Neuronal networks produce reliable functional output throughout the lifespan of an animal despite ceaseless molecular turnover and a constantly changing environment. The cellular and molecular mechanisms underlying the ability of these networks to maintain functional stability remain poorly understood. Central pattern generating circuits produce a stable, predictable rhythm, making them ideal candidates for studying mechanisms of activity maintenance. By identifying and characterizing the regulators of activity in small neuronal circuits, we not only obtain a clearer understanding of how neural activity is generated, but also arm ourselves with knowledge that may eventually be used to improve medical care for patients whose normal nervous system activity has been disrupted through trauma or disease. We utilize the pattern-generating pyloric circuit in the crustacean stomatogastric nervous system to investigate the general scientific question: How are specific aspects of rhythmic activity regulated in a small neuronal network? The first aim of this thesis poses this question in the context of a single neuron. We used a single-compartment model neuron database to investigate whether co-regulation of ionic conductances supports the maintenance of spike phase in rhythmically bursting “pacemaker” neurons. The second aim of the project extends the question to a network context. Through a combination of computational and electrophysiology studies, we investigated how the intrinsic membrane conductances of the pacemaker neuron influence its response to synaptic input within the framework of the Phase Resetting Curve (PRC). The third aim of the project further extends the question to a systems-level context. We examined how ambient temperatures affect the stability of the pyloric rhythm in the intact, behaving animal. The results of this work have furthered our understanding of the principles underlying the long-term stability of neuronal network function.

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Spinal muscular atrophy (SMA) is the leading genetic cause of death of young children. It is an autosomal recessive disease caused by the mutation and/or the deletion within the ubiquitously expressed survival motor neuron 1 (SMN1) gene. SMA pathology is characterized by spinal cord motor neuron degeneration, neuromuscular junction (NMJ) defects and muscular atrophy. Upon disease onset, SMA patients progressively become paralyzed and in the most severe cases, they die due to respiratory complications. Over the years, it has become clear that SMN is a multi-functional protein with important roles in small nuclear ribonucleoprotein (snRNP) assembly, RNA metabolism, axonal outgrowth and pathfinding, mRNA transport as well as in the functional development of NMJs, skeletal muscle and cardiac muscle. However, it remains unclear which of these functions, and the respective perturbed molecular pathways, dictate SMA pathogenesis. Here, we have established Smn-depleted PC12 cells and an intermediate SMA mouse model to characterize a role for Smn in the regulation of actin cytoskeleton dynamics. We find that Smn depletion results in the increased expression of profilin IIa and active RhoA (RhoA-GTP) as well as the decreased expression of plastin 3 and Cdc42. Importantly, the inhibition of rho-kinase (ROCK), a direct downstream regulator of RhoA, significantly increased the lifespan of SMA mice and shows beneficial potential as a therapeutic strategy for SMA. In an addition, we have uncovered a muscle- and motor neuron-independent role for SMN in the regulation of pancreatic development and glucose metabolism in SMA mice and type 1 SMA patients. This finding highlights the importance of combining a glucose tolerance assessment of SMA patients with their existing clinical care management. Thus, our work has uncovered two novel and equally important roles for the SMN protein, both of which contribute significantly to SMA pathogenesis.

