Fibroblast growth factor 2 (FGF2) is a multifunctional protein translated as CUG-initiated, high molecular weight (hi FGF2) or AUG-initiated, low molecular weight (lo FGF2) isoforms with potentially distinct functions. Previous work showed that overexpression of hi- but not lo FGF2 elicited chromatin compaction resulting in cell death, by an intracrine route. A series of studies were undertaken aimed at extending our understanding of the intracrine action of Hi FGF2. Major findings are as follows: a. Hi FGF2 overexpression induces apoptotic cell death, as indicated by increased TUNEL staining, and mitochondrial participation (cytochrome c release to cytosol, rescue of the hi FGF2 phenotype by the anti-apoptotic protein Bcl-2. b. Increased expression of pro-survival signals/proteins that are known to upregulate Bcl-2, such as nuclear Akt; the PIM-1 kinase; and the heat shock protein hsp70, also rescued the hi FGF2-induced phenotype. c. The hi-FGF2 effect was associated with sustained, intracrine, activation of ERK, and was blocked by ERK inhibitors. d. FGF2 isoform specific affinity chromatography followed by mass spectroscopy identified several proteins as potentially interacting with hi FGF2; of these, the p68 RNA helicase and the hsp70 were further confirmed as interacting partners, by co-immunoprecipitation. e. Increased nuclear co-localization, and possibly interaction, between hi FGF2 and overexpressed hsp70 correlated with rescue from hi FGF2 induced cell death. f. Factors associated with cardiac pathology (isoproterenol, angiotensin II, endothelin I) also upregulated endogenous hi FGF2 in cardiac cells in culture. Adriamycin-induced cardiotoxicity in the rat, known to be linked to increased incidence of apoptosis, was also associated with increased endogenous hi FGF2. g. Hi FGF2 is expressed in the human heart (atria) and localizes in both cytosol and nuclei, suggesting a participation in human heart physiology and pathophysiology. Work presented here is consistent with the notion that endogenous hi FGF2 up-regulation may play a role in promoting cell death during prolonged tissue stress and dysfunction. It follows that processes related to hi FGF2 upregulation, hi FGF2-nuclear protein interactions and mechanisms of hi FGF2 induced cell death, represent potential therapeutic targets for modulating cell death.

Fibroblast growth factors (FGFs) lack signal sequences, and are exported through endoplasmic reticulum (ER)-Golgi-independent non-classical routes. FGFs work as modulators of various cellular activities like mitosis, differentiation, survival etc. Among the FGF family, which comprises of 23 different heparin proteins, human FGF-1 (hFGF-1), a potent angiogenic factors are one of the targets in cancer inhibition, as they are involved in blood vessel formation in tissues. There has been intensive research directed at the development of drugs that could effectively inhibit angiogenesis. In this context, the purpose of this study is to fully understand the molecular principles essential to determine probability of inhibition of hFGF-1 signaling transduction by imatinib. Imatinib, a 2-phenyl amino pyrimidine derivative is a tyrosine kinase inhibitor with antineoplastic activity. Imatinib binds to the intracellular pocket located within tyrosine kinases and inhibit the downstream cell proliferation events, but the exact molecular mechanism is still elusive. In this study, expression of hFGF-1 in recombinant E. coli was carried out, and the expressed protein was purified using heparin affinity column chromatography. The structural interactions governing imatinib-hFGF-1 interaction was studied by monitoring its stability, conformation and binding affinity by equilibrium unfolding using steady state fluorescence and proteolytic digestion assay. These data show that imatinib binds to hFGF-1 and enhances its thermal stability and solvent accessibility. In addition, biacore analysis was carried out to determine the binding affinity of imatinib to hFGF-1.

