Invariants of knots coming from twisted signatures have played a central role in the study of knot concordance. Unfortunately, except in the simplest of cases, these signature invariants have proven exceedingly difficult to compute. As a consequence, many knots which presumably can be detected by these invariants are not a well understood as they should be. We study a family of signature invariants of knots and show that they provide concordance information. Significantly, we provide a tractable means for computing these signatures. Once armed with these tools we use them first to study the knot concordance group generated by the twist knots which are of order 2 in the algebraic concordance group. With our computational tools we can show that with only finitely many exceptions, they form a linearly independent set in the concordance group. We go on to study a procedure given by Cochran-Harvey-Leidy which produces infinite rank subgroups of the knot concordance group which, in some sense are extremely subtle and difficult to detect. The construction they give has an inherent ambiguity due to the difficulty of computing some signature invariants. This ambiguity prevents their construction from yielding an actual linearly independent set. Using the tools we develop we make progress to removing this ambiguity from their procedure.

Hepatocellular carcinoma (HCC) is the seventh most prevalent cancer and the third leading cause of cancer-related mortality globally. Metastasis is a major cause of mortality. HCC is also highly chemoresistant which limits treatment options to patients. Understanding the molecular mechanisms involved in these two events is of crucial significance. Deregulation of Rho/ROCK signaling is common in HCC and regulates different cellular events including cell invasion and survival. In this study, we aimed to further investigate how members of the Rho/ROCK pathway regulate HCC cell invasion and chemoresistance. By screening 71 pairs of human HCC samples using real-time qPCR, we identified that RhoE was frequently downregulated in human HCC. RhoE serves as an antagonist of the Rho/ROCK pathway. Clinicopathologically, downregulation of RhoE associated with shorter patient disease-free survival. In virto assays showed that stable knockdown of RhoE enhanced both HCC cell migration and invasion. In vivo mouse models also demonstrated that knockdown of RhoE promoted HCC invasiveness and intra-hepatic metastasis. Mechanically, knockdown of RhoE increased ROCK activity By screening 71 pairs of human HCC samples using real-time qPCR, we identified that RhoE was frequently downregulated in human HCC. RhoE serves as an antagonist of the Rho/ROCK pathway. Clinicopathologically, downregulation of RhoE associated with shorter patient disease-free survival. In virto assays showed that stable knockdown of RhoE enhanced both HCC cell migration and invasion. In vivo mouse models also demonstrated that knockdown of RhoE promoted HCC invasiveness and intra-hepatic metastasis. Mechanically, knockdown of RhoE increased ROCK activity and inhibition of ROCK reversed the effect of RhoE knockdown on cell migration. RhoE overexpression induced disassembly of stress fibers while knockdown of RhoE enhanced formation of plasma membrane blebs. These findings suggested that RhoE acts as a metastatic suppressor in HCC via inhibiting Rho/ROCK signaling. Downregulation of RhoE can increase ROCK activity which is reported to regulate cell survival. Therefore we investigated if the frequent downregulation of RhoE contributes to the high chemoresistance in HCC cells. Stable knockdown of RhoE suppressed cell death/apoptosis induced by chemotherapeutic agents such as cisplatin and doxorubicin. This effect could be reversed by addition of ROCK inhibitor. In vivo mouse model also confirmed that RhoE knockdown augmented HCC chemoresistance. We also observed that combined treatment of cisplatin and ROCK inhibitor profoundly inhibited tumor growth in nude mice. This part of our findings indicated that RhoE/ROCK played an important role in regulating chemoresistance in HCC. We further identified two downstream molecular pathways which were involved in Rho/ROCK-induced chemoresistance. We found that STAT3 and JAK2 were activated by RhoE knockdown but inhibited by addition of ROCK inhibitor. Upon ROCK inhibition, expression of IL-6 and IL-6 receptor were suppressed and the transcription activating activity of STAT3 was also repressed. Finally, ROCK inhibition attenuated Erk1/2 activation. Literature searching suggested nuclear PTEN as a potential candidate for inactivating Erk1/2. We demonstrated that inhibition of ROCK increased the population of nuclear PTEN while overexpressing ROCK2 decreased it. Overexpression of nuclear PTEN alone could already reduce Erk activation in HCC cells. Our findings indicated that RhoE/ROCK may exert their effects on chemoresistance in HCC via regulating the IL-6/JAK2/STAT3 and PTEN/Erk pathways. In conclusion, our study demonstrated the important role of RhoE in HCC. First, aberrant underexpression of RhoE promoted HCC invasion and intra-hepatic metastasis through upregulating the Rho/ROCK signaling. Second, downregulation of RhoE increased activity of the pro-survival IL-6/JAK2/STAT3 and Erk signalings to enhance chemoresistance in HCC cells. Our findings also suggested the Rho/ROCK signaling to be potential therapeutic target in anti-metastatic and chemo-sensitizing therapy. / published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

