Las enfermedades neurodegenerativas cursan con la degeneración y muerte de las neuronas las cuales son incapaces de suplir su propia muerte. Debido a esta característica enfermedades como el Alzheimer, el Parkinson o el Glaucoma no tienen cura en la actualidad. La retina es una proyección del sistema nervioso central (SNC) encapsulada en el globo ocular y aislada del resto del SNC, lo que la hace fácilmente accesible a la manipulación experimental. En este trabajo estudiamos las alteraciones funcionales de la retina con el electrorretinograma de campo completo (ERG), técnica basada en el registro de la respuesta eléctrica retiniana tras la presentación de un estímulo de luz homogéneo. Así hemos estudiado los efectos de la sección del nervio, del aumento de presión intraocular y de la fototoxicidad, dichas lesiones afectan selectivamente a determinadas poblaciones neuronales retinianas e imitan enfermedades neurodegenerativas como el Glaucoma o la Degeneración Macular Asociada a la Edad. / Neurodegenerative diseases are characterized by degeneration and death of neurons which are unable to recover after a given insult, thus impairing functional recuperation. Because of this, there is no cure for diseases such as Alzheimer, Parkinson or Glaucoma. The retina is a projection of the central nervous system (CNS) located in the eye and therefore, isolated from the rest of the CNS. This makes the retina a very good model for experimental manipulation. In this work we have studied the functional changes in the retina by full field electroretinograme (ERG). This technique records the electric response of the retina after presentation of homogeneous light stimuli. We have studied the effects that optic nerve sections, increase of the intraocular pressure and phototoxicity have on ERG recordings. These lesions impair selectively certain retinal neuronal populations and imitate neurodegenerative diseases as Glaucoma or Age-related Macular Degeneration.

Fond : Le substrat de fibrillation auriculaire (FA) vagale et celui secondaire à remodelage par tachycardie auriculaire (RTA) partagent beaucoup des caractéristiques : période réfractaire efficace (PRE) réduite, hétérogénéité accrue de PRE et quelques mécanismes moléculaires communs. Cette étude a comparé les 2 substrats à une abréviation comparable de PRE. Méthodes : Chez chacun de 6 chiens de groupe de stimulation vagal (SV), les paramètres de stimulation cervicale bilatérale de nerves vagaux ont été ajustés pour produire la même PRE moyenne (calculé à 8 sites des oreillettes gauche et droite) avec 6 chiens de groupe de RTA assorti à sexe et poids. Des paramètres électrophysiologiques, la durée moyenne de la fibrillation auriculaire (DAF) et les fréquences dominantes (FD) locales ont étés calculés. Résultats : En dépit des PREs assorties (SV: 80±12msec contre RTA: 79±12msec) la DAF était plus longue (*), l’hétérogénéité de conduction était plus élevée (*), la FD était plus rapide (*) et la variabilité de FD plus grande (*) chez les chiens SV. Les zones de maximum FD qui reflètent les zones d’origine de FA étaient à côté de ganglions autonomes chez les chiens SV. Conclusions : Pour un PRE atriale comparable, la FA secondaire à SV est plus rapide et plus persistante que la FA avec un substrat de RTA. Ces résultats sont consistants avec des modèles de travail suggérant que l'hyperpolarisation SV-induite contribue de façon important à la stabilisation et à l'accélération des rotors qui maintiennent la FA. La similitude de la distribution de FD du groupe vagal avec la distribution des lésions d’ablation après cartographie des électrogrammes atriales fragmentés suggère des nouvelles techniques d’ablation. La distribution des FD entre le SV et le RTA fournit de nouvelles idées au sujet de possible rémodelage neuroreceptorial et indique des différences importantes entre ces substrats de FA superficiellement semblables. / Background: Vagal nerve stimulation (VS) and atrial tachycardia remodeled (ATR) atrial fibrillation (AF) substrates share many features: reduced effective refractory period (ERP), increased ERP heterogeneity and some common molecular mechanisms. This study compared VS and ATR substrates at comparable ERP abbreviation. Methods: In each of 6 VS dogs, bilateral cervical VS parameters were adjusted to produce the same mean ERP as a sex and weight matched ATR dog. Electrophysiological parameters, mean duration of AF (DAF) and local dominant frequencies (DF) were determined (before (CTL) and after VS in VS dogs). Results: Despite matched ERPs (VG: 80±12msec vs ATR: 79±12msec) DAF was greater (*), conduction heterogeneity was greater (*), DF was faster (*) and DF variability greater (*) in VS dogs. AF drivers reflected by maximum DF zones were adjacent to autonomic ganglia in VS dogs; there was a tendency (p<0.07) to faster driver zones in the left atrium comparing to the right in ATR dogs. Conclusions: For a comparable atrial ERP, VS AF is faster and more persistent than AF with an ATR substrate. These results are consistent with modeling work suggesting that VS-induced hyperpolarization is an important contributor to AF-maintaining rotor stabilization and acceleration. Similarities in DF distribution in VS dogs with distribution of ablation lesions performed after Complex Fractionated Atrial Electrograms mapping suggests new curative ablation methods. DF distribution differences between VS and ATR provides new ideas about possible neuroreceptorial remodeling and indicates important differences between these superficially similar AF substrates.

