Cancer immunotherapy aims to use and manipulate the host’s immune system to fight against cancer. The objective of this strategy is to induce specific and persistent immune responses leading to tumor eradication. Heat shock proteins (HSP) purified from cancer tissues have been identified as unique mediators of specific anti-tumor immunity. In our laboratory, we have developed an original vaccine, termed CRCL (Chaperone-Rich Cell Lysate) that consists of multiple HSP complexes enriched from tumor lysates. CRCL immunization leads to an efficient protection against a wide variety of murine cancers by inducing a strong, long-lasting, and specific T and NK-cell dependent immune responses against the tumor from which it has been generated. Tumor-derived CRCL has been shown to be more efficient in triggering DC activation than individual purified HSP or tumor lysates. The immunostimulatory effects of CRCL arise from its superior ability to provide a wide variety of tumor antigens to the immune system and by providing potent adjuvant effects. However, CD4⁺CD25⁺ regulatory T lymphocytes (Treg) critically contribute to the mechanisms of cancer-induced suppression. Data from independent groups including ours suggests they may also restrain the function of antigen presenting cells. The current study was designed to elucidate the molecular signaling events triggered by the tumor-derived CRCL vaccine in antigen presenting cells and evaluate whether CRCL may overcome the inhibitory effects of Treg modulation of DC and macrophage activation. Our results indicate CRCL activates DC and macrophages by inducing proinflammatory cytokine chemokine secretion. CRCL induces iNOS expression and NO production in macrophages. CRCL activation of DC and macrophages results in transcription factor NF-κB activation in vitro and in vivo, and this includes the activation of additional signaling molecules upstream of NF-κB. Following CRCL treatment the phenotypic maturation of DC, the production of DC and macrophage pro-inflammatory cytokines, and the activation of the transcription factor NF-κB are not affected by Treg. Additionally, CRCL induced activation of DC is not diminished by the immunosuppressive cytokine TGF-β 1. Our results indicate tumor-derived CRCL-treated DC and macrophages are refractory to Treg inhibition. These results are important for advancing CRCL-based vaccines in Phase I clinical trials.

Despite the importance of proper synaptogenesis in the CNS, the molecular mechanisms that regulate the formation and development of synapses remain poorly understood. Indeed, the mechanisms through which initial synaptic contacts are established and modified during synaptogenesis have not been fully determined and a precise understanding of these mechanisms may shed light on synaptic development, plasticity and many CNS developmental diseases. The development and formation of spiny synapses has been thought to occur via filopodia shortening followed by the recruitment of proper postsynaptic proteins, however the precise function of filopodia remains controversial. Thus the goal of this study was to investigate the dynamics of dendritic filopodia and determine their role in the development of synaptic contacts. We initially define and characterize short lipidated motifs that are sufficient to induce process outgrowth. Indeed, the palmitoylated protein motifs of GAP-43 and paralemmin are sufficient to induce filopodial extensions in heterologous cells and to increase the number of filopodia and dendritic branches in neurons. We showed that the morphological changes induced by these FIMs (filopodia inducing motifs) require on-going protein palmitoylation and are modulated by a specific GTPase, Cdc42, that regulates actin dynamics. We also show that their function is palmitoylation dependent and is dynamically regulated by reversible protein palmitoylation. Significantly, our work suggests a general role for those palmitoylated motifs in the development of structures important for synapse formation and maturation. We combined several approaches to monitor the formation and development of filopodia. We show that filopodia continuously explore the environment and probe for appropriate contacts with presynaptic partners. We find that shortly after establishing a contact with axons, filopodia induce the recruitment of presynaptic elements. Remarkably, we find that expression of acylated motifs or the constitutively active form of cdc-42 enhances filopodia number and motility, but reduces the recruitment of synaptophysin positive presynaptic elements and the probability of forming stable axo-dendritic contacts. We provide evidence for the rapid transformation of filopodia to spines within hours of imaging live neurons and reveal potential molecules that accelerate this process.

