Hop (the Hsp90/Hsp70 organising protein) is a co-chaperone that acts as an adapter between the major molecular chaperones Hsp90 and Hsp70 during the cellular assembly of the Hsp90 complex. The Hsp90 complex regulates the stability and conformational maturation of a range of important cellular proteins, many of which are deregulated in cancer. In this study, we hypothesised that Hop knockdown inhibits proliferation and migration of cancer cells. We characterised the expression of Hop in cell models of different cancerous status, and provided evidence that Hop was upregulated in tumour cells compared to normal cell counterparts. Using an RNA interference approach, a 60-90% knockdown of Hop was achieved for up to 144 hours in the MDA-MB-231 and Hs578T breast cancer cell lines. Hop knockdown resulted in downregulation of the Hsp90 client proteins, Akt and Stat3, as well as a change in the expression of other Hsp90 co-chaperones, p23, Cdc37 and Aha1, while no change in the levels of Hsp90 or Hsp70 was observed. Silencing of Hop impaired cell proliferation in Hs578T cells but an increase in proliferation in MDA-MB-231, suggesting that the role of Hop in cancer cell proliferation was dependent on type of cancer cell. Hop knockdown in Hs578T and MDA-MB- 231 cells did not lead to any significant changes in the half maximal inhibitory concentrations (IC50) of selected small molecule inhibitors (paclitaxel, geldanamycin and novobiocin) in these cell lines after 72 hours. Hop knockdown cells were however, more sensitive than control cells to the Hsp90 inhibitors geldanamycin and novobiocin at earlier time points and in the presence of the drug transporter inhibitor, verapamil. Hop knockdown caused a decrease in cell migration as measured by the wound healing assay in both Hs578T and MDA-MB-231 cells. Hop was present in purified pseudopodia fractions of migrating cells, and immunofluorescence analysis showed that Hop colocalised with actin at the leading edges of pseudopodia, points of adhesion and at intercellular junctions of cells that have been stimulated to migrate with the chemokine stromal derived factor-1. Hop was able to bind to actin in vitro using actin cosedimentation assays, and silencing of Hop dramatically reduced the capacity of Hs578T cells to form pseudopodia. These results establish a correlation between Hop and actin dynamics, pseudopodia formation and migration in the context of Hop silencing, and collectively suggest that Hop plays a role in cancer cell migration. This study presents experimental evidence for a promising alternative to targeting Hsp90 and Hsp70 chaperones, a novel drug target in cancer therapy.

Breast cancer is one of the most common forms of cancer in not only South African women, but women all over the world. The molecular chaperone heat shock protein 90 (HSP90) is upregulated in cancer and is almost exclusively associated with proteins involved in intracellular signal transduction, thus it plays an important role in signalling pathways within the cell. In cancer, there is an aberrant activation of the Wnt signaling pathway, which results in stabilized β-catenin being able to translocate to the nucleus where it can trigger the transcription of oncogenes found to be involved in the self-renewal of cells. The level of β-catenin is usually kept in check by a destruction complex comprising glycogen synthase kinase 3-beta (GSK-3β), axin1, adenomatous polyposis coli (APC) which phosphorylate β-catenin, resulting in its ubiquitination and degradation. HSP90 has been found to be associated with GSK-3β, but whether this association is only transient is debatable. Very little is known about the association of HSP90 with other members of the Wnt pathway in breast cancer. In this study, we have attempted to further identify the direct associations between HSP90 and GSK-3β, β-catenin, p-β-catenin and axin1. Immunofluorescence and confocal microscopy co-localization studies suggested a potential association between HSP90 and these proteins. Treatment with HSP90 inhibitors, 17-AAG and novobiocin resulted in a shift of axin1 to what appeared to be the plasma membrane. The associations of HSP90 with GSK-3β, β-catenin, p-β-catenin and axin1 were confirmed biochemically by co-immunoprecipitation and inhibition using 17-AAG, geldanamycin and novobiocin. We showed, for the first time that HSP90 is associated in a possible complex with β-catenin, p-β-catenin and axin1 therefore is potentially involved in the modulation of p-β-catenin in the Wnt pathway through the stabilization of the destruction complex.