Esclerose lateral amiotrófica (ELA) é uma doença neurológica progressiva e fatal, caracterizada por perda dos neurônios motores, levando à fraqueza muscular global. As funções sensitivas e mentais são preservadas durante todo o curso da doença. A esclerose lateral amiotrófica tem uma prevalência de 6 por 100.000 pessoas e o início da doença é geralmente entre os 40 a 60 anos de idade. O prognóstico é reservado e em média os pacientes vivem de 3 a 5 anos após o diagnóstico médico. Com a progressão da doença outros sintomas surgem como fraqueza dos membros, comprometimento da fala, aumento da salivação, dificuldades de deglutição, dificuldades para deambular e fadiga muscular. As alterações dos músculos respiratórios levam à falência respiratória, que é a maior causa de óbito nos pacientes com esclerose lateral amiotrófica. Fadiga é definida como a queda da máxima contração isométrica voluntária e falta de tolerância do músculo sob esforço. A máxima contração isométrica voluntária depende de uma cadeia de eventos que se inicia no córtex motor - condutor excitatório dos neurônios motores superior e inferior, e se continua na transmissão pela junção neuromuscular, no acoplamento excitação-contração e na contração da fibra muscular que depende de um suprimento energético metabólico. A fadiga muscular ocorre em pacientes com esclerose lateral amiotrófica prejudicando a função e a qualidade de vida dos pacientes. O objetivo deste trabalho foi: 1) Quantificar a freqüência da fadiga na esclerose lateral amiotrófica; 2) Analisar a evolução da fadiga nos pacientes; 3) Correlacionar a presença da fadiga com fatores como a funcionalidade, a qualidade de vida, a depressão, a dispnéia, e a sonolência, idade e duração da doença em meses. O grupo controle compôs-se de 60 indivíduos (familiares de funcionários do hospital e da equipe multidisciplinar) que não apresentavam história de doenças pregressas. O grupo teste constitui-se de 60 pacientes com diagnóstico de esclerose lateral amiotrófica. O diagnóstico foi realizado por dois neurologistas independentes e baseou-se na presença de história clínica, exame neurológico e estudos neurofisiológicos compatíveis com esclerose lateral amiotrófica, segundo os critérios de El Escorial da Federação Mundial de Neurologia; além disso, houve a investigação complementar por meio de testes hematológicos, bioquímicos, sorológicos, genéticos e radiológicos para excluir outras patologias. Os pacientes dos grupos controle e teste foram entrevistados pela pesquisadora para aplicação de questionários com escalas para verificação da presença de funcionalidade, de qualidade de vida, de depressão, de dispnéia, de sonolência e de fadiga; e os pacientes do grupo teste foram submetidos à avaliação fisioterapêutica no início do estudo e a cada 3 meses, totalizando 12 meses de coletas. O grupo teste apresentou fadiga significantemente maior em relação ao grupo controle, bem como alterações nos questionários de funcionalidade, de qualidade de vida, de depressão, de dispnéia e de sonolência. Percebeu-se que a fadiga foi evolutiva durante os meses de acompanhamento da pesquisa. A fadiga correlacionou-se com a idade, mostrando que os pacientes mais jovens apresentaram maior grau de fadiga que os pacientes mais idosos. Os resultados desta pesquisa sugerem que a fadiga é um dos problemas que afetam os pacientes com ELA; o fato de não ter correlação com outros problemas estudados sugere que a fadiga deve merecer pesquisa e tratamento individualizados no paciente com ELA, principalmente pelo fato de que os resultados sugeriram piora da fadiga no decorrer da evolução da ELA / Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurological disease, characterized by loss of the motor neurons, taking to the global muscular weakness. The sensitive and mental functions are preserved during whole the course of the disease. The ALS has a prevalence of 6 for 100.000 people and the beginning of the disease is usually among the 40 to 60 years of age. The prognostic is reserved and on average the patients live from 3 to 5 years after the medical diagnosis. With the progression of the disease other symptoms they appear as weakness of the members, compromising of the speech, increase of the salivation, deglutition difficulties, difficulties to stroll and it fatigues muscular. The alterations of the breathing muscles take to the breathing bankruptcy, that is the largest death cause in the patients with ALS. Fatigue is defined as the fall of the maxim voluntary isometric contraction and lack of tolerance of the muscle under effort. The maxim voluntary isometric contraction depends on a chain of events that begins in the motor cortex - driver excitatory of the neurons motor superior and inferior, and it is continued in the transmission by the junction neuromuscular, in the joining excitement-contraction and in the contraction of the muscular fiber that depends on a metabolic energy supply. The muscular fatigue happens in patients with ALS harming the function and the quality of the patients\' life. The objective of this work was: 1) to quantify the frequency of the fatigue in the ALS; 2) to analyze the evolution of the fatigue in the patients; 3) to correlate the presence of the fatigue with factors as the functionality, the life quality, the depression, the dispnéa, and the sleepiness, age and duration of the disease in months. The group control was composed of 60 individuals (family of employees of the hospital and of the team multidisciplinar) that didn\'t present history of past diseases. The group test is constituted of 60 patients with diagnosis of ALS. The diagnosis was accomplished by two independent neurologists and he/she based on the presence of clinical history, neurological exam and studies compatible neurophisiologycs with ALS, according to the criteria of El Escorial of the World Federation of Neurology; besides, there was the complement investigation through tests and exams to exclude other pathologies. The patients of the groups control and test were interviewed by the researcher for application of questionnaires with scales for verification of the functionality presence, of life quality, of depression, of dispnéa, of sleepiness and of fatigue; and the patients of the group test they were submitted to the evaluation physiotherapy in the beginning of the study and every 3 months, totaling 12 months of collections. The group test presented fatigue larger significantly in relation to the group it controls, as well as alterations in the functionality questionnaires, of life quality, of depression, of dispnéa and of sleepiness. It was noticed that the fatigue was evolutionary during the months of accompaniment of the research. The fatigue was correlated with the age, showing that the youngest patients presented larger degree of fatigue than the most senior patients. The results of this research suggest that the fatigue is one of the problems that affect the patients with ALS; the fact of not having correlation with other studied problems suggests that the fatigue should deserve research and treatment individualized in the patient with ALS, mainly for the fact that the results suggested worsening of the fatigue in elapsing of the evolution of the ALS