Pathogenic Gram-negative Yersinia species preferentially target and inactivate phagocytic cells of the innate immune defense by translocation of effector Yersinia outer proteins (Yops) into the cells via a type III secretion system. This indicates that inactivation and avoidance of the early innate immune response is an efficient way for Yersinia species to avoid elimination and to cause diseases ranging from mild gastroenteritis (Y. pseudotuberculosis and Y. enterocolitica) to plague (Y. pestis). In this project, we aimed to study the interaction between enteropathogenic Y. pseudotuberculosis and phagocytic cells during early infection. In situ interaction studies on infected intestinal tissues showed that Y. pseudotuberculosis mainly interacts with dendritic cells (DCs) in lymphoid tissues of the intestine during initial infection. After massive recruitment of polymorphonuclear neutrophils (PMNs) to the infected tissues, wild-type (wt) bacteria also interacted with this phagocyte. In contrast to the wt, mutants lacking the anti-phagocytic effectors YopH and YopE are avirulent in mice and unable to spread systemically. Interestingly, our interaction assay showed that these mutants not only interacted with DCs, but also with PMNs during the initial stage of infection. Thus, indicating that Y. pseudotuberculosis can avoid interaction with PMNs during early infection and that this is Yop-dependent. In a phagocytosis assay Y. pseudotuberculosis was demonstrated to inhibit internalization by DCs in a YopE-dependent manner, while both YopH and YopE were shown to be involved in the blocking of phagocytosis by macrophages and PMNs. Thus, indicating that YopH has cell type-specific effects. To further investigate the role of DCs during initial stages of infection, a mouse DC depletion model (CD11c-DTRtg) was applied. However, the DTx-mediated depletion of DCs in CD11c-DTRtg mice induced neutrophilia and the model could not give a definite answer to whether DCs play an important role in either restricting or stimulating progression of Y. pseudotuberculosis infection. To investigate involvement of PMNs during early infection mice were injected with the depleting antibody α-Ly6G. In absence of PMNs wt, as well as yopH and yopE mutants became more virulent, which further supports the importance of these Yops for the ability of Y. pseudotuberculosis to disseminate from the initial infection sites in the intestine to cause systemic disease. In summary, our studies show that inhibiting internalization and maturation of DCs and avoiding phagocytosis by and interaction with macrophages and PMNs during the early stages of infection are important virulence strategies for Y. pseudotuberculosis to be able to colonize tissues, proliferate and disseminate systemically.

In a targeted proteomic screen to identify polarity protein complexes, a number of Scribble (Scrib) -associating proteins were identified; of particular interest was the Nitric Oxide Synthase 1 Adaptor Protein (NOS1AP). NOS1AP contains an N-terminal phosphotyrosine binding (PTB) domain and a C-terminal PSD-95/Dlg homology/ZO-1 (PDZ) binding motif that associates with neuronal NOS (NOS1). We show that the PTB domain of NOS1AP associates with the fourth PDZ domain of Scrib. We identified NOS1AP binding partners including three key regulators of dendritic spine formation, beta-Pix, Git1, and PAK, which require Scrib to associate with NOS1AP. Overexpression of NOS1AP in cultured hippocampal neurons increases dendritic protrusions, a process dependent on the PTB domain. The increase in dendritic protrusions can be blocked by the co-expression of a dominant negative Rac construct. NOS1AP, and the PTB domain of NOS1AP influence Rac activity. Together these data suggest that Scrib and NOS1AP function as important scaffolding proteins in the mammalian synapse and that NOS1AP functions in the dendritic spine by influencing Rho GTPase activity.