Trypanosomes are protozoans, of which many are parasitic, and possess complex lifecycles which alternate between mammalian and arthropod hosts. As is the case with most organisms, molecular chaperones and heat shock proteins are encoded within the genomes of these protozoans. These proteins are an integral part of maintaining the structural integrity of proteins during normal and stress conditions. Heat shock protein 40 (Hsp40) is a co-chaperone of heat shock protein 70 (Hsp70) and in some cases can act as a chaperone. These proteins work together to bind non-native polypeptide structures to prevent unfolded protein aggregrate formation in times of stress, translocate proteins across organelle membranes, and transport unsalvageable proteins to proteolytic degradation by the cellular proteasome. Hsp40s are divided into four types based on their domain structure. Analysis of the nuclear genomes of eight trypanosomatid species revealed that less than 10 of the approximate 70 Hsp40 sequences per genome were Type 1 Hsp40s, many of which contained putative orthologues in the other seven trypanosomatid genomes. One of these Type 1 Hsp40s from T b. brucei, Trypanosoma brucei DnaJ 2 (Tbj2), was functionally characterised in T brucei brucei. RNA interference knockdown of expression in T brucei brucei showed that cells deficient in Tbj2 displayed a severe inhibition of the growth of the cell population. The levels of the Tbj2 protein population in T brucei brucei cells increases after exposure to 42°c and the protein was found to have a generalized cytoplasmic subcellular localization at 37°c. These findings provide evidence that Tbj2 is an orthologue of Yeast DnaJ 1 (Y dj l), an essential S. cerevisiae protein. Hsp40s interact with their partner Hsp70s through their J-domain. The amino acids of the J-domain important for a functional interaction with Hsp70 were examined in Trypanosoma cruzi DnaJ 2 (Tcj2) (the orthologue of Tbj2) and T cruzi DnaJ protein 3 (Tcj3) by testing their ability to substitute for Y dj l in Saccharomyces cerevisae and for DnaJ in Escherichia coli. In both systems, the positively charged amino acids of Helix II and III of the J-domain disrupted the functional interaction of these Hsp40s with their partner Hsp70s. Substitutions in Helix I and IV of the J-domains of Tcj2 and Tcj3 produced varied results in the two different systems, possibly suggesting that these helices serve to define with which Hsp70s a given Hsp40 can interact. The inability of an Hsp40 and an Hsp70 to interact functionally does not necessarily mean a total absence of physical interaction between these proteins. The amino acid substitution of the histidine in the HPD motif (H34Q) of the J-domain of Tcj2 and Tcj3 removed the ability of these proteins to interact functionally with S. cerevisiae Hsp70 (Ssal) in vivo. However, preliminary binding studies using the quartz crystal microbalance with dissipation monitoring (QCM-D) show that Tcj2 and Tcj2(H34Q) both physically interact with M sativa Hsp70 in vitro. This study is the first report to provide evidence that certain trypanosoma! Type 1 Hsp40s are essential proteins. Futhermore, the interaction of these Hsp40s with Hsp70 identified important features of the functional interface of this chaperone machinery.

