No description available.

Rheumatoid arthritis (RA) is a chronic disease characterized by cycles of inflammation and resolution. Previously, it was believed that the resolution of inflammation is simply dissipation of pro-inflammatory signals, although current research indicates that resolution is an active process. Acute inflammation follows defined phases of induction, inflammation and resolution, and resolution occurs by an active process that requires COX-2 activity. This study aims to address whether this paradigm extends to a recognized model of chronic inflammation. We demonstrated in murine collageninduced arthritis that chronic inflammation follows the same sequential course. While there is the normal production of pro-inflammatory cytokines during inflammation and anti-inflammatory mediators such as 15-deoxyΔ12,14PGJ2 (15d-PGJ2) during resolution, interestingly there is sustained production of both COX-2 and the presumably proinflammatory PGE2 during both phases. Blocking COX-2 activity and therefore production of PGE2 during the resolution phase perpetuated instead of attenuated inflammation. Repletion with PGE2 analogs restored homeostasis, and this function is mediated by the pro-resolving lipoxygenase metabolite, lipoxin A4 (LXA4), which is a potent stop signal. Thus, the study provided in vivo evidence for a natural, endogenous link between the cyclooxygenase-lipoxygenase pathways and showed that PGE2 serves as a feedback inhibitor essential for limiting chronic inflammation in autoimmune arthritis. These findings may explain the enigma regarding why COX-2 inhibitors are palliative rather than curative in humans because blocking resolution may mitigate the benefit of preventing induction. / Microbiology and Immunology

Epilepsy is a complex, chronic neurological disorder with diverse underlying etiologies characterized by the spontaneous occurrence of seizures. Epilepsy affects all ages from neonates to elderly adults, with the most recent CDC estimates stating that ~3 million adults and over 400,000 children are currently suffering from active epilepsy in the U.S. alone. In adults, the leading cause of epilepsy worldwide in central nervous system (CNS) infection, while in neonates the most common cause of seizures is hypoxic/ischemic encephalopathy (HIE). However, in both adults and neonates, current antiepileptic drugs (AEDs) are ineffective in 30-50% of patients, despite the availability of over 20 FDA approved AEDs with diverse molecular targets. This disparity highlights a critical need for novel therapeutics in seizure-susceptibility and epilepsy. The microbes that inhabit gut mucosal surfaces, termed the gut microbiota, have been increasingly implicated in the pathology of neurological diseases including epilepsy. This gut-brain axis is an intriguing therapeutic target in epilepsy as gut microbes can affect the CNS through multiple mechanisms including vagus nerve signaling, immune-gut interactions, and through production of microbial-metabolites including neurotransmitters, short chain fatty acids (SCFAs), lactate, vitamins, and S-equol. Furthermore, the gut microbiota is crucial for neurodevelopment, indicating that the gut-brain axis may be involved in pediatric seizure-susceptibility. This dissertation reviews current evidence on the role of gut metabolites in seizure-susceptibility in epilepsy, highlighting the microbial-derived metabolite S-equol as a potential novel AED. We then evaluate gut microbiome alterations in the Theiler's murine encephalomyelitis virus (TMEV) adult mouse model of CNS infection-induced seizures and find decreases in S-equol-producing bacteria in the gut microbiomes of TMEV-infected mice with seizure phenotypes. We characterize the effect of exogenous S-equol on neuronal function in vitro, demonstrating a reduction in neuronal excitation following S-equol exposure. We additionally characterize entorhinal cortex (ECTX) pyramidal neuronal hyperexcitability, and demonstrate the ability of exogenous S-equol to ameliorate CNS-infection-induced ECTX neuronal hyperexcitability ex vivo. Finally, we demonstrate that perinatal and postnatal exposure to antibiotics alters the gut microbiome and increases seizure-susceptibility following HIE exposure in p9/p10 mice, potentially via sex-specific alterations in neuronal function. Together, this dissertation evaluates the gut-brain axis in pediatric and adult mouse models of seizure-susceptibility and identifies the gut metabolite S-equol as a potential target for the treatment of seizures. / Doctor of Philosophy / Epilepsy, a disease defined by the occurrence of two or more spontaneous seizures, affects over 50 million people worldwide. This makes epilepsy one of the most common chronic neurological disorders across the globe. People with epilepsy suffer increased mortality, lower quality of life, and increased social stigma. There is currently a crisis in the