Mantle cell lymphoma (MCL) is a rare lymphoproliferative disorder (LPD) that has very poor survival. Like other LPDs, the neoplastic cells of MCL have an intimate dependence upon accessory cells within haematopoietic tissues. Understanding and exploiting the tissue-relationships of the mantle cells may therefore lead to further new approaches to treatment. This study work has set out to construct an in vitro system to model relevant aspects of the tissue-dependent behaviour of the neoplastic mantle cells, seeking to establish the link between in vitro behaviour and clinical phenotype, and establishing the feasibility of this system to study the effects of different therapeutic interventions. The first experimental chapter employs relevant mouse and human stromal models to mirror the tissue environment of MCL in vivo. Testing relevant agents, the work establishes that the system can identify different behaviour between indolent and aggressive forms of MCL, and demonstrates a particular importance for CD40 ligand both in the proliferation and survival of the neoplastic mantle cells, but shows also how these effects are modulated by the soluble factors interleukin-4 (IL-4) and the toll-like receptor-9 ligand (TLR9-L). The second experimental chapter examines the adhesion molecules expressed on MCL cells. Considerable variation in the level of expression is observed between cases, but overall the cases express particularly high levels of the integrin receptors LFA-1 (detected by alpha chain CD11a) and VLA-4 (detected by alpha chain CD49d). Cases also showed a significant difference in overall adhesion and chemokine-receptor expression between cases that had either a nodal or leukaemic clinical pattern, although no single adhesion molecule was characteristic of clinical phenotype. The final experimental chapter looked at 3-D culture of MCL. Within tissues MCL grows in a 3-D rather than 2-D matrix and it is recognised that cells employ different forms of adhesion and migration within the different spatial environments. This chapter establishes the feasibility of growing cells in 3-D systems and looks at optimal conditions to preserve and examine the cellular characteristics of cells within a 3-D environment. Overall, this thesis demonstrates the feasibility and pathobiological relevance of ex vivo culture of MCL cells giving insights into the factors that drive MCL survival and proliferation and the correlation between in vitro behaviour and clinical phenotype. It is proposed that this work can be expanded to examine therapeutic interventions in the disorder.

Bioengineered skin substitutes have been developed to treat burn and non-healing wounds; however limitations still hinder their clinical success rates. Optimizing these current design strategies requires an understanding of how biochemical and topographical features of the native tissue modulate keratinocyte processes involved in tissue functionality. In this thesis, a novel bioengineered skin substitute was developed that contains a microfabricated basal lamina analog that recapitulates the native microenvironment found at the dermal-epidermal junction (DEJ). In native skin, this microenvironment consists of both biochemical and topographical cues which play critical roles in maintaining tissue architecture and overall homeostasis with the external environment. Therefore, we hypothesize that microfabricated basal lamina analogs with extracellular matrix cues and three-dimensional features that mimics the cellular microenvironment of the DEJ will promote enhanced epithelialization and increase epidermal stem cell clustering on the surface of bioengineered skin substitutes. We determined that the extracellular matrix protein fibronectin (FN) found in the cellular microenvironment of the DEJ enhanced keratinocyte attachment, proliferation, and epithelialization of a collagen based basal lamina analog. It was also found that the collagen material used to create the basal lamina analog as well as the FN conjugation strategy to this material significantly influenced the bioactivity of FN and its ability to modulate keratinocyte functions through integrin based mechanism. To investigate spatial tissue organization and the role it plays in the cellular microenvironment of the DEJ on epithelialization and epidermal stem cell localization, we used photolithography coupled with materials processing techniques to create microfabricated basal lamina analogs. It was determined that epidermal thicknesses found in narrow channels of microfabricated basal lamina analogs (50 µm and 100 µm widths with 200 µm depths) were similar to cultures on de-epithelialized acellular dermis and native foreskin tissues after 7 days of in vitro culture. We also determined that the microfabricated basal lamina analogs created an epidermal stem cell niche that promoted epidermal stem cell clustering in the channels which is critical for longevity of the tissue. Overall, we developed a platform technology that was specifically used to produce a highly functional bioengineered skin substitute with regenerative capacity that mimics native skin. We anticipate through the use of this technology, we can further improve bioengineered skin substitutes by incorporating epidermal structures of native skin including hair follicles and sweat glands as well as improve overall cosmetic appearance. Additionally, this novel bioengineered skin substitute can serve as a model system to further our understanding of pathological conditions and diseases of the skin as well as facilitate robust preclinical screenings of epidermal responses to new therapeutic agents as well as to cosmetic and chemical products.