Mounting evidence suggests that Hsp90 is present and functionally active in the extracellular space. The biological function of extracellular Hsp90 (eHsp90) remains relatively uncharacterized compared to that of intracellular Hsp90. eHsp90 has been shown to interact with a finite number of extracellular proteins, however, despite the identification of eHsp90 interacting proteins, the function of eHsp90 in these complexes is unknown. Several reports suggest a role for eHsp90α in cell migration and invasion. Reported targets for eHsp90 stimulated cell migration include MMPs, LRP-1, tyrosine kinase receptors and possible others unidentified. Limited studies report a role for eHsp90β. Recently, Hsp90α and Hsp90β were isolated in a complex containing fibronectin (FN) on the surface of MDA-MB-231 breast cancer cells. Herein, we report direct binding of Hsp90α and Hsp90β to FN using a solid phase binding assay and surface plasmon resonance (SPR) spectroscopy. SPR spectroscopy showed that Hsp90β bound the 70 kDa amino-terminal fragment of FN (FN70), but that binding of FN to Hsp90β was not limited to FN70. Confocal microscopy showed regions of colocalization of Hsp90 with extracellular FN matrix fibrils in Hs578T breast cancer cell lines. Treatment of Hs578T breast cancer cells with novobiocin (an Hsp90 inhibitor) and an LRP-1 blocking antibody resulted in a loss of FN matrix and FN endocytosis (novobiocin treated). Addition of exogenous Hsp90β was able to recover such effect after both treatments. FN was shown to colocalize with intracellular LRP-1 in novobiocin treated Hs578T cells. Immunoprecipitation of an LRP-1 containing complex showed the presence of Hsp90 and 70 and 120+ kDa FN fragments. Treatment of Hs578T cells with novobiocin increased the level of FN120+ bound in LRP-1 immunoprecipitate. Exogenous Hsp90β decreased the level of low and high molecular weight FN fragments in a complex with LRP-1, despite the fact that higher levels of lower molecular weight FN fragments were detected in this cell lysate compared to the other treatments. We report FN as a novel interacting protein of eHsp90. Taken together, we provide evidence for a direct role of eHsp90β in FN matrix remodeling. We suggest that Hsp90 plays a direct role in FN matrix dynamics through interaction with FN and LRP-1. The identification of FN as a novel interacting protein of eHsp90 suggests a role for Hsp90 in FN matrix remodeling, which is important for a number of fundamental cellular processes including cell migration and metastasis.

Current dogma suggests that the Heat Shock Protein (Hsp) molecular chaperones and associated co-chaperones function primarily within the cell, although growing evidence suggests a role for these proteins on the plasma membrane of cancer cells. Hsp90 does not function independently in vivo, but instead functions with a variety of partner chaperones and co-chaperones, that include Hsp70 and Hsp90/Hsp70 organising protein (Hop), which are thought to regulate ATP hydrolysis and the binding of Hsp90 to its client proteins. Hsp90 on the plasma membrane appears to have distinct roles in pathways leading to cell motility, invasion and metastasis. We hypothesised that Hsp90 on the plasma membrane is present as part of a multi-chaperone complex that participates in the chaperone-assisted folding of client membrane proteins in a manner analogous to the intracellular chaperone complex. This study characterised the membrane expression of Hsp90, Hsp70 and Hop in different cell models of different adhesive and migratory capacity, namely MDA-MB-231 (metastatic adherent breast cancer cell line), MCF-7 (non-metastatic adherent breast cancer cell line), U937 and THP1 (monocytic leukemia suspension cell lines). Membrane expression of the Hsps was analysed using a combination of subcellular fractionation, biotin-streptavidin affinity purification and immunofluorescence. This study provided evidence to suggest that Hsp90, Hsp70 and Hop are membrane associated in MDA-MB-231 and MCF-7 breast cancer cells. Hsp90, Hsp70 and Hop associated with the plasma membrane such that at least part of the protein is located extracellularly. Immunofluorescence analysis showed that Hsp90, Hsp70 and Hop at the leading edge may localize to membrane ruffles in MDA-MB-231 cells, in accordance with the published role of Hsp90 in migration. An increase in this response was seen in cells stimulated to migrate with SDF-1. By immunoprecipitation, we isolated a putative extracellular membrane associated complex containing Hsp90, Hsp70 and Hop. Using soluble Hsp90 and antibodies against membrane associated Hsp90, we suggested roles for soluble extracellular Hsp90 in mediating migration by wound healing assays and inducing actin reorganisation and vinculin-based focal adhesion formation. The effects of extracellular Hsp90 are mediated by signalling through an ERK1/2 dependent pathway. An anti-Hsp90 antibody against an N-terminal epitope in Hsp90 appeared to be able to overcome the death inducing effects of a combination of SDF-1 and AMD3100, while soluble Hsp90 could not overcome this effect. We propose that this study provides preliminary evidence that extracellular Hsp90 functions as part of a multi-chaperone complex that includes Hsp70 and Hop. The extracellular Hsp90 chaperone complex may mediate cell processes such as migration by modulating the conformation of cell surface receptors, leading to downstream signalling.

