Toll-like receptors (TLRs) are evolutionarily conserved pattern recognition receptors that mediate host-responses to pathogens. In mammals, TLR ligands promote cellular activation and the production of cytokines. Several TLR ligands have been employed prophylactically for the control of bacterial or viral diseases in the mouse model. However, the TLR-mediated responses in chickens have not been well described. Importantly, the utility of TLR agonists for the control of viral pathogens, such as avian influenza virus (AIV), has not been fully explored in chickens. To this end, the studies described in this thesis characterized the kinetics of in vivo responses in chickens to the TLR4 ligand lipopolysaccharide (LPS) and the TLR21 ligand CpG ODN. It was demonstrated that both of these ligands induced the up-regulation of several immune system genes in the spleen, including those associated with pro-inflammatory and antiviral responses, as well antigen presentation. By harnessing the immunostimulatory properties of TLR ligands, it was also demonstrated that the prophylactic administration of either poly I:C (a TLR3 ligand), LPS or CpG ODN may confer immunity to a low pathogenic avian influenza virus, as determined by a reduction in both oropharyngeal and cloacal virus shedding in infected birds. Furthermore, transcriptional analysis of genes in the spleen and lungs identified interleukin (IL)-8, interferon (IFN)-α and IFN-γ as correlates of immunity. In conclusion, TLR ligands may modulate several aspects of the chicken immune system to induce an anti-viral state, thereby conferring immunity to AIV.

Ca2+ functions as a second messenger in all eukaryotes. Such Ca2+ signaling is used to coordinate plant responses to numerous stimuli, both developmental and environmental. Ca2+ signals are often transduced via the action of the ubiquitous Ca2+ sensor calmodulin (CaM). CaM-dependent protein phosphorylation forms an important component of such signal transduction pathways, including that regulating the initiation and development of symbiotic rhizobial nodules in legumes such as soybean (Glycine max). To further understand the role of Ca2+/CaM during nodule organogenesis, a nodule cDNA expression library was screened using radiolabeled CaM as a probe to identify novel CaM-binding proteins (CaMBPs). This screen resulted in the identification of a previously uncharacterized receptor-like kinase, termed GmCaMK1. The CaM-binding domain (CaMBD) of GmCaMK1 is located in a 24 residue region of GmCaMK1, which overlaps with the subdomain XI of a conserved Ser/Thr kinase domain. This CaMBD bound CaM in a Ca2+-dependent manner, and with high affinity (Kd = 1.4 nM). Furthermore, two hydrophobic residues (V372 and L375) were identified as critical for GmCaMK1-CaM interaction. Recombinant GmCaMK1 exhibited protein kinase activity in vitro, with autophosphorylation activity unaffected by the presence or absence of Ca2+/CaM. GmCaMK1 expression is enriched in developing nodules and main roots, and highest expression level was observed in lateral roots. While the function of CaM-binding to GmCaMK1 remains unclear, the affinity and Ca2+-dependence of the GmCaMK1-CaM interaction strongly suggests that GmCaMK1 is a physiologically relevant CaM target. The Arabidopsis ortholog of GmCaMK1, AtCaMK1 also bound CaM when expressed as a recombinant protein. GmCaMK1 is part of a multi-member family in soybean, as are putative homologs across taxa, suggesting that this is a novel, conserved family of CaMBPs. / Thesis (Master, Biology) -- Queen's University, 2010-01-28 16:00:48.69

