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Background: Colorectal cancer is the third most common cancer and cause of related deaths worldwide. Early colorectal cancer diagnosis is vital in reducing incidence and mortality. There is a need for the development of non-invasive screening tools for enhancing the detection of the disease. Cancer specific peptides are useful cancer targeting agents that can be used to specifically improve early detection strategies. Several cancer targeting peptides have been identified. Previous work investigated the specific binding of three of these peptides (p.C, p.L and p.14) conjugated to quantum dots and were found to bind to colorectal cancer cell lines (HT-29 and Caco-2). However, their uptake, localization and biodistribution in an in vitro and in vivo colorectal cancer model have not been determined. This is essential in gaining an understanding for future diagnostic or therapeutic based applications. Primary Aim: The aim of this study was investigate the localization of three selected peptides p.C, p.L and p.14 conjugated to gold nanoparticles in an in vitro and in vivo colorectal cancer model using HRTEM. Methodology: The AuNP/peptide conjugates were characterized by HRTEM and DLS. For in vitro studies; HT-29, Caco-2 and C3A cells were exposed to the AuNP-p.C, AuNP-p.L and AuNP-p.14, collected and processed for HRTEM to assess targeting and localization. For in vivo studies; the establishment of a colorectal cancer model using the AOM/DSS model 1 and 2 was conducted. Wistar rats were assigned to 6 groups, five experimental and 1 control group. Group 1 received AOM/DSS method 1 and was treated with AuNP-p.L. Group 2 and 3 received AOM/DSS method 2 and were treated with AuNP-p.C and AuNP-p.14. Group 4 and 5 remained healthy and treated with AuNP-p.C and AuNP-p.14. Group 6 remained healthy receiving no nanoparticle treatment. After treatment, rats were sacrificed and tissue was processed for HRTEM. Tissue chosen for HRTEM analysis included: Group 1 (inflamed colon, rectum, pancreatic and kidney), Group 4 (kidney) and Group 5 (liver). Results: results obtained from nanoparticle characterization suggested that nanoparticles were conjugated to their respective peptides and were stable in dispersion. For in vitro studies, results suggested no AuNP targeting and localization in HT-29 cell lines. For in vivo studies, no colorectal cancer tumours were induced. TEM micrographs did not indicate the presence of nanoparticles in colon, rectum, pancreatic, kidney and liver tissue. However, AuNPs were found in the kidney tissue (group 4). Conclusion: Although the overall objectives were not met, this study provided insight into TEM cell preparation and optimization for future nanoparticle cell interaction research. This study also demonstrated the absence of AuNPs in healthy tissue and the presence of AuNPs in healthy kidney tissue through renal clearance, a favourable quality for diagnostic or therapeutic applications.

Colorectal cancer (CRC) is a common disease with significant morbidity and mortality. In spite of significant advances in understanding the molecular signaling in this disorder, unanswered questions remain. Cyclooxygenase-2 (COX-2) and β-catenin have established roles in colorectal carcinogenesis, with both being upregulated early in the disease course. The role of peroxisome proliferator-activated receptor γ (PPARγ) is less clear, but has been shown to be downregulated in colon cancer models. Butyrate, a short chain fatty acid, produced by colon microbiota and transported into the colonocyte by transporter proteins, appears to be important in early carcinogenesis. The butyrate concentration is reduced in CRC and so are its transporters. Interleukin-17 (IL-17) plays a role in colitis-associated colorectal cancer (CAC), but its function in sporadic CRC is less clear. Similarly, Protein kinase C (PKC) has proven involvement in many solid tumours, including CRC, but its exact mechanistic role is still speculative. AIM: To investigate the role and possible signaling pathways of the major role players, β-catenin, COX-2 and PPARγ in early CRC. Further, to elucidate the mechanistic pathways of butyrate and its transporters, IL-17 and PKC in CRC. METHOD: Informed consent was obtained for all patients. Patients were recruited in various disease categories, including normal, irritable bowel syndrome (IBS), inflammatory bowel disease (IBD) and CRC. Colon biopsy specimens were obtained during colonoscopy and used for immunohistochemistry (IHC) and gene expression analysis of the above genes by quantitative polymerase chain reaction (qPCR). RESULTS: β-catenin mRNA and protein expression was increased in CRC and the IBD groups compared to the normal group, while it was reduced in the IBS groups. COX-2 mRNA expression showed a steady increase from normal, through IBS, IBD and CRC groups to a statistically significant degree. The COX-2 protein expression, however, did not match the mRNA expression with increased COX-2 protein expression in normal and IBS groups and reduced expression in IBD and CRC groups. PPARγ mRNA expression was unchanged in IBD and CRC groups, but significantly increased in the IBS group compared to normal. Butyrate transporter, SLC16A1 mRNA was significantly reduced in CRC, but also in the IBS groups, which was unexpected. In the IBD group, SLC16A1 mRNA was unchanged in Crohn’s disease (CD) but significantly reduced in ulcerative colitis (UC). Similarly, SLC5A8 mRNA expression was significantly reduced in the CRC as well as the IBS groups. In the IBD groups, SLC5A8 was unchanged in UC but significantly increased in CD. IL-17 mRNA expression was significantly reduced in CRC and IBS groups, but unchanged in the IBD groups. PKCε mRNA was significantly increased in CRC as expected. In the IBD groups, PKCε mRNA was unchanged in CD but significantly increased in UC. In the IBS groups, PKCε mRNA in constipation –IBS (C-IBS) was significantly reduced, but unchanged in diarrhoea – IBS (D-IBS). CONCLUSIONS: β-catenin mRNA and protein expression was increased in CRC and the CRC promoting IBD groups. COX-2 protein expression was incongruent with the COX-2 mRNA expression and this may reflect homeostatic control mechanisms. High COX-2 mRNA expression in CRC and CRC promoting IBD groups may be a secondary phenomenon reflecting the inflammatory milieu, rather than a true carcinogenesis-related event. PPARγ does not appear to play a central role in early colon carcinogenesis, in spite of available literature suggesting otherwise. Butyrate transporters showed inconsistent results and for now no firm conclusions can be drawn from this. IL-17 may play a role in CAC as confirmed in this and other studies, but its role in sporadic CRC is tenuous and requires further investigation. Likewise for PKCε, upregulation is associated with increased tumourigenecity as shown in this study, however, the mechanistic pathway(s) involved is still speculative and requires further study.

