Maternal exposure to viral and bacterial infection during pregnancy is associated with fetal dysmorphology and neurodevelopmental disorders including schizophrenia, cerebral palsy, autism and mental retardation. Previous studies in our laboratory using an established mouse model of endotoxin-induced fetal dysmorphology have led to the hypothesis that birth defects caused by infections during pregnancy are the result of fetal zinc deficiency resulting from the induction of a zinc-binding protein, metallothionein (MT) in the maternal liver as part of the maternal inflammatory response. Thus, we predicted that zinc deficiency would exacerbate the negative fetal outcomes caused by bacterial endotoxin lipopolysaccharide (LPS) and that zinc supplementation would protect against LPS-mediated teratogenicity. This premise was investigated herein and was extended to investigate underlying molecular mechanism, including the identification of markers of neurodevelopmental damage following LPS administration in early and late pregnancy, and to determine the influence of zinc treatment on any changes in expression of these markers. In Chapter 2 it was demonstrated that prenatal exposure to LPS on gestational day (GD) 8 resulted in the development of physical birth defects including exencephaly, microcephaly, cleft lip and or palate, and micrognathia in GD 18 fetuses. Dietary zinc supplementation throughout pregnancy was found to prevent the LPS-related abnormalities. Furthermore, low dietary zinc and LPS exposure were found to be synergistic on teratogenicity. In addition, an inverse linear relationship was observed between the concentration of zinc in the diet and teratogenicity with a reduction in the incidence of birth defects observed with increasing concentration of dietary zinc, a finding suggesting that even small increments of zinc above normal dietary intake are likely to have a beneficial impact on teratogenicity. Maternal infection during late pregnancy has also been linked with prenatal brain damage. A major causal link underpinning this relationship is thought to be the cytokines released following a maternal inflammatory response to infection. In Chapter 3, the presence of cytokines released in response to LPS given on GD 16 was demonstrated by an increased number of tumour necrosis factor-alpha (TNF-!)-reactive cells and astrogliosis accompanied by extensive apoptotic cell death in GD 18 fetal brain. Recently our laboratory has reported that dietary zinc supplementation throughout pregnancy, prevented impairments in object recognition memory in offspring from dams exposed to prenatal LPS on GD 8. The question arises as to whether zinc is protective against LPS-exposure in late pregnancy. In Chapter 3, it is further demonstrated that LPS-induced brain injury was prevented by concurrent zinc treatment at the time of LPS exposure. In Chapter 4, the expression of activity-dependent neuroprotective protein (ADNP) mRNA was identified as a marker of changes occurring in the fetus as a result of LPS exposure in early pregnancy. ADNP has been found to be essential for organogenesis and is a sensitive indicator of brain injury. Here it was demonstrated that LPS caused a rapid increase in embryonic ADNP expression, which was highly significant 24 hours after exposure. Whether the elevation in ADNP expression is in response to inflammatory damage or is induced by cytokines released by the maternal inflammatory response is not clear. However, a major finding of the study is that concomitant zinc treatment prevented the LPS-induced increase in ADNP activity. The mechanism of protection by zinc is presumed to be centred on preventing the fall in plasma zinc and associated fetal zinc deficiency caused by LPS induction of MT, but may also include MT-independent actions of zinc including prevention of apoptosis and oxidative damage, or enhance tissue repair processes. Taken together the findings in this thesis support earlier evidence that maternal MTmediated transient fetal zinc deficiency in early pregnancy underpins LPS-induced teratogenicity. This is the first study to demonstrate that this mechanism may also apply to LPS-induced neurodevelopmental damage in early and late pregnancy. However, further studies are warranted to discriminate between the influence of MT and that of other inflammatory reactants (e.g. cytokines) on LPS-mediated damage late in pregnancy. The major finding of the thesis is that zinc treatment (either given subcutaneously with LPS or as dietary zinc supplementation throughout pregnancy) prevents the negative fetal outcomes including neurodevelopmental damage caused by prenatal exposure to LPS. This finding highlights the importance of zinc nutrition in pregnancy and the benefits that might be gained as a potential prophylactic treatment to minimise fetal damage caused by infections during pregnancy. / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2009

As respostas inflamatórias no cérebro são mediadas principalmente pela microglia, mas evidências crescentes sugerem uma importância crucial dos astrócitos. A S100B, uma proteína ligante de cálcio e secretada por astrócitos, tem propriedades neurotróficas e de citocina inflamatória. No entanto, não se sabe se sinais primários que ocorrem durante a indução de uma resposta inflamatória como, por exemplo, lipopolissacarídeo (LPS) modulam diretamente a S100B. A neuroinflamação está implicada na patogênese ou na progressão de uma variedade de distúrbios neurodegenerativos e muitos estudos procuram uma conexão entre S100B e doenças degenerativas, incluindo a doença de Alzheimer e esquizofrenia. O uso terapêutico de fármacos anti-inflamatórios não-esteroidais (AINEs) para estas doenças tem aumentado. No entanto, existem poucos estudos sobre o efeito desses fármacos em relação à proteína S100B. Neste trabalho, nós avaliamos se os níveis de S100B no líquido cefalorraquidiano (LCR) e soro de ratos Wistar são afetados por injeção de LPS administrado por via intraperitoneal (IP) ou intracerebroventricular (ICV), bem como se as culturas primárias de astrócitos respondem diretamente ao LPS. Além disso, nós avaliamos o conteúdo e a secreção de S100B medido por ELISA (bem como o conteúdo de GFAP e secreção de TNF-α) em culturas primárias de astrócitos expostos a dexametasona e quatro classes químicas diferentes de AINEs (ácido acetilsalicílico, ibuprofeno, diclofenaco e nimesulida) durante 24 h. Os nossos dados sugerem que a secreção de S100B no tecido cerebral é estimulada rapidamente e persistentemente (durante pelo menos 24 h) por administração ICV de LPS. Este aumento da S100B no LCR foi transitório quando o LPS foi administrado IP. Em contraste com estes resultados de S100B, observou-se um aumento nos níveis de TNF-α no soro, mas não no LCR, após a administração IP de LPS. Em astrócitos isolados e em fatias de hipocampo frescas, observou-se uma estimulação direta da secreção de S100B por LPS numa concentração de 10 ug/ml. Um envolvimento de TLR4 foi confirmado pelo uso de antagonistas específicos deste receptor. No entanto, baixas concentrações de LPS em culturas de astrócitos foram capazes de induzir uma diminuição na secreção de S100B após 24 h, sem alteração significativa no conteúdo intracelular de S100B. Além disso, após 24 horas de exposição ao LPS, observou-se um decréscimo na glutationa e um aumento na proteína ácida fibrilar glial. Também foi observado que os AINEs apresentam diferentes efeitos sobre parâmetros gliais. O ácido acetilsalicílico e o diclofenaco foram capazes de aumentar a GFAP, enquanto que a nimesulida, um inibidor seletivo de COX-2, e a dexametasona diminuiram a secreção de S100B. No entanto, todos os AINEs reduziram os níveis de PGE2. Juntos, esses dados contribuem para a compreensão dos efeitos de LPS em astrócitos, especialmente sobre a secreção de S100B, e nos ajuda a interpretar mudanças nesta proteína no LCR e soro em