This thesis describes observations, techniques and strategies, which contribute towards the development of nanoparticle based detection and treatment of cancer. Quantum dots and biorecognition molecules were studied towards applications in detection and microgels were used in the rational design of a targeted drug delivery vehicle. The fluorescence intensity of quantum dots was examined in buffers commonly used in molecular biology. The fluorescence intensity of ZnS-capped CdSe quantum dots (QDs) was found to vary significantly, depending on the amount of ZnS capping on the QDs or the concentration, pH and type of buffer the QDs were in. Since fluorescence cannot reliably be used to quantify QDs, an alternative quantification method was developed, which does not rely on their fluorescence. This method employs phage display to identify nanoparticle-specific bacteriophage which were then applied in an assay to quantify QDs in environments where absorbance or fluorescence spectroscopy are ineffective. Biorecognition molecules, which can direct nanoparticles to a molecular target, were also identified through phage display. Phage display on whole cells was used to identify a peptide, which was conjugated with QDs to stain HeLa (cervical cancer) cells. A high-throughput phage display screening strategy was also developed, which could enable the simultaneous identification of multiple biorecognition molecules from a single library. QD-encoded microbead barcodes were conjugated to protein targets and then used to screen a phage display library. The beads and the binding phage were then separated using flow cytometry and fluorescence assisted cell sorting. Finally, biorecognition molecules were combined with nanoparticles to create drug delivery vehicles, which were designed to protect, deliver and then release chemotherapeutic drugs through an intracellular pH trigger. PolyNIPAAm and chitosan hydrogels, under 200 nm in diameter, were loaded with chemotherapeutic drugs, conjugated to transferrin and tested in vitro on HeLa cells. These projects demonstrate the great potential in this growing field as well as some of the many challenges that have yet to be overcome.

Cancer cells display aberrant receptors on their surface that can serve as targets for the development of directed drug therapies. As such, our group has utilized two parallel approaches to redirect the cytotoxic properties of a ribosome-inactivating protein (RIP), Shiga-Like Toxin 1 (SLT 1), by altering its receptor specificity to target and kill cancer cells. The first combinatorial protein library was constructed such that a randomized 7 AA long peptide was inserted within the cytotoxic domain (A chain) of SLT-1. A high-throughput protein-based screening campaign identified a novel A chain toxin variant (named SLT 1AIYSNKLM) capable of targeting and killing human melanoma cells. This variant harbours a peptide insert (IYSNKLM) that directs the A chain to kill human melanoma cell lines. Equilibrium binding studies using 125I-radiolabeled SLT-1AIYSNKLM were conducted to determine the equilibrium binding constant and receptor density on 518-A2 human melanoma cells. When injected into SCID mice bearing a human melanoma xenograft, nanoSPECT/CT imaging as well as the biodistribution profile showed marked tumour uptake and retention of the radiolabeled toxin variant. Furthermore, preliminary experiments have shown that the SLT-1AIYSNKLM receptor is a protein, highlighting the potential for this method to be used in the discovery of novel biomarkers. A second approach was employed to demonstrate that our toxin-based combinatorial library system can be adapted to target known cancer biomarkers. Specifically, SLT-1 A chain variants harbouring 12-residue inserts were expressed in a phage display library. The library was screened against cell lines expressing the human colon cancer marker carcinoembryonic antigen (CEA; CD66e; CEACAM-5) to identify candidates that not only targeted, but internalized into cancer cells within a 1 h period. Variant, CSTA-10, was found to kill CEA-expressing BxPC-3 cells. Overall, the directed evolution of an RIP template such as SLT-1 represents a novel and powerful strategy for the identification of tumour-targeted toxin variants.

