Made available in DSpace on 2016-04-28T20:40:16Z (GMT). No. of bitstreams: 1 Giuliana Gomes Nogueira.pdf: 569220 bytes, checksum: a6fb400334b18bb296f1efb206f7d21c (MD5) Previous issue date: 2010-04-23 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / This paper intend to open the possibility to discuss the Multiple Sclerosis in the hall of the psychosomatic illnesses taking as references the point of view of D.W. Winnicott about the psychosomatic relationship. Thus, this study will allow the reader to get in touch with several aspects of Multiple Sclerosis as it history, etiology, diagnosis, treatments, prognostics and psychological. This study also focus on the basic concepts of matureness theory, elaborated by Winnicott, reflecting about his patients suffering from psychosomatic illness. Not only are the Winnicott´s patients important to try to understand the perturbations of the psycho-soma in the multiple sclerosis as the revision of Jacqueline du Pré´s biography, which died due the aggravation of the illness. The confrontation of her personal history with the theory elaborated by Winnicott helps to illustrated what have been studied in this paper. After these analyses, it is considered the idea what in the base of all psychosomatic disorder there is a split. So we can infer that split is also one of the important points of the Multiple Sclerosis, that way the geneses of the disease may be related to gaps on the process of psycho-soma integration. Finally, the necessity of more studies on that subject is essentially to help those patients to have a better life quality. At last, the main objective of this research is considered achieved as it opened a new door of possibilities to try to understand of the multiple sclerosis / Esta dissertação busca construir um trajeto que possibilite discutir a Esclerose Múltipla no hall das doenças psicossomáticas tomando como referencial a relação psicossomática, abordada sob a ótica de D. W. Winnicott. Assim, realiza-se um estudo que permite ao leitor conhecer de forma ampla a Esclerose Múltipla detendo-se em seus 4 aspectos históricos; sua etiologia; diagnóstico; tratamentos; prognósticos; aspectos psicológicos. Este estudo se preocupa, ainda, em abordar os conceitos fundamentais da teoria do amadurecimento, elaborada por Winnicott e refletir sobre os pacientes com transtorno psicossomático atendidos por ele. Além dos pacientes de Winnicott, para analisar a esclerose múltipla, recorre-se ao relato da vida da personalidade pública Jacqueline du Pré, que morreu em decorrência de agravamento da doença e à confrontação da sua história tanto com a teoria preconizada por Winnicott quanto com o que foi estudado até então. Após essas análises, considera-se a idéia de que na base de todo transtorno psicossomático está uma cisão. Sugere-se que a cisão é uma forte característica da Esclerose Múltipla. Nota-se que há características semelhantes a outros transtornos psicossomáticos abordados pro Winnicott, considerando os indícios de que a gênese da doença possa ter uma origem em questões relativas à integração da psique-soma. Por fim, é ressaltada a necessidade de estudos mais aprofundados sobre o tema, sendo o principal o objetivo do trabalho atingido, que é a abertura de uma nova porta para a compreensão da esclerose múltipla

Amyotrophic Lateral Sclerosis (ALS) is a neurological disease characterized by the degeneration of upper and lower motor neurons. Genetic studies have revealed that many ALS-associated genes are involved in autophagy, but the role of this pathway in motor neurons remains poorly understood. Here, we use the SOD1G93A mouse model to investigate the role of autophagy in ALS. We find neuronal subtype-specific regulation of autophagy over the course of disease progression. Vulnerable motor neurons form large GABARAPL1-positive autophagosomes that engulf ubiquitinated cargo recognized by the selective autophagy receptor p62. Other motor neurons and interneurons do not engulf cargo within GABARAPL1-positive autophagosomes and instead accumulate somatodendritic aggregates. To investigate whether motor neuron autophagy is protective or detrimental, we generated mice in which the critical autophagy gene Atg7 is specifically disrupted in motor neurons. Phenotypic analysis of these mice revealed that autophagy is dispensable for motor neuron survival but plays a key role in regulating presynaptic structure and function. By crossing these mice to the SOD1G93A mouse model, we find that autophagy inhibition accelerates early neuromuscular denervation and neurological dysfunction. However, loss of autophagy in motor neurons eventually leads to an extension of lifespan, and this is associated with reduced pathology in interneurons and glial cells. These data suggest that vulnerable motor neurons rely on autophagy to maintain neuromuscular innervation early in disease. However, autophagy eventually acts in a non-cell autonomous manner to promote disease spread and neuroinflammation. Our results reveal counteracting roles for motor neuron autophagy early and late in ALS disease progression.

Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD) are two devastating neurodegenerative diseases in urgent need of therapeutic intervention. The last seven years has been a period of great progress in understanding these disorders separately and as a disease spectrum. Most notable is the discovery of the hexanucleotide GGGGCC expansion in the C9ORF72 (C9) gene, which is the greatest known cause of inherited and sporadic forms of these two diseases. In response to this groundbreaking discovery, we set out to elucidate the molecular mechanisms of C9 pathogenesis with a focus on the expanded RNA transcripts derived from the C9 expansion. Our two primary goals have been to contribute to the worldwide efforts to understand the primary toxic insults of this mutation that will ultimately shape therapeutic development, and to identify molecular criteria that can be used to define new links between these diseases and undetermined genetic factors. In the introduction, we review the broad conceptual links between RNA binding proteins (RBPs), mRNA regulation, and neurodegeneration. This review contains substantial discussion of ALS, FTD, and C9, as well as related neurodegenerative, neuromuscular and repeat expansion diseases. In addition to providing a detailed history of molecular mechanisms proposed for these disorders, this section serves as a justification for our focus on the C9 RNA, RBP sequestration, and altered splicing that we describe in the following chapters. Chapter two consists of our 2016 Elife paper on sequestration of the RBP hnRNP H and resulting splicing changes in C9ALS-FTD afflicted individuals. In this paper, we sought to identify the most biochemically sound candidate for the proposed RBP sequestration hypothesis. We found that the splicing factor hnRNP H binds with high affinity to the repeat sequence and likely has a role in regulating the transition of the repeat RNA from linear to G-quadruplex (G-Q) conformation. Importantly, we identified functional deficiency of this protein in patient brains, as evidenced by dysregulation of known hnRNP H splicing targets, and loss of soluble hnRNP H. Chapter three consists of recently submitted work on the molecular links between C9ALS/FTD, and sporadic ALS/FTD at large. Building upon our findings in C9ALS-FTD, we have sought to ask whether the changes to hnRNP H we predicted would occur in C9 expansion carriers as a result of the repeat RNA might also occur independent of this expansion. We found that indeed half of all patients in a cohort of 50 sporadic ALS, ALS-FTD, and FTD brains demonstrated hnRNP H sequestration and accompanying splicing changes, a pattern we refer to as like-C9. Like-C9 patients may be thought of as phenocopies of C9 expansion carriers, in that they not only present with similar clinical symptoms, but also possess remarkably similar molecular signatures of RBP dysfunction. While the genomic origins of like-C9 remain unknown, we propose that they are suggestive of repeat expansions analogous to C9, much like what is seen in DM1 and DM2, and HD and HDL2 (discussed in Ch. 1). This work has provided the foundation for our ongoing search for novel genomic expansions that confer increased susceptibility to ALS/FTD.