doenças neuroinflamatórias. Além disso, tecidos periféricos que expressam S100B talvez devam ser regulados diferentemente, uma vez que a administração IP de LPS não foi capaz de aumentar os níveis séricos de S100B. Em relação aos AINEs, a PGE2 parece estar envolvida no mecanismo de secreção de S100B, mas vias adicionais, não claras neste momento, necessitam de uma maior caracterização. O papel inflamatório de S100B em doenças degenerativas, onde também são observados níveis elevados da COX-2 e PGE2, poderia ser atenuado por inibidores de COX-2. / Inflammatory responses in brain are primarily mediated by microglia, but growing evidence suggests a crucial importance of astrocytes. S100B, a calciumbinding protein secreted by astrocytes, may act as a neurotrophic or an inflammatory cytokine. However, it is not known whether primary signals occurring during induction of an inflammatory response (e.g. lipopolysaccharide, LPS) directly modulate S100B. Neuroinflammation has been implicated in the pathogenesis or progression of a variety of neurodegenerative disorders and several studies have looked for a connection of S100B, and degenerative diseases including Alzheimer’s disease and schizophrenia. The therapeutic use of non-steroid anti-inflammatory drugs (NSAID) to these diseases has growth up. However, there are few reports about the effect of these drugs on S100B. In this work, we evaluated whether S100B levels in cerebrospinal fluid (CSF) and serum of Wistar rats are affected by LPS administered by intraperitoneal (IP) or intracerebroventricular (ICV) injection, as well as whether primary astrocyte cultures respond directly to lipopolysaccharide. Moreover we evaluated S100B content and secretion measured by ELISA (as well as GFAP content and TNF-α secretion) in primary astrocyte cultures exposed to dexamethasone and 4 different chemical classes of NSAID (acetyl salicylic acid, ibuprofen, diclofenac and nimesulide) for 24 h. Our data suggest that S100B secretion in brain tissue is stimulated rapidly and persistently (for at least 24 h) by ICV LPS administration. This increase in CSF S100B was transient when LPS was IP administered. In contrast to these S100B results, we observed an increase in in TNFα levels in serum, but not in CSF, after IP administration of LPS. In isolated astrocytes and in acute hippocampal slices, we observed a direct stimulation of S100B secretion by LPS at a concentration of 10 μg/mL. An involvement of TLR4 was confirmed by use of specific inhibitors. However, lower levels of LPS in astrocyte cultures were able to induce a decrease in S100B secretion after 24 h, without significant change in intracellular content of S100B. In addition, after 24 h exposure to LPS, we observed a decrease in astrocytic glutathione and an increase in astrocytic glial fibrillary acidic protein. We also observe that NSAIDs have distinct effects on glial parameters. ASA and diclofenac are able to increase GFAP, while nimesulide, a selective COX-2 inhibitor, and dexamethasone were able to decrease S100B secretion. However, all anti-inflammatories were able to reduce levels of PGE2. Together, these data contribute to the understanding of the effects of LPS on astrocytes, particularly on S100B secretion, and help us to interpret cerebrospinal fluid and serum changes for this protein in neuroinflammatory diseases. Moreover, non-brain S100B-expressing tissues may be differentially regulated, since LPS administration did not lead to increased serum levels of S100. With respect to NSAIDs, PGE2 is possibly involved in the mechanism of S100B secretion but additional pathways, unclear at this moment, demand further characterization. The inflammatory role of S100B in degenerative diseases, where also is observed elevated levels of COX-2 and PGE2, could be attenuated by COX-2 inhibitors in which elevated levels of COX-2.

Systemic inflammation is thought to be an important driver in chronic neurodegeneration. During systemic infection, the inflammatory status of the periphery is communicated to the brain and conserved sickness behaviours initiated. However, in the context of dementia the same inflammatory stimulus might trigger delirium. Delirium is a severe, transient neuropsychiatric condition characterised by altered levels of arousal, inattention, cognitive deficits and psychoses. Delirium and systemic inflammation exacerbate the trajectory of pre-existing dementia, and are associated with increased risk of future dementia. Accumulating experimental studies suggest microglia are “primed” by chronic neurodegeneration, such that a subsequent inflammatory insult – central or systemic – induces an increased inflammatory response which manifests as exaggerated sickness behaviours. To date there have been no studies of microglial priming in the context of pure tau pathology, without amyloid pathology, and none investigating acute sickness behaviour in such a model. The overarching aim of this thesis is to address this gap in the literature and further our understanding of the interactions between systemic inflammation, neuroinflammation and neurodegeneration in the context of tauopathy. The P301S mouse over-expresses human mutant tau protein under the Thy1.2 promoter. It develops hyperphosphorylated and insoluble tau accumulations and progressive neuronal loss. Consequently, P301S mice develop progressive hind limb paralysis. This study identified the horizontal bar task, a test of motor control and coordination, conducted at weekly intervals from 8-22 weeks of age, as a non-invasive measure of disease progression. In addition, a detailed temporal profile of pathological hallmarks at 8, 9, 10, 11, 12, 16 and 20 weeks of age was determined. Key results presented here demonstrate progressive, superficial neuronal loss in the cortex of P301S mice, with associated astrogliosis and surprisingly this occurs in the absence of apparent cortical microgliosis. In stark contrast, there is progressive microgliosis in the spinal cord of P301S mice. On this background, lipopolysaccharide (LPS), a chemical moiety found on the outer surface of gram-negative bacteria, was used to mimic a systemic bacterial infection. P301S mice and C57BL/6 control mice were injected, at 10 or 16 weeks of age, intraperitoneally with 500 μg/kg LPS or saline and were monitored in the following hours and weeks. Acutely, P301S mice showed signs of an exaggerated, longer lasting sickness response. Importantly, exaggerated acute symptoms extended beyond those typically associated with sickness behaviour; LPS induced an exaggerated acute impairment of horizontal bar performance in P301S mice and not C57BL/6 mice – a function which is known to be impaired in P301S mice later in disease. Impairments were age-dependent in terms of timing of injection. These data suggest an interaction between acute infection and existing CNS vulnerability leading to acute neurological dysfunction that is not a feature observed in sickness in a normal animal. LPS-injected P301S mice also showed, again age-dependent, increased rate of decline in motor performance compared with controls. There was no evidence of microglial priming in P301S mice. LPS caused an acute increase in AT8-positive phospho-tau however this did not persist until end stage. At 22 weeks of age there was significant disease-associated cortical neuronal loss in the vehicle-injected P301S mice, and additional superficial cortical neuronal loss in LPS-injected P301S mice and control mice. There was significant IBA1-positive microgliosis in the spinal cord of P301S mice at end stage which was further increased in LPS-injected P301S mice. Taken together these data indicate a clear and clinically relevant interaction between systemic inflammation and tau-associated neuropathology with acute and long-term functional consequences. In the absence of evidence of microglial priming, future work will explore potential mechanisms.

