Orientador: Gisele Fabrino Machado / Banca: Flávia Lombardi Lopes / Banca: Valéria Marçal Felix de Lima / Banca: Sandra Helena Penha de Oliveira / Resumo: A leishmaniose visceral canina (LVC) é uma doença crônica, invariavelmente progressiva, de alta variabilidade clínica, incluindo desordens neurológicas. Para investigar as alterações neurológicas desencadeadas pela LVC, a expressão de genes envolvidos na resposta imune inata e adaptativa em cérebros de cães naturalmente infectados para Leishmania infantum foi realizada. Cães machos/fêmeas, 1 a 5 anos de idade, sem raça definida, com prévia avaliação clínica e laboratorial, avaliação histopatológica cerebral, e ausência das principais co-infecções foram organizados em dois grupos, grupo (Infectados) com 11 cães e grupo (Controle) com 04 cães. A avaliação da expressão gênica foi realizada por PCR Profiler com kit comercial RT² Profiler™ PCR Array Dog Innate & Adaptive Immune Responses (Qiagen®). O p-valor estipulado foi em ≤ 0,05 por Teste-t de Student. Infiltrados inflamatórios, de variação discreta a moderada, foram verificados nos cães infectados. Houve o aumento da expressão de 24 genes de diferentes subclasses comparados ao grupo controle (IRF6, CD4, CD40LG, TLR6, CCR4, TLR5, CCR8, CCL3, IFNG, IFNB1, TNF, IL13, IRF3, CD14, CD209, CD80, TLR4, CD86, CCR6, TRAF6, TLR7, RORC, NOD1, STAT3). Esta informação indica a participação da resposta imune inata e adaptativa no cérebro de cães com LVC, e a alta expressão destes pode ser considerada fator chave para o início, manutenção e persistência da inflamação. / Abstract: Visceral canine leishmaniasis (LVC) is a chronic, invariably progressive disease of high clinical variability including neurological disorders. To investigate the neurological changes triggered by LVC, the expression of genes involved in the innate and adaptive immune response in brains of naturally infected dogs to Leishmania infantum was performed. Male and female dogs, 1 to 5 years of age, with no previous breed, previous clinical and laboratory evaluation, histopathological evaluation, and absence of major coinfections were organized into two groups (Infected) with 11 dogs and group Control) with 04 dogs. The evaluation of gene expression was performed by PCR Profiler with commercial Kit RT² Profiler ™ PCR Array Dog Innate & Adaptive Immune Responses (Qiagen®). The p-value stipulated was ≤ 0.05 per Student's t-Test. Inflammatory infiltrates, mild to moderate, were found in infected dogs. There was an increase in expression of 24 genes of different subclasses compared to the control group (IRF6, CD4, CD40LG, TLR6, CCR4, TLR5, CCR8, CCL3, IFNG, IFNB1, TNF, IL13, IRF3, CD14, CD209, CD80, TLR4, CD86, CCR6, TRAF6, TLR7, RORC, NOD1, STAT3). This information indicates the involvement of the innate and adaptive immune response in the brain of dogs with LVC, and the high expression of these can be considered a key factor for the onset, maintenance and persistence of inflammation. / Doutor

Thesis advisor: Kenneth C. Williams / Thesis advisor: Welkin Johnson / Neuropathogenesis of HIV-associated neurocognitive disorders (HAND) is likely instigated by chronic immune activation in response to residual infection in the central nervous system (CNS), where combined antiretroviral therapy (cART) has limited access. Monocyte/macrophages (Mo/Mϕ) constitute the predominant population of infected cells in the CNS and play a major role in HIV-induced neuropathogenesis. Emergence of compartmentalized HIV subpopulations in the brain corresponds with accumulation of HIV-infected Mo/Mϕ and is consistent with acquired immune deficiency syndrome (AIDS)-related neuropathology. We used a rhesus macaque model of neuroAIDS to elucidate the role of Mo/Mϕ in establishing CNS infection and the emergence of compartmentalized virus in the brain. To do this, we: 1) performed phylogenetic analysis of viral sequences from peripheral and CNS compartments and determined the incidence of Mo/Mϕ infection in CNS tissues to identify sources of CNS viral subpopulations that emerge with AIDS-related neuropathology; 2) optimized a method for obtaining single genome viral sequences from Mo/Mϕ populations extracted from tissues and 3) performed phylogenetic analysis of viral sequences from bone marrow (BM) and CNS Mo/Mϕ and determined the incidence of Mo/Mϕ infection in the BM to assess whether BM Mo/Mϕ are sources of infected Mo/Mϕ that accumulate in the CNS with AIDS-related neuropathology. We found that animals with AIDS-related neuropathology had a higher incidence of Mo/Mϕ infection and compartmentalized SIV subpopulations in CNS tissues compared to animals without neuropathology. Additionally, CSF virus, which is used to assess the presence of CNS virus compartmentalization in living patients, was not compartmentalized even with significant compartmentalization in the brain and severe AIDS-related neuropathology (Chapter 2). Relative to animals without CNS pathology, animals with AIDS-related neuropathology had a higher incidence of Mo/Mϕ infection in the BM and viral sequences from BM and CNS perivascular Mo/Mϕ clustered with sequences from trafficking monocytes and CNS tissues (Chapter 4). The results suggest that infected Mo/MΦ in CNS tissues are sources of compartmentalized virus and that infected Mo/Mϕ in the BM are sources of infected Mo/Mϕ that accumulate in the CNS with AIDS-related neuropathology. In summary, the data in this dissertation suggest that targeting Mo/Mϕ may prevent CNS infection and inflammation associated with HIV-induced neuropathogenesis. / Thesis (PhD) — Boston College, 2018. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Zika virus (ZIKV) has emerged in many regions of the world, with infection outcomes spanning from no apparent illness to crippling nervous system disease. ZIKV and its close relatives, West Nile virus, Japanese encephalitis virus, dengue virus, and yellow fever virus are primarily transmitted by mosquitoes. Three ZIKVs were selected: MR-766 (Uganda, 1947), P6-740 (Malaysia, 1966), and PRVABC-59 (Puerto Rico, 2015), whose place of origin and time of isolation differ substantially. Stable, complementary DNA (cDNA) copies of the three ZIKV RNA genomes were cloned to examine the significance of viral and host genetic variations in directing ZIKV infection outcomes. Using a new toolbox for ZIKV genome engineering and protein analysis, combined with various cell culture and mouse infection model systems, the following were determined: (1) Genome-wide landscape of viral gene products and their related species, with several immuno-reactive gene products identified in the case of all three cloned ZIKVs. (2) Viral replicability in cultured cells, varied significantly depending on the virus strain and host cell type, with one cow cell line being resistant to ZIKV infection. (3) Virus induced neurological disease in mice, differed dramatically depending on the virus dose and strain, mouse age and strain, route of infection, and presence or absence of immune system components. Overall, the findings demonstrate the impact of the viral and host genetic backgrounds on the ability of ZIKV to replicate and cause disease. The ZIKV strain-specific characterizations and molecular instruments described will provide multiple avenues for developing and testing medical countermeasures.

Human immunodeficiency virus type 1 (HIV-1) is known to cause a spectrum of neurological, behavioral and motor deficits collectively termed as HIV-1 associated neurocognitive impairments (HAND). Opiates augment HIV-related CNS complications through both direct and indirect mechanisms that disrupt glial and neuronal function. All CNS macroglia and neurons derive from neural progenitor cells (NPCs) during development, and NPCs in the adult brain contribute to repair processes. Since disruptions in NPC function are known to impact CNS populations and brain function in a number of disease/injury conditions, we determined whether HIV ± opiate exposure affected the maturation and fate of human NPCs (hNPCs). As hNPC infection by HIV has occasionally been reported, we also reexamined this question, and parsed between effects due directly to hNPC infection by HIV, or to hNPC dysfunction caused by the infective milieu. Multiple approaches confirmed the infection of hNPCs by R5 tropic (CCR5 utilizing) HIVBaL, and demonstrated that active infection could be sequentially transferred to naïve hNPCs. Exposure to supernatant from HIVBaL-infected cells (HIVsup) reduced hNPC proliferation and led to premature differentiation into astrocytes and neurons. Morphine co-exposure prolonged hNPC infection and exacerbated functional effects of HIVsup. Neither purified virions nor UV-inactivated HIVsup altered proliferation, indicating that this effect did not require infection. Gene array analysis and RT-qPCR with immunoblot validation suggested that Cdk5 signaling was involved in HIV-morphine interactions. siRNA-mediated knockdown of Cdk5 expression attenuated the effect of HIV-1 and morphine on hNPC proliferation and MAP2 differentiation, but also increased hNPC death. Furthermore, in an attempt to understand the role of mu-opioid receptor (MOR) splice variants on the interactive effect of HIV-1 and morphine on hNPCs, we found that both MOR-1 and MOR-1K are differentially regulated by HIV-1 in these cells. This suggests that these splice variants may have differential actions in the response of hNPCs to HIV-1 and morphine co-exposure. Given the overlap of Cdk5 and MOR signaling, it is likely that MOR-1K and/or MOR-1 converge with Cdk5 in the mechanism underlying HIV-1 and morphine interaction in hNPCs. Overall, dysregulation of hNPC functions by the infectious environment may create cell population imbalances that contribute to CNS deficits in both adult and pediatric patients. Additionally, infected hNPCs may pass virus to their progeny, and serve as an unappreciated viral reservoir. The recent epidemic of opiate/heroin abuse highlights the clinical importance of HIV and opiate interactions.