Thesis (M.Sc.)--Memorial University of Newfoundland, 2003. / Bibliography: leaves 75-86.

Thesis (Ph.D.) - University of Queensland, 2006. / Includes bibliography.

Thesis (Ph. D. in Microbiology and Immunology)--Vanderbilt University, May 2007. / Title from title screen. Includes bibliographical references.

Thesis (Ph. D.)--University of Nevada, Reno, 2008. / "December, 2008." Includes bibliographical references (leaves 257-286). Online version available on the World Wide Web.

Improved understanding of how the liver regenerates would be of great value, particularly given the dearth of therapies for end-stage liver disease. Currently, the only effective treatment for total liver failure is transplantation. Such an invasive, costly and specialised intervention is unable to address the enormous global impact from diseases of the liver. Ironically, the liver has the greatest regenerative potential of any organ in the mammalian body. However, this capacity for repair is overwhelmed in the face of massive or repeated injury. Understanding the key factors driving or inhibiting successful liver regeneration offers the potential for novel, targeted therapies to promote regeneration of a patient’s own liver. Animal models are widely used when studying complex, dynamic, multicellular processes such as liver injury and regeneration. Continued progress in transgenic modification of mice, combined with ongoing advances in microscopy techniques, means that the opportunity now exists to observe labelled cells, and subcellular structures, in real time and in vivo, with previously unobtainable resolution and fidelity. Not only does this afford the opportunity for novel insights into both normal physiology and the response to injury or disease, it can vastly expand the amount of biologically relevant information that can be obtained from each experimental animal. Hence, it is possible to advance scientific knowledge and reduce experimental animal use simultaneously. This thesis examines the role of αv integrins in liver regeneration. Integrins are expressed on the surface of cells and can perform a range of functions, including signalling and extracellular matrix adhesion. The most well-characterised role for αv integrins is activation of transforming growth factor beta, a molecule which has been shown to inhibit hepatocyte proliferation and liver regeneration. Partial hepatectomy was used as an experimental model of liver injury and regeneration. It was performed in mice, in which one or more αv integrins had been genetically depleted from specific cell types in the liver, namely hepatocytes, hepatic stellate cells or liver sinusoidal endothelial cells. These investigations revealed that depletion of integrin αvβ8 from hepatocytes led to increased hepatocyte proliferation and accelerated liver regeneration. The possible mechanisms through which hepatocyte integrin αvβ8 may exert its braking effect on liver regeneration following injury were also explored. In parallel, a novel experimental system to permit intravital multiphoton microscopy of the regenerating liver following partial hepatectomy in mice was developed and validated. Intravital imaging of mouse liver was performed with a range of cellular labels, combined with a fluorescent cell cycle reporter and label-free imaging modalities. This demonstrated the enormous potential of the system to study the dynamics of hepatocytes and non-parenchymal cells in the regenerative niche, reconstruct the sinusoidal vascular network in three dimensions during angiogenesis, and measure sinusoidal blood flow and parenchymal lipid deposition. Advances in experimental animal models such as this drive forward our understanding of the cellular and molecular mechanisms of liver regeneration whilst refining and reducing experimental animal use. Novel insights into the process of liver regeneration will permit the development of innovative therapeutic strategies to allow this remarkable organ to heal itself even in the face of massive or sustained insult.

We have shown herein that β1 integrin stably depleted PC3 sub-clonal cells confer a trend towards increased survival of mice compared to β1 integrin expressing counterparts when tested in an intracardial bone metastasis model. Therefore, we sought to investigate novel factors that mediate β1 integrin-dependent cellular migration and three dimensional growth of prostate cancer PC3 cells in vitro. We show herein that depletion of β1 integrin using siRNA directed techniques results in increased SPARC protein expression. We further show that suppression of SPARC by β1 integrin appears to occur through a JNK dependent mechanism. Moreover, siRNA mediated depletion of β1 integrin results in impaired sphere formation in 3D BME assays. This was mediated in part by the increased production of SPARC. β1 integrin-depleted cells also diminished the enhanced migration of cells on the predominant bone matrix, collagen I. Concomitant SPARC depletion in β1 integrin-depleted cells did not rescue this enhanced migration. These findings suggests that the role of β1 integrin in mediating 3D growth of PC3 cells occurs at least in part through the suppression of SPARC protein expression.