Desde o início a ecofisiologia tenta entender como o comportamento dos animais tampona os desafios ambientais. Anfíbios perdem água através da pele em taxas elevadas e assumem riscos quando buscam por água. Testamos se cinco espécies de anfíbios arborícolas diferem em na adoção de estratégias na busca de água e se diferenças interespecíficas na fisiologia e no comportamento se correlacionam com distribuição temporal e escolha microhabitat na atividade reprodutiva. Estratégias de busca de água foram classificadas como direta ou energética errática de acordo com a atividade de busca de água dos indivíduos em um labirinto. Foram registradas, também, a atividade reprodutiva dos machos durante um ano e sua associação com condições microambientais e de distribuição temporal relacionadas com a disponibilidade de água em um mesmo charco. Observamos grandes diferenças interespecíficas na atividade de busca de água. As espécies do gênero Hypsiboas demonstraram uma clara tendência para a estratégia direta, levando mais tempo para encontrar a fonte de água, em contraposição foram mais precisas. A estratégia das espécies do gênero Scinax foi classificada como energética errática, pois costumavam encontrar a fonte de água mais rápido por meio de uma busca ativa e errática. A espécie Hypsiboas bandeirantes, no entanto, tem um comportamento generalista, exibindo ambas as estratégias. A análise discriminante indicou que a estratégia tinha maior poder discriminativo quando associado à distribuição temporal da atividade reprodutiva (96.24% de classificação correta, 100% de confiabilidade na validação cruzada), sendo que espécies tímidas são mais dependentes da temporada de chuva. Dentro de variáveis de microhábitat, a estratégia energética errática poderia ser diferenciadas por ocupar microhabitats com maior umidade relativa. Estes resultados sugerem que, embora as espécies de estratégia direta concentrem sua atividade no pico da estação chuvosa, elas são independentes das condições do microambiente. Por outro lado, espécies energéticas erráticas se reproduzem quando a umidade relativa é muito alta, independente da temporada. A variação interespecífica na variável fisiológica de resistência da pele à perda de água (RPA) também pode ser discriminada com base na distribuição temporal (89.4% de classificação correta, 94.5% de confiabilidade na validação cruzada). Os valores mais baixos de RPA (H. albopunctatus < S. crospedospilus < S. hayii) são diretamente proporcionais à dependência a estação chuvosa e parece refletir tanto a adaptação à disponibilidade de água no ambiente quanto a herança filogenética, particularmente em espécies caracterizadas por baixo RPAE que tende a vocalizar em períodos de alta disponibilidade de água. Testes em mais espécies serão necessários para confirmar esses padrões / A longstading question in ecophysiology is how behavior buffers environmental challenges in animals. Amphibians loose water at high rates through skin and assume risks when searching for water. We tested if five species of treefrogs differ in water search strategy and whether differences in physiology and in behavior would correlate with temporal distribution and microhabitat choice for reproductive activity. We classified strategies as direct or energetic erratic according to individuals\' activity finding water in a maze. Temporal distribution and microenvironmental conditions related to water availability were recorded associated to males reproductive activity during a year in a same pond. We observed high interspecific differences in treefrogs activity in search for water. Species from genus Hypsiboas demonstrated a clear trend towards direct strategy, taking longer to find water source but being more accurate. Species from Scinax genus were energetic erratic, finding water source sooner by an erratic-active search. Hypsibos bandeirantes, however, had a generalist behavior, exhibiting both strategies. Discriminant analysis indicated that water search strategies had higher discriminative power when associated to temporal distribution of reproductive activity (96.24% correct classification, 100% cross-validated results), with higher dependence of species from direct search strategy to wet season. Within microhabitat variables, energetic erratic strategies could be differentiated by occupying microhabitats with higher humidity. These results suggest that while species with the direct strategy concentrate activity at the wet season peak regardless of microenvironment conditions, energetic erratic species opportunistically call when UR% is very high, independent of the season. Interspecific variation in the physiological variable, the skin resistance to water loss (RWL), can also be discriminated on the basis on temporal distribution (89.4% correct classification, 94.5%. cross-validated results). The lower RWL values (H. albopunctatus < S. crospedospilus < S. hayii) are directly proportional to their dependence to wet season and seems to reflect both adaptation to environmental water availability and phylogenetic inheritance, particularly with species characterized by low RWL tending to call at periods of high water availability. Tests on more species are necessary to confirm this pattern

A mathematical model of the interaction between chronic lymphocytic leukemia (CLL) and CD4+ (helper) T cells was developed to study the role of T cells in cancer survival. In particular, a system of four nonlinear advection diffusion reaction partial differential equations were used to simulate spatial effects such as chemical diffusion and chemotaxis on CLL survival and proliferation.