The American eel (Anguilla rostrata) has suffered a serious population decline in Lake Ontario since the early 1980s due to a decline in recruitment of juveniles migrating from the Sargasso Sea. This has resulted in the closure of the Lake Ontario fishery in 2004 and its listing as endangered under the Ontario Species at Risk act in June of 2008 in Ontario. Due to their complex life cycle, little is known about eels once they leave their freshwater habitats and migrate to the Sargasso Sea to reproduce. Ocean conditions, habitat destruction, disease, reduced lipid content, over-fishing, physical barriers (hydroelectric dams), and chemical contamination are all possible reasons for recruitment decline and may be acting cumulatively. Maternally derived dioxin-like contaminants (DLCs) accumulated during the growth phase of eels in Lake Ontario are toxic to fish embryos, and embryotoxicity is expressed as a series of malformations known as blue sac disease (BSD). I assessed whether these toxicants are in high enough concentrations in sexually maturing, eels to be embryotoxic to their offspring, as assessed by using Japanese medaka (Oryzias latipes), a surrogate species. Medaka embryos were first injected with 2,3,7,8-tetracholordibenzo-p-dioxin (TCDD) to establish their sensitivity to this test chemical expressed as an 11-day EC50 of 3.79 pg/mg, for the induction of BSD. Medaka embryos were injected with eel extracts and their response compared to the TCDD toxicity curve to assess whether extracts caused developmental problems and to estimate the relative concentration of DLCs. Eel extracts from all collection sites caused no dioxin-like toxicity to Japanese medaka embryos. However, significantly higher toxicity at 10 eeq relative to triolein was found for all extracts with no differences among sites, suggesting the presence of non-dioxin-like toxicants. The low level of maternal tissue contamination by DLCs implied by this bioassay is mirrored in chemical monitoring of persistent organic pollutants in Lake Ontario fish. If correct, the low levels of toxicity of extracts to embryos could contribute to the observed increase of eels entering L. Ontario from 2003 to 2008. / Thesis (Master, Biology) -- Queen's University, 2010-09-01 09:31:19.466

Foreign pathogens are recognized by toll-like receptors (TLR), present on various immune cells such as professional antigen-presenting cells (pAPCs). On recognition of its ligand, these receptors activate pAPCs, which may in turn influence naïve CD8+ T cell activation and affect their abilities to clear viral infection. However, how TLR ligands (TLR-L) can regulate CD8+ T cell responses have not been fully elucidated. This thesis will focus on examining how the presence of components from foreign pathogens, e.g. viral or bacterial infection, can contribute to shaping host immunity during concurrent viral infections. Since nitric oxide (NO), an innate effector immune molecule, was recently suggested to regulate proteasome activity; we sought to examine if NO can influence MHC-I antigen presentation during viral infections. The data in this section of the thesis provides evidence that combined TLR engagement can alter the presentation of certain CD8+ epitopes due to NO-induced inhibition in proteasome activity. Taken together, the data demonstrate that TLR ligation can influence the adaptive immune response due to induction of specific innate effector molecules such as NO. Next, the influence of combined TLR engagement on CD8+ T cell immunodominance hierarchies during viral infections was examined. In this section, we established that dual TLR2 and TLR3 stimulation alters immunodominance hierarchies of LCMV epitopes as a result of reduced uptake of cell-associated antigens and reduced cross-presentation of NP396 consequently suppressing NP396-specific CD8+ T cell responses. These findings are significant as they highlight a new role for TLR ligands in regulating anti-viral CD8+ T cell responses through impairing cross-presentation of cell-associated antigens depending on the type of TLR present in the environment during infections. Finally, we addressed TLR ligand induced type I interferon production and the signalling pathways that regulate them in two different mouse macrophage populations – those derived from the spleen or bone marrow. In this study, we observed that concomitant TLR2 stimulation blocked the induction of type I IFN induced by TLR4 in bone marrow-derived macrophages, but not spleen-derived macrophages in SOCS3-dependent manner. Taken together, the data presented in this thesis have defined new facets of how anti-viral responses are regulated by TLR activation, especially if multiple receptors are engaged simultaneously. / Thesis (Ph.D, Microbiology & Immunology) -- Queen's University, 2011-08-18 17:37:18.47