Tanto a luteína quanto o ácido linoleico conjugado (CLA) são nutrientes que já demonstraram efeitos benéficos na modulação do sistema imune em diferentes modelos experimentais, porém o uso de ambos em conjunto ainda não foi explorado. O CLA é incorporado nas membranas celulares e hipotetiza-se que a luteína, com seu potencial antioxidante, possa proteger a estrutura do CLA em situações de estresse oxidativo. Outra razão para se pensar na interação entre estes nutrientes é que já foram observados de forma individual, efeitos modulatórios do CLA e da luteína sobre receptores celulares que atuam em conjunto e possuem papel chave na regulação da resposta imune (receptores PPAR e RXR). Objetivou-se com o presente estudo avaliar o efeito da suplementação dietética de CLA e luteína no desempenho e na resposta imune de frangos de corte de 1 a 22 dias de idade. Foram testados 3 níveis de inclusão de CLA (0, 1 e 2%) em conjunto com luteína (0 e 50 mg/kg) na presença ou ausência de desafio imunológico com LPS. O CLA e a luteína apresentaram efeitos imunomodulatórios positivos, porém, não foi observada interação entre ambos os nutrientes para as avaliações relacionadas ao sistema imune. O CLA, adicionado em 2% na dieta, elevou a produção de IgY em resposta ao estímulo com albumina de soro bovino (BSA) e também foi capaz de aumentar a expressão de RXRα no fígado. A luteína diminuiu o óxido nítrico plasmático e também a expressão de TLR-4 no baço e de IL-1β e IL-12 no fígado apesar de ter aumentado a expressão de TLR-4 hepática. O desafio com LPS estimulou a resposta inflamatória aguda, evidenciado pela queda no ganho de peso, pelo aumento da relação fígado:peso corporal, pelo aumento na expressão de IL-1β e IL-12 hepáticos e diminuição na expressão de PPARα no duodeno e fígado e de PPARγ e RXRα no baço. Entretanto, nem a luteína e nem o CLA foram capazes de reverter ou atenuar os efeitos causados pelo desafio com LPS. Para desempenho, uma forte interação entre CLA e luteína foi observada, de forma que a suplementação com 1 ou 2% de CLA pioraram o peso corporal, o ganho de peso e a eficiência alimentar de 1 a 20 dias de idade, mas estes efeitos foram revertidos quando a luteína foi incluída na dieta com 1% de CLA. Concluiu-se que o CLA teve efeito benéfico sobre a resposta imune humoral, na dependência da sua dose. A luteína se mostrou como nutriente anti- inflamatório e também capaz de reverter o efeito negativo da inclusão dietética de 1% de CLA sobre o desempenho de frangos de corte. / Both lutein and conjugated linoleic acid (CLA) have beneficial effects on the modulation of the immune system. However, the simultaneous supplementation of both lutein and CLA has not yet been examined. CLA is incorporated into cell membranes, and it is hypothesized that lutein, as an antioxidant, protects CLA structure during oxidative stress. Additionally, an examination of the interaction of lutein and CLA is vital because individual effects of CLA and lutein on nuclear receptors that work together and have important roles in the immune response have been observed (PPAR and RXR receptors). The objective of this study was to evaluate the effects of dietary CLA and lutein supplementation on the performance and immune response of 1- to 22-d-old broiler chickens. Three CLA inclusion levels (0, 1 and 2%) and two lutein levels (0 and 50 mg/kg) were tested in the presence and absence of LPS immune challenge. Lutein and CLA showed positive immunomodulatory effects, but no interaction between these nutrients on the immune system was observed. A 2% CLA supplementation increased plasmatic IgY anti-BSA production and hepatic RXRα expression. Lutein decreased plasmatic nitric oxide and TLR-4 in the spleen and IL-1β and IL-12 in the liver in addition to increasing hepatic TLR-4. LPS challenge effectively promoted an acute inflammatory response, as illustrated by decreased body weight gain, increased liver:body weight ratio, increased expression of hepatic IL-1β and IL-12, decreased expression of PPARα in the duodenum and liver and decreased expression of PPARγ and RXRα in the spleen. However, neither lutein nor CLA reversed or attenuated the effects of the LPS challenge. A strong interaction between CLA and lutein was observed on performance: CLA supplementation at 1 or 2% decreased body weight, body weight gain and feed efficiency from 1 to 20 d old. However, these negative effects were reversed when lutein was included in the 1% CLA diet. In conclusion, CLA improved the humoral immune response (depending on CLA dose). Lutein was anti-inflammatory and could reverse the negative effects of dietary 1% CLA supplementation on broiler chicken performance.