Non-polar phase selective solubility of modified poly(4-n-alkylstyrene) supports can be measured using fluorescent dyes as catalyst surrogates with thermomorphic and latent biphasic systems. By modifying the solvent compositions in heptane/ethanol and heptane/N, N-dimethylacetamide, increased non-polar phase selective solubility of modified polystyrene supports can be attained. Likewise, by varying the structure and length of the pendant alkyl chain, an increase in non-polar phase selective solubility is measured. These heptane soluble polymer supports can be useful for applications involving heptane soluble polymer-bound reagents and catalysts. Various polar and non-polar polymer supports were synthesized with an attached solvatochromic catalyst surrogates to determine the solvent accessibility of the supported species in pure and mixed solvents. The results of these studies indicate that in pure solvents, the influence of both polar and non-polar polymer supports on the solvent microenvironment of these polymer-supported probes is minimal. In mixed solvent systems, a polymer-like solvent microenvironment is measured in solvent mixtures comprised of solvents the polymer has unfavorable interactions. Poly(4-n-alkylstyrene) and internally functionalized polyisobutylene supports are two such polymer supports that exhibit this behavior. For terminally functionalized polymers in mixed solvents, the solvatochromic behavior does not indicate a collapsed structure. In mixed solvents, there is minimal influence of the polymer support on the solvent microenvironment of these terminally functionalized polymers. The application of soluble polyisobutylene supported copper complexes in the ATRP polymerization of styrene was investigated. Using the difference in solubility of the product polystyrene and the polyisobutylene copper complex in heptane, a solid/liquid separation of the soluble copper complex from the solid product was achieved. The results of these polymerizations indicate that the polyisobutylene copper complex behaves exactly like a low molecular weight copper complex in terms of control over molecular weight and molecular weight distribution. After the polymerizations, the polyisobutylene complexes could be separated as a heptane solution and recycled in multiple polymerizations of styrene.

The cellular microenvironment consists of soluble and insoluble factors that provide signals that dictate cell behavior and cell fate. Limited characterization has hindered our ability to mimic the physiological or pathophysiological environment. While stem cells have vast promise in the areas of regenerative medicine and disease therapy, harnessing this potential remains elusive due to our limited understanding of differentiation mechanisms. Similarly, many in vitro cardiac disease models lack the critical structure- function relationships of healthy and diseased cardiac tissue. The goal of this work is to induce cardiomyogenesis and pathogenesis in vitro by recapitulating features of the native microenvironment during development and disease. / Engineering and Applied Sciences

Identifying exploitable differences between cancer cells and normal cells has been ongoing since the dawn of cancer therapeutics. This task has proven difficult due to the complex genetic makeup of cancers. Tumours, however, share a low oxygen (hypoxic) microenvironment that selects for malignant cancer cells. It has recently been shown that cells switch from eIF4E to eIF4E2-mediated protein synthesis during periods of hypoxia, similar to those found in tumour cores. We hypothesize that this hypoxic translation complex is required for cell survival in hypoxia and can be targeted by inhibiting the eIF4E2 cap-binding protein. Here, we show that genetically diverse cancer cells require the cap-binding protein eIF4E2 for their growth, proliferation, and resistance to apoptosis in hypoxia, but not in normoxia. Furthermore, in vitro and in vivo eIF4E2-depleted tumour models cannot grow or sustain hypoxic regions without the reintroduction of exogenous eIF4E2. Thus, tumour cells could be targeted over somatic cells by selectively inhibiting their protein synthesis machinery, much like the function of antibiotics that revolutionized medicine.

Making up just over 3% of all new cancer cases in the United States, pancreatic cancer is not inherently a common malignant disease. Yet, it continuously is shown to be one of the most lethal and common causes of cancer death. Early detection is critical among all cancer types. However, oncologists and researchers have struggled to find effective strategies or tests to detect cancer of the pancreas early on in development. Thus, the cancer is often found late stage and requires significant chemotherapy intervention. These multi-drug treatment cocktails have shown benefit, but only in added months and not years to a patient’s life. Significant adverse effects often limit the full effective doses of treatment. In order to limit these adverse effects, as well as increase the effectiveness of treatment, we have designed, optimized, and tested unique drug carriers known as polymersomes. Using characteristics of the localized environment surrounding pancreatic tumors and the cells found therein, we created targeted therapies that are responsive and relatively selective toward cancerous cells. Herein, are found two distinct polymersomes. The first, is a low oxygen reactive drug carrier with an additional small peptide molecule that is able to penetrate dense tumor tissue and has shown decreased tumor growth of as much as 260% as compared to control samples in an animal model of pancreatic cancer. The chemical make-up of this polymersome allows for extended circulation time and a high accumulation at the tumor site. A second design, uses an intracellular enzyme to destabilize the polymersomes’ structure, which in turn, releases a selected chemotherapy drug near its intended site of action. This strategy, has shown a 10 fold increase in potency of the chemotherapy drug, as compared to when the drug is given alone and showed decreased toxicity to non-cancerous cells. It is certain that thoughtful drug delivery strategies and not just drug molecule design will be instrumental in the paradigm shift of pancreatic cancer from likely death to survival. / NIH grant 1 R01GM 114080; Grand Challenge Initiative; Office of the Dean, College of Health Professions