A new class of polymer coated lanthanide fluoride nanoparticle aggregates (NPAs) was developed as potential MRI contrast agents. The NPA synthesis has been perfected to control the size distribution and optimize relaxivities. Polyacrylic acid was used as a stabilizing polymer, and was conjugated to folic acid to improve targeting to SK-BR-3 breast cancer cells. Terbium was incorporated in the synthesis to study the passive and active targeting properties of NPAs. Through a series of microscopy experiments, a significant difference in uptake between NPAs with and without targeting moieties occurs after 48 hours of incubation. The relaxivity of the optimized nanoparticles was measured to be 56 s-1(mg/ml)-1 using a 1.5 T scanner, which may be compared to that of the commercially available Gd3+-DTPA [R1 = 7 s-1(mg/ml)-1]. Abdominal perfusion studies in rats also demonstrated that the NPAs provide better contrast of the vasculature than Gd3+-DTPA does at the same mass concentration.

Catalysts play a vital role in almost every aspect of our lives and are used in the production of fuels, polymers, chemicals, foods, and pharmaceuticals. One challenge facing the heterogeneous catalysis community is the targeted synthesis of dispersed catalyst ensembles. The Barnes research group has developed a general methodology for the synthesis of nanostructured silicate building block supports and heterogeneous catalysts. This methodology provides researchers with the ability to control the dispersion of surface functionality, the dispersion of metal cation centers, the number of linkages from the metal cation center to the support, the surface area of the support, and the porosity of the support. This dissertation describes work aimed at synthesizing and characterizing nanostructured silicate building block supports and heterogeneous catalysts. Nanostructured silicate building block supports were synthesized by reacting SiCl4py2 with Si8O12(OSnMe3)8. The resulting supports contained spatially isolated Me3Sn groups and the density of Me3Sn groups was targeted by varying the stoichiometric ratio of reactants. The stoichiometric ratio of reactants also controlled the surface area and porosity of the supports. Nanostructured heterogeneous catalysts with isolated tungsten(VI) or zirconium(IV) centers were synthesized by reacting a limiting amount of a metal chloride with either Si8O12(OSnMe3)8 or a premade silicate building block support. Two types of catalysts ensembles were targeted: embedded and surface. Embedded ensembles were successfully targeted using WOCl4 and ZrCl4 while the reaction between WCl6 and the building block did not result in the preparation of the targeted ensemble. However the resulting ensemble was thoroughly characterized even though the targeted ensemble was not produced. In all three cases a single type of catalyst ensembles was synthesized and a high surface area silicate support was generated around the embedded ensembles without disrupting the ensemble itself. Surface ensembles were successfully targeted using ZrCl4. The reaction between the tungsten chlorides (WOCl4 and WCl6) and the premade support did not result in the preparation of the targeted ensembles however the resulting ensembles were thoroughly characterized.

Acute myeloid leukemia (AML) is a life-threatening malignant disorder with dismal prognosis. AML is characterized by frequent genetic changes involving tyrosine kinases, normally acting as important mediators in many basic cellular processes. Due to the overexpression and frequent mutations of the FMS-like receptor tyrosine kinase 3 (FLT3) in AML, this tyrosine kinase receptor has become one of the most sought after targets in AML drug development. In this thesis, we have used a combination of high-throughput screens, direct target interaction assays and sequential cellular screens, including primary patient samples, as an approach to discover new targeted therapies. Gefitinib, a previously known inhibitor of epidermal growth factor receptor and the two novel tyrosine kinase inhibitors AKN-032 and AKN-028, have been identified as compounds with cytotoxic activity in AML. AKN-028 is a potent inhibitor of FLT3 with an IC50 value of 6 nM in an enzyme assay, but also displaying in vitro activity in a variety of primary AML samples, irrespective of FLT3 mutation status or quantitative FLT3 expression. AKN-028 shows a sequence dependent in vitro synergy when combined with standard cytotoxic agents cytarabine or daunorubicin, with better efficacy when cells are exposed to standard chemotherapy simultaneously or for 24 hours prior to adding AKN-028. Antagonism is observed when cells are pre-treated with AKN-028, possibly explained by the cell cycle arrest induced by the compound. In vivo cytotoxic activity and good oral bioavailability have made AKN-028 a candidate drug for clinical studies and the compound is presently investigated in an international two-part multicenter phase I/II study. Results from microarray studies performed to further elucidate the mechanism of action of AKN-028, revealed significantly altered gene expression induced by AKN-028 in both AML cell lines and in primary AML cells, with an enrichment of the Myc pathway among the downregulated genes. Furthermore, tyrosine kinase activity profiling shows a dose-dependent kinase inhibition by AKN-028 in all AML samples tested. Interestingly, cells with a high overall kinase activity were more sensitive to AKN-028. Provided conformation in a larger set of samples, kinase activity profiling may give useful information in individualizing treatment of patients with AML.

