Tobacco dependence dramatically increases health burdens and financial costs. Limitations of current smoking cessation therapies indicate the need for improved molecular targets. Nicotine, the main addictive component of tobacco, exerts its dependency effects via nicotinic acetylcholine receptors (nAChRs). The homomeric α7 nAChR is one of the most abundant receptors found in the brain and has unique features in comparison to other nAChR subtypes such as high calcium permeability, low probability of channel opening, and a rapid desensitization rate. α7 nAChR agonists reduce nicotine's rewarding properties in the conditioned place preference (CPP) test and i.v. self-administration. Recently, the peroxisome proliferator-activated receptor type-α (PPARα) has been implicated as a downstream signaling target of the α7 nAChR in ventral tegmental area dopamine cells. It is unknown whether the intrinsic characteristics of the α7 nAChR and PPARα are involved in its attenuation of nicotine reward. Therefore, this dissertation sought to investigate the role of α7 nAChRs in a mouse model of nicotine CPP and nicotine withdrawal by 1) investigating the impact of pharmacological modulation of α7 nAChR function in nicotine dependence and 2) evaluating a possible role for PPARα as a downstream mediator of α7 nAChRs in nicotine dependence. Positive allosteric modulators (PAMs) and a silent agonist were used to investigate the role of α7 nAChR conformations. The utilization of the α7 nAChR Type I PAM NS1738, Type II PAM PNU120596, and silent agonist NS6740 provided insight about the probability of channel opening (NS1738, PNU120596), desensitization (PNU120596, NS6740), and modulation of the endogenous acetylcholine/ choline tone (NS1738, PNU120596) as it relates to the α7 nAChR in nicotine CPP and withdrawal. In addition, this dissertation sought to elucidate the role of the α7 nAChR and PPARα in nicotine dependence using pharmacological interventions. The results suggest that the role of the α7 nAChR in nicotine dependence is conformation-dependent and PPARα-mediated. This dissertation is the first to report PPARα-mediation of the effects of α7 nAChR in nicotine reward and attenuation of nicotine withdrawal signs by PPARα activation. This data supports the development of α7 nAChR agonists and PPARα activators as possible smoking cessation aids.

Neuropeptide B (NPB) and neuropeptide W(NPW) are endogenous neuropeptide ligands for the G protein-coupled receptors NPBWR1 and NPBWR2. Here we report that the majority of NPW neurons in the mesolimbic region possess tyrosine hydroxylase immunoreactivity, indicating that a small subset of dopaminergic neurons coexpress NPW. These NPW-containing neurons densely and exclusively innervate two limbic system nuclei in adult mouse brain: the lateral bed nucleus of the stria terminalis and the lateral part of the central amygdala nucleus (CeAL). In the CeAL of wild-type mice, restraint stress resulted in an inhibition of cellular activity, but this stress-induced inhibition was attenuated in the CeAL neurons of NPW-/- mice. Moreover, the response of NPW-/- mice to either formalin-induced pain stimuli or a live rat (i. e., a potential predator) was abnormal only when they were placed in a novel environment: The mice failed to show the normal species-specific self-protective and aversive reactions. In contrast, the behavior of NPW-/- mice in a habituated environment was indistinguishable from that of wildtype mice. These results indicate that the NPW/NPBWR1 system could play a critical role in the gating of stressful stimuli during exposure to novel environments.

