L'ús extensiu i l'abocament massiu de pesticides en el medi ambient ha portat a alguns autors a preguntar-se sobre la contribució d'aquestes substàncies al desenvolupament de trastorns neuropsiquiàtrics i metabòlics. Actualment, els organofosforats (OP) - i el clorpirifos (CPF) en particular - són els insecticides més emprats al món. Donada la diversitat de fonts d'exposició, la majoria de la població està exposada gairebé permanentment a CPF. Una gran quantitat d’estudis epidemiològics i experimentals han observat dèficits neuroconductuals després de l'exposició al pesticida, i evidències incipients suggereixen vincles entre l'exposició a OPs i el desenvolupament de trastorns metabòlics. Una de les claus consisteix a establir si existeixen subpoblacions particularment susceptibles als efectes perjudicials del CPF. S'ha demostrat que les diferents isoformes de l'apolipoproteïna E (apoE) humana confereixen diferents vulnerabilitats a desenvolupar malalties metabòliques i neurodegeneratives. El principal objectiu d'aquesta tesi va ser avaluar els efectes comportamentals i metabòlics del genotip APOE i del CPF, així com determinar si la interacció entre ambdós factors va contribuir a l'aparició d'aquests efectes. Amb aquest propòsit, ratolins adults portadors de apoE2, apoE3 o apoE4 foren exposats subcrònicament a 2 mg/kg/dia o 3.75 mg/kg/dia de CPF en funció de la fase experimental. Després de l'exposició, els animals foren avaluats conductualment, mentre que els efectes metabòlics del CPF també foren avaluats en paral·lel. En general, el genotip APOE va influenciar el rendiment cognitiu. L'exposició a CPF va alterar el funcionament metabòlic, i va induir dèficits atencionals i motivacionals prolongats en el temps. Els ratolins apoE3 foren els més vulnerables als efectes metabòlics del tòxic, i van mostrar alteracions de la memòria després de l'exposició. A més, el pesticida va invertir el dèficit de control inhibitori característic dels ratolins apoE4. En conclusió, aquests resultats amplien la literatura existent sobre els efectes comportamentals i metabòlics del CPF, i proporcionen informació valuosa sobre interaccions gen - tòxic fins ara desconegudes. / El uso extensivo y el vertido masivo de pesticidas en el medio ambiente ha llevado a algunos autores a preguntarse sobre la contribución de estas sustancias al desarrollo de trastornos neuropsiquiátricos y metabólicos. Actualmente, los organofosforados (OPs) - y el clorpirifos (CPF) en particular - son los insecticidas más utilizados en el mundo. Dada la diversidad de fuentes de exposición, la mayoría de la población está expuesta casi permanentemente a CPF. Una gran cantidad de estudios epidemiológicos y experimentales han observado déficits neuroconductuales tras la exposición al pesticida, y evidencias incipientes sugieren vínculos entre la exposición a OPs y el desarrollo de trastornos metabólicos. Una de las claves consiste en establecer si existen subpoblaciones particularmente susceptibles a los efectos perjudiciales del CPF. Se ha demostrado que las diferentes isoformas de la apolipoproteína E (apoE) humana confieren diferentes vulnerabilidades a desarrollar enfermedades metabólicas y neurodegenerativas. El principal objetivo de esta tesis fue evaluar los efectos comportamentales y metabólicos del genotipo APOE y del CPF, así como determinar si la interacción entre ambos factores contribuyó a la aparición de estos efectos. Así, ratones adultos portadores de apoE2, apoE3 o apoE4 fueron expuestos subcrónicamente a 2 mg/kg/día o 3.75 mg/kg/día de CPF en función de la fase experimental. Tras la exposición, los animales fueron evaluados conductualmente, mientras que los efectos metabólicos del CPF también fueron estudiados en paralelo. En general, el genotipo APOE influenció el rendimiento cognitivo. La exposición a CPF alteró el funcionamiento metabólico, e indujo déficits atencionales y motivacionales prolongados en el tiempo. Los ratones apoE3 fueron los más vulnerables a los efectos metabólicos del tóxico, y mostraron alteraciones de la memoria tras la exposición. Además, el pesticida invirtió el déficit de control inhibitorio característico de los ratones apoE4. En conclusión, estos resultados amplían la literatura existente sobre los efectos comportamentales y metabólicos del CPF, y proporcionan información valiosa sobre interacciones gen - tóxico hasta ahora desconocidas. / The extensive use and massive release of pesticides into the environment has led some authors wonder about the potential contribution of these substances on the emergence of neuropsychiatric and metabolic disorders. Currently, organophosphates (OP) – and chlorpyrifos (CPF) in particular - are the most widely used insecticides in the world. Given the heterogeneity of sources of exposure, most of the population is almost permanently exposed to CPF. Thus, a great body of epidemiological and experimental data have reported neurobehavioural deficits following exposure to the pesticide, and incipient evidence begins to suggest potential links between OPs and metabolic disorders. One of the critical issues is to establish whether there are subpopulations particularly susceptible to the detrimental effects of CPF. It has been proved that human apolipoprotein E (apoE) isoforms confer on their carriers varying vulnerabilities to metabolic diseases and neurodegeneration. The main objective of this thesis was to assess the behavioural and metabolic effects of both APOE genotype and CPF, as well as to determine whether the interaction between both factors contributed to these effects. To test this, adult mice expressing apoE2, apoE3 or apoE4 human isoforms were subchronically exposed to 2 mg/kg/day or 3.75 mg/kg/day CPF depending on the experimental phase. After exposure, animals were behaviourally tested, while the metabolic-disruptor role of CPF was also assessed in paralel. In general, APOE genotype influenced cognitive performance. The exposure to CPF altered metabolic functioning, and induced protracted attentional and motivational deficits. ApoE3 mice were the most vulnerable to the metabolic effects of CPF, and showed memory impairments after exposure. Furthermore, the pesticide reversed the lack of inhibitory control characteristic of apoE4 mice. Taken together, these results expand the existing literature on the behavioural and metabolic effects of CPF, and provide valuable information on gene – toxic interactions hitherto unknown.