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Alzheimer’s disease (AD) is characterized by amyloid-β (Aβ) and tau pathologies, insulin resistance, neuro-inflammation and dysregulation of cholesterol homeostasis, all of which play a role in neuro-degeneration. The main aim of this study was to determine possible relationships between insulin signaling, cholesterol biosynthesis and their effects on Aβ, and inflammatory response in vitro. Insulin treatment increased cholesterol synthesis in human Neuroblastoma SH-SY5Y (SHY) and mouse neuroblastoma 2a (N2a) and N2a transfected with human APP (N2a-APP) by up-regulating biosynthesis enzymes including 24-dehydrocholesterol reductase (DHCR24) and 3-hydroxy-3methyl-glutaryl-CoA reductase (HMGCR) through sterol regulatory element binding protein-2 (SREBP2) up-regulation. Aβ caused insulin resistance in N2a-APP cells by phosphorylating IRS-1 at Ser612, inhibiting signaling to downstream targets. Aβ1-42-treated SHY exhibited similar IRS-1 phosphorylation at Ser612 and inflammatory response of JNK activation. Aβ1-42 caused down-regulation of neuro-protective/anti-inflammatory DHCR24, and an increase in HMGCR levels indicating dysregulation of cholesterol homeostasis in SHY cells. Insulin resistance, Aβ toxicity, neuro-inflammation and dysregulation of cholesterol homeostasis appear to be intertwined processes in AD that should be studied simultaneously.

Intracerebral hemorrhage (ICH) is a devastating form of stroke that results from rupture of a blood vessel in the brain. Tissue inside the hematoma is irreversibly damaged soon after ICH onset and when this thesis research began, there was a dearth of information regarding pathological changes outside the hematoma. Inflammation is often proposed as a mechanism of injury, but very little information was available to show that inflammatory cells were in the right place at the right time to cause secondary brain injury. Using the collagenase-induced model of ICH, this work sought to better define spatial and temporal relationships between secondary brain injury and the inflammatory response after ICH. To test the hypothesis that reducing inflammation can protect the brain from secondary injury, minocycline, an antibiotic with established anti-inflammatory effects, was administered 6 hours after ICH onset. A small number of neurons die in the parenchyma bordering the hematoma between 6 hours and 3 days after ICH onset. This area was not associated with neutrophil infiltration, and most activated microglia/macrophages did not accumulate until after most neuron death had occurred. Despite a pronounced microglial response and prolonged increase in expression of many inflammatory genes, including complement receptor-3, interleukin-1 beta, interleukin-6, and interleukin-1 converting enzyme, no dying neurons were observed further outside the hematoma at any time. Interestingly, less early neuron death was observed in aged than in young animals, without a concomitant difference in the amount of tissue lost at 28 days. However, aged animals had less early microglial activation and a larger residual lesion, which might have resulted from impaired phagocytosis by activated microglia/macrophages. Minocycline was less effective in reducing microglial activation in aged animals, and did not reduce neuron death in either young or aged animals. Edema and BBB disruption was associated with degradation of the basal lamina protein, collagen type IV, and that damaged vessels are associated with tumor necrosis factor-alpha (TNFα)-positive neutrophils and active matrix metalloprotease-12 (MMP-12), all of which were reduced by delayed minocycline treatment. In contrast to ischemic stroke, there is a limited ‘penumbra’ outside the hematoma. Nevertheless, BBB damage in this region appears to be a potential target for protection. Furthermore, the prominent inflammatory response that continues for days after ICH does not appear to be associated with damage to other areas of the brain. Minocycline appears to protect the BBB by reducing neutrophil infiltration and the MMP-12 mediated basal lamina degradation. Future studies should investigate other targets for protection (i.e., white matter injury), and seek drugs that modulate the inflammatory response in aged animals and promote lesion resolution.

Microglia, the resident immune cells of the central nervous system, can become activated following injury, disease, or infection. In vitro, they can be activated by stimuli, which determine the inflammatory phenotype they will develop. In this thesis, stimulating microglia with tumor necrosis factor- and interferon- resulted in classical activation, characterized by proliferation, increased transcription of complement receptor 3 and major histocompatibility class II molecules, and elevated production and transcription of interleukin-1 and nitric oxide. Stimulation with TNF and IFN also changed the intensity of phosphorylated (activated) cyclic adenosine monophosphate response element binding protein immunoreactivity in microglia. Specifically, cells differentiated into populations with high or low pCREB intensity. This was the first example of such a response in microglia and was representative of what occurred in vivo, after ICH. Thus, the characterization of this model will be useful for future studies of this and other intracellular pathways of classically activated microglia.