The discovery that proprotein convertase subtilisin/kexin type 9 (PCSK9) mediates degradation of low-density lipoprotein receptors (LDLR) indicates a critical role in LDL metabolism. PCSK9 is a secreted protein that binds to the epidermal growth factor-like (EGF)-A domain of LDLR and directs the receptor for degradation in lysosomes by an unknown mechanism. A gain-of-function mutation, D374Y, increases binding to LDLR EGF-A >10-fold and is associated with a severe form of hypercholesterolemia in humans. Similar to previous studies, data obtained in my project has established that PCSK9 was capable of promoting robust LDLR degradation in liver-derived cell lines; however, minimal effects on LDLR levels were detected in several lines of fibroblast cells despite normal LDLR-dependent cellular uptake of PCSK9. Importantly, a PCSK9 degradation assay showed that 125I-labeled wild-type PCSK9 was internalized and degraded equally in both hepatic and fibroblast cells, indicating dissociation of wild-type PCSK9 from recycling LDLRs in fibroblasts. Moreover, PCSK9 recycling assays confirmed that no recycling of wild-type PCSK9 to the cell surface could be detected in fibroblast cells. In contrast, more than 60% of internalized PCSK9-D374Y recycled to the cell surface in these cells, and thus had reduced ability to direct the LDLR for lysosomal degradation despite persistent binding. Co-localization studies indicated that PCSK9-D374Y trafficked to both lysosomes and recycling compartments in fibroblast cells, whereas wild-type PCSK9 exclusively trafficked to lysosomes. We conclude that two factors diminish PCSK9 activity in fibroblast cells: i) an increased dissociation from the LDLR in early endosomal compartments, and ii) a decreased ability of bound PCSK9 to direct the LDLR to lysosomes for degradation. Finally, an LDLR variant that binds to PCSK9 in a Ca2+-independent manner could partially restore wild-type PCSK9 activity, but not PCSK9-D374Y activity, in fibroblast cells.

Living organisms are made up of a multitude of individual cells that are surrounded by biomolecules and fluids. It is well known that cells are highly regulated by biochemical signals; however it is now becoming clear that cells are also influenced by the mechanical forces and mechanical properties of the local microenvironment. Extracellular forces causing cellular deformation can originate from many sources, such as fluid shear stresses arising from interstitial or blood flow, mechanical stretching during breathing or compression during muscle contraction. Cells are able to sense variations in the mechanical properties (elasticity) of their microenvironment by actively probing their surroundings by utilizing specialized proteins that are involved in sensing and transmitting mechanical information. The actin cytoskeleton and myosin-II motor proteins form a contractile (actomyosin) network inside the cell that is connected to the extracellular microenvironment through focal adhesion and integrin sites. The transmission of internal actomyosin strain to the microenvironment via focal adhesion sites generates mechanical traction forces. Importantly, cells generate traction forces in response to extracellular forces and also to actively probe the elasticity of the microenvironment. Many studies have demonstrated that extracellular forces can lead to rapid cytoskeletal remodeling, focal adhesion regulation, and intracellular signalling which can alter traction force dynamics. As well, cell migration, proliferation and stem cell fate are regulated by the ability of cells to sense the elasticity of their microenvironment through the generation of traction forces. In vitro studies have largely explored the influence of substrate elasticity and extracellular forces in isolation, however, in vivo cells are exposed to both mechanical cues simultaneously and their combined effect remains largely unexplored. Therefore, a series of experiments were performed in which cells were subjected to controlled extracellular forces as on substrates of increasing elasticity. The cellular response was quantified by measuring the resulting traction force magnitude dynamics. Two cell types were shown to increase their traction forces in response to extracellular forces only on substrates of specific elasticities. Therefore, cellular traction forces are regulated by an ability to sense and integrate at least two pieces of mechanical information - elasticity and deformation. Finally, this ability is shown to be dependent on the microtubule network and regulators of myosin-II activity.