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / This study stems from the findings during the gross and histopathological exam of 3,338 cattle brains as part of the bovine spongiform encephalopathy (BSE) active surveillance program of the Brazilian Ministry of Agriculture, Livestock and Supply (MAPA) from 2001-2005. The work was carried out in the Veterinary Pathology Laboratory (LPV) of the Federal University of Santa Maria (UFSM) which is the national reference laboratory for the diagnosis of BSE and other transmissible spongiform encephalopathies. This work is presented in four parts. Initially the key events about the significance, etiology, epidemiology, clinical signs, pathology, and control of BSE are presented in order to provide the reader with the background that led the MAPA to develop this surveillance program for BSE. In the second part, the mechanics of the surveillance program are explained in detail and most of the written rules and ordinances issued by MAPA in order to strength the surveillance for BSE are included as annexes. The third part deals with the gross and histopathological examinations of the 3,338 cattle brains. Both gross and histopathological methodologies are described. Gross exam considered distribution, color, form, size, consistency and other general aspects of any particular change. Gross examinations included also non-lesions (anatomical normal structures that may be interpreted as lesions), lesions of limited or no clinical significance, postmortem changes and other artifacts. All these are considered pitfalls in the examination of the central nervous system (CNS). Accordingly equivalent pitfalls were described in the histopathological examination; additionally the normal CNS components are described together with their general reactions to injury. In the fourth part of the work, copies of papers that resulted from the systematic examination of 3,338 cattle brains during the BSE active surveillance program are included. The papers are allotted to two groups: the first group includes those primary papers that are a direct part of this thesis and in which the thesis author is the senior author. These papers are: 1) Rech R.R. et al. 2004. Granulomatous meningoencephalitis in cattle grazing vetch (Vicia spp). Pesq. Vet. Bras. 24:169-172, 2) Rech R.R. et al. 2005. Malignant catarrhal fever in cattle in Rio Grande do Sul, Brazil: Epidemiology, clinical signs and pathology. Pesq. Vet. Bras.25:97-105, 3) Rech R.R. et al. 2006. Histomorphology of the Gasserian ganglion, carotid rete mirabile and pituitary gland in cattle: A study of 199 cases. Pesq. Vet. Bras. 26:105-111, 4) Rech R.R. et al. 2006. Poisoning by Solanum fastigiatum (Solanaceae) in cattle: Epidemiology, clinical signs and morphometry of cerebellar lesions. Pesq. Vet. Bras., 26:183-189, 5) Rech et al. 2006. Rabies in herbivores: 27 cases. Proceedings of the Annual National Meetings Veterinary Diagnostic Labs in Brazil, March 2006 (Abstract) and 6) Rech R.R. et al. 2007. Poisonous plants affecting the central nervous system (CNS) of cattle in Brazil, p. 238- 243. In: Panter K.E.; Wierenga T.L. & Pfister J.A. (ed.). Poisonous Plants: Global Research and Solutions. Wallingford: CAB International. The second group of papers includes those that derived from the surveillance BSE program but were developed by another Graduate Student of the LPV/UFSM and in which this thesis author (RR) is a co-author. These papers are: 1) Rodrigues A. et al. 2005. Cerebral babesiosis in cattle: 20 cases. Ciência Rural.35:121-125, 2) Barros R.R. et al. 2006. Ocular squamous cell carcinoma in a cow with cerebral invasion through cranial nerves. Ciência Rural 36:1651-1654, 3) Barros R.R. et al. 2006. Poisoning by Ramaria flavo-brunnescens (Clavariaceae) in cattle. Pesq. Vet. Bras. 26:87-96 and 4) Rissi D.R. et al. 2006. Epidemiology, clinical signs and distribution of the encephalic lesions in cattle affected by meningoencephalitis caused by bovine herpesvirus-5. Pesq. Vet. Bras. 26:123-132. During part of the Graduate Program that led to this Thesis the author stayed for 10 months in a CAPES-Sandwich-Fellowship in The University of Georgia at Athens, USA where a protocol for immunohistochemistry (IHC) for detection of rabies antigen in formalin fixed paraffin blocks from bovine brain was established under the supervision of Prof. Corrie Brown. The description of this protocol and the partial results of the IHC diagnosis for rabies in cattle are included in the text of this Thesis. Most of the conclusions drawn from the work presented here are detailed in the papers listed above. Additionally conclusions are that the MAPA design and implemented an adequate system for BSE surveillance. Weak points in the program are that not enough samples from cattle that tested negative in the official laboratories for rabies diagnosis in Brazil, from imported cattle and from cattle submitted to emergence slaughter in abattoirs under federal Meat Inspection are arriving at the LPV/UFSM. From the material examined, it was also concluded that the more frequent inflammatory diseases in the brain of cattle are rabies, meningoencephalitis by bovine herpesvirus 5 and malignant catarrhal fever and the more frequent degenerative diseases are hepatic encephalopathy and poisoning by Solanum fastigiatum. / Este estudo derivou do exame macroscópico e microscópico de 3.338 encéfalos de bovinos, realizado entre 2001-2005, como parte do programa de vigilância para a encefalopatia espongiforme bovina (BSE) do Ministério da Agricultura, Pecuária e Abastecimento (MAPA). Este trabalho foi realizado no Laboratório de Patologia Veterinária da Universidade Federal de Santa Maria (LPV/UFSM), que é o laboratório de referência nacional para o diagnóstico da BSE e outras encefalopatias espongiformes transmissíveis e foi dividido em quatro partes. Inicialmente, são descritos os principais eventos relacionados à etiologia, epidemiologia, sinais clínicos, patologia e controle da BSE para dar ao leitor uma idéia da situação que levou o MAPA a implantar esse programa de vigilância. Na segunda parte, é explicado