Previous studies have focused on gastric dysmotility and delayed emptying in diabetes mellitus. There is little information about the effects of hyperglycemia on colonic motility and secretion. 5-HT was reported to mediate contractile activity by activating receptors on both enteric neurons and smooth muscle cells. The aim of this study was to investigate and compare the effects of 5-HT on circular contractile activity coordinated with secretion in streptozocin-induced diabetic and non-diabetic rats. Sonomicrometry and voltage clamping techniques were used to measure motility and secretion simultaneously in isolated whole thickness colonic sheets. Male Sprague Dawley rats were injected with streptozocin (65 mg/kg body weight) in 0.1 M sodium citrate buffer, into the tail veins. Glucose levels of 300-400 mg/dl and above were achieved. The control rats were injected with the same volumes of vehicle (0.1 M sodium citrate buffer). Animals were sacrificed 10-12 days following the induction of hyperglycemia. Flat sheets of colon were mounted serosal side up in Ussing chambers. 1 mm piezocrystals were glued to the serosal surface 4-5 mm apart to measure circular contractions as decrease in inter-crystal distances (ICD). Voltage-clamping the tissues at 0 mV was used ix to measure short circuit current (Isc), indicative of chloride secretion. In diabetic rats 50 gM 5-HT caused mean amplitude of contractions of 174 ± 26 gm (n=4), which was significantly reduced as compared to the response in non-diabetic rats of 970 + 243 gm (n=4; p<0.05). The secretory response in diabetic rats paralleled the reduction in ICD (diabetic: 23 +1 gA/cm2, controls: 57 + 18 gA/cm2). Neural blockade with 0.1 gM TTX revealed a decreased myogenic contractile activity in diabetic rats. The mean amplitude of contractions after TTX in diabetic rats was 162 ± 45 gm verses controls of 612 ± 86 gm. These results suggest that the reduction of the 5-HT contractile response in diabetic rats may be a composite of direct effects on the smooth muscle as well as indirect effects on the neurons. / Department of Physiology and Health Science