Mouse cytomegalovirus (MCMV), a β-herpesvirus, encodes the m145 family of glycoproteins. Several members of this family are predicted to adopt the MHC-I fold although their amino acid sequences exhibit less than 30% identity to classical MHC-I (MHC-Ia) proteins. Our aim was to determine how related the viral proteins are to MHCIa and characterize them in terms of cellular expression, structure and function. We studied the cellular localization of FLAG-tagged m17, M37, m145, m151, m152, m153 and m155 in transfected mouse fibroblasts. Flow cytometry analysis of transfected cells showed that M37, m145, m151 and m153 localize predominantly to the cell surface, whereas the majority of m17, m152 and m155 remain inside the cell. MHC-Ia proteins require assembly with β2-microglobulin (β2m) and peptide for stable cell surface expression. Transient transfection studies with β2m- or transporter associated with antigen (TAP)-deficient cell lines revealed that M37, m145, m151 and m153 could be expressed stably at the cell surface in the absence of β2m or TAP expression. To generate protein material for crystallization screening we evaluated both bacterial and insect cell expression systems. Although most m145 family members could be expressed in bacteria in insoluble inclusion bodies, none of the proteins could be accurately refolded. Since M37, m151 and m153 are cell surface molecules with the potential to bind receptors on host cells, we focused our structure determination efforts on them and evaluated their expression in Drosophila S2 insect cells. The extracellular domains of all three proteins expressed at significant levels, however, m151 tended to aggregate and precipitate over time. M37 and m153 were stable and could easily be purified to homogeneity. Size exclusion chromatography and SDS-PAGE analysis of m153 suggested that it forms a non-covalent homodimer. Analytical ultracentrifugation experiments confirmed this observation and provided an estimated molecular mass of 78.8 kDa. Enzymatic and mass spectrometry analyses showed that insect-expressed m153 is highly glycosylated. We tested a wide range of crystallization conditions for m153. It formed very fragile crystals and after optimization we obtained several that diffracted to 2.3 Å. To determine the structure of m153, we prepared a seleno-methionine derivative in insect cells, collected data on a single crystal and solved the phases by the single anomalous dispersion method. The m153 model was refined at 2.4 Å resolution to final Rcryst and Rfree of 23% and 27.9%, respectively. The m153 homodimer is formed by two MHC-I-like heavy chains, each consisting of two α-helices arranged on a platform of seven β-strands and an Ig-like α3 domain. The monomers are arranged “head-to-tail”, with the α1α2 platform domain of one chain interacting with the Ig-like α3 domain of the other. The α1 and α2 helices are closely juxtaposed and do not form a peptide binding groove. Three N-linked carbohydrate residues were visualized in the crystal structure. Major deviations from the MHC-I fold include an extended N-terminus, which originates next to the α3 domain, and an elongated α2 helix (designated H2b) that reaches down towards the α3 domain. In addition, m153 has two unique disulfide bonds, one between strands of the platform domain and another that links the extended N-terminus to the H2b helix. Both unique disulfide bonds were verified by mass spectrometry. The canonical Ig-fold disulfide bond is present in the α3 domain. Alanine mutation of four amino acids involved in interface hydrogen bonds abolished m153 dimer formation, validating the dimer interface visualized in the structure. The crystal structure of m153, together with the recently reported m157 structure, confirms the MHC-I fold for the MCMV m145 family and highlights shared structural features in the m145 family. We have demonstrated dimerization of full-length m153 in mammalian cells by bimolecular fluorescence complementation and co-immunoprecipitation studies. Further, we have shown that m153 is expressed at the surface of MCMV-infected cells at early times after infection. To initiate a search for host ligands of m153, we generated a reporter cell line by introducing an m153-human zeta chain fusion protein into 43.1 cells that contain an NFAT-driven GFP construct. While a variety of mouse cell lines were unable to stimulate the m153-reporter cells, coculture with mouse splenocytes specifically induced GFP production in m153-reporters but not in the parental or control reporter cell lines. We identified conventional CD11c+ and plasmacytoid dendritic cells (DCs) as the most potent m153-reporter cell stimulating populations in the spleen. The stimulation was shown to be m153-specific, dose- and cell contact-dependent. DCs derived from bonemarrow cultures also potently stimulated the m153-reporter cells. Macrophages and NK cells exhibited weaker stimulation of the reporter cells, indicating lower levels of ligand or that only small subsets of the cells express a ligand. DCs from several mouse strains, but not from the rat, stimulated m153-reporter cells. We evaluated DC surface phenotype and migratory capacity after coculture with m153-reporter cells or on m153-coated plates, but could not detect any changes induced specifically by the presence of m153.

Clinical cases of bovine mastitis are most severe in the early stages of lactation. The causes of this increased propensity for severe mastitis during early lactation, compared to mid and late lactation are unclear. In order to better understand the early lactation immune response to mastitis, a murine model of mastitis was employed. Intramammary inoculation of a mastitis causing Escherichia coli strain was performed in lactating mice at various stages of lactation to model the immune response seen in cows during lactation. In our experiments, mice in the early stages of lactation exhibited altered mRNA expression of cytokines IL-1β, IL-6, IL-10, and TNFα over the course of infection when compared to mice at mid lactation. Additionally, increased bacterial growth was observed in the mammary gland of mice infected during early lactation compared to late lactation. These results are consistent with the immune response observed in cows at early lactation. These results suggest that the mouse may provide a useful model to study differences in the immune response seen during different times in lactation.

