Yes / Monolayers of neurons and glia have been employed for decades as tools for the study of cellular physiology and as the basis for a variety of standard toxicological assays. A variety of three dimensional (3D) culture techniques have been developed with the aim to produce cultures that recapitulate desirable features of intact. In this study, we investigated the effect of preparing primary mouse mixed neuron and glial cultures in the inert 3D scaffold, Alvetex. Using planar multielectrode arrays, we compared the spontaneous bioelectrical activity exhibited by neuroglial networks grown in the scaffold with that seen in the same cells prepared as conventional monolayer cultures. Two dimensional (monolayer; 2D) cultures exhibited a significantly higher spike firing rate than that seen in 3D cultures although no difference was seen in total signal power (<50 Hz) while pharmacological responsiveness of each culture type to antagonism of GABAAR, NMDAR and AMPAR was highly comparable. Interestingly, correlation of burst events, spike firing and total signal power (<50 Hz) revealed that local field potential events were associated with action potential driven bursts as was the case for 2D cultures. Moreover, glial morphology was more physiologically normal in 3D cultures. These results show that 3D culture in inert scaffolds represents a more physiologically normal preparation which has advantages for physiological, pharmacological, toxicological and drug development studies, particularly given the extensive use of such preparations in high throughput and high content systems.

Aging is accompanied by dysregulated immune function resulting in increased susceptibility of the elderly to diseases caused by microbial pathogens. There exists a multitude of data suggesting decreased resistance of the elderly to a variety of intracellular pathogens but there is no data relating the effect of aging on the immune response against Brucella. To elucidate the mechanism of immune dysregulation in old, old and young DBA/2 and BALB/c mice were infected with wild-type B. abortus strain 2308. The old and young mice were also vaccinated with vaccine B. abortus strain RB51 over-expressing Cu-Zn superoxide dismutase (SOD) and then challenged with B. abortus strain 2308 to determine the effect of vaccination in old vs. young mice. Specific IgG1 and IgG2a response to Brucella antigens were also evaluated to determine the effect of aging on Th-specificity of the immune response against Brucella infection. The immune response in aged vs. young mice was further assessed using RT-PCR and cytokine antibody array to determine the type of T-helper response. The experimental results indicate that all mice, regardless of age, survived infection ranging from doses of 2 x 104 to - 2 x 108 CFU. Though the older DBA/2 mice had a higher organism burden after 1 week of infection, these mice cleared Brucella infection more efficiently (5 weeks post-infection) than young mice. Vaccination with strain RB51 over-expressing SOD provided significant protection in young DBA/2, young BALB/c and old BALB/c mice but not in old DBA/2 mice after strain 2308 challenge. The results also suggest that old mice produced a different magnitude of IgG1 and IgG2a response to bacterioferritin and SOD of Brucella. The data suggests that both Th17 as well as Th1 responses were accentuated in old mice as compared to young mice following infection with Brucella. How the Th17 and Th1 branches of immune system work together enabling old mice to clear Brucella better than young mice warrants future investigation. / Master of Science

AIDS-related human cytomegalovirus (HCMV) retinitis remains the leading cause of blindness among untreated HIV/AIDS patients worldwide. Understanding the pathogenesis of this disease is essential for developing new, safe, and effective treatments for its prevention or management, yet much remains unknown about the virologic and immunologic mechanisms contributing to its pathology. To study such mechanisms, we use a well-established, reproducible, and clinically relevant animal model with retrovirus-induced murine acquired immunodeficiency syndrome (MAIDS) that mimics in mice the symptoms and progression of AIDS in humans. Over 8 to 12 weeks, MAIDS mice become susceptible to experimental murine cytomegalovirus (MCMV) retinitis. We have found in this model that MCMV infection significantly stimulates ocular suppressor of cytokine signaling (SOCS)1 and SOCS3, host proteins which dampen immune-related signaling by cytokines, including antiviral interferons. Herein we investigated virologic and/or immunologic mechanisms involved in this stimulation and how virally-modulated SOCS1 and/or SOCS3 proteins may contribute to MCMV infection or experimental MAIDS-related MCMV retinitis. Through pursuit of two specific aims, we tested the central hypothesis that MCMV stimulates and employs SOCS1 and/or SOCS3 to induce the onset and development of MCMV retinal disease. MCMV-related SOCS1 and SOCS3 stimulation in vivo occurred with intraocular infection, was dependent on method and stage of immune suppression and severity of ocular pathology, was associated with stimulation of SOCS-inducing cytokines, and SOCS1 and SOCS3 were differentially sensitive to antiviral treatment. In vitro studies further demonstrated that SOCS1 and SOCS3 stimulation during MCMV infection occurred with expected immediate early kinetics, required viral gene expression in cell-type-dependent and virus origin-dependent patterns of expression, and displayed differential sensitivity to antiviral treatment. These data suggest that SOCS1 and SOCS3 are stimulated by divergent virologic, immunologic, and/or pathologic mechanisms during MCMV infection, and that they contribute to the pathogenesis of retinal disease, revealing new insights into the pathophysiology of AIDS-related HCMV retinitis.