Alzheimer's disease (AD) is the most common form of dementia among elderly. The disease is characterized by amyloid-β (Aβ) plaques, neurofibrillary tangles, loss of synapses and neurons and chronic neuroinflammation. The significance of neuroinflammatory processes in disease on-set and progression has been debated since activated microglia and reactive astrocytes have been attributed both protective and damaging properties. However, patients systematically treated with anti-inflammatory drugs have been shown to develop AD to a lesser extent than average. This indicates an important role of neuroinflammation in AD. This thesis focuses on two inflammatory related transcription factors, nuclear factor κB (NF-κB) and CCAAT/enhancer binding protein (C/EBP). Both NF-κB and C/EBP are known regulators of many pro-inflammatory genes and may during certain circumstances dimerize with each other. In paper I we use a new strategy to inhibit NF-κB DNA binding activity in primary astro-microglial cell cultures treated with Aβ and IL-1β. By coupling the NF-κB decoy to a transport peptide both concentration and incubation time can be shortened in comparison to previous studies. Moreover, using the same in vitro model in paper II and III, we show members of the C/EBP family to be dysregulated during AD mimicking conditions. Additional focus was directed towards C/EBPδ, which was shown to respond differently to oligomeric and fibrillar forms of Aβ. Results were also confirmed in vivo using an AD mouse model characterized by high levels of fibrillar Aβ deposits. Finally, in order to get further insight in neurodegenerative processes, induced by Aβ or microglial activation, we present in paper IV a new set of anchored sensors for detection of locally activated caspases in neuronal cells. By anchoring the sensors to tau they become less dynamic and caspase activation can be detected early on in the apoptotic process, in a spatio-temporal and reproducible manner.

Elevated levels of pro-inflammatory cytokines are implicated in the pathogenesis of major depression. Human and animal studies have shown that pro-inflammatory cytokines can induce a behavioural repertoire of symptoms collectively referred to as 'sickness behaviours', which include cognitive and mood symptoms, social withdrawal and sleep disturbance. When likened to clinical depression, these symptoms appear to be strikingly similar. Moreover, subsets of depressed patients have raised inflammatory markers, 30-50% of patients receiving cytokine treatment in the form of interferon-α (IFN-α) therapy develop depressive symptoms, and significantly higher rates of depression are associated with chronic inflammatory conditions, such as rheumatoid arthritis (RA). Converging evidence has led to the hypothesis that chronic, low-grade inflammation could lead to more persistent alterations in neuropsychological function that might be instrumental in the pathophysiology of depression. However, the mechanisms for this potential modulation of mood and cognitive function are unclear. The current thesis therefore aimed to enhance understanding of the neuropsychological underpinnings of the link between inflammation and depression. Negative emotional processing biases are well-recognised in the aetiology of depression; however potential inflammation-induced alterations in emotional processing remain unexplored. Thus, a series of studies were conducted using human models of immune system activation to examine neuropsychological function. The first study demonstrated that IFN-α treatment in patients with hepatitis C produced negative biases in emotional categorisation, attentional vigilance and a specific effect of enhanced recognition of disgust. The subsequent study found a specific effect of false discrimination of disgusted faces in a healthy volunteer model of inflammatory challenge with typhoid vaccination, however further effects on emotional processing were limited. Typhoid vaccination was also shown to disrupt sleep continuity in ways that may be relevant to depression in the third study. Negative biases were not evident, however, in patients with RA. The final study found that neuropsychological effects of the atypical antidepressant tianeptine were similar to effects following IFN-α, which may be of interest considering tianeptine's purported serotonergic re-uptake enhancing properties and the effects of cytokines on serotonin metabolism. This thesis provides intriguing, yet preliminary, evidence that inflammatory pathways may modulate emotional processing - a mechanism which, if supported, may have future implications for improved identification and treatment of subgroups of depressed patients.