em detalhes o funcionamento do programa de vigilância para a BSE no Brasil, com as principais instruções determinadas pelo MAPA relacionadas a esse programa incluídas em anexos. A terceira parte descreve os achados macro e microscópicos observados nos 3.338 encéfalos examinados. No exame macroscópico foram considerados os seguintes aspectos: distribuição, cor, tamanho e consistência e outros aspectos gerais de alterações particulares. A análise macroscópica também incluiu descrições de não-lesões (estruturas anatômicas normais que podem ser interpretadas como lesões), lesões de pouco significado clínico, alterações postmortem e outros artefatos. Todas essas alterações podem confundir o patologista durante o exame do sistema nervoso central (SNC). Essa mesma metodologia foi utilizada para descrever os achados microscópicos, incluindo a descrição de componentes normais do SNC e suas reações à lesão. A quarta seção é constituída pelos trabalhos publicados que resultaram do exame histológico desses 3.338 encéfalos durante o programa de vigilância para a BSE nesses 5 anos. Os trabalhos foram divididos em dois grupos: o primeiro grupo inclui os trabalhos que são parte direta desta tese e nos quais a autora da tese (Rech R.R.) é o primeiro autor. Estes trabalhos são: 1) Rech R.R. et al. 2004. Meningoencefalite granulomatosa em bovinos em pastoreio de ervilhaca (Vicia spp). Pesq. Vet. Bras. 24:169-172, 2) Rech R.R. et al. 2005. Febre catarral maligna em bovinos no Rio Grande do Sul: epidemiologia, sinais clínicos e patologia. Pesq. Vet. Bras. 25:97-105, 3) Rech R.R. et al. 2006. Histomorfologia do gânglio de Gasser, da rete mirabile carotídea e da hipófise de bovinos: estudo de 199 casos. Pesq. Vet. Bras. 26:105-111, 4) Rech R.R. et al. 2006. Intoxicação por Solanum fastigiatum (Solanaceae) em bovinos: epidemiologia, sinais clínicos e morfometria das lesões cerebelares. Pesq. Vet. Bras., 26:183-189, 5) Rech et al. 2006. Raiva em herbívoros: 27 casos. Encontro Nacional de Laboratórios de Diagnóstico Veterinário, Campo Grande, p. 43-44, e 6) Rech R.R. et al. 2007. Poisonous plants affecting the central nervous system (CNS) of cattle in Brazil, p. 238-243. In: Panter K.E.; Wierenga T.L. & Pfister J.A. (ed.). Poisonous Plants: Global Research and Solutions. Wallingford: CAB International. O segundo grupo inclui trabalhos onde foi utilizado material do programa de vigilância da BSE, mas que foram desenvolvidos por outro estudante de pós-graduação do LPV/UFSM e no qual a autora da tese é co-autora. Estes trabalhos são: 1) Rodrigues A. et al. 2005. Babesiose cerebral em bovinos: 20 casos. Ciência Rural 35:121-125, 2) Barros R.R. et al. 2006. Intoxicação por Ramaria flavo-brunnescens (Clavariaceae) em bovinos. Pesq. Vet. Bras. 26:87-96, 3) Rissi D.R. et al. 2006. Epidemiologia, sinais clínicos e distribuição das lesões encefálicas em bovinos afetados por meningoencefalite por herpesvírus bovino-5. Pesq. Vet. Bras. 26:123-132, e 4) Barros R.R. et al. 2006. Carcinoma de células escamosas no olho de bovino com invasão cerebral através dos nervos cranianos. Ciência Rural 36:1651-1654. Durante parte do programa de doutorado, a autora realizou o programa de intercâmbio com bolsa-sanduíche da CAPES por 10 meses (03/2006 a 12/2006) na The University of Georgia em Athens, Estados Unidos. Durante esse período, foi desenvolvido um protocolo de imunoistoquímica para a detecção de antígeno da raiva em blocos de parafina com encéfalos fixados em formol. A descrição desse protocolo e os resultados parciais desse trabalho estão incluídos nesta tese. A maioria das conclusões desta tese estão nos trabalhos acima citados. Conclusões adicionais são que o MAPA programou e colocou em funcionamento um sistema de vigilância para a BSE relativamente eficiente. No entanto, pontos fracos do programa incluem um insuficiente número de encéfalos encaminhados aos laboratórios credenciados de bovinos que testaram negativo para raiva nos laboratórios oficiais de diagnóstico de raiva no Brasil, de bovinos importados e de bovinos submetidos ao abate de emergência em frigoríficos com serviço de inspeção federal. Do material examinado durante esses 5 anos, as doenças inflamatórias mais freqüentes na região de abrangência do LPV/UFSM foram raiva, meningoencefalite por herpesvírus bovino-5 e febre catarral maligna, e as doenças degenerativas mais comuns são encefalopatia hepática e intoxicação por Solanum fastigiatum.

Submitted by Marlene Santos (marlene.bc.ufg@gmail.com) on 2016-10-13T17:13:38Z No. of bitstreams: 2 Dissertação Victor de Melo Caixeta - 2015.pdf: 2750656 bytes, checksum: c7c2c97e0c6537b3d17aa54409152ad3 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Jaqueline Silva (jtas29@gmail.com) on 2016-10-14T20:03:21Z (GMT) No. of bitstreams: 2 Dissertação Victor de Melo Caixeta - 2015.pdf: 2750656 bytes, checksum: c7c2c97e0c6537b3d17aa54409152ad3 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-10-14T20:03:21Z (GMT). No. of bitstreams: 2 Dissertação Victor de Melo Caixeta - 2015.pdf: 2750656 bytes, checksum: c7c2c97e0c6537b3d17aa54409152ad3 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2015-10-15 / Background: Corticobasal Degeneration (CBD) is an rare and heterogeneous disease in its presentations, representing an great diagnostic challenge. There aren´t, in Brasil, large CBD case series, and there aren´t many in the world as well. Its cognitive and behavioural aspects have received little (despite growing) attention. Objectives: To describe sociodemographic, clinic, neuropsychiatric, neuropathological, and neuroradiologic aspects in a large brazilian DCB case series. To perform a literature review on CBD, with special focus on cognitive and behavioural aspects. Methods: 70 patients data was collected, with CBD diagnosis according to the Cambridge criteria modified by Bak and Hodges (2011). The patients underwent clinical, neuropsychiatric, neuroradiologic (structural and functional) and pathologic (in six cases) retrospective analysis. There were studied clinical, sociodemographic, neuropsychiatric, neuroanatomic variables and family history. Results and Discussion: The mean age at onset was 62.8 years (sd=9,5), and both sexes were equally affected (52,9% male). Hemispheric asymmetry was present in 97% of cases, and the left