As immunotherapy grows in popularity as a cancer treatment option, we need to further understand how immune cells interact with the tumor microenvironment and influence tumor progression. The goal of this thesis was to characterize the different immune, cellular, and structural components within the breast tumor tissues of two orthotopic (MCaP0008 and M3C) and one spontaneous (MMTV-PyVT) murine models of immunogenic breast cancer. Identification of the tumor components in question, including CD3+ lymphocytes, CD11b+ myeloid cells, CD31+ endothelial cells, αSMA+ cancer associated fibroblasts, Ki67+ cells, cleaved caspase-3+ cells, collagen-1, and hyaluronan, were done by immunohistochemistry (IHC)-immunofluorescence (IF) staining of frozen tumor tissues with appropriate antibodies and imaging with multispectral confocal microscopy. Quantification and further data analysis were performed using a custom MATLAB program designed by Dr. Mei Rosa Ng. Gaining understanding of these stromal compositions will allow for better utilization of these breast cancer mouse models in future experiments. / 2019-10-31

Cancer is a public health burden which continues to cause many deaths and an economic burden worldwide. New and improved ways of thinking about anti-cancer drug design and development are needed now and in future. Recent reports demonstrate the key role played by the tumour microenvironment (TME) in tumour progression and the development of drug resistance. This study investigated the interactions between cancer cells and the stroma within the TME, specifically fibroblasts, mesenchymal stem cells (MSC), cancer stem cells (CSCs) as well as the extracellular matrix (ECM), with the goal to develop an in vitro model that mimics solid tumours in terms of cellular characteristics and drug response. Mesenchymal stem cells were investigated as potential sources of cancer-associated fibroblasts (CAFs) in solid tumours. The expression of CAFs markers, α-SMA and vimentin, increased significantly in MSCs co-cultured with oesophageal and breast cancer cells indicating conversion of MSCs into cell-like CAFs. WHCO1 (oesophageal) and MDA MB 231 (breast) cancer cells co-cultured with MSCs survived paclitaxel and cisplatin treatments better than cancer cells alone. To assess the prognostic value of CSCs, the expression and malignant behaviour of CSC markers were also examined in clinicopathologically-confirmed oesophageal cancer biopsies and in vitro. Oesophageal cancer biopsies stained strongly for the cancer stem cell markers, CD44 and ALDH1A1, demonstrating the presence of CSCs in these tumours. FACS-isolated side population cells exhibited high levels of cancer stem cell markers, self-renewal markers and drug resistance proteins and were associated with increased drug resistance versus cancer cells. In order to measure how ECM proteins affect oesophageal cancer cell response to chemotherapeutic drugs, 3D cell-derived ECMs was used as a model. The analysis of ECM proteins using qRT-PCR in oesophageal cancer biopsies showed that collagens, fibronectin, and laminins were overexpressed in tumour tissue compared with adjacent normal tissues. The culture of cancer cells on decellularised ECMs reduced the effect of drugs on cancer cells compared to those plated on plastic (control). The reduction of the effects of drugs was associated with significant activation of survival signalling pathways. Knockdown of collagen and fibronectin with siRNA combined with drugs resulted in increased sensitivity of cancer cells to drugs and lower colony formation and cancer cell migration. Lastly, this study utilized multi-cell tumour spheroids (MCTS) from WHCO1 and MDA MB 231 cells co-cultured with WI38 and CT1 fibroblasts to mimic tumour cell-stromal cell interactions as observed within the in vivo tumour microenvironment. The data show that spheroids were more resistant to drugs than monolayer cultures of the same cells. MCTS displayed characteristics similar to in vivo tumours in terms of response to drugs. Associated with these findings were increased levels of CSCs in MCTS compared to monolayer. This study demonstrated that MSCs are a possible source of ‘CAFs' in vivo and can support cancer cell growth. This study also demonstrated the presence of CSCs in tumours and that the targeting of these cells can shrink tumours and prevent potential metastasis and relapse of tumours. This study revealed that ECM proteins play major roles in the response of cancer cells to chemotherapy and suggest that targeting ECM proteins, especially type I collagen and fibronectin, can be an effective therapeutic strategy against chemoresistant tumours. MCTS, as shown in this study, is a valuable tool for the evaluation of the therapeutic effect of drugs. Overall, this study demonstrates the critical role played by the tumour microenvironment in tumour growth and metastasis and provides new insights into cancer treatment.