Introduction: Digital ulcers (DUs) are responsible for much of the pain and disability associated with systemic sclerosis (SSc), and are a biomarker of internal organ involvement and poor prognosis. DUs are often used as the primary end-point in SSc clinical trials, although the reliability of rheumatologists in grading DUs is poor to moderate at best. Fingertip DUs are believed to be ischaemic in aetiology, whereas, extensor DUs are thought to occur due to mechanical factors and recurrent microtrauma. Treatments for DUs are often poorly tolerated due to systemic vasodilation. The overarching aim was to investigate the definition and objective measurement of SSc-related DUs, their pathophysiology, and a new light treatment. Method: Five studies were undertaken. (1) A web-based study in which photographs of digital lesions were graded, all either with or without clinical context. (2) A pilot study to assess the feasibility and tolerability of high-frequency ultrasound (HFUS) imaging to measure DUs. (3) A retrospective study examining whether thermographic abnormalities are associated with DUs. (4) A double-blind, randomised, crossover, controlled study of glyceryl trinitrate (GTN) to explore the pathophysiology of DUs in SSc. (5). A feasibility study of a novel light (red, infrared and blue) device to treat SSc-related DUs. Results: (1) 51 rheumatologists graded ≥ 4500 images. The clinical context (without vs with, weighted kappa statistic) did not significantly improve the intra- (0.32,0.36) or inter-rater (0.64,0.71) reliability. (2) HFUS was performed on 15 DUs and was well tolerated and feasible in the majority. DU measurement was possible in most (n=13) DUs, the mean DU depth and width were 0.99mm and 5.74mm, respectively. (3) Patients (n=138) with abnormal (compared to normal) thermography were more likely (adjusted odds ratio = 2.84) to develop future DUs, including multiple episodes. (4) 16 DUs were studied; the microvessels of the DU centre were responsive to GTN, with an increase in perfusion, with a similar effect in both fingertip and extensor DUs. There was less of a clear signal in the DU periphery. (5) Light treatment was safe, feasible and well tolerated (46 light treatments administered in 8 patients, one studied on three separate occasions). There was a significant improvement (change in visual analogue score per visit) in DUs as assessed by both patient (-7.1, P = < 0.001) and clinician opinion (-5.2, P = < 0.001). DU perfusion (measured by LDI) significantly increased post-treatment. Conclusion: The reliability of DU grading did not improve with clinical context. HFUS was feasible and well tolerated, and measurement was possible in most DUs. Our data suggests that many DUs might have an ischaemic drive, including extensor DUs. A novel light treatment was safe, feasible and well tolerated, with a tentative suggestion of treatment efficacy.

Amyotrophic Lateral Sclerosis (ALS) is a fatal adult-onset paralytic disorder for which there is currently no cure. Underlying the disease mechanism of ALS is the spontaneous pathologic degeneration of motor neurons (MNs). Understanding the molecular mechanisms underlying spontaneous and selective MN demise is critical to the development of rational therapeutic strategies. In the current work, utilizing established in vitro models of ALS, I demonstrate that necroptosis, a form of caspase-independent programmed cell death (PCD), drives MN death. Pharmacologic inhibition and/or genetic silencing of receptor interacting protein kinase-1 (RIPK1), receptor interacting protein kinase-3 (RIPK3), and mixed lineage kinase domain-like-protein (MLKL) rescued MN death in vitro. While this core machinery was conserved, the requirement of nuclear factor kappa-B (NF-κB) and Bcl-2-associated X protein (Bax) deviated from known models of necroptosis. This divergence led me to consider that there may be a MN-specific program of necroptosis. Thus, I then used unbiased approaches, by meta-analyzing a gene expression signature captured from MNs undergoing cell death in vitro, to explore MN cell death drivers that may be engaged upstream or downstream to RIPK1/RIPK3/MLKL. I also explored the relevance of necroptosis to MN disease in vivo, in part by deleting RIPK3 from a genetic mouse model of familial ALS. Overall this approach did not rescue motor neuron loss, and there was no improvement in motor function, disease onset, or survival in these animals. I conclude that while necroptosis machinery drives motor neuron death in in vitro models of ALS, more work needs to be done to (1) assess the motor neuron-specific cell death program, and (2) evaluate the relationship, if any, of necroptosis to motor neuron disease in vivo.