Background: In 2007, the International Agency for Research on Cancer (IARC) classified long-term shift work as a probable human carcinogen; however, the mechanism through which shift work potentially increases cancer risk is not known. One hypothesis is that diminished melatonin production may be involved, possibly as a result of exposure to light during night work. Experimental studies suggest a link between melatonin and peptides in the insulin-like growth factor (IGF) family, also implicated in carcinogenesis. This research aimed to describe the distributions of circulating concentrations of insulin-like growth factor-I (IGF-I) and insulin-like growth factor binding protein-3 (IGFBP-3) and their associations with urinary melatonin as possible intermediates in the pathway between work at night and breast cancer. Methods: A cross-sectional study was conducted among 85 premenopausal nurses working a rotating shift pattern of two 12-hour days, two 12-hour nights, and five days off. Once during both the summer and winter seasons, melatonin metabolites were measured in urine samples and circulating concentrations of IGF-I and IGFBP-3 were determined from serum samples. Weight and height were measured by the study coordinator, while a questionnaire and study diaries were used to collect all other covariate information. Predictors of IGF levels were identified using multivariate mixed effects modeling and relationships between melatonin and the IGFs were investigated using Spearman’s rank correlation and multivariate mixed effects modeling. Results: Both age (β = -3.6, p < 0.0001) and current OC use (β = -40.8, p = 0.003) were associated with decreases in circulating IGF-I, while levels of IGF-I were increased in the winter months (β = 26.3, p = 0.02). A positive relationship between recent alcohol consumption and serum IGFBP-3 was also suggested (β = 197.8, p = 0.05). Neither Spearman’s rank correlations nor mixed effects modeling indicated that urinary melatonin was a determinant of serum IGFs. Conclusions: Age, season, and current OC use were observed to predict circulating IGF-I, while recent alcohol consumption was a determinant of IGFBP-3 levels. A relationship between melatonin and IGFs, theorized as a component of the mechanism linking shift work and cancer, was not supported by the results of this project. / Thesis (Master, Community Health & Epidemiology) -- Queen's University, 2012-05-30 15:29:31.253

Grb10 is a member of the Grb7 family of adaptor proteins that also includes Grb7 and Grb14. These three members contain multiple protein binding domains and lack enzymatic activity. Extensive two-hybrid studies have demonstrated binding of Grb10 to numerous activated tyrosine kinase receptors including the insulin receptor (IR) and insulin-like growth factor-I receptor (IGF-IR), as well as many non-receptor molecules such as MEK1, Raf-1, and Nedd4. Grb10 has been implicated in IGF-I anti-apoptotic signaling regulation through interactions with Raf-1 and the mitochondrial membrane. / In this report the pattern of transient Grb10 translocation following IGF-I cellular stimulation was studied. This report also demonstrates the implication of a short variable amino-terminal region of Grb10 in mitochondrial membrane association. Finally, assays were developed with the goal of identifying new Grb10 binding partners.

TLR3- and TLR4-mediated DC activation lead to distinct function of mature DC, in which MAPKs pathway is involved. JNK-associated leucine zipper protein (JLP) associates with JNK/p38 phosphorylation, however the role of JLP in DC is not well understood. We previously found either LPS or poly(I:C) up-regulated JLP expression in BMDC. Therefore we hypothesized JLP plays a differential role in TLR3- and TLR4-mediated BMDC maturation and functions. We demonstrated JLP facilitated LPS- and poly(I:C)-induced CD86 and CD40 up-regulation, LPS-induced CD80 up-regulation and poly(I:C)-induced MHC-II up-regulation. We found JLP down-regulated IL-12 production in LPS-stimulated BMDC, and up-regulated IL-6 production in poly(I:C)-stimulated BMDC. Our data also showed JLP negatively regulated MHC-II antigen presentation in LPS- and poly(I:C)-stimulated BMDC and JLP was involved in promoting LPS-activated BMDC survival, but not in poly(I:C)-activated BMDCs. Therefore our current data suggested a multi-functional role of JLP in the regulation of TLR3- and TLR4-mediated DC maturation.

