Small cell lung cancer (SCLC) is characterised by rapid growth, early metastatic spread and poor long-term survival. The tumour is initially sensitive to chemotherapy and thus objective response rates are high. Unfortunately, this response is often short-lived and SCLC recurs with acquired drug resistance, resulting in early patient death. Despite intensive chemo- and radiotherapy regimes survival has not improved significantly in 20 years. Prior research suggests a critical role for the tumour microenvironment in the pathogenesis of other cancers. Therefore, investigating interactions between SCLC cells and components of the tumour stroma may identify novel therapeutic targets. This thesis demonstrates that extracellular matrix (ECM) proteins present in the tumour microenvironment protect SCLC cells in vitro from chemo- and radiotherapy induced cell cycle arrest and apoptosis via cell surface β1 integrins. Pharmacological and genetic inhibition of phosphoinositol-3 kinase signalling abrogates this effect, defining a central role for this pathway in SCLC de novo drug resistance. Furthermore, the protective effect of ECM occurs without alteration in chemotherapy-induced DNA damage allowing SCLC cells to survive with new genetic defects. Integrin-mediated drug resistance has been shown to be important in other tumours and thus development of strategies to inhibit this pathway may yield new anti-cancer treatments. The design of targeted agents to down-regulate integrin-ECM interaction requires an in depth understanding of the intracellular signals that modulate integrin affinity. Two such pathways are investigated in this thesis. 1) H-Ras, a dominant suppressor of integrin affinity, acts in part through phosphorylation of Erk. Data presented here demonstrate that H-Ras also suppresses integrins through a phospholipase-C epsilon (PLCε)-dependent pathway, thus explaining discrepancies in prior data and confirming a physiological role for this recently identified phospholipase. 2) The Notch signalling pathway has been shown to have important roles in both development and cancer. It is shown here that activation of Notch signalling increases β1 integrin affinity and can protect SCLC cells from chemotherapyinduced apoptosis. However the mechanisms appear to be different; Notch-1 modulates integrin activation through the small GTPase R-Ras and Notch-2 promotes SCLC cell survival. These results define a new Notch pathway, a novel integrin modulator and a potential therapeutic target in SCLC cells. In addition to ECM proteins, the tumour microenvironment contains immune cells that may contribute to cancer growth. The cellular composition of the SCLC stroma is poorly understood. The data presented here indicate that the microenvironment of SCLC is infiltrated by lymphocytes and macrophages, the degree of which independently predicts patient survival. This suggests that the host immune system may be able to suppress SCLC growth. It is well recognised that patients with SCLC have defects in cellular immunity which correlate with survival. An in vitro coculture model was used to investigate the underpinning mechanisms, showing SCLC cells can suppress CD4+ T-cell proliferation and macrophage CD86 expression. Furthermore, preliminary data suggest a role for a soluble factor released by SCLC cells that up-regulates CD4+ T-cell production of IL-10. The work in this thesis implies a complex interaction between SCLC cells, ECM and immune cells in the tumour microenvironment. Manipulation of these pathways may have important therapeutic implications. Further investigation is required to understand the mechanisms of this interplay, which may in part be aided by prospective analysis of patient tumour samples and an in vivo model of SCLC.

The laminin-binding integrin (LBI) family are cell adhesion molecules that are essential for invasion and metastasis of human epithelial cancers and cell adhesion mediated drug resistance. We investigated whether copy number alteration (CNA) or mutations of a five-gene signature (ITGB4, ITGA3, LAMB3, PLEC, and SYNE3), representing essential genes for LBI adhesion, would correlate with patient outcomes within human epithelial-type tumor data sets currently available in an open access format.