brain hemisphere was the most affected (68,2% of cases). The most frequent initial presentation was “psychiatric” (with changes in behaviour and/or mood), present in 68,1% of cases, followed by motor-extrapyramidal presentation (54.3%). In the course of the disease, the predominant clinical form (phenotype) was the extrapyramidal-motor with 61.4% of cases, followed by "psychiatric" with 51.4%. There were five cases with presentation of Posterior Cortical Atrophy (PCA) In 37.7% there were not found classic CBD syndromes (e.g., alien hand syndrome). We found 18.7% of cases with family DCB, with four families presenting a phenotype not yet described, of CBD with NPH (Normal Pressure Hydrocephalus). We also observed cognitive and functional impairments in the evaluated scales (MMSE, Pfeffer and CDR), and frequent medical and psychiatric comorbidities, especially diabetes mellitus (23.5%), hypothyroidism (22.1%) and Bipolar Spectrum Disorders (46, 4%), the last two significantly more frequent in the sample. There were six cases of rapidly progressive DCB and six confirmed by autopsy CBD cases in the sample. Conclusion: We observed, in agreement with the literature, a wide variety of CBD presentations, including new and little described phenotypes (NPH and PCA). In the characterization of the sample, we observed a great prevalence and importance of cognitive, affective and behavioural presentations. / Introdução: A Degeneração Corticobasal (DCB), é uma doença rara e heterogênea em suas apresentações, representando um grande desafio diagnóstico. Não há descrições de grandes amostras de DCB no Brasil, e poucas no mundo. Seus aspectos cognitivos e comportamentais têm recebido pouca (apesar de crescente) atenção. Objetivos: Descrever aspectos sociodemográficos, clínicos, neuropsiquiátricos, neuropatológicos e de neuroimagem em uma grande série de casos brasileiros com DCB. Realizar uma revisão da literatura sobre a DCB, com especial enfoque em aspectos cognitivos e comportamentais. Metodologia: Foram colhidos dados de 70 pacientes com DCB de acordo com critérios de Cambridge modificados por Bak e Hodges (2011). Os pacientes foram submetidos à avaliação retrospectiva clínica e neuropsiquiátrica, de neuroimagem estrutural e funcional e seis pacientes ao exame neuropatológico. Foram estudadas variáveis sociodemográficas, clínicas, neuropsiquiátricas, neuroanatômicas, e antecedentes familiares. Resultados e Discussão: A idade média de início dos sintomas foi de 62,8 anos (dp=9,5), sendo os dois sexos igualmente afetados (52,9% masculino). Assimetria hemisférica esteve presente em 97% dos casos e o hemisfério esquerdo foi o mais acometido (68,2% dos casos). A apresentação inicial mais freqüente foi a “psiquiátrica” (com alterações do comportamento e/ou humor), presente em 68,1% dos casos, seguida da apresentação motora-extrapiramidal (54,3%). No decorrer da doença, a forma clínica (fenótipo) predominante foi a motora-extrapiramidal, com 61,4% dos casos, seguida da “psiquiátrica”, com 51,4%. Houve cinco casos com apresentação de Atrofia Cortical Posterior (ACP). Em 37,7% não ocorreram achados clássicos da DCB (por exemplo, síndrome da mão estrangeira). Encontramos 18,7% de casos com DCB familiar, com quatro famílias apresentando um fenótipo ainda não descrito, de DCB com HPN (Hidrocefalia de Pressão Normal). Observamos também prejuízos cognitivos e funcionais nas escalas avaliadas (MEEM, Pfeffer e CDR), e comorbidades clínicas e psiquiátricas frequentes, em especial Diabetes Mellitus (23,5%), hipotireoidismo (22,1%) e Transtornos do Espectro Bipolar (46,4%), os dois últimos com frequência na amostra significativamente maior que na população. Houve seis casos de DCB rapidamente progressiva e seis casos confirmados por necropsia na amostra. Conclusão: Observamos, em consenso com a literatura, uma grande variedade de apresentações da DCB, inclusive com formas novas e pouco descritas (HPN e ACP). Na caracterização da amostra, percebemos grande frequência e importância das apresentações comportamentais, afetivas e cognitivas.

L'objectiu principal d'aquesta tesi és el desenvolupament d'un nou mètode de segmentació totalment automàtic capaç de mesurar amb precisió el volum cerebral en imatges de pacients d'EM amb lesions. El mètode que hem proposat s'ha desenvolupat i implementat integrant no només la informació provinent de la intensitat dels vòxels, sinó a través de la incorporació d'atles morfològics i estructurals que guien la segmentació del teixit. Els vòxels candidats de ser lesions són estimats i processats abans de la segmentació del teixit utilitzant un algoritme de post-processat basat en la informació del context local i la informació anatòmica i morfològica prèvia. Aquest mètode de segmentació ha estat avaluat de forma quantitativa i qualitativa utilitzant diferents conjunts d'imatges que contenen lesions de substància blanca. Els resultats mostren que la precisió del mètode proposat és consistent i molt competitiva en tot tipus d'imatges en comparació amb altres tècniques proposades. En aquest sentit, els percentatges d'error obtinguts en els diferents experiments duts a terme mostren que el mètode proposat millora la segmentació del teixit cerebral de les imatges amb lesions / The main goal of this thesis is to develop a novel, fully automated brain tissue segmentation method capable of computing accurate measurements of tissue volume from images of MS patients with lesions. The proposed tissue segmentation method has been designed and implemented using a combination of intensity along with anatomical and morphological prior maps to guide the tissue segmentation. WM outliers have been estimated and filled with signal intensities similar to those of the WM before segmentation using a multi-channel post-processing rule-based algorithm with spatial context, and prior anatomical and morphological atlases. The proposed method has been quantitatively and qualitatively evaluated using different databases of images containing WM lesions, yielding competitive and consistent results in both general and MS specific databases. The percentages of errors obtained in the different experiments carried out show that the proposed algorithm effectively improves automated brain tissue segmentation in images containing lesions.