Le glaucome est la deuxième cause de cécité irréversible dans le monde. La perte de vision qui se produit lors du glaucome s’explique par une dégénérescence du nerf optique et une mort progressive et sélective des cellules ganglionnaires de la rétine (CRG). L'hypertension oculaire est un facteur de risque majeur dans le glaucome, mais des défauts du champ visuel continuent à se développer chez un contingent de patients malgré l'administration de médicaments qui abaissent la pression intraoculaire (PIO). Par conséquent, bien que la PIO représente le seul facteur de risque modifiable dans le développement du glaucome, son contrôle ne suffit pas à protéger les CRGs et préserver la fonction visuelle chez de nombreux patients. Dans ce contexte, j'ai avancé l'hypothèse centrale voulant que les stratégies de traitement du glaucome visant à promouvoir la protection structurale et fonctionnelle des CRGs doivent agir sur les mécanismes moléculaires qui conduisent à la mort des ces neurones. Dans la première partie de ma thèse, j'ai caractérisé l'effet neuroprotecteur de la galantamine, un inhibiteur de l'acétylcholinestérase qui est utilisé cliniquement dans le traitement de la maladie d'Alzheimer. Cette étude s’est basée sur l'hypothèse que la galantamine, en modulant l'activité du récepteur de l'acétylcholine, puisse améliorer la survie des CRGs lors du glaucome. Nous avons utilisé un modèle expérimental bien caractérisé d'hypertension oculaire induite par l’administration d'une solution saline hypertonique dans une veine épisclérale de rats Brown Norway. Les résultats de cette étude (Almasieh et al. Cell Death and Disease, 2010) ont démontré que l'administration quotidienne de galantamine améliore de manière significative la survie des corps cellulaires et des axones CRGs. La protection structurelle des CRGs s’accompagne d’une préservation remarquable de la fonction visuelle, évaluée par l'enregistrement des potentiels évoqués visuels (PEV) dans le collicule supérieur, la cible principale des CRGs chez le rongeur. Une autre constatation intéressante de cette étude est la perte substantielle de capillaires rétiniens et la réduction du débit sanguin associé à la perte des CRGs dans le glaucome expérimental. Il est très intéressant que la galantamine ait également favorisé la protection de la microvascularisation et amélioré le débit sanguin rétinien des animaux glaucomateux (Almasieh et al. en préparation). J'ai notamment démontré que les neuro-et vasoprotections médiées par la galantamine se produisent par iv l'activation des récepteurs muscariniques de l'acétylcholine. Dans la deuxième partie de ma thèse, j'ai étudié le rôle du stress oxydatif ainsi que l'utilisation de composés réducteurs pour tester l'hypothèse que le blocage d'une augmentation de superoxyde puisse retarder la mort des CRG lors du glaucome expérimental. J'ai profité d'un composé novateur, un antioxydant à base de phosphineborane (PB1), pour tester sur son effet neuroprotecteur et examiner son mécanisme d'action dans le glaucome expérimental. Les données démontrent que l'administration intraoculaire de PB1 entraîne une protection significative des corps cellulaire et axones des CRGs. Les voies moléculaires conduisant à la survie neuronale médiée par PB1 ont été explorées en déterminant la cascade de signalisation apoptotique en cause. Les résultats démontrent que la survie des CRGs médiée par PB1 ne dépend pas d’une inhibition de signalisation de protéines kinases activées par le stress, y compris ASK1, JNK ou p38. Par contre, PB1 induit une augmentation marquée des niveaux rétiniens de BDNF et une activation en aval de la voie de survie des ERK1 / 2 (Almasieh et al. Journal of Neurochemistry, 2011). En conclusion, les résultats présentés dans cette thèse contribuent à une meilleure compréhension des mécanismes pathologiques qui conduisent à la perte de CRGs dans le glaucome et pourraient fournir des pistes pour la conception de nouvelles stratégies neuroprotectrices et vasoprotectrices pour le traitement et la gestion de cette maladie. / Glaucoma is the second cause of irreversible blindness worldwide. Loss of vision in glaucoma is accompanied by progressive optic nerve degeneration and selective loss of retinal ganglion cells (RGCs). Ocular hypertension is a major risk factor in glaucoma, but visual field defects continue to progress in a large group of patients despite the use of drugs that lower intraocular pressure (IOP). Therefore, although IOP is the sole modifiable risk factor in the development of glaucoma, its regulation is not sufficient to protect RGCs and preserve visual function in many affected patients. To address this issue, I put forward the central hypothesis that effective therapeutic strategies for glaucoma must interfere with molecular mechanisms that lead to RGC death to successfully promote structural and functional protection of these neurons. In the first part of