Among the complications of current treatments for hemophilia A, the development of anti-FVIII antibodies including “FVIII inhibitors” remains the major clinical problem in treating hemophiliacs. Factor VIII inhibitors work through neutralizing the coagulation cofactor activity of the infused FVIII and preventing the restoration of normal hemostasis. This thesis explains the influence of genetic background on the generation of FVIII inhibitors, introduces a new pre-clinical approach that reduces the immunological response towards FVIII and predicts the in vivo behavior of recombinant and plasma-derived FVIII products in hemophilic patients. First, we studied the influence of the genetic background on the formation of FVIII antibodies by treating hemophilia A Balb/c and C57BL/6 mice with repetitive FVIII infusions. We observed that the C57BL/6 mice developed higher FVIII antibody titers than the Balb/c mice. Our results suggest that differences in the cytokine immune responses due to FVIII in Balb/c and C57BL/6 mice are responsible for the different FVIII antibody titers in each of these strains. Second, we investigated the use of FVIII-pulsed immature dendritic cells in inducing immune tolerance against FVIII prior to the FVIII treatment. We showed that in vivo, FVIII does not induce the activation and proliferation of hemophilic T cells. Furthermore, infusing FVIII-pulsed immature dendritic cells into hemophilic mice resulted in a long-term reduction in immune reactivity towards FVIII. Also, we have proposed methods on how to improve the tolerogenic abilities of dendritic cells. Our results indicate that the immature dendritic cells induced the formation of T regulatory cells and that these T regulatory cells were responsible for the observed reduction in immune reactivity. Finally, we were able to identify the mechanisms behind the immune system activation in mice treated with either recombinant or plasma-derived FVIII products. We showed that plasma-derived FVIII results in reduced FVIII antibody titer formation in hemophilic mice. Our results demonstrate that the differences in antibody formation in hemophilic mice treated with either recombinant or plasma- derived FVIII products are due to the distinct cytokine micro-environment induced by each product. This thesis contributes to the current knowledge on FVIII immunology and the in vivo behavior of FVIII in hemophilic mice. The results generated from this thesis can be used to modify the available FVIII treatments in order to minimize the immunological complications of FVIII and improve the quality of life of hemophilic patients. / Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2008-08-14 18:22:51.56

