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The rise in large-scale cancer genomics data collection initiatives has paved the way for extensive research aimed at understanding the biology of human cancer. While the majority of this research is motivated by clinical applications aimed at advancing targeted therapy, cancer prevention initiatives are less emphasized. Many cancers are not attributable to known heritable genetic factors, making environmental exposure a main suspect in driving cancer risk. A major aspect of cancer prevention involves the identification of chemical carcinogens, substances linked to increased cancer susceptibility. Traditional methods for chemical carcinogens testing, including epidemiological studies and rodent bioassays, are expensive to conduct, not scalable to a large number of chemicals, and not capable of detecting specific mechanisms of actions of carcinogenicity. Thus, there exists a dire need for improvement in data generation and computational method development for chemical carcinogenicity testing. Here, we coin the term "carcinogenome" to denote the complete cancer genomic landscape encompassing both its repertoire of environmental chemical exposures, as well as its germ-line and somatic mutations and epi-genetic regulators. To study the carcinogenome, we analyze both the genomic behavior of real human tumors as well as profiles of the exposome, that is, data derived from chemical exposures in human, animal or cell line models. My thesis consists of two distinct projects that, through the generation and innovative analysis of multi-omics data, aim at advancing our understanding of the molecular mechanisms of cancer initiation and progression, and of the role environmental exposure plays in these processes. First, I detail our effort at data generation and method development for characterizing environmental contributions to carcinogenesis using transcriptional profiles of chemical perturbations. Second, I present the tool iEDGE (Integration of Epi-DNA and Gene Expression) and its applications to the integrative analysis of multi-level cancer genomics data from human primary tumors of multiple cancer types. These projects collectively further our understanding of the carcinogenome and will hopefully foster both cancer prevention, through the identification of environmental chemical carcinogens, and cancer therapy, through the discovery of novel cancer gene drivers and therapeutic targets.

The main objectives of the research presented in this thesis were to understand mechanistic aspects of the photochemistry of ferritin (Ftn) and bioconjugates that consisted of Ftn linked to gold nanoparticles (AuNPs). The photochemistry investigated in this thesis repurposed Ftn from its role in biological systems as an iron-sequestration protein to potential applications in photocatalysis and nanobiomedicine. The first phase of the thesis research developed a mechanistic understanding of the underlying mechanisms involved in the photochemistry of Ftn with relevance to photocatalysis. In particular, research was designed to determine whether the light-induced bandgap excitation of the semiconductor core of horse-spleen ferritin (HSFtn) resulted in electron transfer from the inorganic core to aqueous redox active reactant via electron transport through the 2 nm thick shell of HSFtn. To investigate this mechanistic pathway, 4-5 nm copper (hydr)oxide nanoparticles were mineralized within the internal volume of HSFtn (CuFtn). It was shown that, unlike the native iron oxyhydroxide-bearing (Ferrihydrite; Fh) Ftn, the visible light photoexcitation of the inorganic core of CuFtn (measured optical bandgap to be 3.65 eV) did not exhibit any release of redox-active metal cation from the HSFtn cage into solution. By photoexciting CuFtn in the presence of aqueous chromate (Cr(VI)) it was shown that the Cr(VI) underwent reduction to Cr(III) in solution. The research strategy eliminated the possibility that metal cations escaping from the HSFtn during photoexcitation could be responsible for Cr(VI) reduction. Hence, the research showed for the first time that electrons resulting from a photoexcited metal oxide core of Ftn could transfer through the protein shell to reduce an aqueous redox active reactant. The research also investigated the wavelength-dependent photochemistry of CuFtn to show that bandgap excitation was indeed responsible for the electrons that transfer across the protein shell. In a second project, the research investigated the bioconjugation of anisotropic AuNPs—gold nanorods (AuNRs) and gold nanostars (AuNSs)—to human H-type ferritin (HFtn). After attaching the AuNRs or AuNSs to HFtn, it was shown that the near-infrared (NIR) radiation excitation of the localized surface plasmon resonance (LSPR) of the AuNR or AuNS conjugated to HFtn led to the activation of the Fh core of the protein. This NIR photochemistry (λ = 850 nm light) resulted in the release of Fe(II) from the Ftn and also led to the reduction of Cr(VI) when it was present in the aqueous phase. The novel synthetic protocols to synthesize the bioconjugates focused on attaching the AuNRs and AuNSs to the solvent-exposed cysteines (Cys) on HFtn. The research also developed techniques for the removal of colloidal stabilizing surfactants, such as cetyltrimethyl ammonium bromide (CTAB), and TritonX-100 (TX-100), from anisotropic AuNPs (AuNR/AuNS) before their attachment to HFtn. The removal of the surfactant was not only important for attachment to the HFtn, but it also removed a cytotoxic species so that the bioconjugates could be used in research that had applications to biomedicine. Research also investigated synthetic strategies to form bioconjugates that consisted of spherical gold nanoparticles (AuNSps) attached to HSFtn. In contrast to HFtn, HSFtn contains a few solvent exposed Cys groups. Hence, a challenge that was overcome in this research was to populate the outer surface of HSFtn with thiol groups (-SH) so that AuNSps could be attached. To meet this challenge, the surface primary amine-containing amino acids (Lysine) in HSFtn were modified to active Cys using N-succinimidyl S-acetylthioacetate (SATA). After this chemical modification of HSFtn, it was shown that a relatively high density of AuNSps could be attached to HSFtn. This SATA-modified HSFtn bioconjugate system (AuNSp-HSFtn) exhibited the release of Fe(II) at wavelengths of light where λ > 475 nm. In the absence of AuNSp, HSFtn released Fe(II) during exposure to light at wavelengths of light where λ < 475 nm. The activation of the bandgap at longer wavelengths of light (λ > 475 nm) was due to the excitation of the 532 nm plasmon resonance of AuNSp and the presumed transfer of hot electrons to the inner Fh core of HSFtn. A final project investigated the use of the AuNR-HFtn bioconjugates as a photodynamic strategy utilizing NIR to suppress the growth of cancer cells with the expectation that this process will occur through the mechanism of ferroptosis. We carried out experiments that exposed prostate cancer cells (PC3) to AuNR-HFtn, and during NIR irradiation, they showed the ability to limit the growth of the cells compared to experiments where the cells were exposed to just HFtn or AuNRs. The results suggested that Fe(II) released from the HFtn led to cancer cell death through a process that might be ferroptosis. Future studies will need to investigate this possibility and whether the bioconjugates developed in this thesis will offer a novel therapeutic strategy for cancer/tumor suppression. / Chemistry