my thesis, I characterized the neuroprotective effect of galantamine, an acetylcholinesterase inhibitor that is clinically used for the treatment of Alzheimer’s disease. The specific hypothesis of this study was that galantamine, by modulating acetylcholine receptor activity, can improve the survival of injured RGCs in glaucoma. A well characterized experimental model of ocular hypertension induced by administration of a hypertonic saline into an episcleral vein of Brown Norway rats was used. The results of this study (Almasieh et al. Cell Death and Disease, 2010) demonstrated that daily administration of galantamine significantly improved the survival of RGC soma and axons in this model. Structural protection of RGCs correlated with substantial preservation of visual function, assessed by recording visual evoked potentials (VEPs) from the superior colliculus, the primary target of RGCs in the rodent brain. An interesting finding during the course of my thesis was that there is a substantial loss of retinal capillaries and a reduction in retinal blood that correlates with RGC loss in experimental glaucoma. Interestingly, galantamine also promoted the protection of the microvasculature and improved retinal blood flow in ocular hypertensive animals (Almasieh et al. in preparation). Importantly, I demonstrated that galantamine-mediated neuro- and vasoprotection occur through activation of muscarinic acetylcholine receptors. In the second part of my thesis, I investigated the role of oxidative stress and the use of reducing compounds to test the hypothesis that blockade of a superoxide burst may delay RGC death in experimental glaucoma. I took advantage of a novel phosphinevi borane based antioxidant compound available to us (PB1) to investigate its neuroprotective effect and mechanism of action in experimental glaucoma. The data demonstrate that intraocular administration of PB1 resulted in significant protection of RGC soma and axons. I also explored the molecular pathways leading to PB1-mediated neuronal survival by analyzing the components of survival and apoptotic signaling pathways involved in this response. My results show that PB1-mediated RGC survival did not correlate with inhibition of stress-activated protein kinase signaling, including ASK1, JNK or p38. Instead, PB1 led to a striking increase in retinal BDNF levels and downstream activation of the pro-survival ERK1/2 pathway (Almasieh et al. Journal of Neurochemistry, 2011). In conclusion, the findings presented in this thesis contribute to a better understanding of the pathological mechanisms underlying RGC loss in glaucoma and might provide insights into the design of novel neuroprotective and vasoprotective strategies for the treatment and management of this disease.

O presente texto descreve métodos e apresenta resultados do projeto de pesquisa de mestrado intitulado \"Dois Problemas em Análise de Formas de Estruturas de Ramificação\". Ambos os problemas abordados estão relacionados às sub-áreas da Análise de Formas denominadas Caracterização e Descrição de Formas. O primeiro problema consiste na investigação de um conjunto de características propostas para distingüir, primeiramente, entre estruturas de ramificação de vasos sangüíneos em imagens de retina segmentadas manualmente e automaticamente. A seguir, as mesmas características são aplicadas para discernir entre estruturas de ramificação de vasos sangüíneos em imagens de retina com e sem retinopatia diabética proliferativa (Proliferative Diabetic Retinopathy - PDR). A PDR é uma das patologias associadas à diabetes, que pode culminar na cegueira do indivíduo. Diagnósticos são possíveis por meio de imagens de fundo de olho e, quando efetuados precocemente, viabilizam intervenções oportunas evitando a perda da visão. Neste trabalho, 27 imagens digitais de fundo de olho foram segmentadas por dois processos distintos, isto é, segmentação manual por um especialista e a segmentação automática, mediante a transformada contínua Wavelet - CWT e classificadores estatísticos. Visando à caracterização destas formas, um conjunto de 08 características foi proposto. Este conjunto foi formado por três grupos, a saber: descritores tradicionais geométricos (Área, Perímetro e Circularidade), descritores associados à transformada wavelet ( 2o momento estatístico da distribuição de módulos da CWT, Entropia de Orientação da distribuição de fases da CWT e Curvatura) e um descritor fractal (Dimensão de Correlação - Global e Mediana). Uma Análise Discriminante Linear LDA revelou que as características geométricas tradicionais não detectam o início da retinopatia diabética proliferativa. A maior capacidade discriminante individual foi exibida pela Curvatura, com Área sob a curva ROC de 0.76. Um subconjunto com 6 características apresentou grande capacidade discriminante com Área sob a curva ROC de 0.90. O segundo problema diz respeito à extração de contorno