<p>Development in the field of materials science is propagated by the synthesis of polynuclear metal-containing complexes, that exhibit enhanced chemical and physical properties. This thesis describes the synthesis of new metal-containing linear polymers and dendritic molecules.</p> <p>Chapter 1 presents an overview of the field of metal-containing polymers, with particular attention to the synthesis of polymers via condensation polymerisation. This review includes the various types of metal-containing condensation polymers and the applications of these materials, where available. This discussion is followed by a brief summary of metal-containing dendrimers, which includes a concise description of their structure and applications in general.</p> <p>There are two routes to preparing metal-containing polymers. Chapter 2 describes the synthesis of three bifunctional organometallic monomers, of the general type [M]-O-{2,6-(CH2OH)2-4-CH3-C6H2}, where [M] represents the various metal-containing moieties, (&eta / 5-C5H5)(CO)2 Fe(CH2)3 (25), (&eta / 5-C5H4-CH2CH2CH2-)Re(CO)3 (26) and Fpdendr (27). These monomers were prepared using 2,6-bis(hydroxymethyl)-p-cresol as the key reagent. The monomers were used in classical polycondensation reactions with terephthaloyl chloride using ambient temperature solution techniques. This yielded new low molecular weight oligomeric polyesters, that were characterised using FTIR and 1HNMR spectroscopy, differential scanning calorimetry, thermogravimetric analysis and sizeexclusion chromatography.</p> <p>In Chapter 3, an alternate route to metal-containing polymers is described. In this case, bifunctional organic monomers were polymerised to give preformed organic polymers. Two types of organic polymers were prepared, viz. polyesters (with pendant vinyl moieties) and polyimines (with &alpha / -diimine units along the polymer backbone). Functionalisation of these preformed organic polymers with various metal sources was attempted. Hydrozirconation reactions of the vinyl polyesters with Schwartz&rsquo / s reagent, Cp2Zr(H)Cl, were attempted and were largely unsuccessful. Competing reactions with the ester functionality prevailed, preventing the desired reaction. Reaction of the polyimines with PdCl2(COD) yielded insoluble, intractable metal-containing oligomers. Partial characterisation of the complexes is described.</p> <p>The synthesis of new poly(propylene imine) iminopyridyl metallodendrimers is described in Chapter 4. Schiff-base condensation reaction of the commercially available DAB dendrimers with 2-pyridinecarboxaldehyde, gave the dendrimers 51, 52, and 53, with four, eight and sixteen pyridylimine functionalities respectively on the periphery. Successful complexation reactions with PdCl2(COD), PtCl2(COD) and CuCl2 produced the corresponding metal-containing dendrimers, with either PdCl2 (54, 55, 56), PtCl2 (57) or CuCl2 (58) moieties bound on the periphery. The metallodendrimers were insoluble in the more common organic solvents, and were characterised by IR and 1H-NMR spectroscopy and microanalysis where possible. Dendrimers with salicylaldiminato ligands on the periphery were prepared by reacting the DAB dendrimers with salicylaldehyde. These ligands were reacted with various metal acetates in an attempt to prepare new metalcontaining salicylaldimine dendrimers. This work yielded either paramagnetic metal complexes or insoluble, intractable compounds.</p> <p>Chapter 5 describes the applications of the catalyst precursors (54, 55, 56, 57, 58), discussed in Chapter 4, in the polymerisation of ethylene and the use of complexes 54 and 55 as Heck cross-coupling catalyst precursors. The complexes all showed catalytic activity toward ethylene polymerisation. A discussion of their activity, the polyethylene molecular weight and microstructure is presented in this chapter. The precursors 54 and 55 are also effective catalysts in the Heck reactions, coupling iodobenzene with methyl acrylate, styrene and 1-octene in high conversions.</p> <p>General conclusions are given in Chapter 6.</p>

The current study aimed to determine the selective advantage of lateral amygdala (LA) neurons overexpressing the transcription factor CREB that enables their preferential incorporation into the fear memory trace. I hypothesized that overexpression of CREB drives the formation of dendritic spines, potentially providing these neurons with greater connectivity to sensory inputs at the time of learning. Using viral-mediated gene transfer, CREB tagged with GFP, or GFP as a control, was overexpressed in the LA of wild-type mice. Spine number and morphology were compared in homecage mice at the time when mice are normally trained in fear conditioning. Spine density was increased in neurons with CREB vector compared to neurons with GFP vector whereas spine head diameter and length was not different. Therefore, LA neurons overexpressing CREB have increased spine number at the time of learning, potentially providing these neurons with a selective advantage for incorporation into the fear memory trace.

The current study aimed to determine the selective advantage of lateral amygdala (LA) neurons overexpressing the transcription factor CREB that enables their preferential incorporation into the fear memory trace. I hypothesized that overexpression of CREB drives the formation of dendritic spines, potentially providing these neurons with greater connectivity to sensory inputs at the time of learning. Using viral-mediated gene transfer, CREB tagged with GFP, or GFP as a control, was overexpressed in the LA of wild-type mice. Spine number and morphology were compared in homecage mice at the time when mice are normally trained in fear conditioning. Spine density was increased in neurons with CREB vector compared to neurons with GFP vector whereas spine head diameter and length was not different. Therefore, LA neurons overexpressing CREB have increased spine number at the time of learning, potentially providing these neurons with a selective advantage for incorporation into the fear memory trace.