Spinal muscular atrophy (SMA) is the leading genetic cause of death of young children. It is an autosomal recessive disease caused by the mutation and/or the deletion within the ubiquitously expressed survival motor neuron 1 (SMN1) gene. SMA pathology is characterized by spinal cord motor neuron degeneration, neuromuscular junction (NMJ) defects and muscular atrophy. Upon disease onset, SMA patients progressively become paralyzed and in the most severe cases, they die due to respiratory complications. Over the years, it has become clear that SMN is a multi-functional protein with important roles in small nuclear ribonucleoprotein (snRNP) assembly, RNA metabolism, axonal outgrowth and pathfinding, mRNA transport as well as in the functional development of NMJs, skeletal muscle and cardiac muscle. However, it remains unclear which of these functions, and the respective perturbed molecular pathways, dictate SMA pathogenesis. Here, we have established Smn-depleted PC12 cells and an intermediate SMA mouse model to characterize a role for Smn in the regulation of actin cytoskeleton dynamics. We find that Smn depletion results in the increased expression of profilin IIa and active RhoA (RhoA-GTP) as well as the decreased expression of plastin 3 and Cdc42. Importantly, the inhibition of rho-kinase (ROCK), a direct downstream regulator of RhoA, significantly increased the lifespan of SMA mice and shows beneficial potential as a therapeutic strategy for SMA. In an addition, we have uncovered a muscle- and motor neuron-independent role for SMN in the regulation of pancreatic development and glucose metabolism in SMA mice and type 1 SMA patients. This finding highlights the importance of combining a glucose tolerance assessment of SMA patients with their existing clinical care management. Thus, our work has uncovered two novel and equally important roles for the SMN protein, both of which contribute significantly to SMA pathogenesis.

The diploma thesis is focused on the average monthly flows forecast in the selected hydrometric profile. Aim of this work will be evaluation of the calculated values and the interpretation of the results in understandable form. The next step will be find an appropriate connection between randomly-generated inputs in the form of random real flow series using the standard hydrological prediction models. This models are based on the principles of artificial intelligence and probability model. The result of the work will be verification of procedures and compilation of mean monthly flow stochastic forecast in selected hydrometric profile, which would be used for a reservoirs management, respectively for water systems management.

The transient receptor potential ankyrin 1 (TRPA1) ion channel is expressed in a subset of primary afferent neurones where it is activated by a variety of pungent and chemically reactive compounds such as allyl isothiocyanate or cinnamaldehyde. This voltage- dependent channel is activated through covalent modification of cytoplasmic cysteines and, from the cytoplasmic side, is also critically regulated by calcium ions. Both, amino (N-) and carboxyl (C-) termini have been shown to be involved in these processes. Using electrophysiological and molecular-biology techniques, we explored the role of specific cytoplasmic domains in the activation of TRPA1. By measuring chemically-, voltage-, and calcium-activated membrane TRPA1-mediated currents, we identified highly conserved serine and threonine residues along the N-terminal ankyrin repeat domain, mutation of which strongly affected responses of the channel. In addition, using C-terminally truncated construct previously reported to be involved in calcium regulation, we present a new finding that the distal C-terminal tail contributes to voltage-dependent activation of TRPA1.