de estruturas de ramificação bidimensionais de neurônios tridimensionais. Este trabalho contribui originalmente com uma solução para este problema, propondo dois algoritmos desenvolvidos para Rastreamento de Ramos e Extração do Contorno Paramétrico de estruturas de ramificação, capazes de transpor regiões críticas formadas por cruzamentos ocasionados pela projeção de estruturas 3D no plano das imagens 2D. Grande parte dos métodos baseados em contorno para análise de formas de estruturas de ramificação de células neuronais não produz representações corretas destas formas, devido à presença de sobreposições entre processos neuronais, levando os algoritmos tradicionais de extração de contorno a ignorar as regiões mais internas destas estruturas, gerando representações incompletas. O sistema proposto neste trabalho foi desenvolvido objetivando a solução do problema de extração de contorno, mesmo na presença de múltiplas sobreposições. Inicialmente, a imagem de entrada é pré-processada, gerando um esqueleto 8-conexo com ramos de um pixel de largura, um conjunto de sementes de sub-árvores dendríticas e um conjunto de regiões críticas (bifurcações e cruzamentos). Para cada sub-árvore, o algoritmo de rastreamento rotula todos os pixels válidos de um ramo, até chegar em uma região crítica, onde o algoritmo decide a direção em que deve continuar o rastreamento. Nosso algoritmo mostrou-se robusto, mesmo quando aplicado a imagens com segmentos paralelos muito próximos. Resultados obtidos com imagens reais (neurônios) são apresentados. / This document describes methods and presents results from the Master of Science\'s research project in computer science entitled \"Two Problems in Shape Analysis of Branching Structures\". Both tackled problems herein are related to Shape Analysis sub-fields, namely Characterization and Description of shapes. The former problem consists of an investigation on a proposed set of features aimed at discriminating, firstly, between blood vessels branching structures manually and automatically segmented. In the sequel, the same features are used to assess their discriminative capability in distinguishing between blood vessels branching structures with and withoud proliferative diabetic retinopathy (PDR). The PDR is a pathology related to diabetes, which may lead to the blindness. Diagnosis is possible through optic fundus image analysis, which may allow timely interventions preventing vision loss. In this work, 27 digital optic fundus images were segmented by two distinct segmentation processes, i.e. manual segmentation carried out by an especialist and automated segmentation, through the CWT (Continuous Wavelet Transform) and statistical classifiers. In order to characterize such a shapes, a set of 8 features has been proposed. The aforementioned set was comprised of three features groups, that is: traditional geometric descriptors (Area, Perimeter and Circularity), wavelet-based descriptors (2nd statistical moment from the CWT Modulus distribution, Orientation Entropy from the CWT Phase distribution and Curvature) and a fractal descriptor (Correlation Dimension - global and median). Linear Discriminant Analysis LDA revelead that the traditional geometric features are not able to detect early proliferative diabetic retinopathy. The largest singular discriminant capability was shown by the Curvature, with area under the ROC curve of 0.76. A subset of 6 features presented a good discriminating power with area under the curve of 0.90. The second problem concerns contour extraction from 2D branching structures of 3D neurons. This work contributes with an original solution for such a problem, proposing two algorithms devised for Branches Tracking and Branching Structures Contour Extraction. The proposed algorithms are able to traverse critical regions implied by the projection of 3D structures onto a 2D image plane. Most of contour-based methods intended to shape analysis of neuronal branching structures fall short of yielding proper shape representations, owing to the presence of overlapings among neuronal processes, causing the traditional algorithms for contour following to ignore the innermost regions, thus generating incomplete representations. The proposed framework system was developed aiming at the solution of the contour extraction problem, even in the presence of multiple overlapings. The input image is pre-processed, so as to obtain an 8-connected skeleton with one-pixel wide branches, a set of seeds of dendritic sub-trees and a set of critical regions (bifurcations, crossings and superpositions). For each sub-tree, the Branches Tracking Algorithm labels all valid pixels of a branch, until reaching a critical region, where the algorithm decides about the direction to go on with the tracking. Our algorithm has shown robustness, even in images plenty of very close parallel segments. Results with real images (neurons) are presented.