Immune tolerance to self antigens prevents the onset of autoimmune diseases such as Multiple Sclerosis (MS). There are three branches of tolerance which allow the auto-aggressive potential of T lymphocytes to be limited; these are death, anergy-adaptation and regulation. The main body of this work attempts to clarify a role for adaptation in maintaining the sensitivity of the autoreactive T cell repertoire below a ‘threshold for harm’ in the mouse model of MS, experimental autoimmune encephalomyelitis (EAE). The well defined myelin basic protein (MBP) Ac1-9 epitope altered peptide ligand (APL) system has been used to develop a model allowing the examination of mechanisms underlying the adaptation of cells. Previous data showed immunisation with the 4Lys (wild-type) epitope mediated disease whereas a superagonist APL with a tyrosine substitution at position 4 (4Tyr) did not, despite showing potency in vitro. This was shown to be a result of both activation induced cell death and adaptation. Here an in vitro model was developed using MBP-reactive TCR transgenic cells to make predictions about the mechanisms underlying adaptation. These data lead to the conclusion that T cells can adapt (become less sensitive) either before or after encounter with the wild-type peptide, leading to a reversal of their pathogenic potential. The MBP APL system and MBP reactive transgenic cells were also used to assess the contribution of epitope spreading in a relapsing-remitting (RR) model of EAE induced with proteolipid protein. The cells were tracked and changes in phenotype and behaviour were monitored. The data show that disease induced with one antigen can be manipulated with cells relevant to a different antigen and that bystander suppression may be an effective weapon in controlling the progression to RR-EAE.

Introdução: A esclerose múltipla (EM) é uma doença inflamatória autoimune desmielinizante. Diversos estudos evidenciaram a forte associação entre a EM e a expressão do retrovírus endógeno da família W (HERV-W) e do Epstein Barr Vírus (EBV), sem definir seu real papel no desenvolvimento da doença. Objetivo: Investigar a presença de anticorpos anti EBV e a expressão de HERV-W em pacientes com EM e avaliar a correlação entre a atividade clínica e imagiológica da EM com a avaliação quantitativa do HERV-W e EBV. Métodos: Realizamos avaliações clínicas e de ressonância magnética (RM) por 36 meses de 36 pacientes com EM e a comparamos com a análise quantitativa longitudinal do PCR em tempo real do RNA do HERV-W em PBMC e uma análise transversal por ELISA do anti VCA IgG e IgM de EBV. Foram utilizados dois grupos controles sendo o primeiro com 30 indíviduos saudáveis e o segundo com 26 pacientes com outras doenças neurológicas (ODN) para comparação com os títulos de HERV-W e anti- EBV. Resultados: A dosagem do IgG EBV foi estatisticamente maior no grupo EM quando comparado ao grupo controle saudável (p = 0,024) e a expressão de HERV-W foi estatisticamente maior tanto no grupo EM (p = 0,001) como no grupo ODN (p = 0,022) quando comparados com os controles saudáveis nos grupos de pacientes. Nenhuma sorologia IgM do EBV foi positiva. A avaliação longitudinal da expressão relativa do HERV-W não apresentou correlação com nenhum dos parâmetros clínicos ou imagiológicos de avaliação da EM sendo eles: tipo de EM; medicamento em uso; EDSS; taxa anualizada de surtos; novas lesões em T2/FLAIR pela RM; lesões captando gadolíneo pela RM. Conclusão: Existe uma expressão relativa de HERV-W aumentada em pacientes com EM e em ODN quando comparados com controles saudáveis. Os pacientes com EM apresentam valores superiores de anticorpos IgG anti- EBV. Não encontramos nenhuma correlação na avaliação longitudinal entre a atividade clínica e imagiológica de pacientes com EM e a avaliação quantitativa do HERV-W e do anticorpo anti-EBV. / Introduction: Multiple sclerosis (MS) is a demyelinating autoimmune inflammatory disease. Several studies have demonstrated the strong association between MS and the expression of endogenous retrovirus W (HERV-W) and Epstein Barr Virus (EBV), without defining its true role in the development of the disease. Objective: To investigate the presence of anti-EBV antibodies and HERV-W expression in MS patients and to evaluate the correlation between the clinical and imaging activity of MS with the quantitative evaluation of HERV-W and EBV. Method: We performed clinical and magnetic resonance imaging (MRI) evaluations for 36 months of 36 MS patients and compared it with the longitudinal quantitative real-time PCR analysis of HERV-W RNA in PBMC and a