The purpose of this thesis project was to examine the effects of chronic alcohol on the excitability and molecular adaptation of GABA neurons of the ventral tegmental area (VTA). GABA neurons are of interest with regards to ethanol intoxication, reinforcement, and dependence due to their widespread distribution and connectivity to mesocorticolimbic dopamine (DA) neurons implicated in alcohol reward and addiction. Since we have previously shown adaptation of VTA GABA neuron firing rate to chronic ethanol (Gallegos, Criado et al. 1999) and suppression of gap-junction (GJ) mediated coupling between these neurons by acute ethanol (Stobbs, Ohran et al. 2004), we wanted to further characterize the effects of chronic ethanol on VTA GABA neuron excitability, electrical coupling and molecular adaptation. In particular, we analyzed the GJ mediated coupling and protein regulation of VTA GABA neurons following a three week period of continuous ethanol exposure via liquid diet. Although some animals showed tolerance, there was no significant tolerance to ethanol inhibition of GJ-mediated electrical coupling. In addition, we were able to characterize differences in mRNA expression levels for the DA synthesizing enzyme tyrosine hydroxylase (TH), the DA D2 receptor and the NMDAR2B receptor subunit in DA versus GABA neurons, all three of which were expressed at higher levels in DA neurons. We also determined the effects of chronic ethanol on mRNA levels of these same proteins as well as μ-opioid receptors (μORs) and connexin-36 (Cx36) GJs. Most significantly, we found a down-regulation of the DA D2 receptor, confirming that molecular modification occurs in these VTA GABA neurons with chronic alcohol. While we reject our hypothesis that acute ethanol inhibition of VTA GABA neuron electrical coupling would undergo tolerance to chronic ethanol in these non-dependent rats, which was the focus of this thesis, it remains to be determined if tolerance to chronic ethanol might be obtained in ethanol-dependent rats.

B virus is an alphaherpesvirus, endemic to macaque monkeys, capable of deadly human zoonosis with an 80% mortality rate in untreated cases. The macaque monkey is widely used in biomedical research and the threat of B virus poses an occupational hazard to researchers, veterinarians, and animal handlers. B virus establishes a life-long latent infection in sensory neurons of the peripheral nervous system (PNS) in the natural host. In human infections, B virus readily transits to the central nervous system (CNS) and destroys brain tissues. Identifying immune correlates of B virus infection in the PNS of the natural host is critical in understanding viral lethality in the human host. The lack of an accurate animal model and restrictions on handling potentially infected nervous tissue previously limited studies of B virus infection in macaque ganglia. To address this barrier, a long-lived mixed neuron/glia cell culture model was established from macaque DRG explants using a novel methodology that relied on cellular migration from whole tissues. Utilizing this model, the hypothesis tested was that acute B virus infection of macaque ganglia triggers cellular defense networks to promote leukocyte recruitment and impact leukocyte activation. Chemokines were upregulated in B virus-infected cultures and infected cell media induced leukocyte chemotaxis. Leukocytes were less effectively activated by media from infected cells when compared to media from mock-infected cells. To identify factors responsible for this, focused microarrays were performed and cytokine profiles were quantified from B virus and mock-infected culture supernatants. IL-6 protein levels were significantly reduced in B virus infected cultures. This observation led to the hypothesis that IL-6 downregulation impairs leukocyte activation and, indeed, when IL-6 was added to B virus-infected culture supernatants to control levels, these cultures were far more effective at eliciting leukocyte activation when compared with mock-infected cultures. Collectively, these data support the hypothesis that acute B virus infection of macaque ganglia triggers cellular defense networks to promote leukocyte recruitment and impact leukocyte activation and identifies a potential viral mechanism to impair leukocyte functionality. Additionally, this work presents a novel methodology for establishing long-lived mixed neuron/glia cultures from postnatal/adult macaque DRGs.