Dentre as células ganglionares da retina existe uma pequena população de células que contem melanopsina e respondem diretamente à luz. Estas são as células ganglionares intrinsecamente fotossensíveis (ipRGCs), cujas funções são principalmente não visuais. Dentre as funções não visuais das ipRGCs sua influência na resposta pupilar dependente da luz foi o objeto central desta tese. Tanto a retina interna, através das ipRGCs, quanto a retina externa, através dos bastonetes e cones, fornecem uma informação neural que regula a resposta pupilar à luz (RPL). Este estudo avaliou a integridade das ipRGCs através do RPL em pacientes com glaucoma primário de ângulo aberto (GPAA), leve, moderado e avançado, e em pacientes com síndrome da apnéia obstrutiva do sono (SAOS), moderada e grave. Também foi avaliada a discriminação cromática e a sensibilidade ao contraste espacial de luminância (SC), a perimetria visual e a espessura da retina avaliada por tomografia de coerência óptica (OCT). Foram avaliados 98 participantes: 45 pacientes com GPAA ( 27, 18; idade média = 65,84 + 10,20), 28 pacientes com SAOS ( 14, 14; idade média = 52,93 + 7,13), e 25 controles ( 17, 8; idade média = 54,27 + 8,88). Após o exame oftalmológico foram avaliadas a SC de grades e a discriminação de cores através do Cambridge Colour Test (CCT). A avaliação do RPL foi feita apresentando-se flashes de 470 e 640 nm, de 1s de duração, em 7 luminâncias desde -3 até 2.4 log cd/m2 em um Ganzfeld Q450 SC (Roland Consult). O RPL foi registrado pelo sistema de eye tracker View Point System (Arrington Research Inc.). Os testes foram realizados em ambos os olhos, de forma monocular e no escuro. Para a comparação dos dados entre os grupos, utilizou-se um modelo de equações de estimação generalizada (GEE), para correção da dependência entre os dois olhos. O RPL dos pacientes com GPAA moderado e avançado apresentou redução significativa na amplitude do pico, dependente da severidade do glaucoma, nas diferentes luminâncias tanto para 470 nm quanto para 640 nm, evidenciando redução das contribuições dos cones e bastonetes ao RPL. As contribuições das ipRGCs ao RPL (avaliadas pela amplitude da resposta sustentada entre 6-8 s) foram também significativamente menores em GPAA moderado e avançado. No estado inicial do GPAA as contribuições das ipRGCs para o RPL encontram-se preservadas. No entanto, o GPAA parece afetar o processamento espacial desde o inicio da doença. Nos pacientes com GPAA leve foi observada uma perda acentuada nas faixas baixas de frequência espacial, compatível com prejuízo seletivo das células ganglionares do tipo M. A SC de pacientes com GPAA moderado e avançado mostrou perdas nas faixas baixas e altas de frequência espacial, apontando um prejuízo nas vias parvo- e margnocelulares. Uma perda significativa da discriminação de cores no eixo azul-amarelo foi observada em todos os estágios do GPAA. O RPL nos pacientes com SAOS está parcialmente preservado, não obstante, as respostas da amplitude do pico para o flash de 470 nm diminuem conforme aumenta a severidade da SAOS. As contribuições dos fotorreceptores da retina externa ao RPL, foram significativamente menores em algumas das luminâncias. Não foram observadas diferenças significativas de SC ou discriminação de cores nos pacientes com SAOS. Em conclusão, no estágio moderado e avançado do glaucoma tanto as contribuições das ipRGCs ao RPL quanto as vias M e P, se encontram mais afetadas do que no inicio do GPAA, quando a via parvocelular e as contribuições das ipRGCs ao RPL parecem estar mais preservadas / Among the retina ganglion cells there are a small population of cells containing melanopsin and which respond directly to light. They are the intrinsically photosensitive ganglion cells (ipRGCs), whose functions are mainly non-visual. Among these non-visual functions of the ipRGCs, their influence on the pupillary response as a function of light was the central subject of this thesis. Both the inner retina through the ipRGCs and the outer retina through the rods and cones, provide neural information that regulates the pupillary light response (PLR) to light. This study evaluated the integrity of ipRGCs through PLR in patients with Primary Open Angle Glaucoma (POAG), mild, moderate and advanced, and in patients with Obstructive Sleep Apnea Syndrome (OSAS), moderate and severe. We evaluated also the color discrimination and achromatic spatial contrast sensitivity (CS), visual perimetry and retinal thickness evaluated by Optical Coherence Tomography (OCT). 