Abstract HIV/AIDS is a global epidemic with the greatest burden in terms of prevalence, morbidity and mortality in sub-Saharan Africa, parts of Asia and, more recently, the Caribbean. Immigrants from these regions of birth now make up a significant proportion of people living with HIV in many high-income countries, including Australia. The higher priority accorded to people from culturally and linguistically diverse (CALD) backgrounds in Australian national and local HIV/AIDS strategies generates a broad question on ‘how’ to implement HIV prevention interventions with immigrants to address what are often atypical modes of HIV transmission and observed disparities in areas such as later presentation with HIV. HIV prevention in Australia has included whole-of-population approaches alongside targeted approaches, which address HIV prevention with specific groups – usually those disproportionately affected by HIV/AIDS such as gay men or injecting drug users. Targeted health promotion interventions for immigrants have also formed part of the HIV response in Australia. Immigrants in Australia may have acquired HIV prior to their first arrival in Australia, on subsequent travel abroad, or within Australia. A key gap in our evidence base in Australia includes what we can learn from interventions implemented in other high-income countries to guide new, or strengthen existing, approaches to culturally appropriate primary and secondary HIV prevention with immigrants locally. Typically it is taken as a given that prevention interventions will be more effective if they are culturally appropriate to the population they serve, and a range of strategies and activities are used to achieve this. However, there is rarely an examination of what mechanisms – the ‘change elements’ or program theories of the intervention – contribute to culturally appropriate interventions. This research, in the form of a realist review of evidence, sought to ‘unpack’ the mechanisms for achieving cultural appropriateness in HIV prevention interventions with immigrants that have been implemented in contexts similar to Australia. Thus the broad question the research sought to answer was ‘How and why do interventions work (or not), for which groups of immigrants, and in what contexts?’ The review of evidence in HIV prevention included a span of interventions from community-level approaches using mass media through to interventions delivered at a group level to immigrants. Systematic searches were carried out on major public health databases (PubMed, CINAHL, Sociological Abstracts, PsychInfo) and Google Scholar to find peer-reviewed and grey literature relevant to HIV prevention among immigrants. Two types of studies contributed to the review of evidence – studies of interventions and qualitative studies of immigrants’ views on HIV/AIDS prevention – in order to bring together ‘expert’ and ‘lay’ understandings of HIV prevention among immigrants. Simultaneously, a scan of the literature mapped preliminary mechanisms contributing to cultural appropriateness in HIV prevention interventions with immigrants. This preliminary set of seven mechanisms – ‘authenticity’, ‘understanding’, ‘consonance’, ‘specificity’, ‘embeddedness’, ’endorsement’ and ‘framing’ – were theorised as the key, rather than the only, interrelated mechanisms contributing to cultural appropriateness in interventions with immigrants. These preliminary mechanisms were then tested, revised and refined against evidence – 74 ‘grey’ and peer-reviewed studies and reports relevant to HIV prevention with immigrants – found in systematic searches. The evidence indicates that the pivotal mechanisms contributing to cultural appropriateness in HIV prevention interventions with immigrants are ‘understanding’ and ‘consonance’ – ensuring that language (usually the ‘mother tongue’) and cultural values are included as key elements in the development and implementation of the intervention. ‘Authenticity’, ‘specificity’ and ‘embeddedness’were moderately important in contributing to cultural appropriateness – mechanisms that dealt with staffing, targeting through ethnicity and using settings for interventions – from the evidence included in the review. Finally, there was mixed evidence for the roles of ‘endorsement’ and ‘framing’, which suggests that gaining community endorsement or partnering initiatives with immigrants or immigrant community institutions were the least critical mechanisms in contributing to cultural appropriateness in terms of HIV prevention interventions. Further research is needed to examine the relationships between these seven mechanisms and any impacts they contribute to the effectiveness of interventions and HIV-related health outcomes among immigrants.

