Whole-cell patch recordings were made from immature (six- to 12-day- old) rat rostral ventrolateral medulla neurons in brainstem slices. GABA or the specific GABA(B) receptor agonist (-)baclofen (10-50 μM) by superfusion or by pressure ejection induced an outward current or a hyperpolarization, which persisted in a tetrodotoxin (0.3 μM)-containing Krebs' solution in nearly every cell tested. The GABA(B) receptor antagonists 2-hydroxy saclofen (50-200 μM) and CGP 35348 (50-200 μM) dose-dependently suppressed baclofen- currents. Baclofen-currents were suppressed by barium (1 mM) but not by tetraethylammonium (20 mM), low Ca2+ (0.24 mM) solution or in a solution containing the Ca2+ chelator BAPTA-AM (10 μM). The outward current had an estimated reversal potential of -98, -77 and -52 mV in 3.1, 7 and 15 mM [K+](o). Pre-incubation of slices with pertussis toxin (500 μg/ml for 5-7 h) or intracellular dialysis with GDP-β-S (500 μM) markedly reduced baclofen-currents. Baclofen in low concentrations (1-3 μM) that caused slight or no change of holding currents and of inward or outward currents induced by exogenously applied glutamate or glycine/GABA, decreased excitatory and inhibitory postsynaptic currents by an average of 86.5 ± 4.3% and 78.4 ± 2.7%. The GABA(B) antagonist CGP 35348 (100 μM) increased the excitatory postsynaptic currents by an average of 64%, without causing a significant change in holding currents in 10/18 cells tested. Our results indicate the presence of post- and presynaptic GABA(B) receptors in the rostral ventrolateral medulla neurons. Activation of postsynaptic GABA(B) receptors induces an outward K+ current which is barium-sensitive, Ca2+- independent and may be coupled to a pertussis-sensitive G-protein. Activation of presynaptic GABA(B) receptors attenuates excitatory or inhibitory synaptic transmission. More importantly, the observation that CGP 35348 enhanced excitatory synaptic currents implies a removal of tonic activation of presynaptic GABA(B) receptors by endogenously released GABA (disinhibition), supporting the hypothesis that these receptors may have a physiological role in regulating the input and output ratio in a subset of rostral ventrolateral medulla neurons in vivo.

The speech auditory brainstem response (sABR) is a promising technique for assessing the function of the auditory system. This non-invasive technique has shown utility as a marker of central processing disorders, some types of learning difficulties in children, and potentially for fitting hearing aids. However, the sABR needs a long recording time to obtain a reliable signal due to the high background noise, which limits its clinical applicability. The objective of this work is to develop methods to detect the sABR in high background noise and enhance it based on a modeling approach and through experimental testing. First, sABR noise estimation based on LQ/QR decomposition is derived, and its mathematical proof is shown. Second, an autoregression model is used to estimate the single-trial sABR which is then used to test several sABR detection and enhancement methods. Third, a novel Artificial Neural Network (ANN) based detection approach is proposed and compared using modeled and recorded data to other detection methods in the literature: Optimal Linear Filter (LF), Online Estimator (OE), Mutual Information (MI) and Artificial Neural Network based on the Discrete Wavelet Transform and Approximate Entropy (ANN DA). Finally, comprehensive evaluation of several sABR enhancement methods is performed, based on the Wiener Filter (WF), Maximum-SNR Filter (Max-SNR), Adaptive Noise Cancellation (ANC) with Least-Mean-Square (LMS), Affine Projection (AP) and Recursive-Least-Square (RLS) adaptation algorithm. The results show that the developed LQ/QR decomposition estimated noise is similar to the actual noise, and the modeled data are statistically similar to the recorded data. Moreover, the proposed ANN-based detection method is more accurate and requires less processing time than other methods, and the comprehensive evaluation of enhancement methods shows that RLS has best overall performance in enhancing the sABR. Therefore, the methods developed and evaluated in this work have the potential to reduce the required recording time for the sABR, and thus make it more practical as a clinical tool.

No description available.

No description available.