To date, a significant portion of research has been devoted to understanding the biological role of the molecular chaperone, heat shock protein 90 (Hsp90), in cancer development and metastasis. Studies have alluded to over 300 clients for intracellular Hsp90, many of which are involved in oncogenic signaling pathways, making Hsp90 a bone fide drug target with several inhibitors already in clinical trials. In recent years, a limited number of extracellular Hsp90 clients have been elucidated with roles in cancer cell migration and invasion. Examples of such clients include matrix metalloproteinase-2 (MMP-2), LRP-1/CD91 and HER-2. Inhibition of extracellular Hsp90 using cellimpermeable inhibitors has been shown to reduce cancer cell migration and metastasis by a hitherto undefined mechanism. Using surface biotinylation and an enzyme linked immunosorbent assay, we provided evidence to support that Hsp90 was found extracellularly in cancers of different origin, cell type and malignancy. Next, we isolated extracellular Hsp90-containing complexes from MDA-MB-231 breast cancer cells using a cell impermeable crosslinker followed by immunoprecipitation and identified by mass spectrometry that the extracellular matrix protein, fibronectin, co-precipitated with Hsp90β. This interaction between Hsp90β and fibronectin was confirmed using pull down assays and surface plasmon resonance spectroscopy with the purified proteins. The ability of exogenous Hsp90β to increase the insoluble fibronectin matrix in Hs578T breast cancer cells indicated a role for Hsp90 in fibronectin matrix stability or fibrillogenesis. Hsp90 knockdown by RNA interference or inhibition with the small molecule inhibitor, novobiocin, resulted in a dose and time-dependent reduction of the extracellular fibronectin matrix. Furthermore, novobiocin was shown to cause the internalization of a fluorescently-labeled exogenous fibronectin matrix incorporated into the extracellular matrix by Hs578T cells. This suggested endocytosis as a possible mechanism for fibronectin turnover. This was supported by the colocalization of fibronectin with key vesicular trafficking markers (Rab-5 and LAMP-1) in small, intracellular vesicles. Furthermore, treatment with the vesicular trafficking inhibitor, methyl-β-cyclodextrin, resulted in a dose-dependent recovery in the extracellular fibronectin matrix following treatment with novobiocin. Taken together, these data provided the first evidence to suggest fibronectin as a new client of Hsp90 and that Hsp90 was involved in regulating extracellular fibronectin matrix dynamics.

Active Galactic Nuclei (AGN) are cores of distant protogalaxies, with a supermassive blackhole at the center surrounded by an accretion disk, and bipolar jets. Blazars, a subset of AGN, have their jets aligned with our line of sight. Emission from blazars is highly variable on all timescales and frequencies. Microvariability refers to rapid continuum variations that arise within the jet. Bhatta et al. (2013) suggest a modified Kirk, Rieger, \& Mastichiadis (1998) model (KRM) to explain microvariability. The KRM model assumes that when shock waves passes though the jet, each turbulent cell encountered produces a pulse of emission characterized by cell size, local density enhancement, and magnetic field strength. NASA's \kepler\ has monitored optical emission from four AGN. We use the modified KRM model to analyze micro-variations in these \kepler\ data. The distribution of cell sizes computed from these data is consistent with the distribution expected from a turbulent plasma.