cross-sectional analysis by anti-VCA IgG and EBV IgM ELISA. Two control groups were used, the first with 30 healthy subjects and the second with 26 patients with other neurological diseases (OND) for comparison with HERV-W and anti-EBV titers. Results: IgG EBV was statistically higher in the MS group when compared to the healthy control group (p = 0.024). HERV-W expression was statistically higher in the MS group (p = 0.001) and the OND group (p = 0.022) when compared to healthy controls. No IgM EBV serology was positive. The longitudinal evaluation of the relative expression of HERV-W did not present any correlation with the clinical or MRI of the MS group following parameters: type of MS; medication in use; EDSS; annualized rate of relapses; new MRI T2/FLAIR lesions; MRI gadolinium enhancing lesions. Conclusion: There is a relative increased HERV-W expression in patients with MS and in OND when compared with healthy controls. Patients with MS have higher values of anti-EBV IgG antibodies. We found no correlation in the longitudinal evaluation between the clinical and imaging.

Axonal degeneration is the major cause of disability in progressive multiple sclerosis (MS). It has been shown that in MS and relevant disease models, demyelinated axons harbor an increased number of mitochondria, which is reflected in bigger stationary sites of mitochondria, increased mitochondrial activity and increased transport speed of mitochondria. This axonal response of mitochondria to demyelination (ARMD) is protective, as there is an increase in energy demand due to the redistribution of sodium channels along the axon following demyelination. However, it remains to be determined how this ARMD is mounted and how mitochondrial dynamics are involved. By using in vivo and in vitro systems we are determined to elucidate the transport and fusion dynamics of the ARMD and where these additional mitochondria come from. Using a cerebellar slice culture system with lysolecithin induced demyelination, we show that the increase in mitochondrial occupancy of the axon already occurs at 24 hours after demyelination and plateaus around 3 to 4 days after demyelination. At 24 hours, there was a steep increase in the mitochondrial numbers inside the axon, which is then followed by an increase in mitochondrial size over the following days. All parameters decrease again over the following days, but remain elevated compared to baseline even 12 days after demyelination. To determine the source of these additional mitochondria and to assess the fusion dynamics within the axon, we used a lentivirus expressing a mitochondrial targeted photoconvertible dye (mEOS2) to label mitochondria in Purkinje cells. The mitochondria that are labelled green in the Purkinje cell axons are then photoconverted to red by illuminating the initial part of the axon with a 405-nm laser and imaged over the following 20 minutes to determine the transport and fusion dynamics. This showed an increased number of mitochondria moving from the cell body into the axon, as well as an increase in retrograde transport of mitochondria in the demyelinated compared to the myelinated axons. Furthermore the size of newly transported mitochondria and their speed was increased in the anterograde direction. Furthermore, the fusion rate of newly transported mitochondria with stationary converted mitochondria was increased in the demyelinated axons compared to myelinated control. These changes can also be observed in unmyelinated axons, as well as axons of cerebellar slices of the dysmyelinating shiverer mutant with or without lysolecithin treatment. The manipulation of mitochondrial dynamics after demyelination with the fission inhibitor mdivi-1 and the ATPase inhibitor oligomycin both showed an increasing or decreasing effect on the mitochondrial parameters after demyelination respectively. The effect on the axonal health after demyelination was detrimental with both of these treatments. Increasing mitochondrial biogenesis with pioglitazone increased axonal mitochondrial parameters, as well as ameliorated axonal damage after demyelination with lysolecithin. As the neuronal cell bodies in MS harbour mitochondrial DNA deletions, which affects their physiology, including energy production efficiency, another aim of this thesis was to model this deficiency in vitro. As it was not possible to model these mitochondrial defects in vitro within the experiments of this thesis, the characterization of a mitochondrial mutant in vivo model was done as a contribution to a greater set of experiments performed by other members of the Mahad lab.