As respostas inflamatórias no cérebro são mediadas principalmente pela microglia, mas evidências crescentes sugerem uma importância crucial dos astrócitos. A S100B, uma proteína ligante de cálcio e secretada por astrócitos, tem propriedades neurotróficas e de citocina inflamatória. No entanto, não se sabe se sinais primários que ocorrem durante a indução de uma resposta inflamatória como, por exemplo, lipopolissacarídeo (LPS) modulam diretamente a S100B. A neuroinflamação está implicada na patogênese ou na progressão de uma variedade de distúrbios neurodegenerativos e muitos estudos procuram uma conexão entre S100B e doenças degenerativas, incluindo a doença de Alzheimer e esquizofrenia. O uso terapêutico de fármacos anti-inflamatórios não-esteroidais (AINEs) para estas doenças tem aumentado. No entanto, existem poucos estudos sobre o efeito desses fármacos em relação à proteína S100B. Neste trabalho, nós avaliamos se os níveis de S100B no líquido cefalorraquidiano (LCR) e soro de ratos Wistar são afetados por injeção de LPS administrado por via intraperitoneal (IP) ou intracerebroventricular (ICV), bem como se as culturas primárias de astrócitos respondem diretamente ao LPS. Além disso, nós avaliamos o conteúdo e a secreção de S100B medido por ELISA (bem como o conteúdo de GFAP e secreção de TNF-α) em culturas primárias de astrócitos expostos a dexametasona e quatro classes químicas diferentes de AINEs (ácido acetilsalicílico, ibuprofeno, diclofenaco e nimesulida) durante 24 h. Os nossos dados sugerem que a secreção de S100B no tecido cerebral é estimulada rapidamente e persistentemente (durante pelo menos 24 h) por administração ICV de LPS. Este aumento da S100B no LCR foi transitório quando o LPS foi administrado IP. Em contraste com estes resultados de S100B, observou-se um aumento nos níveis de TNF-α no soro, mas não no LCR, após a administração IP de LPS. Em astrócitos isolados e em fatias de hipocampo frescas, observou-se uma estimulação direta da secreção de S100B por LPS numa concentração de 10 ug/ml. Um envolvimento de TLR4 foi confirmado pelo uso de antagonistas específicos deste receptor. No entanto, baixas concentrações de LPS em culturas de astrócitos foram capazes de induzir uma diminuição na secreção de S100B após 24 h, sem alteração significativa no conteúdo intracelular de S100B. Além disso, após 24 horas de exposição ao LPS, observou-se um decréscimo na glutationa e um aumento na proteína ácida fibrilar glial. Também foi observado que os AINEs apresentam diferentes efeitos sobre parâmetros gliais. O ácido acetilsalicílico e o diclofenaco foram capazes de aumentar a GFAP, enquanto que a nimesulida, um inibidor seletivo de COX-2, e a dexametasona diminuiram a secreção de S100B. No entanto, todos os AINEs reduziram os níveis de PGE2. Juntos, esses dados contribuem para a compreensão dos efeitos de LPS em astrócitos, especialmente sobre a secreção de S100B, e nos ajuda a interpretar mudanças nesta proteína no LCR e soro em doenças neuroinflamatórias. Além disso, tecidos periféricos que expressam S100B talvez devam ser regulados diferentemente, uma vez que a administração IP de LPS não foi capaz de aumentar os níveis séricos de S100B. Em relação aos AINEs, a PGE2 parece estar envolvida no mecanismo de secreção de S100B, mas vias adicionais, não claras neste momento, necessitam de uma maior caracterização. O papel inflamatório de S100B em doenças degenerativas, onde também são observados níveis elevados da COX-2 e PGE2, poderia ser atenuado por inibidores de COX-2. / Inflammatory responses in brain are primarily mediated by microglia, but growing evidence suggests a crucial importance of astrocytes. S100B, a calciumbinding protein secreted by astrocytes, may act as a neurotrophic or an inflammatory cytokine. However, it is not known whether primary signals occurring during induction of an inflammatory response (e.g. lipopolysaccharide, LPS) directly modulate S100B. Neuroinflammation has been implicated in the pathogenesis or progression of a variety of neurodegenerative disorders and several studies have looked for a connection of S100B, and degenerative diseases including Alzheimer’s disease and schizophrenia. The therapeutic use of non-steroid anti-inflammatory drugs (NSAID) to these diseases has growth up. However, there are few reports about the effect of these drugs on S100B. In this work, we evaluated whether S100B levels in cerebrospinal fluid (CSF) and serum of Wistar rats are affected by LPS administered by intraperitoneal (IP) or intracerebroventricular (ICV) injection, as well as whether primary astrocyte cultures respond directly to lipopolysaccharide. Moreover we evaluated S100B content and secretion measured by ELISA (as well as GFAP content and TNF-α secretion) in primary astrocyte cultures exposed to dexamethasone and 4 different chemical classes of NSAID (acetyl salicylic acid, ibuprofen, diclofenac and nimesulide) for 24 h. Our data suggest that S100B secretion in brain tissue is stimulated rapidly and persistently (for at least 24 h) by ICV LPS administration. This increase in CSF S100B was transient when LPS was IP administered. In contrast to these S100B results, we observed an increase in in TNFα levels in serum, but not in CSF, after IP administration of LPS. In isolated astrocytes and in acute hippocampal slices, we observed a direct stimulation of S100B secretion by LPS at a concentration of 10 μg/mL. An involvement of TLR4 was confirmed by use of specific inhibitors. However, lower levels of LPS in astrocyte cultures were able to induce a decrease in S100B secretion after 24 h, without significant change in intracellular content of S100B. In addition, after 24 h exposure to LPS, we observed a decrease in astrocytic glutathione and an increase in astrocytic glial fibrillary acidic protein. We also observe that NSAIDs have distinct effects on glial parameters. ASA and diclofenac are able to increase GFAP, while nimesulide, a selective COX-2 inhibitor, and dexamethasone were able to decrease S100B secretion. However, all anti-inflammatories were able to reduce levels of PGE2. Together, these data contribute to the understanding of the effects of LPS on astrocytes, particularly on S100B secretion, and help us to interpret cerebrospinal fluid and serum changes for this protein in neuroinflammatory diseases. Moreover, non-brain S100B-expressing tissues may be differentially regulated, since LPS administration did not lead to increased serum levels of S100. With respect to NSAIDs, PGE2 is possibly involved in the mechanism of S100B secretion but additional pathways, unclear at this moment, demand further characterization. The inflammatory role of S100B in degenerative diseases, where also is observed elevated levels of COX-2 and PGE2, could be attenuated by COX-2 inhibitors in which elevated levels of COX-2.