Zugl.: Mainz, Univ., Diss., 2004 / Hergestellt on demand

The typical response of cancer patients to treatment is only temporary, and is often followed by relapse. The failure of various therapeutic strategies is commonly attributed to the emergence of drug resistance. The response patterns for patients under such treatments indicate that complex dynamics regulate the response of the tumour to the therapy. The environment in which the tumour lives (the stroma) is known to be a modulator of multiple mechanisms that lead to drug resistance and seems to be a likely candidate for explaining some of this complexity. Understanding the role of stromal cells in the promotion of drug resistance is critical for the design of optimal treatment strategies, and for the development of novel therapies that selectively target both the tumour and the stroma. In this thesis we design two novel mathematical models that describe cancer growth within its environment and the evolution of drug resistance within spatially complex and temporally dynamic tumours. A compartment model captures clinically observed dynamics and allows direct comparison with experimental data, facilitating model parametrisation and the understanding of inter-tumour heterogeneity. An individual cell-based model highlights the key role of local interactions, determining heterogeneity at the tissue scale, that will eventually determine treatment outcome. A non-spatial approximation of this second model allows us to find analytic guidelines for the design of effective therapy. These tools allow the simulation of a range of treatment strategies (including combination of different drugs and variation of schedule) as well as the investigation of therapy response based on patient- or organ-specic parameters. The work developed in this dissertation is based on the paradigmatic biology of melanoma and non-small cell lung cancer. Its results are therefore applicable to a variety of cancer treatments that target similar processes, and whose therapeutic failure can be attributed to environment-mediated drug resistance.