Human norovirus (NoV) causes gastroenteritis worldwide and has been associated with a number of produce related outbreaks. The design of effective inactivation and prevention procedures requires an understanding of virus survival in environments applicable to the production and processing of fresh produce. To evaluate the extent of NoV risk from farm to fork, the survival of murine norovirus (MNV), a surrogate for human NoV, was studied on stainless steel disks, soil and in bottled water for 42 days and on lettuce for 15 days in the laboratory. Stability experiments were then conducted on farm during one lettuce planting/harvest cycle, for 4 weeks. MNV stability was tested at room temperature in the laboratory or under ambient conditions on the farm. A one log reduction in virus titre was achieved after 30 days in water, 4 days on lettuce, 15 days on stainless steel disks, 12 days on loamy and sandy soil. For farm testing, infectious virus was recovered from both soil and lettuce on the day of inoculation. Although infectious virus was not recovered at later time points, the viral genomes were detected for up to four weeks. The observed long-term persistence of NoV, under both laboratory and field conditions, provides valuable information for developing risk assessments and control procedures to limit the possibility for NoV transmission in the food supply.

Human norovirus (NoV) causes gastroenteritis worldwide and has been associated with a number of produce related outbreaks. The design of effective inactivation and prevention procedures requires an understanding of virus survival in environments applicable to the production and processing of fresh produce. To evaluate the extent of NoV risk from farm to fork, the survival of murine norovirus (MNV), a surrogate for human NoV, was studied on stainless steel disks, soil and in bottled water for 42 days and on lettuce for 15 days in the laboratory. Stability experiments were then conducted on farm during one lettuce planting/harvest cycle, for 4 weeks. MNV stability was tested at room temperature in the laboratory or under ambient conditions on the farm. A one log reduction in virus titre was achieved after 30 days in water, 4 days on lettuce, 15 days on stainless steel disks, 12 days on loamy and sandy soil. For farm testing, infectious virus was recovered from both soil and lettuce on the day of inoculation. Although infectious virus was not recovered at later time points, the viral genomes were detected for up to four weeks. The observed long-term persistence of NoV, under both laboratory and field conditions, provides valuable information for developing risk assessments and control procedures to limit the possibility for NoV transmission in the food supply.

Human norovirus (NoV) causes gastroenteritis worldwide and has been associated with a number of produce related outbreaks. The design of effective inactivation and prevention procedures requires an understanding of virus survival in environments applicable to the production and processing of fresh produce. To evaluate the extent of NoV risk from farm to fork, the survival of murine norovirus (MNV), a surrogate for human NoV, was studied on stainless steel disks, soil and in bottled water for 42 days and on lettuce for 15 days in the laboratory. Stability experiments were then conducted on farm during one lettuce planting/harvest cycle, for 4 weeks. MNV stability was tested at room temperature in the laboratory or under ambient conditions on the farm. A one log reduction in virus titre was achieved after 30 days in water, 4 days on lettuce, 15 days on stainless steel disks, 12 days on loamy and sandy soil. For farm testing, infectious virus was recovered from both soil and lettuce on the day of inoculation. Although infectious virus was not recovered at later time points, the viral genomes were detected for up to four weeks. The observed long-term persistence of NoV, under both laboratory and field conditions, provides valuable information for developing risk assessments and control procedures to limit the possibility for NoV transmission in the food supply.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 4 uppsatser.

Human norovirus (NoV) causes gastroenteritis worldwide and has been associated with a number of produce related outbreaks. The design of effective inactivation and prevention procedures requires an understanding of virus survival in environments applicable to the production and processing of fresh produce. To evaluate the extent of NoV risk from farm to fork, the survival of murine norovirus (MNV), a surrogate for human NoV, was studied on stainless steel disks, soil and in bottled water for 42 days and on lettuce for 15 days in the laboratory. Stability experiments were then conducted on farm during one lettuce planting/harvest cycle, for 4 weeks. MNV stability was tested at room temperature in the laboratory or under ambient conditions on the farm. A one log reduction in virus titre was achieved after 30 days in water, 4 days on lettuce, 15 days on stainless steel disks, 12 days on loamy and sandy soil. For farm testing, infectious virus was recovered from both soil and lettuce on the day of inoculation. Although infectious virus was not recovered at later time points, the viral genomes were detected for up to four weeks. The observed long-term persistence of NoV, under both laboratory and field conditions, provides valuable information for developing risk assessments and control procedures to limit the possibility for NoV transmission in the food supply.