The mammary gland contains a subset of cells with regenerative capacity that is able to generate both luminal and myoepithelial mammary epithelial lineages. Those cells are described as mammary epithelial stem cells. The fate of stem cells is tightly controlled by their microenvironment and adhesion receptors on the stem cells play a vital role in the microenvironment–stem cell communication. They facilitate the interaction of stem cells with the extracellular matrix as well as adjacent cells, and they regulate stem cell homing to their niches, as well as stem cell proliferation, self-renewal, and differentiation. Stem cells express high levels of ECM binding adhesion receptors such as β1 and α6-integrins. Those integrins were used to isolate stem cells from the rest of the differentiated epithelial cells within the mammary gland. However, little is known about the role of those integrins in stem cell self-renewal and differentiation. This project aimed to understand how β1-integrin receptors contribute to stem cell behavior. To achieve this, FACS sorting method of stem cells, the organoid assay, and lentivirus knockdown of β1-integrin using shRNA were optimised. The organoid assay was used as an in-vitro test to assess for the frequency of bi-lineage and luminal progenitor cells in a given mammary epithelial population. It is known that bi-lineage cells produce solid organoids in culture while luminal progenitors produce hollow organoids. The frequency of solid and hollow organoids might therefore be an indication of the stem cells and luminal progenitor frequency respectively. My results showed that cells with the highest solid organoid forming ability were within the basal population, which is high for β1- and α6-integrin. The β1-integrin signaling pathway was shown to be important for maintaining the organoid-forming population in basal and luminal populations. Knocking out β1-integrin in MECs resulted in abolishing their solid and hollow organoid-forming activity. Downstream of β1-integrin, I found that Rac1 but not ILK is important in β1-integrin maintenance of solid organoid-forming cells. Active Rac1 was able to rescue solid organoid formation but was not able to rescue hollow organoids in the β1-integrin knockdown cells. β1-integrin and Rac1 deletion resulted in the down regulation of Wnt/β-catenin signaling, which is important for stem cells. This down regulation was rescued using active Rac1. Activating Wnt/ β1-catenin signaling in primary cells (using Wnt3a ligand or GSK3β inhibitor) resulted in an increase in solid organoid and a decrease in hollow organoid formation. When activating Wnt signaling using GSK3I in β1-integrin knockdown cells, the solid organoid activity was rescued. However, Wnt3a did not rescue solid organoid formation in the β1-integrin knockdown cells. When active Rac1 was overexpressed in β1-integrin null cells, Wnt3a was able to activate solid organoid formation. When inhibiting Rac1 in primary MECs, solid but not hollow organoid activity was significantly decreased. Wnt3a or GSK3I addition did not rescue this reduction. Taken these results together, it can be concluded that β1integrin-Rac1 signaling play a role in controlling stem cells and this is might be achieved through controlling Wnt/β-catenin signaling. These studies are important in understanding the role of integrins in mammary stem cells. They will also provide new insight on how integrins might be controlling breast cancer and thereby, help in providing new targets for cancer therapy.

Oligodendrocytes in the central nervous system (CNS) are responsible for wrapping axons with myelin in order to insulate them and allow for a faster conduction of the nervous impulse. The axonal signals that determine whether an axon is myelinated, and what regulates the number of wraps is still not fully understood. The importance of signals that initiate myelination is significant because they may point to novel therapies for Multiple Sclerosis, where remyelination prevents the axon degeneration that is thought to underlie chronic disease. Neuregulin 1 (Nrg1) has been identified as a key axonal signaling molecule that regulates myelin thickness and glial fate in the peripheral nervous system (PNS). In the PNS, neuregulin I type III is a necessary and sufficient signal that regulates axoglial interaction. The role of neuregulin in the CNS remains unclear and controversial. Integrins, the major family of extracellular matrix (ECM) receptors are involved in the regulation of many fundamental cellular functions. Interaction with a wide range of receptors including growth factor receptors is well described. Our lab showed that α6β1 integrin regulates oligodendrocyte survival signaling by amplification of neuregulin activity. We have found that mice expressing a dominant-negative β1 integrin (that reduces β1 integrin signaling independently of ligand binding) in myelinating oligodendrocytes require a larger axon diameter to initiate myelination. These results suggest that there are other signals in the axon that also contribute to initiation of myelination. We therefore hypothesized that β1 integrin and neuregulin act in concert and play a role in axoglial interactions that sense the axon size and initiate myelination. By crossing the dominant negative β1 integrin mice with heterozygous mice for neuregulin 1 and analyzing myelination, we have found that neuregulin does not enhance the phenotype previously described. This result together with previous reports that mice lacking NRG1, ErbB3 or ErbB4 (the neuregulin receptors expressed on oligodendrocytes) have normal CNS myelin sheaths demonstrates that neuregulin 1 is not required for CNS myelination. Interestingly, neuregulin 1 has been associated as a susceptibility gene in schizophrenia, a disease independently associated with myelin abnormalities (Davis et al., 2003; Hakak et al., 2001). Post-mortem brains of schizophrenic patients showed an increased level of neuregulin 1 type IV. We have analysed mice overexpressing neuregulin 1 type IV (Nrg1 type IV) and show that increased levels of neuregulin 1 type IV does not alter the brain morphology or myelin pattern and integrity. A possible explanation is that since neuregulin 1 type IV is human specific, the mice lack species-specific receptors or other neuregulins have compensatory equilibrium mechanism that are not destabilized by overexpression of neuregulin 1 type IV.

