complexity and to address CAR T cell products for a greater number of patients and a variety of malignant diseases.

No description available.

Systemic chemotherapy remains the backbone of many cancer treatments. Due to its untargeted nature and the severe side effects it can cause, numerous nanomedicine approaches have been developed to overcome these issues. However, targeted delivery of therapeutics remains challenging. Engineering microrobots is increasingly receiving attention in this regard. Their functionalities, particularly their motility, allow microrobots to penetrate tissues and reach cancers more efficiently. Here, we highlight how different microrobots, ranging from tailor-made motile bacteria and tiny bubble-propelled microengines to hybrid spermbots, can be engineered to integrate sophisticated features optimised for precision-targeting of a wide range of cancers. Towards this, we highlight the importance of integrating clinicians, the public and cancer patients early on in the development of these novel technologies.

Thesis (M.Sc. (Chemistry)) -- University of Limpopo, 2021 / Breast cancer is a malignant tumour that starts in the cells of the breast. Many studies revealed aromatase (CYP19A1) and cyclin-dependent kinase 2 (CDK2) as possible therapeutic targets regarding breast cancer treatment, because they play crucial roles in anti-apoptotic processes during cell proliferation. Quinoxaline derivatives have attracted a great deal of attention due to their biological activities against fungi, virus, bacteria and cancer. Computer modelling was employed in order to reduce time and cost by searching the library of molecules and identifying those which are likely to bind to the drug target. A library of new one hundred (100) nitro and amino quinoxaline alkyne derivatives were successfully designed and screened against target proteins (CYP19A1 and CDK2) using virtual screening technique and thirteen (13) molecules were identified to be hit compounds against both targets with the docking score ranging from -6.143 to -8.372 kcal/mol as a measure of binding affinity. The hit compounds were subjected to IFD in order to identify tight binding through intermolecular interactions with active site residues of the binding pocket of the target proteins. All identified nitro and amino quinoxaline alkyne derivatives were successfully synthesised in a multi-step reaction sequence and their spectroscopic analysis (NMR, FTIR and MS) were in good agreement with the proposed structures in a good to moderate yield. The newly synthesised novel amino and nitro-quinoxaline derivatives were evaluated for anti-proliferative activity against breast cancer (MCF-7). Compound 59 showed to possess good inhibition against MCF-7 with an IC50 of 9.102 μM, whereas compounds 34, 54, 56 and 61 showed promising activity against MCF-7 with an IC50 value of < 50 μM. However, the MTT assay results showed that 59 was found to be toxic with an IC50 value of 0.205 μM against Raw 264.7 cell line. The dose response investigations showed that 31 and 34 have the promising anti-cancer activity against CYP19A and the correlation between molecular modelling (in-silico) and CYP19A inhibition activities (in- vitro), was established as compounds 31 and 34 were identified to bind to the drug target (CYP19A) with the docking score of -8.372 and 7.630 kcal/mol respectively. All the synthesized compounds were evaluated for the antitubercular activity against Mtb H37Rv strain as a secondary study. Compounds 57-62 with nitro-quinoxaline derivatives exhibited stronger inhibitory effects on Mtb H37Rv strain. In addition, compounds 60 and 62 were found to be most active against Mtb H37Rv with the high activity at MIC90 of <0.65 and <0.64 μM respectively. All active compounds are currently investigated for their cytotoxicity which have not been investigated before. / National Research Foundation (NRF) and Sasol Inzalo Foundation

Thesis (M.Med. (Radiation Oncology)) -- University of Limpopo, 2022 / Background Breast cancer is known to be a heterogeneous disease that demands patient centered care. Establishing the clinicopathological characteristics of breast cancer patients is a vital step in an effort to individualize their treatment. Aim The aim is to evaluate the clinicopathologic features of the different subtypes of breast cancer when classified according to immunohistochemistry markers in women attending Pietersburg hospital. Methods A retrospective review of medical records of women treated at Pietersburg hospital between 2010 and 2011 was done. Data collection was extracted on a customized data collection sheet. Chi square was used to determine association between clinicopathologic features and molecular subtypes. Analysis of variants was used to assess association between molecular types and age. Results The mean age of the population was 55.3 years (+/-14 standard deviation). The majority of patients were in stage III (46.9%) and IV (33.5%). The ER, PR, HER2/neu positive rate was 50.6%, 30% and 14,3 % respectively with a negative rate of 13,4%, 19,5% and 23,4% respectively. ER, PR and HER2/neu was unknown in 18%, 19, 5% and 23,4% respectively. The most common molecular subtype was luminal A (53,6%) followed by triple negative (27.2%), HER2/neu (11, 4%) and luminal B (7. 9%).There was no association between the subtypes and tumour stage (p=0.578).The rate of distant metastasis was similar across the subtypes being 37,9%,35%, 32,4% and 31,9% in HER2/neu, luminal B ,luminal A and TNBC, respectively. All four molecular subtypes had high rate of axillary lymph node involvement (p=0.886) Luminal A had the least percentage of high grade tumours with TNBC having the highest. Five-year overall survival for the cohort was 25, 6% with luminal A and B having a better 5 year overall survival of 27,2% and 25% respectively, whereas HER2/neu and TNBC had lower 5 year OS of 24% and 23,3%. Conclusion The findings of this study suggest that luminal A subtype is the most predominant and the majority might benefit from hormonal therapy. However, some patients could not be classified due to missing IHC marker test results. The outcome across all four subtypes is poor and more effort should be put towards improving the diagnosis and treatment individualization and follow-up in these patients.

<p>The image-guided radiation therapy (IGRT) protocol used at Juravinski Cancer Center for post-prostatectomy patients involves acquiring a kV cone beam computed tomography (CBCT) image at each fraction and shifting the treatment couch to align surgical clips. This IGRT strategy is promising but its dosimetric impact is unknown, it requires significant resources, and delivers non-negligible doses to normal tissues. The objective of this work is to evaluate this IGRT protocol and investigate possible alternatives.</p> <p>IGRT delivered dose is reconstructed by deforming the planning CT to the CBCT images acquired at each fraction, computing dose on the deformed images, and inversely transforming the dose back to the original geometry. The treatments of six patients were evaluated under four scenarios: no guidance (Non-IGRT), daily guidance as performed clinically (IGRT), guidance on alternating days (Alt-IGRT), and daily automated guidance (Auto-IGRT). For one patient, the impact of reducing the planning target volume (PTV) margin to five (IGRT-5) and eight (IGRT-8) mm isotropic was also evaluated.</p> <p>With the standard clinical PTV margin of ten/seven mm, the evaluated alternatives produced similar results. The minimum dose to the CTV was decreased by 1.6±1.0, 1.2±0.7, and 0.8±0.8 Gy for Non-IGRT, Alt-IGRT, and IGRT, respectively. IGRT with manual shifting did not appear to significantly improve the delivered treatment dose compared to Auto-IGRT (difference in CTV minimum dose was 1.2±2.1Gy). Doses to the organs at risk varied but in general, an increased volume of the bladder and rectum received low doses while smaller portions received high doses. The IGRT-5 and -8 analyses showed the same CTV dose can be delivered with significant reduction in normal tissue exposure. Overall, the desired doses are delivered during IGRT although much of this may be attributed to the large PTV margins currently employed clinically.</p> / Master of Science (MSc)