Fibroblast growth factor 2 (FGF2) is a multifunctional protein translated as CUG-initiated, high molecular weight (hi FGF2) or AUG-initiated, low molecular weight (lo FGF2) isoforms with potentially distinct functions. Previous work showed that overexpression of hi- but not lo FGF2 elicited chromatin compaction resulting in cell death, by an intracrine route. A series of studies were undertaken aimed at extending our understanding of the intracrine action of Hi FGF2. Major findings are as follows: a. Hi FGF2 overexpression induces apoptotic cell death, as indicated by increased TUNEL staining, and mitochondrial participation (cytochrome c release to cytosol, rescue of the hi FGF2 phenotype by the anti-apoptotic protein Bcl-2. b. Increased expression of pro-survival signals/proteins that are known to upregulate Bcl-2, such as nuclear Akt; the PIM-1 kinase; and the heat shock protein hsp70, also rescued the hi FGF2-induced phenotype. c. The hi-FGF2 effect was associated with sustained, intracrine, activation of ERK, and was blocked by ERK inhibitors. d. FGF2 isoform specific affinity chromatography followed by mass spectroscopy identified several proteins as potentially interacting with hi FGF2; of these, the p68 RNA helicase and the hsp70 were further confirmed as interacting partners, by co-immunoprecipitation. e. Increased nuclear co-localization, and possibly interaction, between hi FGF2 and overexpressed hsp70 correlated with rescue from hi FGF2 induced cell death. f. Factors associated with cardiac pathology (isoproterenol, angiotensin II, endothelin I) also upregulated endogenous hi FGF2 in cardiac cells in culture. Adriamycin-induced cardiotoxicity in the rat, known to be linked to increased incidence of apoptosis, was also associated with increased endogenous hi FGF2. g. Hi FGF2 is expressed in the human heart (atria) and localizes in both cytosol and nuclei, suggesting a participation in human heart physiology and pathophysiology. Work presented here is consistent with the notion that endogenous hi FGF2 up-regulation may play a role in promoting cell death during prolonged tissue stress and dysfunction. It follows that processes related to hi FGF2 upregulation, hi FGF2-nuclear protein interactions and mechanisms of hi FGF2 induced cell death, represent potential therapeutic targets for modulating cell death.

Background: Airway fibroblasts have become a critical addition to all facets of structural lung tissue changes such as in human asthma and chronic obstructive pulmonary disease, but little is known about their role in the equine recurrent airway obstruction, a disease that resembles to the human asthma. Since the equine bronchial fibroblasts (EBF) have not been isolated and characterized yet, the use of defined medium was investigated. Results: Primary EBF were cultured on non-collagen coated flasks without serum or in the presence of feta bovine serum (FBS) or horse serum (HS) or in serum depleted medium. EBF cultured in serum-free basal media and those serum deprived were not able to proliferate and even exhibited considerable cell death. In media containing FBS or HS, proliferation of the cells was reproducible between different primary cultures and cells demonstrated expression of vimentin. Large variations were found in the ability of FBS and HS to support growth and differentiation of EBF in monolayer culture. Indications of growth-promoting actions, increasing passage number as well as maintaining fibroblast morphology were found rather in FBS than in HS. EBF culturing in HS needed longer doubling and confluence time. The protein content of the cell pellets was higher in EBF cultured in medium containing HS than FBS. Alpha-smooth muscle actin seemed to be less expressed in EBF cultured in medium containing FBS than those in HS. Conclusions: In sum, serum addition to basal EBF medium enhanced EBF differentiation into myofibroblasts, and these findings are useful to develop in vitro fibroblast culture models that mimic in vivo physiological processes and to study airway disease mechanisms and remodeling.

The Snail1 transcriptional repressor is a key factor responsible in triggering epithelial to mesenchymal transition. Although Snail1 is widely expressed in early development, it is limited in adult animals to a subset of mesenchymal cells where it has a largely unknown function. In this project we have demonstrated that Snail1 is required to maintain mesenchymal stem cells (MSCs). This effect is associated to the responsiveness to TGF-[beta]1 which showed a strong Snail1 dependence. Snail1-depletion in conditional knock-out adult animals caused a significant decrease in the number of bone marrow-derived MSCs. In culture, Snail1-deficient MSCs prematurely differentiated to osteoblasts or adipocytes and, in contrast to controls, were resistant to the TGF-[beta]1-induced differentiation block. TGF-[beta]1 was unable to up-regulate most of its targets in Snail1 KO MSCs, an effect that was related, but not limited, to defective PTEN repression and Akt activation. Correspondingly, an analysis of human sarcomas also showed enhanced expression of Snail1 in undifferentiated tumors, which was strongly associated with high expression of TGF-[beta] and poor outcome. These results not only demonstrate a new role for Snail1 in TGF-[beta] response and MSC maintenance but also suggest the involvement of MSCs in sarcoma generation. / El repressor transcripcional Snail1 ha estat descrit principalment com el responsable de la inducció de la transició epiteli mesènquima. Encara que Snail1 s’expressa durant les etapes més primerenques del desenvolupament embrionari, la seva expressió en adults es veu limitada en un conjunt de cèl•lules mesenquimals sense saber-se la seva funció. En aquest projecte hem demostrat que Snail1 es requereix per mantenir el fenotip més indiferenciat de les cèl•lules mare del mesènquima. Aquesta funció la fa en part, per la capacitat de resposta de la citoquina TGF-[beta] la qual mostra una força dependència amb Snail1. Quan s’elimina Snail1 en ratolins adults provoca una clara disminució en el nombre de cèl•lules mare de la medul•la òssia. Aquestes cèl•lules en cultiu presenten una clara diferenciació prematura a osteoblasts i adipòcits. Pel contrari, tractaments amb TGF-[beta]1 aturen la diferenciació. El TGF-[beta]1 es incapaç de incrementar moltes dianes en cèl•lules mare del mesènquima aïllades del ratolí deficient per snail1, aquest efecte en part es degut a la repressió de PTEN i l’activació de AKT. L’anàlisi de sarcomes humans ens ha mostrat una alta expressió de Snail1, el qual també es troba associada amb una alta expressió de TGF-[beta] i baixa supervivència. Aquests resultats no només demostren una nova funció per Snail1 en resposta a TGF-[beta] i el manteniment de les MSC, sinó que també suggereix que Snail1 podria participar en la generació del sarcoma.