Hyperglycemia associated with diabetes has been recognized for adverse neurodegenerative effects it has on the central nervous system (CNS). However, few cerebral histopathological studies have been completed to adequately define the neuropathology of type 2 diabetes. The aim of the study was to conduct a neuropathological survey of diabetic Zucker Diabetic Sprague Dawley (ZDSD) rat brains that included a wide variety of potential pathologies. Ten ZDSD rat brains (diabetic: n=6 non-diabetic obese: n=4) were collected for neuropathological assessments. Specific measures include assessments of gray and white matter atrophy, neurodegeneration, astrocyte activation, blood brain barrier integrity, inflammation, and amyloid protein deposit. After brain sectioning, formal thionin, immunoglobulin G (IgG), glial fibrillary acidic protein (GFAP), giemsa, congo-red, and flourojade (FJ) stains were performed for analysis. Of the several neuropathological assessments, two revealed significant differences between diabetic and non-diabetic groups. Diabetic ZDSD rats had a relative decrease in the amount of white matter in the corpus callosum underlying the cingulate cortex of the brain. Secondly, higher numbers of lymphocytes were observed in the hypothalamus of the diabetic rats compared to non-diabetic rats. Enhanced expression of GFAP was not present. No measurable differences were observed in analysis of amyloid, FJ intensity levels or immunoglobulin G (IgG) extravisation into the brain. These results suggested that ZDSD rats do not exhibit neuropathology excepting white matter atrophy and increased lymphocyte infiltration into the hypothalamus, or that the duration of 6-7 month old diabetic ZDSD rats may be insufficient to support most of our hypotheses. Future work is required to determine profiles of neuropathology in longer term of diabetic ZDSD rats.

Neuromyelitis optica is a severe autoimmune neuroinflammatory disorder characterised by longitudinally extensive myelitis and severe optic neuritis. An oft-neglected symptom of the condition is severe, intractable chronic pain that can be detrimental to quality of life and is marked out by its severity and prevalence in comparison to the related disorder, multiple sclerosis. The experiments within this thesis make use of both conventional and advanced MRI techniques applied to the spinal cord and brain, and ¹H NMR spectroscopy of blood plasma to explore the mechanisms driving chronic pain in NMO. The principle findings are: i. Thoracic lesions are associated with greater pain, irrespective of lesion length or cervical lesion volume. They are associated with spinothalamic tract damage in the cervical cord that correlates with the severity of pain. A possible autonomic aetiology is proposed. ii. Periaqueductal grey (PAG) to dorsolateral prefrontal cortex and to pregenual anterior cingulate cortex functional connectivity is correlated with pain severity, conversely PAG to rostroventromedial medulla connectivity is negatively associated with pain severity. Disruption of descending pain modulatory circuits is considered. iii. PAG glutamate concentration is negatively correlated with pain scores and is higher in low pain patients compared to controls and high pain patients. The discussion includes consideration of pain vulnerability. Chronic pain in NMO can devastate lives. The studies undertaken in this thesis are some of the earliest MRI imaging studies directed at understanding pain in NMO and the association of pain with thoracic lesions and the aberrations in the descending pain modulatory network are novel findings. There is more to be done and my hope is that this body of work will serve as a stepping-stone to a better understanding of chronic pain in NMO and the future development of effective treatments.

Infections are a common co-morbidity in Alzheimer's disease (AD), and evidence suggests that infections can exacerbate neuroinflammation and increase cognitive decline in AD patients. In AD, immune changes are observed both in the central nervous system (CNS) and in the rest of the body. However, only a few studies have investigated immune responses to infection in AD. Here, two extensively studied infections, Toxoplasma gondii (T. gondii) and Trichuris muris (T. muris) were used to investigate infection in AD. T. gondii is a protozoan parasite which is common globally, including in the developed world where AD cases are increasing dramatically. Infection with T. gondii starts in the gut, before becoming systemic and then infecting the CNS, where the parasite forms a chronic cyst infection. In contrast, T. muris is a nematode parasite, which remains localised to the gut. Notably, T. gondii is known to alter neuroinflammation and behaviour. T. gondii forms cysts preferentially in the areas of the brain commonly affected by AD, such as the hippocampus, which therefore makes it an interesting model to study co-morbidity. AD is often associated with advanced age. As we age, our immune system declines, and an important unanswered question is whether age impacts on the immune response to infection. This is of particular significance when considering chronic infections such as T. gondii, which require immune surveillance to prevent parasite recrudescence. Therefore, the aim of this thesis was to investigate infection in AD by determining: whether the immune response to an infection is altered in AD; whether the immune response to an infection in AD differs with age; what the effects of infection are on neuroinflammation, pathology and behaviour in AD; what are the effects of chronic infection with T. gondii. Immune responses to infection were altered in both the 3xTg-AD and the APP PS1 mouse models of AD, including increased inflammation and weight loss in AD mice following infection. Although older (eleven to twelve-month-old) 3xTg-AD mice showed some alterations in cytokine responses following infection, overall there were no major difference compared to younger (five to six-month-old) animals. Additionally, infection was found to alter neuroinflammation in both 3xTg-AD and APP PS1 mice, though differently. In 3xTg-AD mice, microglia activation increased following infection with T. gondii and T. muris, showing that infection did not need to be in the brain to alter neuroinflammation. In APP PS1 mice, a decrease in microglia activation occurred after infection with T. gondii, which was accompanied by an increase in IL-1alpha production and increased amyloid beta levels in APP PS1 mice following infection. However, no changes were found in behaviour following infection with T. gondii or T. muris in AD mouse models. Finally, chronic T. gondii infection was investigated in the TgF344-AD rat, which was established as a suitable AD model with both amyloid and tau pathology in which to study chronic infection. This work adds to a growing body of literature to suggest that infections are detrimental to AD patients, and that future measures to decrease morbidity could focus on further study of infections in AD, and the development of strategies to better prevent infections in AD patients.