Ubiquitin fold modifier 1 (Ufm1) is a member of the ubiquitin like protein (UBL) family. Like other UBLs, Ufm1 can be conjugated to protein substrates via specific E1 (Uba5), E2 (Ufc1) and E3 (Ufl1) enzymes, and removed from these substrates via the action of Ufm1-specific proteases. While Ufm1 has been implicated in endoplasmic reticulum (ER) function, its biological roles remain poorly understood. By identifying; (a) Ufm1 binding proteins, (b) protein interactors of the Ufm1 conjugation/deconjugation system, (c) Ufm1 conjugates, as well as (d) the intracellular localization of Ufm1 and its main interactors, I aimed to better characterize the biological role(s) of this poorly understood UBL.

Blood contacting devices are frequently limited by complications such as surface-induced thrombosis. This thesis investigated the feasibility of using a family of recombinant elastin-like polypeptides (ELPs), namely ELP1, ELP2 and ELP4 that differ by molecular weight and sequence length, as potential thromboresistant coatings. The ELP coatings were prepared by physical adsorption onto the surface of Mylar, with surface modification confirmed by goniometry, X-ray photoelectron spectroscopy (XPS), and chemical force microscopy (CFM). Both surface wettability and hydrophilic adhesion force increased as the ELP sequence length decreased. The ELP adsorption process monitored by using quartz crystal microbalance with dissipation (QCM-D) showed that the ELPs adsorbed within a monolayer. Additionally, ELP surface coverage was found to increase with the polypeptide sequence length. The QCM-D studies also revealed that the longer polypeptides (ELP2 and ELP4) exhibited higher specific dissipation values indicating that they established adsorbed layers with greater structural flexibility and associated water content compared to ELP1. Exposure of the ELP coatings to flowing reconstituted blood demonstrated that both the ELP2 and ELP4 coatings reduced the quantity of adsorbed fibrinogen (Fg), with the ELP4 coating resulting in the lowest levels of adherent platelets. Energy dissipation versus frequency shift plots obtained from QCM-D studies indicated that adsorbed Fg on the ELP4 coating maintained a softer, more flexible film then on the other ELPs. The ELP4 coating also demonstrated an altered binding activity for GPIIb/IIIa where only the AGDV motif in the adsorbed Fg gamma-chain appeared to be exposed and bioactive. Conversely, on the other ELP coatings both the AGDV and RGD motifs (found within the Fg alpha-chain) were available for binding, suggesting that a different Fg conformational state exists on the ELP1 and ELP2 coatings. Moreover, both the ELP2 and ELP4 coatings displayed minimal bulk platelet reactivity following extended whole blood shear exposure (up to an hour) compared to Mylar. This was not observed with the ELP1 coating. Overall, the results suggest that the structural flexibility and associated water content of the ELP coatings appear to be important criteria influencing their thrombogenicity, with ELP4 displaying the most favourable blood-material response compared to ELP1 and ELP2.

Glucagon-like peptide-2 (GLP-2) augments intestinal adaptation in animal models of short bowel syndrome (SBS) and in adult patients with SBS. However, GLP-2 has not been used as a therapy for pediatric SBS. In this thesis, it is hypothesized that exogenous GLP-2 therapy will improve outcomes of intestinal adaptation in proximal intestinal resection (JI) and distal intestinal resection (JC) neonatal piglet models of SBS. Improvements in morphological parameters (increased small intestinal length) and histological parameters (increased jejunal villus length or jejunal crypt depth) of intestinal adaptation in JI and JC neonatal piglets treated with GLP-2 were observed. However, improved clinical outcomes (fewer days of diarrhea, fewer days on parenteral nutrition, more days on enteral nutrition alone) were only observed in GLP-2 treated JC animals. Since the JC anatomical subtype (no remnant ileum) represents the majority of clinical cases of neonatal SBS, these results support a potential role for GLP-2 therapy in pediatric SBS.