Neurogenesis occurs in exclusive regions in the adult nervous system, the subventricular zone and dentate gyrus in the brain, and olfactory epithelium (OE) in the periphery. Cell replacement after death or injury, occurs to varying degrees in neural tissue, and is thought to be dependent upon the biological responses of stem and/or progenitor cells. Despite the progress made to identify adult OE and central nervous system (CNS) progenitors and lineage trace their progeny, our spatial and temporal understanding of embryonic OE neuroglial progenitors has been stalled by the paucity of identifiable genes able to distinguish individual candidate progenitors. In the developing CNS, radial glia serve as both neural progenitors and scaffolding for migrating neuroblasts and are identified by the expression of a select group of antigens, including nestin. Here, I show that the embryonic OE contains a novel radial glial-like progenitor (RGLP) that is not detected in adult OE. RGLPs express the radial glial antigens nestin, GLAST and RC2, but not brain lipid binding protein (BLBP), which, distinct from CNS radial glia, is instead found in olfactory ensheathing cells, a result confirmed using lineage tracing with BLBP-cre mice. Nestin-cre-mediated lineage tracing with three different reporters reveals that only a subpopulation of nestin-expressing RGLPs activate the “CNS-specific” nestin regulatory elements, and produce spatially restricted neurons in the OE and vomeronasal organ. The dorsal-medial restriction of transgene-activating cells is also seen in the embryonic OE of Nestin-GFP transgenic mice, where GFP is found in a subpopulation of GFP+ Mash1+ neuronal progenitors, despite the fact that endogenous nestin expression is found in RGLPs throughout the OE. In vitro, embryonic OE progenitors produce three biologically distinct colony subtypes, that when generated from Nestin-cre/ZEG mice, produce GFP+ neurons, recapitulating their in vivo phenotype, and are enriched for the most neurogenic colony subtype. Neurogenesis in vitro is driven by the proliferation of nestin+ progenitors in response to FGF2. I thus provide evidence for a novel neurogenic precursor, the RGLP of the OE, that can be regulated by FGF2, and provide the first evidence for intrinsic differences in the origin and spatiotemporal potential of distinct progenitors during OE development.

Wound healing is governed by a complex cascade of related processes, involving cells, extracellular matrix and cytokines. In adults this always results in a scar whilst embryonic wound healing is scarless and extensive research worldwide is aimed at reducing scarring in adults. A mathematical framework for problems in dermal wound healing is developed that incorporates models of the individual processes involved. Cells are modelled as discrete individuals. Cytokines and other biologically active factors are modelled as continua. A novel tensorial approach is taken to modelling the extracellular matrix. The numeric and computational challenges associated with combining models for the individual processes are identified and investigated. These include the development of data structures and numeric methods for the continuous and discrete species. Effective visualisation methods for the large amounts of data generated by the model are also discussed. The possibilities offered by high performance computing in mathematical biology are highlighted in this work. The final part of this thesis gives an example of a combined model of the inflammatory and proliferative phases of dermal wound healing using the new computational framework. Both quantitative and qualitative methods are used to analyse the information-rich data sets generated by the model, offering insight into the dynamic systems that can be modelled using the new approach.

Bibliography: leaves 105-136. / vi, 136, [73] leaves, [13] leaves of plates : ill. (some col) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1992