Matrix metalloproteinase-2 (MMP-2, gelatinase A, type IV collagenase) is a member of a family of zinc-dependent metalloendopeptidases that functions in the degradation of elastin, collagens, and other components of extracellular matrix (ECM). Both secretion and activation of MMP-2 are elevated in human atherosclerotic lesions and abdominal aortic aneurysms (AAA). In this dissertation project, we sought to test the hypothesis that MMP-2 plays a critical role in both atherosclerosis and AAA. We also sought to determine the detailed mechanism. We first examined the atherosclerosis and AngII-induced AAAs development in MMP-2-/- x apolipoprotein (apoE)-/- mice in vivo. It was surprising that MMP-2 deficiency did not reduce the incidence of AngII-induced AAAs or the size of atherosclerosis in apoE-/- mice. However, the cellular and ECM content of atherosclerotic plaques were modified in MMP-2-/- x apoE-/- mice as compared to MMP-2+/+ x apoE-/- control mice. To explain the apparent paradox between this result and the hypothesis, we investigated the morphological characteristics of the aortic wall of MMP-2-/- mice. We detected an enhanced MMP-9 level in the aortic wall of MMP-2-/- x apoE-/- mice compared with MMP-2+/+ x apoE-/- mice. Interestingly, we also observed more branching of the elastin fibers in aortic wall of MMP-2-/- mice as compared with aorta of wild type mice. We also examined the behavior of macrophages from MMP-2-/- mice. Reduced adhesion, migration, and expression of integrin beta 3 were detected in MMP-2 deficient macrophages compared with wild type macrophages. Lastly, we examined whether MMP-2 deficiency in bone marrow-derived cells may influence AAAs and atherosclerosis using bone marrow transplantation technique. There was a significant reduction of both atherosclerosis development and AAAs formation in mice that were reconstituted MMP-2-/- bone marrow cells. In conclusion, the findings in this dissertation suggest that MMP-2 might play an important role in atherosclerosis and aneurysm through influencing inflammatory cell infiltration.

Sunlight is the main aetiological agent in the development of non melanoma skin cancer (NMSC), the most commonly diagnosed cancer in the fair skinned population. Mounting epidemiological and molecular data suggest that infection with cutaneous Human papillomavirus (HPV) may act as a co-factor in the early stages of NMSC development. The viral E6 protein can inhibit the apoptotic response to UV induced damage, partly through proteasomal degradation of the pro-apoptotic protein BAK. Upon UV damage, BAK undergoes a series of activating modifications that are linked to changes in phosphorylation status. BAK activation commits a cell to apoptosis through mitochondrial permeabilisation. The aim here was to elucidate the underlying mechanism of HPV5 E6-mediated BAK degradation. Mutation of lysine 113 in BAK and siRNA knockdown of a novel HPV5 E6 associated ubiquitin ligase, HERC1, prevented HPV5 E6 mediated-BAK degradation. A proximity ligation in situ assay showed an interaction between HERC1 and BAK in cells, which was dependent on the presence of the E6 protein and UV irradiation. Probing the BAK conformation and phosphorylation status suggests E6 targets a dephosphorylated BAK monomer before mitochondrial permeabilisation occurs, leading to the proposal of a mechanism for the interaction of BAK with HERC1 which depends on the activation status of BAK and a putative BH3 domain identified in HERC1. Additional work identifies β1-integrin as an interacting protein of a conserved YHDW amino acid motif at the C-terminus of certain β1 HPV E6 proteins. Only expression of E6 proteins with the YHDW motif (HPV5 and 8) in keratinocytes disrupted β1 integrin membrane localisation, altered expression of β1 integrin downstream effectors, such as focal adhesion kinase, and increased cell migration. Mutation of W157A in the HPV5 E6 YHDW motif reduced these effects. Together, this work furthers the understanding of mechanisms by which infection with HPV may promote the early stages of NMSC development.

Pathological angiogenesis is an essential component of tumor growth, development, and metastasis for which few effective therapeutic options exist. Though many cancer therapies target the function of cell surface receptors, mechanisms regulating membrane receptor crosstalk remain unclear. Two important families of receptors in angiogenesis, the Ties and Integrins, respond to the extracellular environment via outside-in and, in the case of Integrins, also inside- out signaling. Recent reports showed that the endothelial specific tyrosine kinase receptor, Tie2, forms complexes with two of the endothelial Integrin heterodimers, α5β1 and αVβ3, providing a convenient mechanism for the integration of extracellular stimuli. Our data confirm the interaction between Integrins and Tie2 and additionally indicate an interaction with the orphan co-receptor Tie1. To elucidate the biological role of these macromolecular complexes, biochemical and biophysical methods including co-immunoprecipitation, FRET microscopy, and cellular based assays were used to follow receptor/Integrin association in response to the Tie2 ligands Angiopoietin-1 and -2 as well as the Integrin ligand fibronectin. Furthermore, structural analysis by small angle x-ray scattering of Tie2-ligand complexes and specific Integrin and Tie complexes are being used to identify the basis for growth factor receptor and Integrin signal transduction.