Glioblastoma (GB) is the highest grade and most common form of primary adult brain tumors, characterized by a highly invasive cell population. GB tumors develop treatment resistance and ultimately recur; the median survival is nearly fifteen months and importantly, the invading cell population is attributed with having a decreased sensitivity to therapeutics. Thus, there remains a necessity to identify the genetic and signaling mechanisms that promote tumor spread and therapeutic resistance in order to develop new targeted treatment strategies to combat this rapidly progressive disease. TWEAK-Fn14 ligand-receptor signaling is one mechanism in GB that promotes cell invasiveness and survival, and is dependent upon the activity of multiple Rho GTPases including Rac1. Here, we show that Cdc42 is essential in Fn14-mediated Rac1 activation. We identified two guanine nucleotide exchange factors (GEFs), Ect2 and Trio, involved in the TWEAK-induced activation of Cdc42 and Rac1, respectively, as well as in the subsequent TWEAK-Fn14 directed glioma cell migration and invasion. In addition, we characterized the role of SGEF in promoting Fn14-induced Rac1 activation. SGEF, a RhoG-specific GEF, is overexpressed in GB tumors and promotes TWEAK-Fn14-mediated glioma invasion. Moreover, we characterized the correlation between SGEF expression and TMZ resistance, and defined a role for SGEF in promoting the survival of glioma cells. SGEF mRNA and protein expression are regulated by the TWEAK-Fn14 signaling axis in an NF-kB dependent manner and inhibition of SGEF expression sensitizes glioma cells to TMZ treatment. Lastly, gene expression analysis of SGEF depleted GB cells revealed altered expression of a network of DNA repair and survival genes. Thus TWEAK-Fn14 signaling through the GEF-Rho GTPase systems which include the Ect2, Trio, and SGEF activation of Cdc42 and/or Rac1 presents a pathway of attractive drug targets in glioma therapy, and SGEF signaling represents a novel target in the setting of TMZ refractory, invasive GB cells.

Stat3 (signal transducer and activator of transcription-3) is activated by a number of receptor and non-receptor tyrosine kinases, while a constitutively active form of Stat3 alone is sufficient to induce neoplastic transformation. Results presented in this thesis reveal that Stat3 can also be activated through homophilic interactions by the epithelial (E)-cadherin and cadherin-11, two members of the classical type I and II cadherin family of surface receptors, responsible for the formation of cell to cell junctions. Indeed, by plating cells onto surfaces coated with fragments encompassing the two outermost domains of these cadherins, we definitively demonstrate that cadherin engagement can activate Stat3, even in the absence of direct cell to cell contact. At the same time, levels of the extracellular signal regulated kinase (Erk)1/2, which is often coordinately activated by growth factor receptors and oncogenes, remain unchanged upon cadherin ligation. Most importantly, we report, for the first time, an unexpected surge in total Rac1 and Cdc42 protein levels, triggered by cadherin engagement, and an increase in Rac1 and Cdc42 activity, which is responsible for the Stat3 stimulation observed. Inhibition of cadherin interactions reduced Rac/Cdc42 and Stat3 levels and induced apoptosis, pointing to a significant role of this pathway in cell survival signalling, a finding which could also have important therapeutic implications. To better understand the role of Rac/Cdc42 in the cadherin-mediated Stat3 activation, we compared Stat3 activity in mouse HC11 cells before and after expression of the mutationally activated, RacV12. We demonstrate a dramatic increase in protein levels and activity of both the endogenous Rac and RacV12 with cell density, which was due to inhibition of proteasomal degradation. Moreover, we clearly show that RacV12 expression can activate Stat3 through an increase in expression of members of the IL6 family of cytokines, known potent Stat3 activators. In fact, knockdown experiments indicate that gp130 receptor function, and Stat3 activation, are essential for the migration and proliferation of RacV12-expressing cells, thereby demonstrating that the gp130/Stat3 axis represents an essential target of activated Rac in the regulation of both of these fundamental cellular functions. / Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2010-02-18 10:38:29.549

The purpose of this project was to determine how Dlc-2 and Rho signaling modulate the ceramide induction of PGP synthase. This induction was studied at the transcriptional, post-transcriptional, and post-translational levels using cell culture, Real-Time RT-PCR, protein purification, phage display, and western blotting techniques. We have demonstrated that the PGP synthase gene is not controlled at the transcriptional level by ceramide and Rho, nor is the mRNA stability of PGP synthase affected. However, ceramide and Rho do seem to exhibit translational or post-translational control over the PGP synthase protein. The relationships between Dlc-2 (and Rho), ceramide, and PGP synthase (and CL) are important to understand. All three are involved in cancer and apoptotic responses. The knowledge gained by the experiments discussed in this thesis will contribute to an understanding of how these proteins and lipids interact. This knowledge may then be used in the future to develop cancer treatments.