98 participants were evaluated, 45 patients with POAG ( 27 18; mean age = 65.84 + 10.20), 28 with OSAS ( 14 14; mean age = 52.93 + 7.13) and 25 controls ( 17 8; mean age = 54.27 + 8.88). After the ophthalmological exam it was evaluated the contrast sensitivity and color discrimination measures using the Cambridge Colour Test (CCT). Pupil responses were elicited by Ganzfeld (Q450 SC, Roland Consult) presentation of 1-sec flashes of 470- and 640-nm at 7 luminance from -3 to 2.4 log cd/m2. PLR was measured with the eye tracker system View Point (Arrington Research Inc.). The tests were performed monocularly, on both eyes, in a darkened room. In order to compare data across groups, we used a General Estimating Equations (GEE) to adjust for within subject inter-eye correlations. Patients with moderate and advanced POAG had a significantly decreased PLR that depends on the severity of the glaucoma, for both the 470- and 640-nm stimuli, making evident the reduction of the contributions of the cones and rods to the PLR. The contributions of ipRGCs to PLR (assessed by the amplitude of the sustained response between 6 8 sec) were also significantly lower in patients with moderate and advanced POAG. In the initial and mild stages of POAG the contribution of ipRGCs to the PLR is preserved. However, POAG appears to affect spatial processing from the early stages of the disease. Mild-POAG patients showed a marked loss in the low spatial frequency bands, compatible with selective loss of magnocellular ganglion cells. The CS of patients with moderate and advanced POAG showed losses at both low and high spatial frequencies, suggesting a loss in both parvo- and margnocellular channels. A significant loss of color discrimination along the blue-yellow axis was observed in all stages of POAG. The PLR in patients with OSAS is partially preserved, however the peak amplitude responses for the 470-nm flash decreased with increased severity of OSAS. The contributions of the photoreceptors of the outer retina to the PLR were significantly lower at some of the luminance. Significant differences in CS or color discrimination were not observed in patients with OSAS. In conclusion, in moderate and advanced stages of glaucoma, both the contributions of ipRGCs to PLR as well as the M- and P channels, were found more affected than at the beginning of POAG, in contrast the parvocellular channel and the contributions of ipRGCs on the PLR would be more preserved

Abstract Herpes simplex virus type 1 (HSV-1) is a common pathogen with an age-standardized seroprevalence of 52% in Finland. The most common manifestation of HSV-1 infection is labial herpes, but recently HSV-1 has emerged as the most common cause of primary genital herpes in Finnish women. HSV-1 can also lead to severe conditions such as encephalitis. After the primary lytic HSV-1 infection at the epithelia, the progeny viruses infect the innervating sensory neurons. The neuronal infection may lead to a quiescent infection form, called latency. Periodically, the virus may reactivate, which can lead to recurrent infection at the epithelia. During different phases of the viral life cycle the host cells try to restrict the infection. This study set out to investigate the roles of two HSV-1 proteins, γ134.5 and Us3 during different phases of the HSV-1 life cycle. The aim of the first study was to investigate how the deletion of Us3 affected host responses, especially Toll-like Receptor (TLR) signaling, in monocytic U937 cells. TLR3 expression was increased during Us3 deletion virus infections. This also led to increased activation of IRF-3 and increased expression of type I interferons (IFN) and an interferon stimulated protein. This study shows that TLR3 is involved in controlling the HSV-1 infection and that Us3 regulates IRF-3 activation. The second study focused on the role of the γ134.5 protein in HSV-1 latency. Embryonic mouse dorsal root ganglion (DRG) cultures were used as a cell culture model for HSV-1 latency and reactivation. In this model γ134.5 deletion viruses did not reactivate as efficiently as wild-type viruses, even though they replicated well and established latency in the neurons. Stress granules are part of the host response. In the third study, the roles of the innate immunity effectors HSV-1 Us3 and human Z-DNA binding protein 1 (ZBP1) in stress granule formation (SG) were studied. Wild-type HSV-1 efficiently prevented the formation of SGs. The overexpression of ZBP1 resulted in accumulation of smaller but more abundant SGs during oxidative stress. Overexpression of Us3 did not significantly affect the size or number of SGs, but during Us3 deletion virus infection, SG proteins localized to cis-Golgi. This work shows that HSV-1 uses Us3 to evade and modulate host responses and that the γ134.5 protein is required for reactivation in mouse DRG cultures. / Tiivistelmä Herpes simplex virus tyyppi 