Les chordomes sont des tumeurs rares. Elles représentent 1-4% de toutes les tumeurs osseuses. Ces tumeurs à histogénèse notochordienne sont d'origine mésodermique exprimant des marqueurs épithéliaux. Elles sont dans la majorité des cas de bas grade et d’évolutions insidieuses, mais toujours infiltrantes et destructrices pouvant donner des métastases. Au plan thérapeutique, ce sont des lésions réputées pour leur résistance aux cytotoxiques, aux rayonnements ionisants et aux thérapies ciblées actuelles. Leur prise en charge standard consiste en une chirurgie première suivie d'une radiothérapie complémentaire à haute dose. Le taux de contrôle local est de 70% et 50% à 5 ans et 10 ans respectivement et la survie médiane est de 6 ans. Les futurs challenges consistent à mieux définir le volume cible par l’imagerie multimodalité pour une radiothérapie personnalisée voire adaptative tenant compte des modifications tumorales morphologiques et métaboliques en cours de traitement. L’hypoxie est connue depuis longtemps comme un facteur de radiorésistance aux traitements. En effet, l'hypoxie induit un phénotype agressif avec un haut potentiel métastatique, favorisant la progression tumorale. Ces caractéristiques sont intimement liées à certaines modifications biologiques comme l’angiogenèse tumorale et le dérèglement du métabolisme cellulaire du glucose. Une meilleure connaissance de ces facteurs régulateurs des cibles biologiques constitue un vrai challenge pour le clinicien et les industriels dans le cadre du développement de nouvelles thérapeutiques. Dans ce cadre, mon projet de thèse a été construit en trois étapes : (i) nous avons récemment rapporté, pour la première fois, la présence de composantes cellulaires hypermétaboliques et de composantes cellulaires très hypoxiques quiescentes par l'imagerie TEP/CT, (ii) mettre en évidence sur des prélèvements de chordomes provenant de patients, les facteurs régulateurs de l'hypoxie (HIF-1, HIF-2) du métabolisme (GLUT1 ; MCT1 ; MCT4 ; Basigine, CAIX - CAXII) et de l’autophagie (BNIP3) par des techniques d'immunohistochimie ; (iii) quantification de ces marqueurs par des techniques de Western Blot. La corrélation de ces résultats biologiques aux résultats cliniques obtenus, montre que les patients présentant des lésions arborant un métabolisme glycolytique et un processus de survie autophagique présentent un phénotype agressif et radiorésistant. La présence de ces mécanismes de survie est fortement corrélée avec un contrôle local faible et une survie réduite. La mise en évidence de ces mécanismes et de leurs facteurs régulateurs nous permet d’envisager des traitements ciblés en association avec la chirurgie et la radiothérapie. / Chordomas are rare tumors. They represent 1-4% of all bone tumors. These tumors are notochordal histogenesis and mesodermal origin expressing epithelial markers. They are in most cases low grade and have insidious development with destructive and invasive power. Therapeutically, they are known for their resistance to cytotoxic drugs, ionizing radiation and targeted therapies. "Gold standart" treatment for these lesions is a primary surgery followed by high doses of radiotherapy. The local control rate is 70% and 50% at 5 years and 10 years respectively and median survival is 6 years. Future challenges are to define better the target volume by multimodality imaging for radiation therapy or adaptive personalized taking into account changes in tumor metabolic and morphological processing. Hypoxia has long been known as a radioresistance factor. Indeed, hypoxia induces an aggressive phenotype with a high metastatic potential, promoting tumor progression. These characteristics are closely related to certain biological changes such as tumor angiogenesis and disruption of cellular metabolism of glucose. A better understanding of these regulators factors of biological targets is a real challenge for clinicians and industry in the development of new therapies. In this context, my thesis project was built in three steps: (i) we recently reported, for the first time, the presence of quiescent and hypoxic cellular components and hypermetabolic cellular components by PET / CT, (ii) highlight on samples from patients with chordoma, the hypoxia regulatory factors (HIF-1, HIF-2) metabolism (GLUT1, MCT1, MCT4; Basigine, CAIX - CAXII) and autophagy (BNIP3) by immunohistochemistry, (iii) quantification of these markers by immunoblotting techniques. The correlation of these laboratory findings to clinical results showed that patients with lesions displaying a glycolytic metabolism and survival autophagic process have an aggressive and radioresistant phenotype. The presence of these mechanisms is strongly correlated with poor local control and survival reduced. The highlight of these mechanisms and their regulatory factors allow us to consider targeted therapies in combination with surgery and radiotherapy.

abstract: This study utilized targeted messages and expert and referent sources in an effort to promote physical activity behavior in college students. College students aged 18-25, excluding collegiate athletes, were randomized into three conditions using their current physical activity level. Two of the conditions received targeted messages highlighting three primary components of social cognitive theory - self-efficacy, outcome expectations, and goals - while the third condition received no messages. In addition, the experimental conditions received the messages from either an expert (i.e., a personal trainer) or a referent (i.e., a close friend). In this way, this experiment analyzed whether receiving social cognitive theory messages increased physical activity indicators compared to the control condition, as well as if the message source caused differences in the physical activity indicators. Moreover, participants completed Time 1 and Time 2 measures to determine if receiving messages or not caused changes over a one week time period. Seven physical activity indicators were assessed: self-efficacy, positive outcome expectations, negative outcome expectations, attitudes, response-efficacy, intentions, and physical activity behavior. Results revealed that both the personal trainer and close friend conditions had significantly higher scores than the control condition for intentions at Time 1 and Time 2, as well as physical activity behavior at Time 2. Moreover, the personal trainer condition had significantly higher positive outcome expectations compared to both the friend and control conditions. No other significant differences were found across conditions for Time 1 attitudes, response-efficacy, negative outcome expectations, self-efficacy, and Time 2 attitudes, and self-efficacy. Overall, targeted messages were effective in increasing physical activity intentions and behavior regardless of the message source. / Dissertation/Thesis / Ph.D. Communication 2012