iv Abstract Crosstalk of integrin and mTOR signalling is an essential process that monitors cellular interaction with extracellular matrix and transmits these inputs to cell growth signalling. Although adhesion status of the cell monitored by integrin signalling is clearly important for regulation of cellular growth, a little is known about the crosstalk of integrin and mTOR signalling. In this study, we employed two different approaches to describe and elucidate character of this crosstalk. p130Cas is an adaptor protein phosphorylated by Src kinase and focal adhesion kinase upon integrin ligand binding and implicated in cell adhesion, motility and survival in both Src-transformed and untransformed cells. Recently, p130Cas was also described in cellular pathology, mainly by its ability to stimulate cell invasion and metastasis. In this study, we described that p130Cas affects mTOR signalling in Src-transformed cells. Substrate domain of p130Cas was found to be indispensable for this effect and is also responsible for serum-induced activation of mTOR signalling. In addition, we prepared cell lines overexpressing various Rheb protein versions and characterized them in context of mTOR signalling, integrin signalling and cell cycle progression. Interestingly, a cell line overexpressing constitutively active...

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In dieser Arbeit sollen die funktionellen Zusammenhänge zwischen Integrinrezeptoren und myokardialer Hypertrophieentwicklung am Maus-Model nach aortic-banding untersucht werden. Durch den Einsatz eines Integrin-alphaV/Beta3 und alphaV/beta5-Inhibitors (EMD 270179 der Fa. Merck) konnten gezielt diese Integrinrezeptoren gehemmt werden. Mittels morphologischen, histologischen, immunhistochemischen, biochemischen und bildgebenden Verfahren konnten bekannte Signalwegskaskaden reproduziert und eben auch eine mangelhaft einsetzende Hypertrophieentwicklung unter Integrin-Blockade nachgewiesen werden. Innerhalb von 2-7 Tagen (Untersuchungszeitraum) setzte in der Gruppe der behandelten Tiere eine myokardiale Dilatation ein, die mit einer deulich eingeschränkten linksventrikulären Funktion einherging. Nahezu die Hälfte der Tiere verstarben konsekutiv, während die unbehandelten Tiere deutlich bessere Überlebenschancen zeigten. Ein zur transthorakalen Aortenkonstriktion vergleichbares Ereignis beim Menschen wäre eine langjährig bestehende, unbehandelte arterielle Hypertonie oder eine Aortenstenose. Beides sind jedoch Ereignisse, die in der Regel nicht derart akut einsetzen, sondern sich entwickeln. Demnach wäre ein möglicher Einsatz beim Menschen nach entsprechenden Studien zur Ermittlung des optimalen Therapie-Fensters zu diskutieren. / We examined the functional connections between integrin-receptors and cardiac hypertrophy development in mouse model after aortic-banding. By application of an alphaV/Beta3 and alphaV/beta5-inhibitor (EMD 270179 Fa Merck) these integin-receptors could be restrained. By means of morphological, histologic, immunhistologic, biochemical and picture-giving procedures known signal way cascades could be reproduced. Also a faultily starting hypertrophy development under integrin-inhibition could be proved. Within 2-7 days (investigation period) in the group of the treated animals, cardiac dilatation started which was associated with a notable depressed leftventricular function. Nearly half of the animals died consecutively, while the untreated animals showed clearly better survival chances. One to aortic-banding comparable event in human would be a for many years existing, untreated arterial hypertension or an valvular aortic stenosis. Nevertheless, both are events which start as a rule not so urgently, but develop. Therefore, a possible application in humans would be to be discussed according to suitable studies to the inquiry of the optimum therapy window.