treatment and management of epilepsy, because although over 20 different anti-epileptic drugs (AEDs) are available to patients, these drugs only work in ~70% of individuals with epilepsy, leaving 30% of patients with uncontrolled seizures. Currently available AEDs are designed to target classical central nervous system (CNS) components. However, a growing body of evidence suggests that epilepsy is related to complex systems throughout the body. Therefore, in this manuscript we explore novel therapeutic targets outside of the CNS for the management of seizures. Over 1000 species of bacteria live in the in the human gut, and are termed the gut microbiota. Gut microbes produce a variety of chemicals that circulate through the body and can even reach the brain. Interaction of chemicals produced by the gut microbiota and brain chemistry have been shown to affect disease outcomes in Autism Spectrum Disorder, Parkinson Disease, and other brain disorders. However, very few studies have examined the possibility of a role for the gut microbiota in epilepsy. In this dissertation, we review chemicals produced by the gut microbiota that may alter epilepsy biology. We additionally examine gut microbiota alterations in a rodent model of epilepsy, and identify a novel chemical, S-equol, that is produced by the gut microbiota and impacts epilepsy biology in our rodent model. Lastly, we explore how altering the maternal gut microbiota in rodents can influence seizure-susceptibility in infants.

<I>Neospora hughesi</I> is a recently described cause of equine protozoal myeloencephalitis (EPM). In the present study, we examined the susceptibility of BALB/c gamma-interferon gene knockout (gamma-INFKO), BALB/c, CD-1, and C57BL/6 strains of mice and gerbils to infection with tachyzoites of the Nh-A1 strain of <I>N. hughesi</I>. Only the gamma-IFNKO mice developed severe clinical disease following infection with <I>N. hughesi</I>. The most severe lesions were in the hearts of these mice. Two dogs fed the brains of mice, shown to contain <I>N. hughesi</I> tissue stages by cell culture and g-IFNKO mouse bioassay, did not shed <I>N. hughesi</I> oocysts over a 23 day observation period. We report important differences between the nucleotide and deduced amino acid sequences of the dense granule proteins GRA6 and GRA7 of <I>N. hughesi and N. caninum</I>. The newly defined proteins of <I>N. hughesi</I> are referred to as NhGRA6 and NhGRA7. From analysis of the sequences we found that there is a 14.8% difference in deduced amino acid sequence between NhGRA7 and NcGRA7, and a 4% difference between NhGRA6 and NcGRA6 in areas that could be compared. This thesis supports the identification of <I>N. hughesi</I> as a separate species from <I>N. caninum</I> and describes novel methods of distinguishing between the two. / Master of Science

No description available.

Anxiety disorders commonly emerge during adolescence, and girls are diagnosed with these disorders more frequently than boys. Understanding why anxiety disorders emerge and why non-clinical anxiety symptoms increase during adolescence is important for understanding this sex difference and how to treat adolescent sufferers. Potential mechanisms, such as puberty or cognitive biases, can be investigated both in humans and in rodent models of anxiety. This thesis aimed to characterise sex differences and changes in anxiety-like behaviour across adolescence and into adulthood in the rat, and to examine anxiety and interpretive bias in adolescent humans. In rats, we examined performance on common tests of anxiety-like behaviour: the emergence test, open field and elevated plus-maze. Exploration on these tests increased from mid-adolescence into adulthood, and greater exploration by females was apparent from late adolescence. While the behaviours themselves remain interesting, caution on interpreting these behaviours as anxiety-related warranted and is discussed throughout the thesis. Potential effects of the ovarian cycle and testing order on EPM performance were also examined. In humans, 12-14 year old adolescents complete visual and written interpretive bias tasks, this bias being considered to be a cognitive vulnerability for anxiety. The results showed that, when imagining themselves in ambiguous scenarios, girls were more negative in their interpretations than boys. Additionally, both sexes also interpreted social scenarios more negatively than non-social scenarios. As puberty is thought to be important to the emergence of disorder during adolescence, interpretive bias could potentially mediate the puberty-anxiety relationship. While more interpretive bias work is needed in both species, the recent development of interpretive bias tasks for rodents provides an opportunity to move away from difficult to interpret tests of anxiety-like behaviour in rodents, and should allow for greater convergence of the human and rodent anxiety research.