O Transtorno Bipolar (TB) é uma doença psiquiátrica grave, altamente incapacitante que está associada com diversas comorbidades médicas e altas taxas de suicídio. Embora sua fisiopatologia não esteja completamente elucidada, inúmeros estudos têm mostrado alterações no sistema imune de indivíduos com TB. A resposta crônica destes indivíduos ao estresse parece gerar um aumento da inflamação sistêmica bem como da neuroinflamação. A micróglia ativada devido aos estímulos inflamatórios contínuos deve ocasionar diferentes prejuízos tanto bioquímicos quanto funcionas. Os macrófagos, primeira linha de defesa, são células de característica plástica de extrema importância do sistema imune e podem ser estimulados a polarizar para diferentes formas com liberação de fatores pró e antiinflamatórios, estimulando ou mantendo a homeostase no ambiente agredido de alguma forma. Desta forma, nosso trabalho buscou investigar a resposta fenotípica dos macrófagos contra o meio ambiente pró-inflamatório sistêmico observado no plasma de pacientes bipolares eutímicos, maníacos e depressivos em comparação aos controles. A amostra incluiu 5 controles saudáveis, 8 pacientes bipolares remetidos, 5 pacientes maníacos e 5 pacientes depressivos. As citocinas e quimiocinas de RNAm em células U937 tratadas com plasma mostraram um padrão de expressão diferente relativo entre controles saudáveis e pacientes com TB. As citoquinas inflamatórias tais como IL-1β e TNF-α, em pacientes bipolares maníacos e depressivos demonstram maiores quantidades de IL-1β mRNA do que os pacientes eutímicos e pacientes depressivos induziram maiores quantidades de RNAm de TNF-α do que os pacientes eutímicos em células U937. Já a expressão das quimiocinas CXCL9 e CXCL10 no plasma de pacientes com TB depressivos, demostraram ser de menor expressão significativa no grupo de pacientes maníacos quando comparados a controles e pacientes bipolares eutímicos. Nossos resultados sugerem que as citocinas periféticas devem modular a polarização M1 ou M2 de macrófagos no TB. / Bipolar Disorder (BD) is a severe and highly incapacitating psychiatric disorder which is associated with the presence of medical comorbidities. The progression of BD is related to an important cognitive deficit and also to biological and clinical manifestations that lead to treatment resistance and worse prognosis. Immune disturbances have been widely observed and investigated in BD patients. Chronic inflammatory responses induce neuroinflammation, mainly by pro-inflammatory microglial activation, and result in biochemical and functional impairment. Macrophages are the first line of defense of the immune system and exhibit cell plasticity. As well, microglia represents the resident macrophage of the central nervous system been responsible for its protection. Both cells can be stimulated to polarize into two different phenotypes, mainly pro- and anti-inflammatory, maintaining the homeostasis under physiologic and pathologic conditions. Therefore, we aimed to investigate macrophages phenotypical response when submitted to BD patients plasma in different episodes, which is considered a pro-inflammatory environment, and healthy controls plasma. Subjects included healthy controls (n=5), remitted BD patients (n=8), manic patients (n=5) and depressive patients (n=5). The mRNA expression of chemokynes and cytokines from U937 cells treated with BD patients plasma were different from those submitted to healthy controls plasma. Higher mRNA expression of IL-1β was observed in those cells submitted to manic and depressive BD patients plasma when compared to euthymic patients. Also, depressive BD patients plasma induced higher expression of TNF-α compared to euthymic patients. However, chemokynes expression, such as CXCL9 and CXCL10, were reduced in depressive BD patients. However, chemokynes expression, such as CXCL9 and CXCL10, were reduced in depressive BD patients. Inflammatory cytokines such as IL-1β and TNF-α in bipolar manic and depressive patients demonstrate higher amounts of IL-1β mRNA that euthymic patients and depressive patients induced higher amounts of TNF-α mRNA levels than the patients in euthymic U937. Since the expression of CXCL9 and CXCL10 chemokines in plasma from patients with depressive TB, proved less significant expression in the group of manic patients when compared to controls and euthymic bipolar patients.