Los movimientos voluntarios del cuerpo son controlados por el sistema nervioso central y periférico a través de la contracción de los músculos esqueléticos. La contracción se inicia al liberarse un neurotransmisor sobre la unión neuromuscular, iniciando la propagación de un biopotencial sobre la membrana de las fibras musculares que se desplaza hacia los tendones: el Potencial de Acción de la Unidad Motora (MUAP). La señal electromiográfica de superficie registra la activación continua de dichos potenciales sobre la superficie de la piel y constituye una valiosa herramienta para la investigación, diagnóstico y seguimiento clínico de trastornos musculares, así como para la identificación de la intención movimiento tanto en términos de dirección como de potencia. En el estudio de las enfermedades del sistema neuromuscular es necesario analizar el nivel de actividad, la capacidad de producción de fuerza, la activación muscular conjunta y la predisposición a la fatiga muscular, todos ellos asociados con factores fisiológicos que determinan la resultante contracción mioeléctrica. Además, el uso de matrices de electrodos facilita la investigación de las propiedades periféricas de las unidades motoras activas, las características anatómicas del músculo y los cambios espaciales en su activación, ocasionados por el tipo de tarea motora o la potencia de la misma. El objetivo principal de esta tesis es el diseño e implementación de protocolos experimentales y algoritmos de procesado para extraer información fiable de señales sEMG multicanal en 1 y 2 dimensiones del espacio. Dicha información ha sido interpretada y relacionada con dos patologías específicas de la extremidad superior: Epicondilitis Lateral y Lesión de Esfuerzo Repetitivo. También fue utilizada para identificar la dirección de movimiento y la fuerza asociada a la contracción muscular, cuyos patrones podrían ser de utilidad en aplicaciones donde la señal electromiográfica se utilice para controlar interfaces hombre-máquina como es el caso de terapia física basada en robots, entornos virtuales de rehabilitación o realimentación de la actividad muscular. En resumen, las aportaciones más relevantes de esta tesis son: * La definición de protocolos experimentales orientados al registro de señales sEMG en una región óptima del músculo. * Definición de índices asociados a la co-activación de diferentes músculos * Identificación de señales artefactuadas en registros multicanal * Selección de los canales mas relevantes para el análisis  Extracción de un conjunto de características que permita una alta exactitud en la identificación de tareas motoras Los protocolos experimentales y los índices propuestos permitieron establecer que diversos desequilibrios entre músculos extrínsecos del antebrazo podrían desempeñar un papel clave en la fisiopatología de la epicondilitis lateral. Los resultados fueron consistentes en diferentes ejercicios y pueden definir un marco de evaluación para el seguimiento y evaluación de pacientes en programas de rehabilitación motora. Por otra parte, se encontró que las características asociadas con la distribución espacial de los MUAPs mejoran la exactitud en la identificación de la intención de movimiento. Lo que es más, las características extraídas de registros sEMG de alta densidad son más robustas que las extraídas de señales bipolares simples, no sólo por la redundancia de contacto implicada en HD-EMG, sino también porque permite monitorizar las regiones del músculo donde la amplitud de la señal es máxima y que varían con el tipo de ejercicio, permitiendo así una mejor estimación de la activación muscular mediante el análisis de los canales mas relevantes. / Voluntary movements are achieved by the contraction of skeletal muscles controlled by the Central and Peripheral Nervous system. The contraction is initiated by the release of a neurotransmitter that promotes a reaction in the walls of the muscular fiber, producing a biopotential known as Motor Unit Action Potential (MUAP) that travels from the neuromuscular junction to the tendons. The surface electromyographic signal records the continuous activation of such potentials over the surface of the skin and constitutes a valuable tool for the diagnosis, monitoring and clinical research of muscular disorders as well as to infer motion intention not only regarding the direction of the movement but also its power. In the study of diseases of the neuromuscular system it is necessary to analyze the level of activity, the capacity of production of strength, the load-sharing between muscles and the probably predisposition to muscular fatigue, all of them associated with physiological factors determining the resultant muscular contraction. Moreover, the use of electrode arrays facilitate the investigation of the peripheral properties of the active Motor Units, the anatomical characteristics of the muscle and the spatial changes induced in their activation of as product of type of movement or power of the contraction.The main objective of this thesis was the design and implementation of experimental protocols, and algorithms to extract information from multichannel sEMG signals in 1 and 2 dimensions of the space. Such information was interpreted and related to pathological events associated to two upper-limb conditions: Lateral Epicondylitis and Repetitive Strain Injury. It was also used to identify the direction of movement and contraction strength which could be useful in applications concerning the use of biofeedback from EMG like in robotic- aided therapies and computer-based rehabilitation training.In summary, the most relevant contributions are:§The definition of experimental protocols intended to find optimal regions for the recording of sEMG signals. §The definition of indices associated to the co- activation of different muscles. §The detection of low-quality signals in multichannel sEMG recordings.§ The selection of the most relevant EMG channels for the analysis§The extraction of a set of features that led to high classification accuracy in the identification of tasks.The experimental protocols and the proposed indices allowed establishing that imbalances between extrinsic muscles of the forearm could play a key role in the pathophysiology of lateral epicondylalgia. Results were consistent in different types of motor task and may define an assessment framework for the monitoring and evaluation of patients during rehabilitation programs.On the other hand, it was found that features associated with the spatial distribution of the MUAPs improve the accuracy of the identification of motion intention. What is more, features extracted from high density EMG recordings are more robust not only because it implies contact redundancy but also because it allows the tracking of (task changing) skin surface areas where EMG amplitude is maximal and a better estimation of muscle activity by the proper selection of the most significant channels.