It is important to understand the mechanism of endocytic invasion into the host cell by Staphylococcus aureus. Activation of phosphoinositide-3-kinase (PI3K) is essential to S. aureus invasion. In a normal cell, the p85 subunit of PI3K is bound at the Rho GTPase activating protein (RhoGAP) domain to small guanosine triphosphate binding proteins (GTPases), which are attached to the cell membrane by a prenyl group. This association anchors PI3K near the cellular membrane. PI3K must be anchored near the membrane in order to phosphorylate its substrate. The hypothesis for this project is that deletion of the binding domain between PI3K and small GTPases will block endocytic bacterial invasion by sequestering PI3K in the cytosol. To investigate this hypothesis, the RhoGAP binding domain of PI3K p85 was mutated using site-directed mutagenesis and S. aureus invasion was reduced by up to 86% (p<0.05), which shows that this domain is important to bacterial invasion. / Department of Biology

Access to abstract restricted until May 2015 / Access to thesis restricted until May 2015 / Department of Biology

Staphylococcus aureus infections have become a widespread problem. Simvastatin decreases S. aureus invasion. Simvastatin use reduces prenylation of target proteins, including CDC42. Prenylated CDC42 is active at the cell membrane. Our hypothesis is that CDC42 activity at the cell membrane is needed for endocytic S. aureus invasion. The prenylation site on CDC42 was deleted and mutant CDC42 (CDC42C5O7V/V5) was transfected into mammalian cells, which were exposed to S. aureus. Decreased bacterial infection of up to 90% was seen in cells stably expressing CDC42C507V/V5. Mammalian cells were treated with secramine A, an inhibitor of CDC42 activity, and exposed to S. aureus. Decreased bacterial invasion of 70% in these cells was seen. These findings suggest that CDC42 activity at the cell membrane is needed for S. aureus cell invasion. These findings increase understanding of the mechanism of S. aureus cell invasion and could be used to develop new treatment or prevention methods. / Department of Biology

The purpose of this project was to determine how Dlc-2 and Rho signaling modulate the ceramide induction of PGP synthase. This induction was studied at the transcriptional, post-transcriptional, and post-translational levels using cell culture, Real-Time RT-PCR, protein purification, phage display, and western blotting techniques. We have demonstrated that the PGP synthase gene is not controlled at the transcriptional level by ceramide and Rho, nor is the mRNA stability of PGP synthase affected. However, ceramide and Rho do seem to exhibit translational or post-translational control over the PGP synthase protein. The relationships between Dlc-2 (and Rho), ceramide, and PGP synthase (and CL) are important to understand. All three are involved in cancer and apoptotic responses. The knowledge gained by the experiments discussed in this thesis will contribute to an understanding of how these proteins and lipids interact. This knowledge may then be used in the future to develop cancer treatments.

<p>Essential and diverse biological processes such as cell division, morphogenesis and migration are regulated by a family of molecular switches called Rho GTPases. These proteins cycle between active, GTP-bound states and inactive, GDP-bound state and this cycle is regulated by families of proteins called Rho GEFs and GAPs. GAPs are proteins that stimulate the intrinsic GTPase activity of Rho-family proteins, potentiating the active to inactive transition. GAPs target specific spatiotemporal pools of GTPases by responding to cellular cues and utilizing protein-protein interactions. By dissecting these interactions and pathways, we can infer and then decipher the biological functions of these GAPs.</p><p>This work focuses on the characterization of a novel Rho-family GAP called srGAP2. In this study, we identify that srGAP2 is a Rac-specific GAP that binds a Formin-family member, Formin-like 1 (FMNL1). FMNL1 is activated by Rac and polymerizes, bundles and severs actin filaments. srGAP2 specifically inhibits the actin severing of active FMNL1, and the assembly of an srGAP2-FMNL1 complex is regulated by Rac. Work on FMNL1 shows that it plays important roles in regulating phagocytosis and adhesion in macrophages. To learn more about srGAP2 and its role in regulating FMNL1, we studied macrophages isolated from an srGAP2 KO mouse we have recently generated. This has proven quite fruitful: loss of srGAP2 decreases the ability for macrophages to invade through extracellular matrix but increases phagocytosis. These results suggest that these two processes might be coordinated in vivo by srGAP2 and that srGAP2 might be a critical regulator of the innate immune system.</p> / Dissertation