Liu Bonnie Pui-ling. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 134-163). / Abstracts in English and Chinese. / Acknowledgments --- p.i / Abbreviations --- p.ii / Abstract / English --- p.1 / Chinese --- p.3 / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Colorectal Cancer Statistics --- p.5 / Chapter 1.2 --- Colon Cancer --- p.5 / Chapter 1.3 --- Treatment --- p.6 / Chapter 1.4 --- Effects of Cytotoxic Drug Treatment --- p.7 / Chapter 1.5 --- Cell Cycle --- p.8 / Chapter 1.6 --- Oxidases --- p.9 / Chapter 1.7 --- Chemistry of Novel β-carboline: Tangutorine --- p.11 / Chapter 1.8 --- Aim of Study --- p.14 / Chapter Chapter 2 --- Cytotoxicity / Chapter 2.1 --- Introduction --- p.15 / Chapter 2.2 --- Materials and Methods --- p.18 / Chapter 2.3 --- Results --- p.23 / Chapter 2.4 --- Discussion --- p.44 / Chapter Chapter 3 --- Oxidase Activity and Protein Oxidation / Chapter 3.1 --- Introduction --- p.48 / Chapter 3.2 --- Materials and Methods --- p.54 / Chapter 3.3 --- Results --- p.60 / Chapter 3.4 --- Discussion --- p.80 / Chapter Chapter 4 --- Cell Cycle / Chapter 4.1 --- Introduction --- p.89 / Chapter 4.2 --- Materials and Methods --- p.93 / Chapter 4.3 --- Results --- p.96 / Chapter 4.4 --- Discussion --- p.118 / Chapter Chapter 5 --- General Discussion --- p.126 / References --- p.134

published_or_final_version / Medicine / Master / Master of Medical Sciences

Thesis (M.D.)--University of Adelaide, Dept. of Surgery, 1999. / Includes bibliographical references (leaves 249-291).

Thesis (Ph. D.)--Ohio State University, 2007.

Thesis (M.Sc.)--Memorial University of Newfoundland, 2004. / Bibliography: leaves 134-158.

Bioengineered probiotics enable new opportunities to improve colorectal cancer (CRC) prevention, screening, and treatment strategies. With CRC incidence on the rise in younger populations, there is an increased need to engineer technologies that enhance patient access to diagnostic exams and disease management. This dissertation presents the development of an orally-delivered probiotic to screen for and treat early CRC lesions with a particular emphasis on translatability factors including: safety of probiotic use, exploration of oral delivery, and testing in clinically relevant models. At the interface of immunology, synthetic biology, and the microbiome fields is the overarching concept that microbes play a critical role in the tumor microenvironment (TME). The innate ability of bacteria to seek out tumor-specific signatures and proliferate within their necrotic cores due to reduced immune surveillance enables the precise immunoengineering of the local TME. Here, we will design, characterize, and test a probiotic encoded with a lysis mechanism to aid in biocontainment and maximize the release of recombinantly-produced diagnostic and immunotherapeutic cargo. In this lysis circuit, bacteria grow to a critical density within tumors and synchronously lyse, locally releasing their payload. A small fraction of bacteria remains to reseed the population and the cycle continues, resulting in repeated and sustained drug delivery. Drawing from advancements in immunology, we engineered bacteria to produce immune checkpoint inhibitors. Monoclonal antibodies targeting immune checkpoints have revolutionized cancer therapy, but only work in a subset of patients and can lead to a multitude of toxicities, suggesting the need for more targeted delivery systems. Due to their preferential colonization of tumors, bacteria are a natural chassis for the localized delivery of such therapeutics. Therefore, we engineered a commercially available probiotic, E.coli Nissle 1917 (EcN), for the controlled production and intratumoral release of nanobodies targeting programmed cell death protein – ligand 1 (PD-L1) and cytotoxic T- lymphocyte-associated protein-4 (CTLA-4) using the described lysing release mechanism. We demonstrate that a single injection of this engineered probiotic enhanced therapeutic response compared to analogous clinically-relevant antibodies, resulting in tumor regression in syngeneic mouse models. In an effort to create a more effective therapeutic for poorly immunogenic cancers, we utilized the modularity of our platform to slow tumor growth in mouse models of established CRC by combining it with a probiotically-produced cytokine, granulocyte-macrophage colony stimulating factor (GM-CSF). We sought to expand upon the relevance of this approach for early-stage CRC screening and treatment, by characterizing the platform in CRC precancerous lesions, or adenomas. When orally-delivered, EcN robustly colonized adenomas in genetically-engineered and orthotopic murine models of CRC, and human CRC patients. Leveraging adenoma-specific colonization, we probed for EcN presence in fecal matter, demonstrating its utility as a non-invasive screen for adenomas. For more accessible testing, we engineered EcN to produce salicylate and showed that it could be detected in the urine of tumor-bearing mice for days after oral delivery of the probiotic. Moreover, we demonstrated that the therapeutic effectiveness of our previously engineered therapeutic strain, producing PD-L1, CTLA-4 and GM-CSF, was maintained when delivered orally, ultimately resulting in significant adenoma reduction. Altogether, this dissertation aims to highlight the potential for engineered EcN to be used as a safe, orally-deliverable screening and therapeutic platform for early-stage CRC disease. While we have chosen to focus on CRC here, we will conclude by discussing efforts to adapt this platform to work in combination with other cellular therapies and therapeutic indications, ultimately engineering a platform to impact a broader patient population.