O Histoplasma capsulatum (H.capsulatum) é um fungo dimórfico patogênico e responsável por graves lesões pulmonares, as quais se caracterizam pelo acúmulo de leucócitos ao redor do fungo, resultando na formação de granulomas. A infecção ocorre principalmente pela inalação de conídios ou pequenos fragmentos de micélio que alcançam os alvéolos, onde se transformam em leveduras, que é a forma patogênica do fungo. Na resposta imune do hospedeiro, as integrinas participam nos mecanismos fagocíticos, essenciais na resposta à histoplasmose. As 2integrinas contêm uma cadeia 2, também conhecida como CD18, comum a várias moléculas de adesão, e uma cadeia variável. Até o momento foram identificadas quatro cadeias distintas: L, a qual forma o dímero L2, também conhecido como LFA-1 (do inglês leukocyte function antigen-1) ou CD11aCD18; m, formando m2, chamado Mac-1 (do inglês macrophage differentiation antigen 1) ou CR3 (do inglês complement receptor 3) ou CD11bCD18; x, formando x2, CD11cCD18, gp150, 95 ou CR4 (do inglês complement receptor 4) e a cadeia d, formando d2, CD11dCD18. Neste trabalho, investigamos o papel da molécula CD18 em macrófagos alveolares (MAs) e macrófagos peritoneais (MPs) nas funções efetoras contra H. capsulatum e a relação do leucotrieno B4 (LTB4) nestas respostas. Inicialmente confirmamos que MAs e MPs provenientes dos animais CD18low, expressam baixa porcentagem de CD11bCD18 (CR3). Demonstramos que, como esperado, MAs e MPs de ambos os grupos fagocitam mais leveduras opsonizadas com complemento do que não opsonizadas. Surpreendentemente, MAs de animais CD18low fagocitam 136% mais leveduras opsonizadas do que MAs de C57BL/6. Também, MPs destes animais fagocitam aproximadamente 240% mais leveduras quando infectados com H. capsulatum e opsonizados, quando comparados aos MPs de C57BL/6. A adição de LTB4 aumenta a atividade fagocítica em 520% por MAs de animais C57BL/6 e 200% por MAs de CD18low, enquanto que a adição de LTB4 aumentou a fagocitose dos MPs de animais C57BL/6 em 600% vezes quando comparado aos MPs de CD18low. Este fenômeno foi inibido pela pré-incubação destas células com antagonista específico do receptor BLT1 apenas em animais C57BL/6. A adição de LTB4 na cultura de MPs reduziu a porcentagem de morte das leveduras apenas nos animais C57BL/6. Os animais CD18low produzem espontaneamente mais LTB4 e apresentaram um grande aumento na produção de óxido nítrico quando comparados aos animais C57BL/6. Pacientes acometidos pela Doença Granulomatosa Crônica (DGC) possuem deficiência congênita da molécula CD18. Células fagocíticas isoladas do sangue periférico de pacientes com DGC foram incubadas com leveduras opsonizadas e assim como macrófagos de animais deficientes de CD18, fagocitam mais leveduras opsonizadas (900%) ou não (300%), quando comparado com células de indivíduos sadios. Sugerimos que a molécula CD18 tem importante participação nos mecanismos efetores da imunidade inata, por mecanismo dependente de mediadores lipídicos, como o LTB4, no controle dos mecanismos de defesa contra H. capsulatum. / Histoplasma capsulatum (H. capsulatum) is a pathogenic dimorphic fungus and its infection is characterized by accumulation of leukocytes and granuloma formation. Infection occurs mainly by fungal inhalation that reaches the alveoli, which became yeast (the pathogenic form). in the immune response of host, integrins participate in phagocytic mechanisms, fundamental in the response against histoplasmosis.,8^2-integrin has a 02 chain known as CD18, usual to many adhesion molecules, and a variable a chain. Until the moment, it was identified four variable a chains: aL, that constitutes the dimer aL,82, also known as LFA-1 (leukocyte function antigen) or CD11aCD18; am forming the a^m,B^2 or Mac-1 (macrophage differentiation antigen 1) and CR3 (complement receptor 3) and CD11bCD18; ax, constituting the dimer Able CD11cCD18, gp150, 95 or CR4 (complement receptor 4) and ad chain, that constitutes a^d,8^2, CD11dCD18. In the present study, we sought to investigate the effect of CD18 in alveolar (AMs) and peritoneal macrophages (PMs) effecter