L’équipe a identifié une nouvelle GAP de RhoA, ARHGAP19, majoritairement exprimée dans le système hématopoïétique. Le projet a consisté à étudier la régulation de cette protéine dans des lymphocytes T humains. Pour cela, les analyses se sont portées sur la phosphorylation d’ARHGAP19 et sur sa localisation au cours de la division des lymphocytes T. ARHGAP19 est phosphorylée par l’effecteur de RhoA, la protéine kinase ROCK, sur la Sérine 422 et par la protéine kinase mitotique CDK1 sur les Thréonines 404 et 476. La phosphorylation par ROCK permet à ARHGAP19 d’interagir avec la famille de protéines 14-3-3 qui la protège des déphosphorylations pouvant avoir lieu au cours de la division cellulaire. L'ensemble des phosphorylations est primordial pour la régulation de la localisation cellulaire d'ARHGAP19 et contribue à une division cellulaire correcte. En effet, en absence de phosphorylation, on observe des défauts lors de la cytodiérèse entrainant la formation de cellules multinucléées. De plus, des dérégulations de RhoGTPases comme l’absence de GAP, sont aujourd’hui mises en évidence dans les cancers. C’est pourquoi nous avons généré des souris arhgap19 KO pour étudier les conséquences de l’absence du gène codant pour ARHGAP19, dans le système hématopoïétique murin. L’ensemble des cellules progénitrices et matures intervenant dans l’hématopoïèse murine a été analysé. Par ce modèle d’invalidation conditionnelle d’arhgap19, aucun rôle majeur de la protéine n'a été mis en évidence mais les résultats suggèrent une implication aux différents stades de la différenciation hématopoïétique et un impact sur l'ensemble des populations de ce système. / The team identified a new GAP of RhoA, ARHGAP19, mostly expressed in the hematopoietic system. The project consisted in studying the regulation of this protein in human T lymphocytes. For this, the analyzes focused on the phosphorylation of ARHGAP19 and on its localization during the division of the T lymphocytes. ARHGAP19 is phosphorylated by the effector of RhoA, the protein kinase ROCK, on the Serine 422 and by the protein CDK1 mitotic kinase on Threonines 404 and 476. ROCK phosphorylation allows ARHGAP19 to interact with the 14-3-3 family of proteins that protects it from dephosphorylation that occur during cell division. All phosphorylations are essential for regulating the cellular localization of ARHGAP19 and contribute to correct cell division. Indeed, in the absence of phosphorylation, defects are observed during cytodiérèse resulting in the formation of multinucleate cells. In addition, deregulation of RhoGTPases, such as the absence of GAP, are now highlighted in cancers. This is why we generated arhgap19 KO mice to study the consequences of the absence of the gene coding for ARHGAP19, in the murine hematopoietic system. All progenitor and mature cells involved in murine hematopoiesis were analyzed. By this model of conditional invalidation of arhgap19, no major role of the protein has been demonstrated but the results suggest an involvement at different stages of hematopoietic differentiation and an impact on all populations of this system.

APOBEC3 (A3) proteins are a family of host-encoded cytidine deaminases that protect against retroviruses and other viral intruders. Retroviruses, unlike other viruses, are able to integrate their genomic proviral DNA within hours of entering host cells. A3 proteins hinder retroviral infectivity by editing retroviral replication intermediates, as well as by inhibiting retroviral replication and integration through deamination-independent methods. These proteins thus constitute the first line of immune defense against endogenous and exogenous retroviral pathogens. The overall goal of my Master's project was to better understand the critical role A3 proteins play in restricting inter- and intra-host transmission of retroviruses. There are two specific aspects that I focused on: first, investigating the role of mouse APOBEC3 (mA3) in limiting the zoonotic transmission of murine leukemia retroviruses (MLVs) in a rural environment; second, to identify the molecular features in MLVs that confer susceptibility or resistance to deamination by mA3. For the first part of my project, we collected blood samples from dairy and production cattle from four different geographical locations across Canada. We then designed a novel PCR screening strategy targeting conserved genetic regions in MLVs and Mouse Mammary Tumor Virus (MMTV) and MMTV-like betaretroviruses. Our results indicate that 4% of animals were positive for MLV and 2% were positive for MMTV. Despite crossing the species barrier by gaining entry into bovine cells, our study also demonstrates that the bovine A3 protein is able to potently inhibit the spread of these murine retroviruses in vitro. The next question we asked was whether mA3 could also mutate and restrict murine endogenous retroviruses and thereby partake in limiting zoonotic transmission. Moloney MLV and AKV MLV are two highly homologous murine gammaretroviruses with opposite sensitivities to restriction by mA3: MoMLV is resistant to restriction and deamination while AKV is sensitive to both. Design of MoMLV/AKV hybrid viruses enabled us to map the region of mA3 resistance to the region encoding the glyco-Gag accessory protein. Site-directed mutagenesis then allowed us to correlate the number of N-linked glycosylation sites with the level of resistance to deamination by mA3. Our results suggest that Gag glycosylation is a possible viral defence mechanism that arose to counteract the evolutionary pressure imposed by mA3. Overall, my projects show the important role A3 proteins play in intrinsic immunity, whether defending the host from foreign retroviral invaders or endogenous retroviral foes.