1 (HSV-1) on yleinen taudinaiheuttaja, jonka ikävakioitu seroprevalenssi Suomessa on 52&#160;%. HSV-1 tunnetaan yleisimmin huuliherpeksen aiheuttajana, mutta myös kasvava osuus genitaaliherpeksistä on HSV-1:n aiheuttamia. HSV-1 voi johtaa myös vakaviin ilmentymiin, kuten aivotulehdukseen. Epiteelisolujen infektion tuottamia viruksia siirtyy aluetta hermottaviin tuntohermosoluihin, mikä voi johtaa piilevään infektiomuotoon eli latenssiin. Latentti virus voi ajoittain reaktivoitua, mistä voi seurata uusintainfektio. Isäntäsolu pyrkii rajoittamaan infektiota sen eri vaiheissa. Tämän tutkimuksen tarkoituksena oli selvittää kahden HSV-1:n virulenssiproteiinin, γ134.5:n ja Us3:n, merkitystä HSV-1:n elinkierrossa. Osatyössä I tutkittiin, miten Us3:n poisto vaikuttaa luontaisen immuniteetin vasteisiin, keskittyen etenkin Tollin kaltaisten reseptorien (TLR) signaalivälitykseen U937-monosyyttisoluissa. Us3-poistogeenisillä viruksilla suoritetuissa infektioissa TLR3:n ilmentyminen lisääntyi merkittävästi. Tämä johti myös lisääntyneeseen IRF-3-aktivaatioon sekä tyypin I interferonien ja interferonistimuloituvan proteiinin lisääntyneeseen ilmentymiseen. Tämä osoittaa, että TLR3 osallistuu HSV-1-viruksen tunnistukseen ja että Us3 säätelee IRF-3:n aktivaatiota. Osatyössä II keskityttiin γ134.5-proteiinin merkitykseen HSV-1:n latenssissa. Hiirialkioiden takajuuren hermoganglioita käytettiin soluviljelymallina HSV-1:n latenssin ja reaktivaation tutkimisessa. Tässä mallissa γ134.5-poistogeeniset virukset kasvoivat hyvin ja asettuivat latenteiksi, mutta eivät silti reaktivoituneet kuten luonnonkannan virukset. Stressijyväset ovat osa luontaista immuniteettia. Osatyössä III määritettiin HSV-1:n Us3-proteiinin ja ihmisen Z-DNA:han sitoutuvan proteiini 1:n (ZBP1) merkitystä stressijyvästen muodostumisessa. Luonnonkannan virus kykeni tehokkaasti estämään jyvästen muodostumisen. ZBP1:n yli-ilmentäminen oksidatiivisen stressin aikana johti suureen määrään pienikokoisia stressijyväsiä. Us3:n yli-ilmentäminen ei vaikuttanut stressijyväsiin, kun taas Us3-poistogeenisellä viruksella suoritetuissa infektioissa stressijyväsproteiinit paikantuivat Golgin laitteeseen. Tämä tutkimus osoittaa, että HSV-1 käyttää Us3-proteiinia luontaisten immuunivasteiden muunteluun ja että γ134.5-proteiini on välttämätön reaktivaatiossa hiiren hermoganglioissa.

The aim of the work described in this thesis is to investigate the nature and mechanisms of action of repellent cues for growing axons. In particular I try to resolve the controversy in the literature regarding the need for protein synthesis in the growth cone in response to external guidance cues. My results resolve the conflicting data in the literature on Semaphorin-3A signalling, where differing labs had shown that inhibiting protein synthesis either blocks or has no effect upon repulsion. They demonstrate the presence of at least two independent pathways, protein synthesis-dependent mTOR activation and -independent GSK3? activation. The higher sensitivity of the synthesis-dependent pathway, and its redundancy at higher concentrations where synthesis-independent mechanisms can evoke a full collapse response alone, resolve the apparent conflict. My experiments also demonstrated that Nogo-?20, a domain of Nogo-A, requires local protein synthesis to cause collapse. Unlike Semaphorin-3A, the dependence of collapse upon protein synthesis is concentration-independent and does not involve guanylyl cyclase, but it does share a dependence upon mTOR activity and the synthesis of RhoA, sufficient to cause collapse downstream of Semaphorin-3A. The other axon-repelling domain of Nogo-A, Nogo-66, is partially dependent upon the proteasome instead. It does not share a common pathway with Nogo-?20, except that both are RhoA-dependent. I further attempted to identify the nature of a repulsive activity found in grey matter, ruling out a previously suggested candidate identity. Finally, I examined the phenomenon of nitric oxide-induced growth cone collapse. My experiments revealed that S-nitrosylated glutathione causes growth cone collapse through the activity of protein disulphide isomerase. This mechanism shows only a partial dependence upon soluble guanylyl cyclase, but I argue that it has total dependence upon an S-nitrosylated donor. Coupled with its apparent relation to S-palmitoylation, the reciprocal of S-nitrosylation, I propose that nitric oxide causes collapse by crossing the cell membrane to inhibit S-palmitoylation-determined localisation of proteins. These results reveal some of the many pathways involved in growth cone collapse, whose further characterisation may provide new targets for the treatment of injuries of the central nervous system.