Brainstem Auditory Evoked Potentials (BAEP) contain valuable information about the condition of the neural fibers associated with the auditory pathways. Extraction of this information is a difficult task due to contamination by on-going scalp EEG. This thesis reviews the current processing techniques and introduces adaptive noise cancellation (ANC) using systolic arrays as an alternative to existing technology. Q-R decomposition theory is reviewed and an explanation of the mechanics of systolic adaptive noise cancellation (SANC) is presented. A modified Given's rotation algorithm is derived resulting in a saving of up to 2/3 in memory requirements. Real data were collected in the laboratory. Real and simulated data were processed to determine the characteristics and effectiveness of adaptive noise cancellation strategies. Successful ANC of BAEP was performed on simulated data using a number or signal-to-noise ratios (S/N), data sequence lengths, reference signals and filter parameter values. We conclude that systolic arrays are a very powerful and appropriate technique for the extraction or BAEPs. Correlation studies indicated that the pre-stimulus EEG signal is inadequately correlated to the primary signal for successful ANC or BAEP in real data. A multi-channel collection scheme is outlined for future collection or Evoked Potential data. A summary or experimental results is presented to address the problem or data collection and signal processing optimization. / Thesis / Master of Engineering (MEngr)

O Tronco encefálico é uma estrutura singular do sistema nervoso central, pois nele passam tratos sensoriais ascendentes da medula espinal, tratos sensoriais da cabeça e do pescoço, os tratos descendentes motores originados no prosencéfalo, as vias ligadas a centros de movimento dos olhos, contemos núcleos dos nervos cranianos, e também está envolvido na regulação do nível de consciência através de projeções ao prosencéfalo oriundas da formação reticular. Tudo isto compactado em um espaço muito exíguo o que faz deste um local particularmente sensível às alterações patológicas, mesmo que pequenas, que acabam cursando com uma riqueza de sinais neurológicos devido a presença muito próxima das estruturas já citadas. Compreender a anatomia interna do tronco encefálico é essencial para o diagnóstico neurológico e a prática da medicina clínica. Por tudo que foi exposto, o tronco encefálico é um terreno fértil para o estudo através do diagnóstico por imagem, principalmente quando realizado por novas tecnologias, como exames em aparelho de ressonância magnética de alto campo (3 teslas). No entanto, pouco se sabe sobre as correlações existentes entre a microscopia e as imagens de ressonância magnética do tronco encefálico. Sendo assim, o objetivo deste estudo foi analisar e correlacionar as diversas estruturas encontradas no tronco encefálico, visualizadas em peças microscópicas de encéfalos humanos post mortem, com as imagens de ressonância magnética dos mesmos, antes da dissecção, mapeandoas e discernindo-as, contribuindo assim para diagnósticos mais precisos e topográficos das patologias que acometem o tronco encefálico, justificando o presente estudo. O estudo foi de caráter observacional exploratório e descritivo, adotando as seguintes técnicas para coleta da informações: os encéfalos humanos (n=3) foram submersos em recipiente contendo água e então lacrados de forma que o ar ambiente não entrasse no recipiente. As imagens de RM foram adquiridas em sequência gradiente echo (FFE) 2D em equipamento de campo 3T (PHILIPS ACHIEVA), com bobina de 8 canais de encéfalo. Foi utilizado TE=9,0 ms, TR=1000 ms e o ângulo de flip 90°; número de médias igual 10 e BW por pixel igual a 72 Hz/pixel. O Fator EPI utilizado foi igual a um e a resolução espacial de 0,219x0,219x2,0 mm3 com FOV de 210x210x90mm3. O tempo total de aquisição foi de 3 horas e 01 minuto e 96 segundos. Imagens histológicas utilizadas no presente estudo são do banco de dados do departamento de patologia da UNICAMP e foram comparadas às imagens obtidas na ressonância magnética. Demonstramos que foi possível a identificação das estruturas visíveis, histologicamente, nas imagens obtidas, com definição e resolução suficiente para a geração de um atlas de imagens de ressonância magnética de cortes do tronco encefálico / The Brainstem is a unique structure of the central nervous system, because in it pass ascending sensory tracts of the spinal cord, sensory tracts of head and neck, descending tracts originated in the forebrain, the pathways linked to eye movement centers, contains nuclei of cranial nerves, and is also involved in regulating the level of consciousness through projections to the forebrain that arise from the reticular formation. All these estructures are packed into a very small space which makes the brainstem a particularly sensitive place to pathological changes, that bring up a large amount of neurological signs due to very close packing of the aforementioned structures.Understanding the internal anatomy of the brainstem is essential for the neurological diagnosis and the clinical medicine practice. Thus, the brainstem is fertile ground for the study through diagnostic imaging, especially when performed by new technologies such as high-field (3 tesla) MRI machines. However, little is known about the correlation between the microscopy and magnetic resonance imaging of the brainstem. The aim of this study was to analyze and correlate the various structures found in the brainstem, viewed in microscopic slides of human brains post mortem, with the magnetic resonance imaging thereof, prior to dissection, mapping them and defining them, thus contributing to more accurate diagnoses and surveying of pathologies that affect the brainstem. Human brains (n = 3) were submerged in a container containing water, and then sealed so that the ambient air does not enter the container. MRI images were acquired in gradient echo sequence (FFE) 2D 3T field equipment (PHILIPS ACHIEVA) with coil 8- channel brain. It was used TE = 9.0 ms, TR = 1000 ms and flip angle 90°; number of averages equal to 10 and BW per pixel equal to 72 Hz/pixel. Factor PPE used was equal to one and the spatial resolution of FOV with 0,219x0,219x2,0 mm3 210x210x90mm3. The total acquisition time was 3 hours, 01 minute and 96 seconds. Histological images used in this study are from the pathology department of State University of Campinas (UNICAMP) database and compared to images obtained in MRI. We demonstrated that it was possible to identify histologically visible structures in images acquired with sufficient resolution and definition to generate an magnetic resonance imaging atlas of the brainstem sections