Pancreatic ductal adenocarcinoma (PDAC) is the 3rd deadliest cancer in the United States with a projected 12% 5-year survival rate. Acinar cells have been proposed as a potential cell-of-origin for PDAC after undergoing acinar to ductal metaplasia (ADM). In the absence of oncogenic mutations (e.g. Kras), ADM lesions form as an adaptive response and eventually resolve to regenerate the acinar compartment, which we term as adaptive ADM. However, in the presence of oncogenic Kras mutations, the ADM lesions can transform to a pre-invasive state called pancreatic intraepithelial neoplasia (PanIN). Thus, a normally adaptive metaplastic response becomes maladaptive, which we term as oncogenic ADM. The mechanisms that drive PanIN formation in the context of injury and oncogenic mutations are poorly understood, resulting in an absence of targets to combat persistent ADM. This thesis investigates the role of FRA1 (gene name Fosl1) in acinar cell de-differentiation, PanIN transformation, and eventual PDAC tumorigenesis. Through CUT&RUN sequencing of mice undergoing recovery from caerulein-induced acute pancreatitis, we identify FRA1 as the most active transcription factor during KrasG12D mediated acute pancreatitis- mediated injury. We have elucidated a functional role of FRA1 by generating an acinar-specific Fosl1 knockout mouse expressing KrasG12D. Using a gene regulatory network and pseudotime trajectory inferred from single nuclei ATAC-seq and bulk-RNA seq, we hypothesize a regulatory model of the acinar-ADM-PanIN continuum and experimentally validate that Fosl1 knockout mice are delayed in the onset of ADM and PanIN. Furthermore, deletion of Fosl1 in an autochthonous PDAC mouse model revealed that this ADM-initiated delay eventually culminates in a significant survival advantage and a less aggressive tumor phenotype. Through investigation of upstream regulators of FRA1, we identified G-CSF as an ADM-promoting cytokine. Fosl1 depletion prevented the pro-inflammatory effects of G-CSF, indicating that the G-CSF/FRA1 signaling axis can modulate ADM. Using ex vivo acinar cultures, we also showed that G-CSF can induce FRA1 through MEK/ERK signaling. Our findings reveal that FRA1 is a mediator of acinar cell plasticity and contributes to acinar cell de-differentiation and malignant transformation. Although the majority of this thesis focuses on oncogenic ADM, we also include a chapter on the role of Prrx1 in adaptive ADM. Our comprehensive and unbiased approach identified previously the Paired-Related homebox1 (Prrx1) as the most upregulated transcription factor in the intersection of pancreatic ductal development, regeneration, and evolution of PanIN. We have demonstrated previously that Prrx1 can promote a ductal phenotype by binding the Sox9 promotor and inducing its expression during pancreatitis. In this body of work, we present a novel mechanism by which Prrx1 regulates maintenance of adaptive ADM. Using novel mouse models and ex vivo acinar culture systems, we demonstrate that Prrx1 can induce TGFβ signaling and reduce E-Cadherin expression to promote ADM. We do not know if there is any potential epistatic interaction between FRA1 and PRXX1. Overall, we reveal the rippling effects of FRA1 can have during the early stages of pre-neoplasia, and we unveil an alternative function of PRRX1 for stimulating an adaptive response to stress. This thesis presents a new understanding of how acinar cell de-differentiation occurs in the pancreas by revealing novel roles of two transcription factors, FRA1 and PRRX1, and furthers our understanding of tissue regeneration in an injured pancreas.