O objetivo deste estudo foi verificar o comportamento anatomopatológico e expressão imunohistoquímica das proteínas GFAP, Vimentina, COX-2 e Amilóide β em 14 casos de encefalopatias não infecciosas em cães, mais especificamente Meningoencefalite Granulomatosa (MEG), Meningoencefalite Necrotizante (MEN) e Angiopatia Amilóide Cerebral (CAA). Foram coletadas informações clínicas (gênero, raça, idade) e morfológicas (necrose, presença de proteína amilóide, infiltrado inflamatório) dos animais. Para a expressão das proteínas por imunohistoquímica foram confeccionadas lâminas próprias para tal com amostras dos tecidos, juntamente com controles positivos das reações. A avaliação da expressão das proteínas foi de como positivo ou negativo para a marcação para GFAP, Vimentina e COX-2, e para o Aβ seguiu-se a classificação proposta por Olichney (1995), com quatro graduações. Animais SRD (4/14, 28%), de raça Maltês (2/14, 14%), Labrador (2/14, 14%), Poodle (3/14, 21%), Fox Terrier (1/14, 7%), Pug (1/14, 7%) e Bichon Frisè (1/14, 7%) fizeram parte deste estudo, na maioria machos (9/14, 63%). As lesões em todos os casos foram características, sendo que em MEG foi observado manguitos perivasculares abundantes com infiltrado inflamatório disperso pelo parênquima encefálico, caracterizando os quatro casos como de MEG disseminada. Em MEN foi observado o infiltrado inflamatório acompanhado de áreas de necrose, sinais característicos da doença. Nos casos de CAA foi observado agregados proteicos junto aos vasos sanguíneos do encéfalo, confirmados como sendo material amilóide por meio da coloração Vermelho Congo. Quanto a expressão dos antígenos por imunohistoquímica, houve a marcação de todos anticorpos em todos os casos, e, para os casos de CAA, houve uma maioria de casos com classificação 1 (5/6, 83%). Em conclusão: 1. os dados clínicos obtidos reproduzem o comportamento biológico destas doenças em cães; 2. a análise imunohistoquímica das lesões de MEG, MEN e CAA apresentou resultados dentro dos quadros descritos na literatura; 3. a marcação positiva para COX-2 indica um aumento na atividade macrofágica no encéfalo; 4. as marcações de GFAP e Vimentina indicam uma reação das células da glia frente a lesão apresentada; 5. a utilização da imunohistoquímica como ferramenta de diagnóstico para estas doenças é válida. / The goal of this study was to analyze the anatomopathological and immunohistochemical profile for GFAP, Vimentin, COX-2 and β-amyloid in 14 cases of noninfectious encephalopathies in dogs, more specifically Granulomatous Meningoencephalitis (GME), Necrotizing Meningoencephalitis (NME) and Cerebral Amyloid Angiopathy (CAA). Clinical (gender, breed, age) and morphological (necrosis, presence of amyloid protein, inflammatory infiltrate) informations were collected regarding such animals. Microscopy slides with paraffin-embedded sections of tissue were elaborated for the immunohistochemical analysis, as well as positive controls for the reactions. The expressions of the proteins were graded as positive or negative for GFAP, Vimentin and COX-2, and the grading system described by Olichney was used for the β-amyloid. Animals of mixed breed (4/14, 28%), malteses (2/14, 14%), Labrador (2/14, 14%), Poodle (3/14, 21%), Fox Terrier (1/14, 7%), Pug (1/14, 7%) and Bichon Frisè (1/14, 7%) featured in this study, and most of them were males (9/14, 63%). All of the lesions observed were characteristic of such diseases, as perivascular cuffing with an abundant inflammatory infiltrate was observed in GME cases, being all 4 cases considered as disseminated GME. Necrosis, as well as the inflammatory infiltrate, was observed in all NME cases, which is the hallmark of this disease. The deposition of amyloid protein was observed in cases of CAA, which were confirmed by the Congo Red special staining. As for the immunohistochemical expression, all antibodies performed positive staining, and for the β-amyloid there was a predominance of grade 1 cases (5/6, 83%). In conclusion: 1. the clinical data reproduce the biological behavior of such diseases in dogs; 2. the immunohistochemical analysis of the lesions from GME, NME and CAA presented results coherent with those found in the literature; 3. positive staining for COX-2 presented an increased macrophage activity in the brain of those animals; 4. GFAP and Vimentin positive staining indicate a reaction by the glial cells against the presented lesion; 5. immunohistochemistry is a valid diagnostics tool for such diseases.

O objetivo deste estudo foi verificar o comportamento anatomopatológico e expressão imunohistoquímica das proteínas GFAP, Vimentina, COX-2 e Amilóide β em 14 casos de encefalopatias não infecciosas em cães, mais especificamente Meningoencefalite Granulomatosa (MEG), Meningoencefalite Necrotizante (MEN) e Angiopatia Amilóide Cerebral (CAA). Foram coletadas informações clínicas (gênero, raça, idade) e morfológicas (necrose, presença de proteína amilóide, infiltrado inflamatório) dos animais. Para a expressão das proteínas por imunohistoquímica foram confeccionadas lâminas próprias para tal com amostras dos tecidos, juntamente com controles positivos das reações. A avaliação da expressão das proteínas foi de como positivo ou negativo para a marcação para GFAP, Vimentina e COX-2, e para o Aβ seguiu-se a classificação proposta por Olichney (1995), com quatro graduações. Animais SRD (4/14, 28%), de raça Maltês (2/14, 14%), Labrador (2/14, 14%), Poodle (3/14, 21%), Fox Terrier (1/14, 7%), Pug (1/14, 7%) e Bichon Frisè (1/14, 7%) fizeram parte deste estudo, na maioria machos (9/14, 63%). As lesões em todos os casos foram características, sendo que em MEG foi observado manguitos perivasculares abundantes com infiltrado inflamatório disperso pelo parênquima encefálico, caracterizando os quatro casos como de MEG disseminada. Em MEN foi observado o infiltrado inflamatório acompanhado de áreas de necrose, sinais característicos da doença. Nos casos de CAA foi observado agregados proteicos junto aos vasos sanguíneos do encéfalo, confirmados como sendo material amilóide por meio da coloração Vermelho Congo. Quanto a expressão dos antígenos por imunohistoquímica, houve a marcação de todos anticorpos em todos os casos, e, para os casos de CAA, houve uma maioria de casos com classificação 1 (5/6, 83%). Em conclusão: 1. os dados clínicos obtidos reproduzem o comportamento biológico destas doenças em cães; 2. a análise imunohistoquímica das lesões de MEG, MEN e CAA apresentou resultados dentro dos quadros descritos na literatura; 3. a marcação positiva para COX-2 indica um aumento na atividade macrofágica no encéfalo; 4. as marcações de GFAP e Vimentina indicam uma reação das células da glia frente a lesão apresentada; 5. a utilização da imunohistoquímica como ferramenta de diagnóstico para estas doenças é válida. / The goal of this study was to analyze the anatomopathological and immunohistochemical profile for GFAP, Vimentin, COX-2 and β-amyloid in 14 cases of noninfectious encephalopathies in dogs, more specifically Granulomatous Meningoencephalitis (GME), Necrotizing Meningoencephalitis (NME) and Cerebral Amyloid Angiopathy (CAA). Clinical (gender, breed, age) and morphological (necrosis, presence of amyloid protein, inflammatory infiltrate) informations were collected regarding such animals. Microscopy slides with paraffin-embedded sections of tissue were elaborated for the immunohistochemical analysis, as well as positive controls for the reactions. The expressions of the proteins were graded as positive or negative for GFAP, Vimentin and COX-2, and the grading system described by Olichney was used for the β-amyloid. Animals of mixed breed (4/14, 28%), malteses (2/14, 14%), Labrador (2/14, 14%), Poodle (3/14, 21%), Fox Terrier (1/14, 7%), Pug (1/14, 7%) and Bichon Frisè (1/14, 7%) featured in this study, and most of them were males (9/14, 63%). All of the lesions observed were characteristic of such diseases, as perivascular cuffing with an abundant inflammatory infiltrate was observed in GME cases, being all 4 cases considered as disseminated GME. Necrosis, as well as the inflammatory infiltrate, was observed in all NME cases, which is the hallmark of this disease. The deposition of amyloid protein was observed in cases of CAA, which were confirmed by the Congo Red special staining. As for the immunohistochemical expression, all antibodies performed positive staining, and for the β-amyloid there was a predominance of grade 1 cases (5/6, 83%). In conclusion: 1. the clinical data reproduce the biological behavior of such diseases in dogs; 2. the immunohistochemical analysis of the lesions from GME, NME and CAA presented results coherent with those found in the literature; 3. positive staining for COX-2 presented an increased macrophage activity in the brain of those animals; 4. GFAP and Vimentin positive staining indicate a reaction by the glial cells against the presented lesion; 5. immunohistochemistry is a valid diagnostics tool for such diseases.