functions against H. capsulatum and the relation of LTB^4 in those responses. We confirm that AMs and PMs of CD18\'°^W mice have low expression of ,32-integrin compared to wild type mice (WT). We demonstrate that, as expected, AMs and PMs from WT and CD18\'°^W, phagocytosed more complement (C)-opsonized yeasts than the unopsonized yeasts. Surprisingly, AMs from CD18\'°^Wmice phagocytosed 136% more (C)-opsonized yeasts than AMs obtained from WT. Also, PMs of CD18^b°^W mice phagocytosed 240% more (C)-opsonized yeasts than PMs of WT. The addition of LTB^4, increases the phagocytic activity by AMs of WT mice in 520% and by AMs from CD18\'°^W mice in 200%, while the addition of LTB^4 only increased the phagocytosis of C-opsonized H. capsulatum by PMs of C57BL/6 mice in 600%, when compaired with PMs from CD18\'°^W mice. This phenomenon was inhibited by pretreatment of these cells with an especific BLT1 receptor antagonist only in PMs from C57BU6 mice. The addition of LTB^4 in the culture of MPs reduced the percentage of death of yeasts in animals C57BL/6. CD18\'°^W mice, spontaneously produce more LTB^4 and showed a large increase in the production of nitric oxide when compared to C57BU6. Patients affected by Chronic Granulomatous Disease (DGC) have congenital deficiency of the CD18 molecule. Phagocytic cells isolated from peripheral blood of patients with DGC were incubated with C-opsonized yeasts and as well as macrophages from CD18\'°^W, phagocytosed more C-opsonized yeasts (900%) or not (300%) when compared with cells from healthy individuals.Therefore, we suggest that the CD18 molecule has important participation in the effector mechanisms of innate immunity, a mechanism dependent on lipid mediators such as LTB^4, to control these mechanisms in defense against H. capsulatum.

Infection with human gastrointestinal (GI) parasites, such as Trichuris trichiura, affects more than billion people worldwide, causing significant morbidity and health problems especially in poverty-stricken developing countries. Despite extensive research, the mechanisms of induction and regulation of effector immune responses against these parasites are incompletely understood, which hinders the development of anti-parasite therapies. Infection with GI parasite is usually chronic suggesting that parasites are capable of modulating immune responses of their host to prevent expulsion. However, mechanisms by which parasites control host immunity to allow infection are still ill-defined. The aim of this PhD study was to characterise the role of different immunoregulatory mechanisms in immunity to GI parasite infection, with a focus on dendritic cells (DCs), regulatory T cells (Tregs) and the regulatory cytokine transforming growth factor β (TGF-β).Here we showed for the first time that loss of TGF-β-activating integrin alphavβ8 specifically on DCs resulted in protection from chronic infection with Trichuris muris, a mouse model of T. trichiura infection in man. Accelerated expulsion was immune-mediated and although increased levels of protective Th2 cytokines were observed very early during infection, elevated levels of non-protective Th1 cytokines were also detected. Partial depletion of CD4+ or FcεRI+ cells had no effect on the observed phenotype. Since deletion of alphavβ8 on DCs results in decreased numbers of Tregs in the gut, we tested whether depletion of Tregs using a mouse model that allows conditional ablation of Foxp3+ Tregs (DEREG mice) would alter infection development. Although transient Treg depletion at the beginning of infection had no major effect on expulsion kinetics, we observed a tendency for enhanced Th2 responses in DEREG mice. Moreover, even though DC-mediated TGF-β activation via alphavβ8 integrin was essential for T. muris infection development, transient depletion of DCs had no effect on the induction of Th2 responses or parasite expulsion. These data indicate a novel role for the TGF-β-activating integrin alphavβ8 and DCs in regulating effector immune responses during T. muris infection and may contribute to the development of new anti-parasite therapies.