Orientadora: Profa. Dra. Elizabeth Teodorov / Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Neurociência e Cognição, 2016. / O sistema nervoso central desenvolve-se, em humanos, a partir da terceira semana gestacional e e muito sensivel as influencias ambientais, como, por exemplo, as infeccoes, resultando em danos em longo prazo e muitas vezes irreversiveis, dentre elas o autismo. Devido a incapacidade de expressao de dor, associada a alta taxa de injurias e automutilacoes sofridas por esses pacientes, o mundo cientifico que passou a questionar a veracidade da existencia de hipoalgesia. Buscando-se uma melhor compreensao da dinamica nociceptiva, tanto em aspectos comportamentais quanto moleculares, optou-se pela utilizacao de um modelo animal autista por meio da administracao de LPS no 9,5 dias da gestacao de ratas, a qual induz neuroinflamacao, comprometendo o ambiente neurologico da prole irreversivelmente. Assim, as ratas gestantes tratadas com LPS foram avaliadas quanto a performance reprodutiva, comportamento maternal, sem alteracoes, e expressao de receptores opioides tipo ¿Ê quanto ao gene (Oprm1) e conteudo proteico (MOR) na PAG, onde observou-se um aumento significativo da expressao de Oprm1 e MOR na PAG das mesmas. As proles (machos e femeas) foram avaliadas quanto ao play behavior aos 21 dias de idade, a hiperalgesia termica e mecanica por meio dos testes Hot Plate e Von Frey, respectivamente, aos 21, 45 e 90 dias de idade e, quanto a expressao de Oprm1 e MOR na PAG aos 90 dias de idade. Em ambas as proles do grupo LPS, ocorreu diminuicao do play behavior, aumento na latencia da resposta de retirada da pata e da intensidade da forca nos testes nociceptivos, e aumento de expressao molecular de Oprm1 e MOR. Conclui-se que a administracao de LPS no 9,5 dias da gestacao de ratas promove prejuizo no desenvolvimento cerebral das proles, que pode ser evidenciada por alteracoes comportamentais e ativacao de plasticidade no sistema opioidergico. / The central nervous system develops in humans from the third week of gestation and is very sensitive to both genetic and environmental influences such as, for example, the infections, resulting in long term or irreversible damage, among them the autism. Due to the inability of pain expression, associated with increased rates of dammages and self mutilation suffered by these patients, the scientific world began to question the veracity of the existence of hypoalgesia. Seeking for a better understand over the nociceptive dynamics, in both behavioral and molecular aspects, it was decided by the use of an animal model for autism through the administration of LPS on gestational day 9.5 of rats, which induces neuroinflammation, compromising irreversibly the neurological environment of the offspring. Thus, the pregnant rats treated with LPS were evaluated about the reproductive performance, maternal behavior, with no changes, and expression of opioid receptors type ì for gene (Oprm1) and protein product (MOR) in the PAG, with a significant increase in Oprm1 and MOR expression in their PAG. The offspring derived from these mothers were evaluated for the play behavior at 21 days old, thermal and mechanical hyperalgesia through the Hot Plate test and Von Frey test, respectively, at 21, 45 and 90 days old and over the expression of Oprm1 and MOR in PAG at 90 days old, and both males and females of LPS group showed a decrease of play behavior, increased response latency and intensity of strength in nociceptive tests, and increased molecular expression for Oprm1 and MOR in PAG. It can be concluded that the LPS administration at 9.5 days of rat gestation promotes cerebral injury of the offspring, which can be evidenced by behavior changes and plasticity enabling the opioid system.

Made available in DSpace on 2016-06-02T19:23:01Z (GMT). No. of bitstreams: 1 6356.pdf: 1279539 bytes, checksum: bba9a716eb38004d198f80a231efcc1b (MD5) Previous issue date: 2014-08-29 / Universidade Federal de Sao Carlos / Low and high LPS doses (endotoxin) have been used as models to study fever and endotoxic shock, respectively. In rats, systemic nitric oxide (NO) directly affects thermogenesis, but does not mediate those responses. Even though few studies in birds suggest NO as a pyrogenic signalling to brain, there is not enough evidence for confirming this suggestion. We hypothesise that, compared to mammals, systemic NO presents similar thermogenic action during endotoxin challenge in birds, even in early life. As precocial birds, chickens have a developed thermoregulatory capacity immediately after hatching, which constitutes an interesting model to study thermoregulation in early life. Thus, we investigated the role of NO in LPS-induced fever and endotoxic shock in 5-day-old chicks. The doses of 2 (LPS2) and 10 g kg-1 of LPS induced fever 3-5 hours after intramuscular (IM) injection, while 50 and 100 (LPS100) g kg-1 decreased Tc within the first hour followed by fever at 4-5 hours postinjection. Plasma nitrate levels increased 4 hours, but not 1 hour, after treatment with LPS2 and LPS100 (higher increase). There was no correlation between plasma nitrate concentrations and Tc at both LPS doses. L-NAME (non-selective NOS inhibitor; 50 mg kg-1; IM) inhibited LPS2- and LPS100-induced fever and increased the magnitude of the fall in Tc caused by LPS100. In contrast, no effect of L-NAME was observed in LPS-treated chicks in warm conditions. L-NAME decreased oxygen consumption in LPS2- and LPS100-treated chicks, but the response was more pronounced with LPS100. Moreover, LPS-induced huddling (heat conservation behaviour) was accentuated by LNAME. Our results seem to indicate that, like in rats, systemic NO is not a mediator of fever and endotoxic shock in chicks, but does affect thermogenesis in these animals, which may constitute a common effect of NO in the endothermic vertebr. / Doses baixas e altas de LPS (endotoxina de bactéria Gram negativa) são utilizadas como modelo para o estudo da febre e do choque endotoxêmico, respectivamente. Em ratos, o óxido nítrico (NO) sistêmico afeta diretamente a termogênese, mas não constitui um mediador da resposta febril. Alguns poucos estudos em aves, sugere uma sinalização pirogênica do NO no encéfalo, mas não há evidencias suficientes para confirmar essa sugestão. Nossa hipótese é que, em comparação com os