La presente tesis examina el proceso de adaptación psicológica en padres de niños con discapacidades congénitas. Para investigar el proceso de adaptación al estrés crónico de los padres se utilizó el Modelo de Personalidad de Millon (1990, 1994, 2004) y el modelo de afrontamiento al estrés de Folkman y Lazarus (1985). El estudio incluye 166 adultos, padres de niños con espina bífida severa o Mielomeningocele, Parálisis Cerebral y Fisura Labio-Velo-Palatina. Los resultados identifican estilos de personalidad, indicadores de la forma en que los padres responden y hacen frente a la discapacidad de un hijo, y sugieren un impacto diferencial en hombres y mujeres. Se discuten cuestiones derivadas de los condicionamientos género, así como la especial repercusión en la mujer. Finalmente, se sugiere un modelo de intervención interdisciplinar para una mejor adaptación de padres y madres a esta difícil experiencia vital. / This thesis analyzes the psychological adaptation of parents with children having congenital disabilities. Millon’s Personality Model (1990, 1994, 2004) and Lazarus and Folkman ‘s stress coping model , were used to investigate and analyze the process of adaptation of parents to chronic stress. The study included 166 adults, who were parents of children with severe spina bífida or Mielomeningocele, cerebral palsy, and cleft lip and cleft palate. The identified personality styles may serve as indicators of the way parents respond to and cope with a child's disability. The results also suggest that a child’s disability has a differential impact on men and women. Gender issues are evaluated, as well as any repercussions these may have on women. Finally, a model of interdisciplinary professional intervention for a better adaptation of fathers and mothers to this difficult life experience is suggested.

Stroke and traumatic brain injuries are common causes of disability, with loss of nerve tissue due to secondary degeneration, gliosis, and often the formation of cavities that inhibit neural cell growth. Recent attempts at neural cell regeneration have therefore focused on the use of engineering materials that mimic the adult neural stem cell (NSC) niche, in order to establish an adequate environment for neurogenesis and differentiation. However, because the adult mammalian NSC niche has limited regenerative capacities, effective regeneration of the central nervous system (CNS) requires the reconstitution of its embryonic counterpart. Radial glia are bipolar cells with 1-2 µm-thick shafts that form a palisade and span the entire CNS parenchyma serving as substrates for neuronal migration. They contain high levels of glycogen and release L-lactate. Cerebral energy metabolism is a highly compartmentalized and complex process. The adult brain normally uses glucose as its primary energy source. However, before and immediately after birth, lactate is also an important energy source because at this time the level of glucose is low. Over the past decade, a role for lactate in fuelling the energetic requirements of neurons has emerged, not only during the perinatal period but also in adulthood. Initial evidence suggests that the metabolisms of NSC, neurons and astrocytes differ and that energy-dependent processes may influence the balance between NSC self-renewal and differentiation. The main goal of this thesis was to design an implantable biomaterial scaffold that reproduces the organization and supportive function of embryonic radial glia. Here we tested two types of poly L/DL lactic acid (PLA95/5 and PLA70/30), a biodegradable material permissive for neural cell adhesion and growth, as materials for nerve regeneration. PLA95/5 films were highly crystalline, stiff (GPa), and did not degrade significantly in the period analyzed in culture. In contrast, PLA70/30 films were more amorphous, softer (MPa) and degraded faster, releasing significant amounts of lactate into the medium. PLA70/30 performed better than PLA95/5 for primary cortical neural cell adhesion, proliferation and differentiation, maintaining the pools of neuronal and glial progenitor cells in vitro. Finally, for in vivo studies, we designed 3D cell-free biomimetic scaffolds consisting of electrospun PLA70/30 nanofibers. Radially aligned scaffolds released L-lactate and reproduced the 3D organization and supportive function of radial glia. These scaffolds implanted into cavities made in mouse brain fostered complete implant vascularization, sustained neurogenesis, and allowed the long-term survival and integration of the newly generated neurons. Our results suggest that PLA70/30 scaffolds mimic some of the physical and biochemical characteristics of the NSC niche. Overall, our results show that the endogenous CNS is capable of regeneration through the in vivo dedifferentiation induced by biophysical and metabolic cues, with no need for exogenous cells, growth factors, or genetic manipulation. / Las lesiones cerebrales son causas comunes de discapacidad que conllevan pérdida de tejido nervioso debido a la degeneración secundaria, la gliosis, y con frecuencia la formación de cavidades que inhiben el crecimiento neuronal. Recientemente, las terapias neuroregenerativas se han centrado en el uso de la ingeniería de materiales. Durante el desarrollo del SNC, las células de glia radial son las principales CMN que generan neuronas y glía y son retenidas en el cerebro adulto de especies que regeneran. Durante la neurogénesis temprana, los vasos sanguíneos invaden el SNC e interactúan con las CMN, dando lugar al nicho neurovascular. En el cerebro adulto, la glia radial puede ser recuperada al menos en cierta medida después de una lesión, lo que indica un intento endógeno en la reconstitución del nicho embrionario. Las células de glía radial son bipolares con 1-2 micras de espesor que forman una empalizada que abarca todo el parénquima del SNC sirviendo como sustrato para la migración neuronal. Esta glía radial contiene altos niveles de glucógeno liberando al exterior L-lactato. El metabolismo energético cerebral es un proceso altamente compartimentado y complejo. El cerebro adulto normalmente usa glucosa como fuente de energía primaria. Sin embargo, antes e inmediatamente después del nacimiento, el lactato es también una fuente de energía importante, debido a que los niveles de glucosa son bajos. En la última década, se ha visto que el