A avaliação da mecânica respiratória (AMR) busca descrever o comportamento mecânico do sistema respiratório, para melhor entender a sua fisiologia e a sua patofisiologia. O método quasi-estático é caracterizado pela AMR em condições nas quais a aceleração dos tecidos pode ser considerada desprezível. Esta dissertação de mestrado teve como objetivo avaliar a mecânica respiratória pelo método quasi-estático em roedores, utilizando um insuflador pulmonar para pequenos roedores (IPPR) construído no Laboratório de Engenharia Biomédica (LEB) da Escola Politécnica da Universidade de São Paulo (EPUSP). Uma extensa revisão da literatura sobre fisiologia e técnicas de AMR em humanos e roedores é apresentada. Uma documentação bastante detalhada sobre a implementação e o funcionamento do IPPR é fornecida, com o intuito de permitir que o dispositivo seja reproduzido. Testes de bancada foram executados, para avaliar o funcionamento do IPPR, e um protocolo experimental com modelo animal de doença, em ratos Wistar envenenados por Paraquat, foi realizado. Os resultados dos testes de bancada mostraram que o equipamento ainda carece de alguns ajustes, pois apresenta erros na indicação de volume, da ordem de 5%, mas que podem ser compensados na análise dos dados experimentais obtidos. No futuro, pretende-se sanar essa imprecisão, tornando o IPPR independente de um microcomputador com sistema operacional multitarefa. Os resultados da aplicação do modelo matemático exponencial, nos dados obtidos de curvas pressão-volume no protocolo experimental, trouxeram informações que estão de acordo com as características observadas no modelo animal de envenenamento por Paraquat. / The assessment of respiratory mechanics (AMR) aims to describe the mechanical behavior of the respiratory system in order to better understand its physiology and pathophysiology. The quasi-static method is characterized by the AMR under conditions where the tissue\'s acceleration can be considered negligible. The purpose of this master degree dissertation was to assess the respiratory mechanics by quasistatic method in rats using a lung insufflator for small rodents (IPPR) implemented in the Biomedical Engineering Laboratory (LEB) of Escola Politécnica of University of São Paulo (EPUSP). Abroad literature review on AMR techniques and on physiology in humans and rodents was presented. A fairly detailed documentation of the assembly and the operation of the IPPR was provided in order to allow the device to be reproduced. Bench tests were conducted to evaluate the behavior of the IPPR, and an animal disease model by Paraquat poisoning protocol was performed in Wistar rats. The bench tests results showed that the equipment still needs some tweaking, because it presents errors in volume indication of the order of 5%, but these errors can be compensated in the analysis of the experimental data. In the future, there is an intention to correct this inaccuracy turning the IPPR independent of a multitask microcomputer operating system. The results of the mathematical exponential modeling applied on the pressure-volume curves in the experimental protocol are in good agreement with the characteristics observed on Paraquat poisoning animal model.