When I first arrived in Liège, the scientific activities of our laboratory focused on the identification of candidate-genes whose different alleles interfere with the resistance/susceptibility of animals against infectious diseases. Two genes were being intensively studied (Mx and OAS) due to their theoretical potential in interfering with the replicative cycle of several viruses responsible for bovines viral pneumonias (Baise et al., 2004 ; Gerardin et al., 2004 ; Leroy et al., 2005 ; 2006). My work consisted then, in the identification of other genes potentially implicated in the resistance against the Paramyxoviruses. The Paramyxoviridae family includes some of the great and ubiquitous disease-causing viruses of animals, including the bovine parainfluenza type 3, the bovine respiratory syncytial virus, the Newcastle disease virus, the distemper virus, etc. Evidence was accumulating that genetic factors were involved both in the control of infectious diseases (Abel et al., 1991 ; 1995 ; Alcais et al., 1997 ; Jin et al., 1999 ; Martinson et al. 1997 ; Shaw et al., 1995) and in the regulation of infection levels and clinical presentation (Garcia et al., 1999 ; Plancoulaine et al., 2000 ; 2003). Thus, identifying genes that control the organism response to paramyxoviruses was a crucial step in elucidating how they might affect the pathophysiological processes underlying the severity of the disease induced. Other experiences done in this laboratory showed us how risky and difficult was any extrapolation of the mouse results to another species if these results were brought through infection with a heterologous virus, so we decided to implement this strategy with Sendai virus (SeV), the archetype organism of the Paramyxoviridae family, from which most of the basic biochemical, molecular and biologic properties of the whole family were derived from (Chanock et al., 2001). With this goal in mind my thesis was divided in five successive steps: In order to establish a standard model of SeV infection, the first step consisted in determining the best volume of inoculum that was needed to achieve a safe, reproducible pulmonary deposition of Sendai virus in the mice lungs. Secondly we developed a murine model of SeV infection using a series of different and sophisticated procedures that allowed a quantitative assessment of disease severity and progression. Then we compared SeV infections among 6 strains of mice that were deliberately chosen because they originated from different lineages, as deduced from known genealogical and phylogenetic data. Applying these procedures to distinct inbred strains of mice, revealed highly significant differences in susceptibility between them. More specifically 129/Sv were highly susceptible while BALB/c were particularly resistant, BALB/c exhibiting a benign and asymptomatic affection of the epithelium of the airways, with no functional impact, generating slight mononucleated cell infiltration, in which viral replication is repressed and the virus swiftly eliminated. As a result, in the fourth part of my study we discussed a series of hypotheses that should be tested in the future to improve our understanding of why BALB/c is so resistant to SeV infection. Practically speaking, our studies led to the gathering of a genomic DNA collection from the parental extreme lines in terms of susceptibility (129/Sv) and resistance (BALB/c) and their F1 and F2 offspring. Within this bank, each of the 263 DNA sample is associated with a portfolio of phenotypic values that are estimators of the resistance each mouse opposed to the SeV. We hope that, between expert hands, this bank will allow the detection of genes of which the alleles contribute, at least in part, to the spectacular resistance of BALB/c. The last part of my thesis consisted in applying our model to establish if the receptor TLR4 influenced the pathophysiology of the Paramyxoviridae in general. Because the role of this receptor had already been excluded for SeV, we tested another virus of the same family and homologous for the mice, the PVM (standing for pneumonia virus of mice). This work showed, in contradiction of what had been found in heterologous models in the past, that TLR4 is not involved in host defense against respiratory tract infection with the Paramyxoviridae.

No description available.