Acute spinal cord injury is often caused by collisions with motor vehicles, falls or violence. This injury could potentially lead to paraplegia or tetraplegia, causing great economic and personal loss. The patophysiology is biphasic, with primary and secondary mechanisms. Regarding secondary spinal cord injury, glutamate and interleukin-1 beta (IL-1<img src="http://www.diva-portal.org/cgi-bin/mimetex.cgi?%5Cbeta" data-classname="equation" data-title="" />) activates N-methyl-d-aspartate (NMDA)-receptors leading to prolonged excitotoxity, causing neuronal death and subsequently glial scarring. Cross-linked-hyaluronic acid gel and interleukin-1 receptor antagonist (IL-1RA) are believed to have a neuroprotective effect. The major aim of this study was to evaluate neuroprotection in the central neural system. Briefly, spinal cord slice cultures from mice (p9-12) were chemically injured with NMDA and treated with two hyaluronic acid-based gels with integrated, or added, IL1RA. RNA was extracted and transcripted to cDNA. The gene expression of Neuronal nuclear protein, <img src="http://www.diva-portal.org/cgi-bin/mimetex.cgi?%5Cbeta" data-classname="equation" data-title="" />-aktin and IL-1<img src="http://www.diva-portal.org/cgi-bin/mimetex.cgi?%5Cbeta" data-classname="equation" data-title="" /> were studies with RT-qPCR. Results showed that gel integrated with IL1RA had significant therapeutic effect, resembling undamaged cultures. Furthermore, axonal outgrowth was investigated in dorsal root ganglion (DRG) in which two preparations of the method were evaluated. Results demonstrated that changing medium every other day was more preferred, compared to adding 20 µl medium every day.  In conclusion, gels integrated with IL1RA have neuroprotective properties and in DRG preparations, medium should be changed every other day for optimal results.

La sclérose en plaques est une maladie dégénérative qui peut affecter la vision ainsi que différentes structures du système visuel afférent. La partie de l'oeil plus souvent affectée par la sclérose en plaques est le nerf optique, sous forme de névrite optique. Une technologie, nommée TCO (tomographie par cohérence optique), permet de prendre une image du nerf optique et de ses fibres nerveuses qui s'étendent sur la rétine. Dans cette thèse, la TCO a permis d’obtenir une épaisseur des fibres nerveuses autour du nerf optique, ainsi qu’une épaisseur totale de la macula et de la couche de cellules ganglionnaires chez les patients atteints de sclérose en plaques, avec et sans histoire de nérite optique, et chez un groupe de patients contrôle. Les résultats démontrent que seule l’épaisseur de la couche de cellules ganglionnaires permet de différentier les patients avec sclérose en plaques sans histoire de névrite optique des patients contrôle. Une deuxième étude a évalué la qualité visuelle en mesurant la sensibilité aux contrastes ainsi que la qualité de vie reliée à la vision avec un questionnaire de qualité de vie. Les résultats démontrent qu’une nouvelle charte de sensibilité aux contrastes, plus facile à administrer en clinique, permet aussi de différentier les patients sans névrite optique du groupe contrôle. De plus, la qualité de vie des patients ayant eu un épisode de névrite optique semble significativement affectée, même si le pronostic est considéré très favorable et que l’acuité visuelle est « bonne » suite à une névrite optique. En conclusion, l’utilisation de l’OCT en plus de mesures sensibles de fonction visuelle, telle la sensibilité aux contrastes, et de qualité de vié peuvent contribuer à mieux détecter des dysfonctions oculo-visuelles subtiles, mais importantes chez les patients atteints de sclérose en plaques. / Multiple sclerosis (MS) is the most common neurological condition causing disability in working-age adults. The hallmark of MS related disability is axonal loss. Through new technologies, such as optical coherence tomography (OCT), the retinal nerve fibre layer (RNFL), composed of ganglion cell axons, can be visualized and studied non-invasively in cross-section. Furthermore, recent OCT advances allow precise retinal layer segmentation and macular imaging of the ganglion cell layer. In this thesis, these different OCT parameters were measured to see which layers would be most affected in MS patients without previous optic neuritis. Results show that macular ganglion cell layer thickness is the only OCT parameter that can differentiate this sub-group of patients from healthy controls. Visual function was then assessed using a newly available, easy to use contrast sensitivity chart that can be self-administered by patients. Results show that this chart is also capable of differentiating MS patients without optic neuritis from controls, but usually gives better contrast sensitivity scores than the Mars chart. Lastly, vision-specific quality of life was assessed and proved to be reduced in MS patients with prior optic neuritis, despite supposed favorable recovery and good visual acuity in patients with this diagnosis. In sum, the use of OCT imaging, as well as sensitive visual function and quality of life measures, could help detect subtle, yet important structural or functional visual changes in patients with MS. This could ultimately help better screen, manage and counsel this subset of patients.

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