lactato juega un papel importante como factor energético para las neuronas, no sólo durante el período perinatal sino también en la edad adulta. Nuevas evidencias sugieren que el metabolismo de las CMN, neuronas y astrocitos son diferentes y que los procesos dependientes de energía pueden influir en el equilibrio entre la auto-renovación y diferenciación de las CMN. El objetivo principal de esta tesis ha sido diseñar un andamio implantable que reprodujera la organización y función de soporte de la glía radial embrionaria. Para ello, hemos probado dos tipos de ácido poli L/DL láctico (PLA95/5 y PLA70/30), material biodegradable y permisivo para la adhesión y el crecimiento neuronal. Las películas de PLA95/5 eran cristalinas, rígidas (GPa), y no se degradaban significativamente en el período analizado durante el cultivo in vitro. Sin embargo, las películas de PLA70/30 eran más amorfas, menos rígidas (MPa) y se degradaban más rápido, liberando cantidades significativas de lactato en el medio. A diferencia del PLA95/5, en el 70/30 había una mejor adhesión neuronal, así como la proliferación y el mantenimiento de los progenitores neuronales y gliales in vitro. Finalmente, para los estudios in vivo, diseñamos andamios biomiméticos en 3D libres de células que consistían en nanofibras de PLA70/30. Los andamios radialmente alineados reproducían la organización 3D y la función de apoyo de la glía radial. Estos andamios implantados en la corteza cerebral del ratón ayudaron a la completa vascularización del implante, permitiendo la supervivencia y la integración a largo plazo de las neuronas recién generadas. Nuestros resultados sugieren que los andamios de PLA70/30 imitan algunas de las características físicas y bioquímicas del nicho neurovascular. En conclusión, nuestros resultados muestran que el SNC es capaz de regenerar de manera endógena a través de la des-diferenciación in vivo inducida por las señales biofísicas y metabólicas, sin necesidad de células exógenas, factores de crecimiento o manipulación genética

The type of serotonin receptor 1A (5-HT1A), the galanin type 1 (GalR1) and the orphan receptor 39 (GPR39) belong to the superfamily The serotonin receptor type 1A (5-HT1A), the galanin receptor type 1 (GalR1) and the orphan receptor 39 (GPR39) belong to the superfamily of G-protein-coupled receptors. All three GPCRs share, among other features, a relationship with the pathophysiology of unipolar depression associated with presence of zinc. The current paradigm with respect to these functional unities is that these receptors are not acting as monomeric forms but through complexs involving specific interactions with themselves or other receptors from the same family. This allows them to increase their functional possibilities exponentially, allowing versatility from a fixed number of receptors, and consequently also increases the number of pharmacological approachmes. The heterodimerization between 5-HT1A receptor and GalR1 has been previously described as an antagonistic interaction that could lead to unipolar depression. In this thesis we have shown that this interaction is avoided in the presence of zinc, giving a rational explanation for the antidepressant effect widely described for this cation. Although there was no published evidence regarding the interaction of these two receptors with GPR39, a receptor that is activated by zinc and whose expression depends on the concentration of this cation, in this thesis we have demonstrated the ability to interact of these three receptors in both forms GPR39-5-HT1A, 5-HT1A-GalR1 and the trimer GPR39-5-HT1A-GalR1. It has also been found that the functional capacity of the receptors is modified according the type of interaction in which these receptors are involved. So that monomeric and oligomeric forms have different signalling capacities. This would suggest that in the human brain all putative receptor configurations could be present, and that the presence of one or other would be regulated by zinc concentration. Deepening in the detailed molecular mechanism of the effect of zinc on these interactions should allow the development of new drugs for a disease with high prevalence in the nowadays society. / El receptor de serotonina del tipus 1A (5-HT1A), el de galanina de tipus 1 (GalR1) i el receptor orfe 39 (GPR39) pertanyen a la superfamília dels receptors acoblats a proteïna G. Tots tres receptors comparteixen, entre d'altres característiques, una relació amb la fisiopatologia de la depressió unipolar associada amb la presència de zinc. El paradigma actual, respecte a la unitat funcional d'aquests receptors, és que no actuen en forma monomèrica sinó mitjançant complexos que involucren interaccions específiques amb ells mateixos o amb altres receptors de la mateixa família. Això els hi permet augmentar les seves possibilitats funcionals exponencialment, permetent una gran versatilitat a partir d'un nombre fix de receptors, i en conseqüència també augmenta el nombre de possibilitats d'abordatge farmacològic. L'heterodimerització entre el receptor 5-HT1A i el GalR1 s'ha descrit prèviament com una interacció antagònica que podria donar lloc a la depressió unipolar. En aquesta tesi s'ha demostrat que aquesta interacció s'evita en presència de zinc, donant una explicació racional a l'efecte antidepressiu àmpliament descrit per a aquest catió. Tot i que no hi havia cap evidència publicada respecte a la interacció d'aquests dos receptors amb el GPR39, un receptor que és activat pel zinc i l'expressió del qual depèn de la concentració del mateix, en aquesta tesi s'ha demostrat la capacitat d'interacció d'aquests tres receptors, tant en les formes GPR39-5-HT1A i 5-HT1A-GalR1 com en el trímer GPR39-5-HT1A-GalR1. A més, s'ha trobat que la capacitat funcional dels receptors es veu modificada segons el tipus d'interacció en la que aquests receptors participen, de manera que les formes monomèriques i oligomèriques presenten una capacitat de senyalització diferent. Això faria pensar que en el cervell humà podrien trobar-se totes les configuracions potencials de receptors, i que la presència d'unes o altres estaria regulada per la concentració de zinc. L'aprofundiment en el mecanisme molecular detallat de l'efecte del zinc en aquestes interaccions hauria de permetre el desenvolupament de nous fàrmacs per a una malaltia d'alta prevalença en la població mundial.