O Tronco encefálico é uma estrutura singular do sistema nervoso central, pois nele passam tratos sensoriais ascendentes da medula espinal, tratos sensoriais da cabeça e do pescoço, os tratos descendentes motores originados no prosencéfalo, as vias ligadas a centros de movimento dos olhos, contemos núcleos dos nervos cranianos, e também está envolvido na regulação do nível de consciência através de projeções ao prosencéfalo oriundas da formação reticular. Tudo isto compactado em um espaço muito exíguo o que faz deste um local particularmente sensível às alterações patológicas, mesmo que pequenas, que acabam cursando com uma riqueza de sinais neurológicos devido a presença muito próxima das estruturas já citadas. Compreender a anatomia interna do tronco encefálico é essencial para o diagnóstico neurológico e a prática da medicina clínica. Por tudo que foi exposto, o tronco encefálico é um terreno fértil para o estudo através do diagnóstico por imagem, principalmente quando realizado por novas tecnologias, como exames em aparelho de ressonância magnética de alto campo (3 teslas). No entanto, pouco se sabe sobre as correlações existentes entre a microscopia e as imagens de ressonância magnética do tronco encefálico. Sendo assim, o objetivo deste estudo foi analisar e correlacionar as diversas estruturas encontradas no tronco encefálico, visualizadas em peças microscópicas de encéfalos humanos post mortem, com as imagens de ressonância magnética dos mesmos, antes da dissecção, mapeandoas e discernindo-as, contribuindo assim para diagnósticos mais precisos e topográficos das patologias que acometem o tronco encefálico, justificando o presente estudo. O estudo foi de caráter observacional exploratório e descritivo, adotando as seguintes técnicas para coleta da informações: os encéfalos humanos (n=3) foram submersos em recipiente contendo água e então lacrados de forma que o ar ambiente não entrasse no recipiente. As imagens de RM foram adquiridas em sequência gradiente echo (FFE) 2D em equipamento de campo 3T (PHILIPS ACHIEVA), com bobina de 8 canais de encéfalo. Foi utilizado TE=9,0 ms, TR=1000 ms e o ângulo de flip 90°; número de médias igual 10 e BW por pixel igual a 72 Hz/pixel. O Fator EPI utilizado foi igual a um e a resolução espacial de 0,219x0,219x2,0 mm3 com FOV de 210x210x90mm3. O tempo total de aquisição foi de 3 horas e 01 minuto e 96 segundos. Imagens histológicas utilizadas no presente estudo são do banco de dados do departamento de patologia da UNICAMP e foram comparadas às imagens obtidas na ressonância magnética. Demonstramos que foi possível a identificação das estruturas visíveis, histologicamente, nas imagens obtidas, com definição e resolução suficiente para a geração de um atlas de imagens de ressonância magnética de cortes do tronco encefálico / The Brainstem is a unique structure of the central nervous system, because in it pass ascending sensory tracts of the spinal cord, sensory tracts of head and neck, descending tracts originated in the forebrain, the pathways linked to eye movement centers, contains nuclei of cranial nerves, and is also involved in regulating the level of consciousness through projections to the forebrain that arise from the reticular formation. All these estructures are packed into a very small space which makes the brainstem a particularly sensitive place to pathological changes, that bring up a large amount of neurological signs due to very close packing of the aforementioned structures.Understanding the internal anatomy of the brainstem is essential for the neurological diagnosis and the clinical medicine practice. Thus, the brainstem is fertile ground for the study through diagnostic imaging, especially when performed by new technologies such as high-field (3 tesla) MRI machines. However, little is known about the correlation between the microscopy and magnetic resonance imaging of the brainstem. The aim of this study was to analyze and correlate the various structures found in the brainstem, viewed in microscopic slides of human brains post mortem, with the magnetic resonance imaging thereof, prior to dissection, mapping them and defining them, thus contributing to more accurate diagnoses and surveying of pathologies that affect the brainstem. Human brains (n = 3) were submerged in a container containing water, and then sealed so that the ambient air does not enter the container. MRI images were acquired in gradient echo sequence (FFE) 2D 3T field equipment (PHILIPS ACHIEVA) with coil 8- channel brain. It was used TE = 9.0 ms, TR = 1000 ms and flip angle 90°; number of averages equal to 10 and BW per pixel equal to 72 Hz/pixel. Factor PPE used was equal to one and the spatial resolution of FOV with 0,219x0,219x2,0 mm3 210x210x90mm3. The total acquisition time was 3 hours, 01 minute and 96 seconds. Histological images used in this study are from the pathology department of State University of Campinas (UNICAMP) database and compared to images obtained in MRI. We demonstrated that it was possible to identify histologically visible structures in images acquired with sufficient resolution and definition to generate an magnetic resonance imaging atlas of the brainstem sections