In this study, two semi-analytical models of microsegregation were developed to predict the concentration fields of solute in the liquid and solid regions for dendritic solidification of binary metallic alloys. Both models assume that the growing dendrites are cylindrical in shape. This assumption is more realistic compared to the common assumptions of plate-like dendrites that most of the earlier researchers employed in their microsegregation modeling study. The solute redistribution profile, in the developing solid layer, necessary to determine the back diffusion parameter was derived from Fick's second law for the model without coarsening. The application of this parameter in a wide range of conditions and the use of its basic form in the model with coarsening was verified. The concept of coordinate transformation and enhancement of back-diffusion Fourier number were used in deriving the model which took into account the coarsening of dendrites. The models are then extended to deal with rapid solidification, peritectic transformations and multi-component systems and the results were compared with relevant experimental data. A good agreement between the model predictions and experimental results was found. / The second part of this study was focused on developing a fully numerical microsegregation model. The numerical model built upon a previously proposed phase change model which relied upon a coordinate transformation technique. The model was extended to deal with moving boundaries with solute diffusion. A suitable computational procedure was developed to solve the model equations which are strongly coupled to each other. To verify the accuracy of the present algorithm with regard to the capability of tracking the moving interfaces, analytical solution of the Stefan problem was used for verification purposes. A good agreement between the model predictions and the analytical solution was found. Evolution of concentration fields during solidification was calculated in the growing solid as well as in the shrinking liquid regions for rectangular, cylindrical and spherical dendrite geometries. The effects of various cooling conditions and relevant parametric values on microsegregation were analyzed and discussed.

Dendritic cells (DCs) are specialized members of the innate immune system that are responsible for the initiation of primary adaptive immune responses whose purpose is to resolve infection and inflammation. During most viral infections, mature dendritic cells present critical viral antigens to naïve T-cells within secondary lymphoid organs, resulting in the generation of an antigen-specific adaptive immune response and clearance of the virus. During infection with HIV-1 however, the virus is not cleared and a chronic systemic infection develops characterized by immune dysfunction, CD4+ T-cell depletion, systemic inflammation, and opportunistic infections. A growing body of evidence indicates that HIV-1 subversion of DCs contributes to both HIV-1 pathologies and viral dissemination. A number of similar effects by accessory HIV-1 peptides on DC physiology have also been reported. In vitro studies demonstrate that HIV-1 inhibits DC maturation and function. Ex vivo studies on the other hand describe partially mature, dysfunctional DCs collecting in secondary lymphoid organs. In vitro studies examining the effects of HIV-1-Tat and HIV-1-Vpr have described opposing effects on DC maturation. Therefore we undertook experiments to comprehensively describe the effects of HIV-1 and the Tat and Vpr accessory peptides on DC maturation and function. To understand the contributions of individual viral proteins to DC dysfunction we infected DCs with a dual tropic HIV-1 and examined phenotypic and functional changes after maturation with inflammatory cytokines. Following this we examined the influence of exogenous and endogenous HIV-1-Tat and HIV-1-Vpr on MDDC maturation and function using recombinant proteins and deletion mutant lab adapted HIV-1 strains. Live dual tropic HIV-1 was found to selectively inhibit aspects of phenotypic maturation as well as antigen capture and presentation functions. MDDC MAPK responsiveness to bacterial LPS remained intact however. Exogenous accessory HIV-1 Tat and Vpr did not affect MDDC phenotype but inhibited dextran endocytosis and viral peptide presentation. HIV-1-gp120 increased iMDDC maturation while blunting cytokine induced decreases in MDDC antigen capture abilities. The deletion of HIV-1-Tat did not affect MDDC phenotype, but was found to affect antigen capture decreases by R5 tropic HIV-1BaL. Deletion of HIV-1-Vpr likewise did not affect MDDC phenotype, however it was found to be influential in HIV-1 induced decreases in MDDC antigen presentation to autologous T-cells. These accumulated results indicate that HIV-1 subverts DC maturation and function through whole virus effects and individual accessory peptide influences. Understanding the mechanisms of DC dysfunction in HIV infection may provide some insight into infection prevention strategies and therapies leading to adaptive immune system activation and viral clearance.