Consumption of fish oils (FO) enriched with the n-3 polyunsaturated fatty acids (PUFA), docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), is beneficial to a variety of inflammatory disorders due, in part, to the alteration of membrane composition of T-lymphocytes and other immune cells. We previously observed that down-regulation of proliferation and cytokine synthesis by CD4+ T-cells in mice fed diets rich in n-3 PUFA was dependent on the involvement of CD28, a co-stimulatory molecule necessary for T-cell activation. Since the co-receptor homologues, CD28 and CTLA-4, have opposing effects on T-cell activation, we hypothesized that the balance of costimulatory and downregulatory properties of CD28 and CTLA-4, respectively, would be altered by diet. A significant increase (p<0.05) in CD28 and CTLA-4 surface expression was observed in CD4+ T-cells post-stimulation with phorbol ester and calcium ionophore (PMA/Iono) or anti-CD3 and anti-CD28 (&#945;CD3/CD28) antibodies in all diet groups. A significant increase (p<0.01; 20%) in the number of CD28 molecules was observed in n-3 PUFA vs. CO-fed mice after 48 h of in vitro CD4+ T-cell activation, and both CTLA-4 mRNA transcript and protein levels were upregulated by 50% at 72 h post-activation (p<0.01). Treatment with anti-CTLA-4 mAb in vivo in Mycobacterium bovis (BCG)-vaccinated mice did not alter the suppressive effects of dietary n-3 PUFA on antigen (PPD)-induced lymphocyte proliferation or delayed hypersensitivity reactions. T-cells from both the C57BL/6 and IL-10mice fed dietary n-3 PUFA after 72 h of in vitro stimulation with &#945;CD3/CD28. CD4T-cells from C57BL/6 mice fed DHA produced significantly less IFN&#947; and IL-10, while CD4T-cells from IL-10Ligation of CD28 upregulates IL-10 receptor (IL-10R) expression on CD4+ T-cells. Therefore, we hypothesized that dietary n-3 PUFA would suppress T-cell function through the effects of IL-10. Surprisingly, the proliferation of purified splenic CD4+ T-cells activated in vitro with &#945;CD3/CD28 was suppressed by dietary n-3 PUFA in both conventional mice (C57BL/6) and IL-10 gene knockout (IL-10(-/-)) mice. Furthermore, IL-10R cell surface expression was significantly down-regulated on CD4+ T-cells from both the C67BL/6 and IL-10(-/-) mice fed dietary n-3 PUFA produced significantly more IFN&#947; compared to the CO-fed group.

No description available.

Control of blood flow to skeletal muscle is essential to maintain the overall homeostasis of an organism. The primary route that skeletal muscle uses to accommodate an increased metabolic demand associated with physical activity is to increase its blood flow through functional hyperemia. The importance of functional hyperemia in ensuring proper skeletal muscle function spurred 130 years of investigation into the mechanism(s) regulating its occurrence.Despite not identifying the essential factor(s) for controlling skeletal muscle blood flow, the last century of investigation has uncovered much about the process; including the observation that skeletal muscle functional hyperemia is impaired with ischemic disease. In patients, this can result in immobility, chronic ulcerations, gangrene, and at worst, amputation. To develop efficacious therapies, we as scientists must develop a better understanding of the molecular mechanisms underlying impaired vascular function during ischemia.The goal of this work was to lay the foundation for investigations examining the role of specific gene products involved in modulating blood flow control during ischemic revascularization by assessing vascular function in the mouse following an ischemic event. Unique among research animals, the mouse is routinely accessible for targeted genetic disruption, which allows investigators to assess the requirement of specific gene-products in a physiological process. Unfortunately, to date, no publication that I am aware of describes blood flow measurement to contracting mouse skeletal muscle following an ischemic/revascularization event. Therefore, the primary objective of this work was to assess vascular function in genetically unaltered animals.I found that unlike other species thus far examined, vascular dysfunction is not an obligatory response to hindlimb ischemic revascularization in the mouse. Ex vivo vasodilation responses to acetylcholine were statistically significantly impaired in the muscular branch artery 14 days following an ischemic event. However, using a newly developed fluorescent microsphere-based approach for determining skeletal muscle blood flow, I found that functional hyperemia was similar for the gracilis posterior muscle between non-ischemic and day-14 ischemic animals. In light of the primary literature, these findings suggest that vascular growth, and not ischemia per se is the primary regulator of vascular function during health and disease.

Multiple-mouse MRI (MMMRI) accelerates biomedical research by imaging multiple mice simultaneously. To date, MMMRI has been explored in three ways: shielded transmit-receive coils, shielded transmits coil with separate unshielded receive coils; and finally shielded transmit-receive coils with independent gradient coils. However alternative transmit coil configurations and possible benefits of eliminating shielding have not yet been explored. The goal of this thesis is to test possible radiofrequency configurations with and without shielding for the purpose of improving image quality for MMMRI. Results demonstrate that using an unshielded transmit-receive coil array provided a 20% improvement over an identical shielded coil. A new unshielded 7-coil MMMRI array is presented, minimizing the ghosting between image overlap using mutual inductance minimization and a sensitivity encoding (SENSE) reconstruction. The final array provided high resolution images (90µm) of up to seven live mice simultaneously with appropriate signal-to-noise for automated analysis.