Background: The efferent dorsal motor nucleus of the vagal nuclei complex may degenerate early in the course of Parkinson’s disease (PD), while efferent nucleus ambiguous, the principal source of parasympathetic vagal neurons innervating the heart, and afferent somatosensory nuclei remain intact.

<p>Brain tumors are an incredibly diverse group of neoplasms, as evidenced by their varied locations in the brain, histological characteristics, and genetic alterations. Brain tumor heterogeneity can be potentially explained by distinct oncogenic events or cells-of-origin, or by region-specific intrinsic or extrinsic factors. Brainstem Glioma (BSG) is a particularly deadly brain tumor, afflicting 200-300 children in the United States each year. High-grade BSG (also known as Diffuse Intrinsic Pontine Glioma, DIPG) cannot be surgically removed, and the standard treatment of radiation therapy provides only temporary relief from symptoms. The past 5 years has witnessed a dramatic increase in knowledge regarding the biological basis of this disease along with the realization that BSG is distinct from other more common types of glioma, such as cerebral cortex glioma (CG). It was the goal of this study to investigate the regional differences in gliomas arising in the brainstem versus the cerebral cortex, using mice as a model system, and to begin to understand the contributions of the various possible sources of heterogeneity.</p><p> </p><p>In doing so, we have uncovered region-specific gene expression patterns in these two types of pediatric gliomas that are apparent even when the initiating genetic alterations and cell-of-origin are kept constant. Focusing on the <italic>paired box 3</italic> (Pax3) gene, which is expressed at higher levels in BSG than CG, we have found that Pax3 expression not only characterizes mouse BSGs driven by PDGF signaling, Ink4aARF-loss, p53-loss, and H3.3-K27M expression, but also identifies a novel subset of human BSGs that are associated with <italic>PDGFRA</italic> alterations and wild type <italic>ACVR1</italic> and that commonly harbor <italic>TP53</italic> alterations and the H3.3-K27M mutation. </p><p>As Pax3 plays a pro-tumorigenic role in other types of cancer, we hypothesized that Pax3 expression contributes to the brainstem gliomagenesis process as well. By utilizing mouse models, we found that Pax3 inhibits apoptosis and promotes proliferation of Nestin-expressing brainstem progenitor cells <italic>in vitro</italic> and enhances PDGF-B-driven BSG <italic>in vivo</italic>. Furthermore, we speculate that Pax3 expression may be a marker for Wnt pathway activation in BSG, which is targetable via pharmacologic agents. Indeed, a subset of Wnt inhibitors tested effectively slowed the growth of BSG cells <italic>in vitro</italic>, however cross talk with the Shh pathway might indicate that dual Wnt and Shh inhibition is necessary.</p><p>In addition, the regional expression pattern of Pax3 in gliomas correlates with its expression in normal murine brain development, leading us to hypothesize that Pax3 progenitor cells in the neonatal brainstem can serve as a cell-of-origin for BSG. We discovered that targeting Pax3 progenitors with PDGF-B overexpression and Ink4aARF- or p53-loss induces high-grade BSG that physiologically resemble the human disease. This novel and distinct model of BSG may be utilized in the future for preclinical studies.</p><p>The identification of Pax3 as a regional marker of mouse and human BSG has led to the discovery of a novel subset of the human disease, the identification of a novel oncogene contributing to pathogenesis, and the characterization of a novel cell-of-origin with the potential to give rise to the disease. This information contributes significantly to the current understanding of the mechanisms and cellular origins of BSG, and will hopefully instruct future investigations into how to better treat this disease.</p> / Dissertation