A síntese dos hormônios esteroides é feita através da ação de um complexo enzimático chamado citocromo P450. As enzimas 3&#946;-HSD (3&#946;-hidroxiesteroide desidrogenase), P450c17 (citocromo P450 17&#945;-hidroxilase/17,20-liase) e P450arom (citocromo P450 aromatase), fazem parte desse complexo e são responsáveis pela síntese dos hormônios progestágenos, andrógenos e estrógenos, espectivamente. Essas enzimas já foram identificadas em inúmeros tecidos, inclusive em embriões em período pré-implantação. Por isso, acredita-se que o blastocisto seja uma fonte de produção de progesterona e estrógeno, hormônios essenciais para implantação e manutenção da gestação. Em roedores, as informações sobre a capacidade esteroidogênica dos embriões são controversas e também restritas apenas a estudos com espécies laboratoriais. Para trazer mais informações sobre a capacidade esteroidogênica em blastocistos de roedores utilizamos o preá (Galea spixii), uma espécie de roedor silvestre encontrada nas regiões de Caatinga e Semiárido do Nordeste brasileiro e muito utilizado na alimentação de famílias de baixa renda como fonte proteica. Diante o exposto, o objetivo deste trabalho é fazer um apanhado histórico sobre os dados publicados a cerca da capacidade dos embriões de diversas espécies de produzir os hormônios esteroides além de identificar e quantificar a expressão das enzimas esteroidogênicas em embriões de preá através das técnicas de imunohistoquímica e PCR em tempo real. As análises morfológicas indicam que o desenvolvimento inicial do preá se assemelha com os demais roedores, porém o tempo de desenvolvimento difere das espécies usadas em laboratório. As marcações da imunohistoquímica foram positivas nos tecidos maternos, mas os embriões não tiveram marcação para nenhuma das três enzimas do estudo. No entanto, os dados obtidos pelo PCR indicam que os genes CYP11, CYP17 e CYP19 foram expressos nos embriões e, portanto, o concepto de G. spixii tem capacidade de produzir hormônios esteroides, assim como visto em várias outras espécies que já foram estudadas / The synthesis of steroid hormones is made through the action of an enzyme complex called cytochrome P450. 3&#946;-HSD (3&#946;-hydroxysteroid dehydrogenase), P450c17 (cytochrome P450 17 &#945;-hydroxylase/17,20-lyase) and P450arom (cytochrome P450 aromatase), are present in this complex and are are responsible for the synthesis of progestins, androgens and estrogens hormones, respectively. These enzymes have been identified in numerous tissues, including embryos in pre-implantation period. Therefore, it is believed that the blastocyst is a source of production of progesterone and estrogen, hormones essential for the implementation and maintenance of pregnancy. In rodents, the information about the steroidogenic capacity of embryos are controversial and confined to studies with laboratory species. To get more information about the steroidogenic capacity in blastocysts of rodents we used the spix yellow-toothed cavy (Galea spixii), a species of wild rodent found in the northeastern of Brazil, and used in the feeding of low-income families as a source of protein. On the above, the aim of this work is to make a historic about the data published about the ability of embryos of various species to produce steroid hormones and identify and quantify the expression of steroidogenics enzymes in embryos of spix yellow-toothed cavy by immunohistochemical and real-time PCR techniques. The morphological analyses indicate that the G. spixii early development resembles other rodents, but development time differs from the species used in laboratory. The immunohistochemical stainning was positive in maternal tissues, but the embryos did not have stainnig for any of the three enzymes of the study. However, the data obtained by PCR indicate that CYP11, CYP17 and CYP19 genes were expressed in embryos and, therefore, the concept of G. spixii has capacity to produce steroid hormones, as seen in several other species that have been studied