Epithelial cell adhesion to the surrounding extracellular matrix (ECM) is necessary for their proper behaviour and function. During pregnancy and lactation mammary epithelial cells (MECs) require signals imparted by specific β1 integrin-laminin interactions for their functional differentiation in response to Prolactin (Prl) and for the correct formation of polarised secretory acini. Downstream of β1 integrin (β1Itg), the scaffold protein Integrin Linked Kinase (ILK) has been identified as the key signal transducer that is required for both Prl driven lactational differentiation and the establishment of apico-basal polarity in MECs. ILK is a multifunctional adaptor protein that links integrins to the actin cytoskeleton and Rho GTPases such as Rac1. ILK forms a ternary IPP (ILK-PINCH-Parvin) complex with PINCH and Parvins, which are central to its adaptor functions. However, it is not known which of ILKs interacting partners are important for controlling tissue-specific gene expression, or what acts downstream of the IPP complex. In this thesis I have now established that inducible ILK deletion in MECs from ILKfl/flCreER mice, prevents phosphorylation of Stat5 leading to a failure of Prl induced milk expression. In addition I have established a 3-dimensional culture model using the EpH4 mammary epithelial cell line, which respond to Prl treatment and form polarised acini similar to primary cells. In these cells knocking down β1Itg and ILK by lentiviral shRNA delivery was confirmed to have a profound effect on β-Casein production. Expression of ILK mutants that disrupt its protein-protein interactions, showed that mutation of K220 and E359 in the kinase domain also reduced milk production. This means that ILKs kinase domain is important for MEC differentiation, and suggests that Parvin binding (which is disrupted by these mutations) is key in mediating ILKs differentiation functions. Using a complimentary shRNA approach, knockdown of the βParvin binding Rac guanine nucleotide exchange factor αPix also prevented MEC differentiation. This identified for the first time that αPix is required for differentiation and suggests a route by which ILK, via it’s interaction with Parvin, can link integrins to αPix and Rac activity. Interestingly, αPix depletion did not disrupt the IPP complex or polarity, suggesting that αPix represents a differentiation specific bifurcation point in β1Itg-ILK adhesive signalling. Together, this work has helped to establish how ILK is involved in MEC differentiation and has identified a new role for the downstream Rac GEF αPix. In addition, this work contributes to our understanding of the molecular mechanisms by which cell adhesion regulates fundamental cell biological behaviours.

Luminal epithelial cells in the mammary gland require two types of signals to proliferate: soluble signals (growth factor signals) and signals from the extracellular matrix (ECM). The composition of the ECM is sensed by adhesion receptors such as integrins. Integrins modulate cell behaviour and play a key role in cell cycle entry. Altered integrin expression and signalling has been associated with breast cancer and studies using mouse mammary epithelial cells (MECs) have shown that the absence of β1-integrin induces growth arrest. However, it is not completely understood how integrins transduce the signals from the plasma membrane to the nucleus to induce cell cycle entry. Thus, the first aim of this project was to determine how β1-integrin controls proliferation in MECs. I established a model to study the effects of depleting β1-integrin using the FSK7 mammary epithelial cell line. The proliferation defect observed in this β1-integrin knockdown model was rescued by expressing a constitutively active Rac1 or Pak. Moreover, inhibiting Rac1 or Pak prevented normal proliferation in MECs in a similar fashion as β1-integrin depletion. Furthermore, in this thesis I have identified the complex comprised of Src, paxillin and p130Cas as a potential link between β1-integrin and Rac1. These results provide an insight into the mechanism that regulates proliferation downstream of β1-integrin. During breast cancer initiation, β1-integrin signals are disrupted. This indicates that additional signals must be driving proliferation during tumorigenesis. Therefore, the second aim of this project was to test whether expression of breast oncogenes can overcome the proliferation defect present in β1-integrin null cells. In order to do so, an oncogenic ErbB2, a constitutively active form of Akt (myrAkt) and the Notch1 intracellular domain (NICD) were transfected in the β1-integrin knockdown MECs. The results showed that ErbB2 overcomes the need for β1-integrin by signalling to Pak. NICD does not require β1-integrin to drive proliferation by an unknown mechanism. Expression of myrAkt did not restore normal levels of proliferation in β1-integrin depleted MECs. This finding suggests that Akt is not sufficient to induce cell cycle entry by itself and instead, both Akt and Erk signalling are needed to exert this function. This work has further delineated the specific signals controlling proliferation downstream of β1-integrin, and has provided a model to test the dependence of oncogenes for β1-integrin to drive proliferation in MECs. These studies are important to understand the role of β1-integrin in breast cancer formation and to define the types of breast cancer where β1-integrin can be used as an effective therapeutic target.