Le gliome infiltrant du tronc cérébral (en anglais “diffuse intrinsic pontine glioma”, DIPG) est une tumeur pédiatrique rare et très agressive. La durée moyenne de survie après diagnostic est inférieure à un an. Une caractéristique génétique majeure des DIPG est la mutation de l’histone H3 (H3K27M). L’évolution des connaissances en épigénétique a permis de concevoir des inhibiteurs de régulateurs épigénétiques capables de modifier, voire de contrebalancer, l’effet de cette mutation. Ainsi, le panobinostat (PS), un inhibiteur des histone-désacétylases, diminue la croissance cellulaire et conduit à la mort des cellules de DIPG in vitro et in vivo. Son efficacité est en cours d'évaluation dans des essais cliniques. Mon projet de thèse avait pour objectif de déterminer le rôle d’EBP50 et d’IRSp53, deux protéines spécifiquement dérégulées dans les lignées de DIPG après traitement des cellules par le PS. EBP50 est connue pour intervenir dans la progression tumorale mais sa dualité de fonction, à la fois oncogène et suppresseur de tumeur, nous a conduits à étudier plus précisément son rôle dans les cellules de DIPG. IRSp53 a été peu étudiée dans les cancers solides, bien qu'elle semble jouer un rôle important dans la motilité cellulaire et l’invasion. La diminution de l’expression d’IRSp53 et d’EBP50 par ARN interférence dans des lignées DIPG induit la mort des cellules par apoptose et bloque leur croissance ainsi que leur motilité cellulaire, ce qui suggérerait que ces deux protéines sont oncogéniques dans ce modèle. De plus, la localisation cytoplasmique et nucléaire d’EBP50 semble en accord avec son rôle pro-oncogénique dans les cellules de DIPG. En étudiant in vitro l’effet d’un traitement combinatoire du PS avec des inhibiteurs de l’expression d’EBP50 ou d’IRSp53, j’ai mis en évidence une augmentation de la sensibilité des cellules de DIPG au traitement par le PS. Enfin, j’ai validé le traitement ciblant EBP50 in vivo dans un modèle préclinique d’embryon de poulet. En conclusion, ces deux protéines constituent de nouvelles cibles thérapeutiques dans les DIPG et un moyen d’augmenter l’efficacité du PS. / Diffuse Intrinsic Pontine Glioma (DIPG), is a rare and highly aggressive pediatric tumor. The average survival time after diagnosis is less than one year. A major genetic characteristic of this disease is the mutation of histone H3 (H3K27M). The evolution of knowledge in epigenetics has made it possible to design epigenetic regulatory inhibitors able to modify, or even offset, the effect of this mutation. For example, panobinostat (PS), a histone deacetylase inhibitor, reduces cell growth and induces DIPG cell death, both in vitro and in vivo. Its effectiveness is currently being evaluated in clinical trials. My thesis project aimed at determining the role of two proteins, EBP50 and IRSp53, deregulated in different DIPG cell lines after treatment with PS. EBP50 has already been described as involved in tumor progression but its dual function, both oncogenic and tumor suppressor, has led us to further investigate its role in the DIPG cells. IRSp53 has been poorly studied in solid cancers, though it plays an important role in cell motility and invasion. Down-regulation by RNA silencing of these two proteins in DIPG cell lines induces apoptosis, decreases cell growth and motility, leading us to the hypothesis that these two proteins are oncogenic proteins. In addition, the cytoplasmic and nuclear localization of the EBP50 protein is consistent with its oncogenic role in DIPG cells. Then, I investigated the effect of combinatorial therapy that associates PS with EBP50 or IRSp53 expression inhibitors. My results show an increase in the antitumor effect in vitro for both proteins but also in vivo for EBP50, in a preclinical model, the chicken embryo. In conclusion, these two proteins could be the targets of new treatments for DIPG tumors in combination with PS to enhance its efficacy.