Ritmos biológicos e sua sincronização por ciclos ambientais geralmente são estudados em roedores, mantidos em laboratório. Nosso grupo vem estudando ritmos de atividade/repouso de roedores subterrâneos sul-americanos conhecidos como tuco-tucos (Ctenomys aff. knighti) em laboratório e campo. Na natureza, sabemos que os tuco-tucos emergem à superfície diariamente em episódios de até uma hora. Registros curtos da atividade de superfície dos tuco-tucos, anteriormente obtidos pelo grupo, haviam sugerido que o padrão diário das saídas variava sazonalmente, sendo crepusculares durante o verão e concentrados no meio do dia, durante o inverno. Esse mesmo padrão sazonal já foi observado em populações de outros roedores de deserto e foi atribuído a reações dos roedores às variações diárias de temperatura, no verão e inverno. Por outro lado, a sazonalidade dos padrões de atividade de várias espécies de mamíferos e aves também pode ser explicada pela sincronização diária do ritmo de atividade a diferentes fotoperíodos. O objetivo desse trabalho é caracterizar a variação sazonal dos ritmos diários de atividade dos tuco-tucos, com uso de bio-logging, e verificar a contribuição dos fatores ambientais para a sazonalidade desses padrões. Ao longo do ano, acelerômetros e luxímetros foram presos a 29 animais recentemente capturados (15 fêmeas e 14 machos). Cada animal foi mantido individualmente em arenas semi-naturais por um mês. Variáveis ambientais foram registradas simultaneamente. Outros 18 animais (9 fêmeas e 9 machos) foram mantidos em laboratório, expostos ao fotoperíodo natural e em ambiente com temperatura constante. Os resultados confirmaram a sazonalidade do padrão temporal de atividade de superfície dos tuco-tucos. Os acelerômetros se mostraram mais precisos e sensíveis a pequenos movimentos corporais do que os registros de telemetria encontrados na literatura. Esses sensores também revelaram padrões de atividade subterrânea e noturna, em ambiente semi-natural. Durante o verão, a atividade de superfície diária dos tuco-tucos foi melhor prevista pelo horário do dia (fator endógeno) do que pelas variações de temperatura ambiental (fator exógeno). Durante o inverno, entretanto, a temperatura ambiental teve maior contribuição no padrão de saídas dos tuco-tucos, indicando complexas contribuições dos fatores endógenos e exógenos no delineamento dos padrões de atividade / Biological rhythms and their synchronization by environmental cycles are generally studied in rodents, inside laboratory. Our group studies activity/rest rhythms of South American subterranean rodents known as tuco-tucos (Ctenomys aff. knighti), in laboratory and field. In nature, it is known that tuco-tucos emerge daily to the surface, in brief episodes. Short records of tuco-tucos\' individual surface activity, previously collected by our group, had suggested that daily temporal pattern of emergence varied seasonally, being crepuscular in summer and concentrated around midday in winter. The same seasonal pattern was observed in populations of other desert rodents and was attributed to daily variations of environmental temperature, in summer and winter. At the same time, seasonality of activity patterns of various species of mammals and birds can be explained by daily synchronization of the activity rhythm by different photoperiods. Our aim is to characterize seasonal variation of daily activity rhythms of tuco-tucos, using bio-logging, and to verify the contribution of environmental factors in the seasonality of these patterns. Throughout the year, accelerometers and luximeters were tied to 29 freshly caught animals (15 females and 14 males). Each animal was kept inside semi natural enclosures for one month. Environmental variables were recorded simultaneously. Other 18 animals (9 females and 9 males) were kept inside laboratory, exposed to natural photoperiod, in a room with constant temperature. We confirmed the seasonality of temporal pattern of surface activity of tuco-tucos. The accelerometers resulted to be more precise and sensitive to small body movements than telemetry records found in literature. They also revealed nocturnal and subterranean activity patterns inside the enclosures. During summer, daily surface activity of tuco-tucos was better predicted by time of day (endogenous factor) than by environmental temperature (exogenous factor). During winter season, however, environmental temperature has a stronger contribution on the emergence pattern in tuco-tucos, indicating complex contributions of endogenous and exogenous factors that outline activity patterns