mamíferos, o NO sistêmico apresenta ação termogênica similar durante desafio com endotoxinas em aves, mesmo no início da vida Sendo aves de desenvolvimento precoce, os frangos têm capacidade termorreguladora por mecanismos comportamentais e autonômicos imediatamente após a eclosão, o que constitui um modelo interessante para estudar a termorregulação no início da vida. Desse modo, nós investigamos o papel do NO na febre e no choque endotoxêmico induzidos por LPS em pintainhos de 5 dias de idade. LPS nas doses de 2 (LPS-2) e 10 g kg-1 induziu febre 3-5 horas após a injeção, enquanto que 50 e 100 (LPS-100) g kg-1 de LPS causaram queda da Tc na primeira hora seguida por febre 4-5 horas após a injeção. A menor (LPS-2; induz febre) e a maior (LPS-100; induz queda de Tc seguida por febre) doses de LPS foram escolhidas para a realização dos protocolos seguintes. As concentrações plamáticas de nitrito + nitrato aumentaram 4 horas, mas não 1 hora, após as injeções de LPS-2 e LPS-100 (maior aumento com a dose mais elevada). Não houve correlação entre os aumentos de Tc e as concentrações de nitrato nas duas doses de LPS. Injeções intramusculares de 50 mg/kg de L-NAME (inibidor não seletivo de NOS) inibiu a febre induzida por LPS-2 e LPS- 100, e aumentou a magnitude da queda de Tc provocada pelo LPS-100. L-NAME reduziu o consumo de oxigênio dos animais tratados com LPS-2 e LPS-100, sendo a queda mais acentuada nesse último grupo. Além disso, o comportamento de agrupamento (redução da perda de calor) induzido por LPS-100 foi acentuado pelo pre-tratamento com L-NAME. Por outro lado, nos pintainhos mantidos em ambiente quente, que não interferiu com a Tc dos animais, os efeitos do LPS-2 e LPS- 100 sobre a TC não foram afetados pelo L-NAME. Juntos esses resultados indicam que o NO parece não ser um mediador de febre e choque endotóxêmico em pintainhos, mas afeta a termogênese nesses animais, o que pode constituir um efeito comum do NO nos vertebrados endotérmicos.

Submitted by PATRICK DA SILVA null (patrick_dasilva@ymail.com) on 2016-09-01T14:44:37Z No. of bitstreams: 1 Dissertação Patrick da Silva.pdf: 3098856 bytes, checksum: 3fb93fb0061cbc5d3f1d4f4066945e07 (MD5) / Approved for entry into archive by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br) on 2016-09-01T18:20:53Z (GMT) No. of bitstreams: 1 silva_p_me_arafcf.pdf: 3098856 bytes, checksum: 3fb93fb0061cbc5d3f1d4f4066945e07 (MD5) / Made available in DSpace on 2016-09-01T18:20:53Z (GMT). No. of bitstreams: 1 silva_p_me_arafcf.pdf: 3098856 bytes, checksum: 3fb93fb0061cbc5d3f1d4f4066945e07 (MD5) Previous issue date: 2016-07-19 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Sinalização química em bactérias patogênicas é um mecanismo empregado para interagir com o hospedeiro e sua respectiva microbiota. Através desta interação ocorre a regulação dos mecanismos de virulência. Estudando o mecanismo de sinalização química do sistema de 2-componentes QseBC em Salmonella enterica serovar Typhimurium foi aberta uma nova perspectiva para desvendar os mecanismos de patogenicidade. Dentre estes, uma nova proteína VisP (Virulence and stress-related Periplasmic protein) foi reportada. Seu papel inicial na interação com a enzima LpxO em S. Typhimurium foi anteriormente demonstrada. O antígeno-O da camada de LPS fornece proteção contra as defesas do hospedeiro e, particularmente, o comprimento de sua cadeia exerce um papel essencial. A montagem do antígeno-O possui o sistema Wzz, o qual determina o comprimento final de sua cadeia polissacarídica, e também apresenta uma distribuição tri-modal. Forma cadeias curtas (S-OAg), longas (L-OAg) e muito longas (VL-OAg) de antígeno-O. As proteínas WzzST e WzzfepE regulam, respectivamente, a síntese das cadeias L-OAg e VL-OAg. Neste estudo, os genes wzzST, wzzfepE, visP e lpxO foram mutados, via mutagênese λ Red, obtendo simples e duplos mutantes. Dados preliminares evidenciaram que há um aumento nas cadeias VL-OAg e L-OAg no mutante ΔvisP, e reciprocamente há diminuição de L-OAg em ΔlpxO. Foram feitos ensaios para avaliar a motilidade, invasão em células epiteliais e sobrevivência intracelular em macrófagos com as amostras obtidas pelos ensaios de mutagênese. Foi evidenciado uma redução de 1,2 e 1,0 ordem de magnitude nos processos de invasão e sobrevivência intracelular, respectivamente, no mutante ΔvisP em relação à amostra selvagem, conforme já descrito, e também uma redução de 70,61% na motilidade comparada com à amostra selvagem. Observou-se que a deleção dos genes wzzfepE e lpxO no mutante ΔvisP faz com que a bactéria retorne ao fenótipo da amostra selvagem nos processos de motilidade, invasão em células epiteliais e sobrevivência intracelular em macrófagos. Este efeito de complementação fenotípica não ocorre no mutante duplo ΔwzzST, apresentando os mesmos níveis do mutante simples ΔvisP, apesar deste possuir um aumento na expressão de genes envolvidos na motilidade e invasão celular comparado com o mutante ΔvisP. Aparentemente, há a atenuação destes processos patogênicos em bactérias com um alto nível de VL-OAg, levando-nos a hipotetizar que este tipo de cadeia de antígeno-O possui relevância na patogênese de S. Typhimurium. WzzfepE e LpxO apresentaram uma evidente participação nos processos de motilidade, invasão celular e sobrevivência intracelular via VisP, e possivelmente o antígeno-O de comprimento muito longo (VL-Oag) apresenta um papel de inibição nestes processos, além disso, evidenciou-se que VisP desempenha uma função relevante na montagem das cadeias de antígeno-O e na patogênese de S. Typhimurium. / Chemical signaling is a mechanism employed by several bacterial species to interact within surrounding microbiota and their host. Upon this interaction the pathogenic bacteria regulate their virulence traits. The two-component system QseBC was described on chemical signaling in Salmonella enterica serovar Typhimurium, and a novel branch of pathogenic cascade regulation was revealed. Among these mechanisms a novel protein was described, VisP (Virulence and stress related Periplasmic protein). VisP interacts with LpxO enzyme on the periplasm. The O-antigen of the LPS layer provides protection against host defenses, and particularly its chain’s length plays an essential role. The O-antigen assembly has the Wzz system, which determines the O-antigen final chain length, and also