This dissertation explores motivation in decision-making and action in science and technology, through the lens of a case study: scientific data sharing in cancer research. The research begins with the premise that motivation and emotion are key elements of what it means to be human, and consequently, are important variables in how individuals make decisions and take action. At the same time, institutional controls and social messaging send a variety of signals intended to motivate specific actions and behaviors. Understanding the interplay between personal motives and social influences may point to strategies that better align individual and social perceptions and discourse. To explore these dynamics, this research centers on a large-scale cancer research program led by the National Institutes of Health's National Cancer Institute. The goal of the program is to encourage interoperability and data sharing between diverse and highly autonomous cancer centers across the U.S. Housed in an organization focused on biomedical informatics, the program has a technologically-focused mission; the goal is to facilitate institutional data sharing to connect the cancer research enterprise. This focus contrasts with the more relationship-based point-to-point data sharing currently reported by researchers as the norm. Researchers are motivated to share data with others under specific conditions: when there is a foundation of trust with the person or community being shared with; when the perceived reward of sharing is well-defined and of value to the person sharing; and when there is perceived to be a lower risk or cost than the benefit received. Without these conditions, there are often determined to be insufficient incentives and rewards for sharing. Data sharing is both a personal decision and a social level problem. Data is both subjective and personal; it is often an extension of researcher's identity, and serves as a measure of his or her value and capability. In the search for standards and interoperable data sets, institutional and technologically-mediated forms of data sharing are perceived to ignore the subjective and local knowledge embodied in the data being shared. To explore these dimensions, this study considers the technology, economics, legal elements, and personal sides of data sharing, and applies two conceptual frameworks to evaluate alternatives for action. / Ph. D.

1. The acid catalyzed cyclodehydration method was extended to the 9-aryl-1,2-benzanthracene and 9-naphthylanthracene series. 2. A new method of cyeclodehydration was described. 3. The series of 10-mono- and 10-dimethylphenyl-1,2-benzanthracenes was completed. All the isomeric 9-mono- and 9-dimethylphenyl-1,2-benzanthracenes were prepared together with the corresponding ketones. Some highly hindered ketones were prepared and their cyclization realized. A total of 47 compounds, not previously reported in the literature, were synthesized. 4. The spectra of 24 hydrocarbons were recorded and the theory of nonplanarity of these compounds substantiated. / Ph. D.

Hypoxia inducible factor (HIF)-1α is a heterodimerically-activated transcription factor central to the cellular response to hypoxic environments and is often upregulated in cancer. Binding of HIF-1α to the co-activator p300 is necessary for the hypoxia-induced transcription of many oncogenic proteins. The aim of this project was to develop novel small molecule inhibitors of the HIF-1α/p300 protein-protein interaction (PPI). Initial work focused on designing, validating and optimising two high-throughput competition binding assays to screen for inhibitors of the PPI (Chapter 2). Alongside these, zinc ejector assays for both p300 and KDM4A proteins were developed to probe the mechanism of action and selectivity. Analysis of hits from a natural product high-throughput screen (HTS) revealed two compound classes; benzoquinones and 2-substituted indandiones, which modulate the PPI. The potency of these series correlated with the reactivity of the core functional groups, which act as electrophiles to covalently modify reactive cysteines, ejecting structural zinc and disrupting the p300/KDM4A protein fold (Chapter 3). Conjugating electrophilic groups to putative HIF-1α/p300 inhibitors did not replicate the activity of the zinc ejecting HTS hits (Chapter 4). Further work focused on non-covalent inhibitors of the HIF-1α/p300 interaction, first with peptide truncates, and then rationally designed α-helix peptidomimetics. An 11mer truncate showed encouraging activity (IC50 ≈ 70 μM), and corresponded to a key α-helix in the HIF-1α C-terminal transactivation domain. Three distinct double-sided scaffolds were used to imitate up to five hot-spot ampiphilic residues on this α-helix (Chapter 6 and 7). Of the 35 compounds screened, only modest inhibition was observed (IC50 ≈ 200-500 μM). Future work will look to conjugate electrophilic functionality onto the 11mer peptide in an attempt to gain potency from zinc ejection, while maintaining selectivity for p300.