As variáveis biológicas de muitos seres vivos apresentam ritmos diários, em paralelo ao dia e à noite do ambiente. A maioria desses ritmos não é uma mera reação aos estímulos cíclicos do ambiente: eles são ritmos circadianos gerados endogenamente por um temporizador interno - o oscilador circadiano - que sustenta uma ritmicidade com período diferente de 24 horas na ausência de pistas ambientais, mas se sincroniza para um período de 24 horas quando exposto ao ambiente cíclico. Em mamíferos, o oscilador circadiano se localiza no cérebro e pode ser sincronizado pelo ciclo diário de claridade e escuridão (ciclo CE) do dia e da noite transmitido através das retinas. Animais subterrâneos passam boa parte do tempo abaixo da terra, onde as condições ambientais variam menos que na superfície. Em razão disso, questiona-se até que ponto estes animais mantêm ritmos circadianos e se seus osciladores circadianos se baseiam no ciclo CE para se sincronizar com o ambiente. Procuramos responder essas questões nos roedores subterrâneos da espécie Ctenomys cf. knighti (tuco-tucos). Em campo, realizamos observações visuais diretas para registrar o padrão temporal de exposição à luz. Em laboratório, construímos a Curva de Resposta de Fase (CRF), que consiste em medir indiretamente a resposta do oscilador circadiano a estímulos luminosos em diferentes momentos do dia. Por fim, recorremos a simulações computacionais de um modelo matemático de oscilador, para integrar os dados anteriores. Verificamos, em campo, que os tuco-tucos se expõem, diariamente, a pulsos de luz irregularmente distribuídos nas horas claras do dia levantando alguns questionamentos sobre sua capacidade como sincronizador dos ritmos em campo. Contudo, a CRF nos indicou que o sistema circadiano dos tuco-tucos responde a estímulos luminosos de forma semelhante aos animais não-subterrâneos. Nós verificamos 3 hipóteses: ou a informação da exposição à luz em campo é suficiente para sincronizar o oscilador circadiano, apesar da aparente irregularidade; ou a CRF do tuco-tuco possui alguma característica que facilita a sincronização pelo regime observado de exposição à luz; ou seu sistema circadiano se baseia em outra pista temporal do ambiente para se manter sincronizado. Nossas simulações computacionais indicaram que a primeira hipótese é a mais provável, visto que os padrões de exposição à luz simulados, como aqueles observados em campo, com o mínimo de informação temporal, ainda assim são suficientes para sincronizar os osciladores. Em conjunto, nossos dados indicam que o ciclo claro-escuro tem um papel importante na sincronização dos ritmos circadianos dos tuco-tucos na natureza. / Daily rhythms are found in the biological variables of many organisms, in parallel to the environmental day and night. Most of those rhythms are not mere reactions to the cyclic stimulus of the environment. They are actually circadian rhythms, endogenously generated by an internal timer, the circadian oscillator, which sustains a non-24-hour rhythm under constant conditions, but is synchronized to a 24-hour period when exposed do a cyclic environment. In mammals, this oscillator is located in the brain, and can be synchronized by the daily light-dark cycle (LD cycle) of day and night. Subterranean animals remain most of the time in an underground habitat, where environmental conditions vary less markedly than aboveground. Therefore, there are doubts whether these animals maintain circadian rhythms and whether their circadian oscillators rely on the LD cycle for environmental synchronization. We attempted to answer those questions in subterranean rodents of the species Ctenomys cf. knighti (tuco-tucos). In the field, we performed direct visual observations to assess their temporal pattern of light-exposure. In the lab, we build the Phase Response Curve (PRC), which consists in an indirect measurement of the circadian oscillator responses to light stimuli applied at different times of the day. Finally, computer simulations of an oscillator model were used to integrate these previous data. We verified that tuco-tucos expose themselves to light pulses that are irregularly distributed through day-light hours, raising some questions about its validity as an environmental synchronizer. However, the PRC results indicated that the tuco-tucos circadian system responds to light stimuli in a way similar to non-subterranean animals. We then verified three hypothesis: either the light-exposure temporal information was enough for the synchronization of the circadian system, despite its apparent irregularity; or the tuco-tuco\'s PRC present some feature that facilitates the synchronization by the observed light-exposure regimen; or the tuco-tuco\'s circadian system rely on another environmental temporal cue to maintain its synchronization. Our computer simulations support the first hypothesis, given that the simulated regimens, despite being little informative in the temporal sense, were still sufficient to synchronize the simulated oscillators. Together, our data indicate that the light-dark cycle plays an important role in the synchronization of the tuco-tucos\' circadian rhythms in nature.