presents a tri-modal distribution. It forms short (S-OAg), long (L-OAg) and very-long (VL-OAg) O-antigen chains. The WzzST and WzzfepE proteins respectively regulate the L-OAg and VL-OAg synthesis. In this study, wzzST, wzzfepE, visP and lpxO genes were mutated via λ Red mutagenesis, obtaining single and double-mutants. Our preliminary data have shown that VisP increases VL-OAg and L-OAg, conversely LpxO diminishes L-OAg chain length. The motility, epithelial cell invasion and macrophage intracellular survival and replication were assessed with the mutants obtained. The ΔvisP presented 1,2 and 1,0 order of magnitude reduction in cell invasion and intracellular macrophage replication, respectively, comparing with WT S. Typhimurium, as described, and 70,61% less motility rate than WT. Mutating wzzfepE or lpxO genes in ΔvisP recovers the motility, epithelial cell invasion and macrophage intracellular survival and replication WT phenotypes. Nonetheless, this effect does not occur with wzzST gene deletion in ΔvisP, as this double-mutant presents the same phenotypes of ΔvisP single-mutant, although this deletion raises expression rates of motility and cell invasion related genes significantly. Apparently, these pathogenic processes are attenuated within samples with VL-Oag high levels, taking us to hypothesize that there is a correlation between this O-antigen chain length and S. Typhimurium pathogenesis. WzzfepE and LpxO presented an important role on motility, epithelial cells invasion and macrophage intracellular survival through VisP, and probably the VL-OAg inhibits these processes. Furthermore, VisP plays a relevant function on O-antigen chain assembly and pathogenesis of S. Typhimurium. / CNPq: 134434/2014-5

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2018-03-12T21:07:03Z No. of bitstreams: 1 ErickSharllsRamosDePontes_DISSERT.pdf: 5142026 bytes, checksum: c5d7c498155fa9693ffcb28bbc0c9c56 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2018-03-19T12:35:22Z (GMT) No. of bitstreams: 1 ErickSharllsRamosDePontes_DISSERT.pdf: 5142026 bytes, checksum: c5d7c498155fa9693ffcb28bbc0c9c56 (MD5) / Made available in DSpace on 2018-03-19T12:35:22Z (GMT). No. of bitstreams: 1 ErickSharllsRamosDePontes_DISSERT.pdf: 5142026 bytes, checksum: c5d7c498155fa9693ffcb28bbc0c9c56 (MD5) Previous issue date: 2017-08-25 / Uma Linha de produto de software (LPS) representa uma fam?lia de sistemas relacionados que compartilham similaridades e variabilidades visando atender ?s necessidades de um segmento de mercado espec?fico. A ado??o de LPS tem sido aplicada em diversas ?reas na ind?stria de software devido aos benef?cios alcan?ados, tais como, redu??o dos custos no desenvolvimento, aumento da qualidade e redu??o do tempo de comercializa??o. No entanto, cen?rios distintos podem ser encontrados para implementa??o de uma linha de produtos, caracterizando 3 abordagens para ado??o de LPS: (1) abordagem proativa: n?o existe softwares em produ??o, e uma LPS ? desenvolvida do zero; (2) abordagem reativa: j? existe uma LPS em produ??o que vai sofrer incremento para atender novos requisitos; (3) abordagem extrativa: a LPS ? desenvolvida a partir dos artefatos de um sistema ou conjunto de sistemas relacionados que j? est?o em produ??o. No contexto de abordagens extrativa e reativa, este trabalho prop?e um m?todo de extra??o e evolu??o de LPSs a partir de sistemas existentes implementados na linguagem Java. O m?todo foi extra?do a partir da condu??o de um estudo emp?rico de desenvolvimento de uma LPS para o dom?nio de sistemas de controle de espa?os f?sicos utilizados em diferentes centros da Universidade Federal do Rio Grande do Norte (UFRN) e define tr?s atividades que apresentam um conjunto de diretrizes para refatora??o e modulariza??o de features em sistemas implementados em Java: (i) Modelar features da LPS, (ii) Projetar e implementar LPS atrav?s da refatora??o de um sistema existente, e (iii) Realizar testes para cada um dos produtos atuais existentes. Em seguida, o m?todo foi avaliado por meio da sua aplica??o durante evolu??es da LPS para atender novos requisitos demandados pelos clientes. Por fim, foi constatado um aumento de linhas de c?digo dos produtos da LPS, no entanto, o n?cleo da LPS possui uma quantidade de linhas de c?digo menor que qualquer produto antes e depois da extra??o da LPS. Com isso, os artefatos da LPS ficaram melhor modularizados em termos de features, o que pode facilitar a evolu??o tanto do c?digo do n?cleo quanto dos artefatos variantes de cada aplica??o. / A software product line (SPL) represents a family of related systems that share commonalities and variabilities to address the needs of a specific market or mission. The adoption of SPL has been applied in several areas in the software industry due to the benefits achieved, such as reduction of development costs, quality improvement and reduction of time to market. However, distinct scenarios can be found when developing a SPL, which lead to 3 approaches for adopting a SPL: (1) proactive approach: there are no previous software implementation and a SPL is developed from scratch; (2) reactive approach: there is a SPL available which is evolved to address new features and products; (3) extractive approach: SPL is developed from the assets of a system or a set of related systems that already exists. In the scenarios of the extractive and reactive approaches, this dissertation proposes a method of extraction and evolution of SPLs from existing systems implemented in the Java language. The method was extracted from an empirical study of an LPS for the domain of systems that manage physical spaces from different Federal University of Rio Grande do Norte (UFRN) departments and defined three activities that present a set of guidelines for refactoring and modularizing features in systems implemented in Java: (i) Model SPL features, (ii) Design and Implement LPS by refactoring an existing system, and (iii) Perform tests for each of the existing products. The method is evaluated through its application during SPL evolutions to address new requirements demanded by the customers. As a result of the study, we found an increase in the number of lines of code of the products of the products, however, the SPL core had lower lines of code than any product before and after the LPS extraction. Thus, the SPL assets have become better modularized in terms of features, which may facilitate the evolution of core and variant implementations of each application.