Ammonium nitrate (AN) is commonly used as an explosive and as a fertilizer. In both roles it is provided as prills or pellets, approximately spherical and a few millimetres in diameter. The microstructures of several commercially-available AN compositions were investigated usingenvironmental scanning electron microscopy (ESEM) and X-ray microtomography. Those intended for explosive use were found to bemore porous than those intended for fertilizer use. The pores in explosiveprills were also found to form a connected network. The elemental composition of pellets of mixed AN and dolomite was investigated using energy-dispersive X-ray spectroscopy (EDX); the dolomite additivewas found to take the form of grains roughly 50 μm in size. The compaction behaviour of confined cylindrical beds of these prillsand pellets was studied at strain rates between 4 x 10-4 s-1 and 200 s-1. Quasi-static experiments were performed using a screw-driven instrumented press, while higher-rate experiments used a drop weight,instrumented with a line laser and load cell. The resistance of a bed to compaction was found to depend on the microstructure of its prills in most cases. Denser prills offered greater resistance to compaction. The exception to this rule was a pellet, rather than prill, formulation. Beds were also found to offer more resistance to compaction at higher strain rates. The Kawakita compaction model was found to agree well with the experimental data. A commercial fertilizer, not containing any AN, was assessed for use as an inert mock for AN prills and pellets. Prills of a suitable size for this purpose were found using EDX to consist of P2O5, with a coatingof unknown composition. They were supplied mixed with smaller K2CO3 and urea prills. The mixture was found to have comparablecompaction behaviour to AN compositions, indicating that it was useful as a mock for those compositions. In a plate impact experiment on a single layer of P2O5 prills, very little light was observed. Thisindicated that these prills were sufficiently inert for these purposes. The light produced by shocked granular ammonium nitrate beds and single prill layers was investigated using high-speed framing photography, photodiodes and gated visible-light spectroscopy. Framing photography of prill layers suggested that reaction in prill beds was dominatedby effects internal to prills. This was further supported by the similarity between photodiode recordings of prill beds and beds of inert prills containing a single reactive prills. Framing photography of drop weight experiments searching for a mechanism for initiation of reaction by interaction between prills found nothing. Decay of the light output of the beds suggested that in both granularand prill beds this light output was due to small regions heated to thousands of kelvin, which then cooled. Spectroscopic study confirmed this. These regions were found to reach a peak temperature of 6660 ± 20 K, well in excess of the approximately 2000 K predicted by a simple chemical model. Investigation of spectral lines observedduring this study indicated that the exothermic reaction that led to heating of these emitting regions involved NO.

Numerical simulations of Mach shock reflections have shown that the Mach stem can bifurcate as a result of the slip line jetting forward. Numerical simulations were conducted in this study which determined that these bifurcations occur when the Mach number is high, the ramp angle is high, and specific heat ratio is low. It was clarified that the bifurcation is a result of a sufficiently large velocity difference across the slip line which drives the jet. This bifurcation phenomenon has also been observed after triple point collisions in detonation simulations. A triple point reflection was modelled as an inert shock reflecting off a wedge, and the accuracy of the model at early times after reflection indicates that bifurcations in detonations are a result of the shock reflection process. Further investigations revealed that bifurcations likely contribute to the irregular structure observed in certain detonations.

Recent attacks and accidental explosions have demonstrated the necessity of ensuring the blast resistance of critical buildings and infrastructure in Canada such as federal and provincial offices, military buildings and embassies. Of particular importance is the blast resistance of ground-story columns in buildings which must be properly detailed to provide the necessary strength and ductility to prevent progressive collapse. There exists a need to explore the use of innovative materials that can simultaneously improve the performance of such columns, while also allowing for a relaxation of required detailing to ease construction. Advancements in concrete material science have led to the development of ultra-high performance fiber reinforced concretes (UHPFRC) which show superior mechanical properties when compared to conventional concrete, such as increased compressive strength, tensile resistance and toughness. These enhanced properties make UHPFRC an attractive material for use in the blast design of reinforced concrete columns. This thesis presents the results of a research program examining the performance of UHPFRC columns under simulated blast loads. As part of the experimental program twelve half-scale UHPFRC specimens, six built with regular grade steel reinforcement and six built with steel high-strength steel reinforcement, are tested under blast loading using the University of Ottawa shock tube. The specimens were designed according to CSA A23.3 standard requirements for both seismic and non-seismic regions, using various fibre types, fibre amounts and longitudinal reinforcement ratios, allowing for an investigation of various design parameters on blast behaviour. The results demonstrate that the use of UHPFRC improves the blast performance of columns by reducing displacements, increasing resistance and enhancing damage tolerance. The results also indicate that fiber content, fiber properties, seismic detailing, longitudinal reinforcement ratio and longitudinal reinforcement strength are factors which can affect the behaviour and failure mode of UHPFRC columns. As part of the analytical study the response of the UHPFRC columns is predicted using dynamic inelastic analysis. The dynamic responses of the columns are predicted by generating dynamic load-deformation resistance functions for UHPFRC and conducting single-degree-of-freedom (SDOF) analysis using software RC-Blast.