In biological systems, small changes can have significant impacts. It is, therefore, very important to be able to identify these changes in order to understand what is occurring in the organism. In many cases, this is not an easy task. Mass spectrometry has proven to be a very useful tool in elucidating biological changes even at a very small scale. Several different mass spectrometry based techniques have been developed to discover and investigate complex biological changes. Some of these techniques, such as proteomics, have been through years of development and have advanced to the point that anyone can complete complex analyses of global protein identification and measurement with relative ease. Other techniques are still developing and still have some ground to cover in terms of experimental outcome and ease of execution. Herein we show improvements we have made in high-throughput high-resolution mass spectrometry based techniques to identify and quantify small molecules that are involved in significant biological changes. To begin, we show that our improved high-resolution mass spectrometry based lipidomics techniques are capable of identifying small changes in diseased states that are associated with inflammation, mitochondrial shape and function, and cancer. With our techniques we have been able to extract, identify, and quantify several thousand unique lipid species from complex samples with confidence. Our initial studies looked at global lipidome profiles of differing tissue types from human and mouse biopsies. This was then adapted to compare the global lipidomes of diseased states against healthy states in asthmatic lung tissue, cigarette smoke treated cells, high fat high sugar (HFHS) stressed animals (with and without additional treatment), and in signaling lipids associated with cell death resistance and growth signaling in pancreatic cancer. As a result of our success with lipidomic method improvement we then adapted our techniques and knowledge for use in elucidating small molecule signaling peptides and oxidation changes in proteins. We were able to show that our improved liquid chromatography mass spectrometry based small molecule assays are capable of identifying and quantifying small peptides and protein modifications that would otherwise be undetectable using traditional techniques. This work resulted in the development of a scalable method to detect and quantify the small iron-regulatory hormone known as hepcidin from a variety of samples such as blood, urine, and cell-culture media. We were also instrumental in evaluating and revising a new ultra-high pressure liquid chromatography (UHPLC) system that allows for better separation of analytes from complex mixtures for identification and quantification. Through these advances we hope to aid researchers and clinicians to enable them to use mass spectrometry to further our knowledge about the small but significant changes that regulate complex biological systems.

The present work has endeavored to survey the occurence of adenosine deaminase as well as its multiple forms in normal and some cancerous human tissues. A recent report by Akedo, Nishihara, Shinkai and Komatsu concerning the appearance of the C form adenosine deaminase in cancerous tissues is investigated. The thesis reports, however, the occurence of both A and C forms of adenosine deaminase in almost all normal and cancerous tissues investigated. An increase in C form adenosine deaminase does seem to occur in cancerous tissues, and a ratio method is developed to monitor such an increase in the C form enzyme.Fundamental catalytic and physical parameters are used to characterize the A and C forms of adenosine deaminase in several normal human tissues. Little difference is noted between the two forms of the enzyme. Only substrate specificity for cordycepin is of significant value in differentiating between the A and C forms of the enzyme.Ball State UniversityMuncie, IN 47306

Medical research is not only a necessary part in the quest to improve the quality of life for people by finding new diagnostic procedures and treatment; it is also a multi billion rand endeavour. Africa itself poses a huge challenge in providing facilities to respond to the global network engaged in medical research. South Africa has responded in part to this need, and is a pioneer in medical research for the continent. In essence, the continent not only provides great challenges, but also great opportunities for research. Many of its facilities engage in collaborative research with global institutions, but these established ties do not adequately fulfil the capacity required. In addition to this, the research environment is constantly evolving. Not only is the process constantly changing, but also the environments in which the research is conducted and the attitude as to how research should be conducted. To stay as current in the field of medical research, new institutions need to respond to the technical, practical and philosophical changes in the field. The National Health Laboratory Services, a South African chapter 21 institution involved in research and diagnosis, is the proposed client for the cancer research institute to be designed. It has established research credentials in cancer; pioneering the national cancer register; and has established links to other national organisations, such as the Medical Research Council of South Africa. / Thesis (M.Arch.)-University of KwaZulu-Natal, Durban, 2008.