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2017-07-17T13:05:36Z No. of bitstreams: 1 JuliaJensenDidonet_TESE.pdf: 2583824 bytes, checksum: 051c963d447673a358cc7bee29ee8629 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2017-07-18T13:26:56Z (GMT) No. of bitstreams: 1 JuliaJensenDidonet_TESE.pdf: 2583824 bytes, checksum: 051c963d447673a358cc7bee29ee8629 (MD5) / Made available in DSpace on 2017-07-18T13:26:56Z (GMT). No. of bitstreams: 1 JuliaJensenDidonet_TESE.pdf: 2583824 bytes, checksum: 051c963d447673a358cc7bee29ee8629 (MD5) Previous issue date: 2017-03-15 / A depress?o reduz a qualidade de vida do indiv?duo, compromete a funcionalidade profissional e social e ? considerada a principal causa para incapacidade em termos de anos perdidos no curso da doen?a. Apesar da severidade relatada, ainda n?o h? uma compreens?o clara dos substratos neurais alterados na depress?o, por isso o estudo de modelos animais que investiguem a etiologia deste transtorno torna-se extremamente necess?rio. Este trabalho buscou comparar altera??es bioqu?micas no soro, c?rtex pr?frontal (CPF) e hipocampo de camundongos submetidos a dois modelos animais de depress?o: desamparo aprendido e administra??o do lipopolissacar?deo de E.Coli (LPS). O teste de desamparo aprendido resultou em m?dia 70 % de animais desamparados, verificado pela falha em escapar aos choques 24 h e 48 h ap?s a sess?o de indu??o do desamparo. Os 30 % restantes foram considerados resilientes. Os animais desamparados apresentaram mais dano oxidativo no CPF e soro, quando comparados aos animais controles. N?o houve diferen?a entre desamparados e resilientes, por?m, foi observada correla??o positiva entre o dano oxidativo no soro e CPF e o comportamento desamparado. A concentra??o das citocinas pr?-inflamat?rias IL-1?, TNF?, IL-6 e antiinflamat?ria IL-10 no CPF e hipocampo dos animais submetidos ao desamparo e controles n?o foi diferente entre os grupos, por?m houve correla??o positiva entre a citocina IL-6 no hipocampo e o comportamento desamparado dos animais. A atividade da enzima indolamina 2,3-dioxigenase (IDO) n?o apresentou diferen?a significativa nos animais submetidos ao modelo do desamparo e controles. A administra??o sist?mica de LPS (0,8 mg/kg i.p.) induziu um comportamento doentio nos animais, caracterizado por diminui??o da ingest?o de ?gua e comida, perda de peso e altera??o da temperatura retal 6 h ap?s a inje??o. Em 24 h o estado doentio diminuiu, por?m, os animais que receberam LPS apresentaram imobilidade aumentada no teste de suspens?o pela cauda em compara??o aos animais que receberam salina. Foi observado mais dano oxidativo no soro, CPF e hipocampo do grupo LPS em compara??o aos grupos salina e controle. As citocinas IL-?, TNF? no soro, CPF e hipocampo n?o apresentaram nenhuma altera??o, indicando que a inflama??o induzida pela administra??o de LPS foi transit?ria. A citocina IL-6 mostrou-se elevada no CPF do grupo que recebeu LPS em compara??o ao grupo salina, correlacionada positivamente com o comportamento do tipo depressivo dos animais. Os n?veis de IL-10 no hipocampo correlacionaram-se negativamente com o comportamento do tipo depressivo e a atividade da IDO foi aumentada no CPF e diminu?da no hipocampo do grupo LPS. Os resultados apresentados corroboram a hip?tese da ativa??o do sistema imune no evento depressivo e consequente dano oxidativo, verificado em dois modelos animais de depress?o. A ativa??o da IDO variou entre as ?reas analisadas em cada modelo animal. / Major depression has a great impact on an individual?s quality of life and it is considered the leading cause of burden in terms of years lost due to disability. However, despite the severity of depression, the pathophysiology of the disease is still elusive. In this regard, the use of animal models plays an important role in research for the etiology of depression. This work compared biochemical alterations occurring on serum, prefrontal cortex (PFC) and hippocampus in two animal models of depression: learned helplessness and administration of lipopolyssaccharide from E.Coli (LPS). Learned helplessness protocol used in this work resulted in 70 % of helpless mice, assessed by the inability to escape from electroshocks given 24 h or 48 h after the helpless-induction session. The other 30 % of mice were considered resilient. Helpless animals showed more oxidative damage in PFC and serum when compared to controls. No difference was seen between helpless and resilient groups, but there was a positive correlation between the oxidative damage on serum and PFC and helpless behavior. There was no difference in the concentration of IL-1?, TNF?, IL-6 and IL-10 cytokines on PFC and hippocampus of the animals exposed to the learned helplessness test, but there was a significant positive correlation between IL-6 concentration and depressive-like behavior on hippocampus. Indoleamine 2,3-dioxygenase (IDO) enzyme activity was not altered on learned helplessness model. Systemic administration of LPS (0,8 mg/kg) induced sickness behavior on animals characterized by decreased food and water intake, bodyweight loss and altered body temperature 6 h after administration. Sickness behavior is over after 24 h, but LPS-treated mice displayed higher immobility time in the tail suspension test when compared to saline. There was more oxidative damage in serum, PFC and hippocampus of LPS group when compared to saline and controls. No differences on IL-1? and TNF? concentration on serum, PFC and hippocampus of the animals were detected, suggesting a transient nature of the LPS-induced inflammation. LPS-treated group displayed higher concentrations of IL-6 on PFC when compared to saline group, and IL-6 concentration positively correlated to depressive-like behavior. IL-10 concentrations on hippocampus were negatively correlated to depressive-like behavior and IDO activity was increased on PFC and decreased on hippocampus of LPS-treated mice. Data presented here corroborate for the hypothesis of immune activation during depressive episodes, then resulting in oxidative damage assessed in two animal models of depression. IDO activity behaved with some specificity in each animal model depending on the brain or systemic area.