Amyotrophic Lateral Sclerosis (ALS) is a rapid and fatal neurodegenerative disease marked by progressive muscle weakness and wasting. Approximately 50% of people with ALS experience changes in cognition and behaviour. Previous research has been mixed as to whether cognition declines over the course of ALS, or whether it is related to proxies of disease progression (e.g., functional disability scales). However, this research has suffered from limitations including the use of inappropriate measures of cognition, imprecise measures of disease progression, high attrition, practice effects, and biased analytic approaches. Fortunately, recent advances in clinical assessment have provided accurate measures of neuropsychological functioning and disease progression, namely, the Edinburgh Cognitive and Behavioural ALS Screen (ECAS) and the King's Clinical Disease Staging. The present study aims to utilise recent advances in ALS disease metrics to overcome previous limitations and explore the evolution of cognitive and behavioural dysfunction over the course of ALS. Specifically, the aims of the present project are to 1) develop alternate forms of the ECAS to accommodate repeated longitudinal assessment; 2) examine how cognition and behaviour relate to clinical disease stages in ALS; 3) evaluate how cognitive and behavioural symptoms evolve over the course of the disease in ALS; and 4) explore clinicians' attitudes toward cognitive and behavioural screening in ALS. To achieve Aim 1, two new versions of the ECAS (ECAS-B and ECAS-C) were developed and administered to a group of age, education, and gender matched controls to that of the original ECAS-A validation study. Results demonstrate that the alternate forms of the ECAS (B and C) were equivalent to the original ECAS-A, reducing practice effects and possessing excellent inter-rater reliability. The ECAS forms were administered longitudinally to a separate group of healthy controls. Over an interval of 4 months, the ECAS-A-B-C showed no evidence of practice effects and excellent test-retest reliability validating their utility in the longitudinal monitoring of cognition in ALS. The ECAS forms were then used in an international multi-centre clinical sample of 161 ALS patients and 80 matched controls to achieve Aim 2. Patients were grouped into their King's Clinical Disease Stage at time of testing. Analysis revealed a significant cross-sectional relationship between disease stage and ALS-Specific cognitive functions, driven by a decline in verbal fluency performance. A significant relationship was also observed between disease stage and behavioural features. By end-stage disease 80% of patients demonstrated neuropsychological impairment. Participants were followed up longitudinally to explore the progression of cognitive and behavioural symptoms. Latent Growth Curve models of the ECAS subdomains (utilising the alternate versions) demonstrated a significant decline in ALS Specific cognitive, but not behavioural, functioning over time. This decline was explained by advancing disease stage, the presence of the C9orf72 repeat expansion, and years of education. Rate of change in ALS Non-Specific functions was dependent on baseline performance. Visuospatial functions and perseveration declined at similar rates and were distinct from language, fluency, apathy, and disinhibited behaviour. Cluster analysis of patients revealed a three-cluster solution with one group demonstrating no significant decline, a second group with mild cognitive and behavioural decline, and a third group with more severe neuropsychological decline. When data was restructured by diseases stage, rather than time, longitudinal results were similar to cross-sectional findings. To examine clinician's attitudes to cognitive and behavioural screening. Fourteen Health Care Professionals (HCP) working in ALS (Neurologists, Psychologists, and Clinical Care Specialists) were interviewed. Thematic analysis revealed that HCPs recognised the importance of cognitive and behavioural screening in ALS, but that it is not common practice. Important barriers to screening were reported including other members of staff, a lack of resources, and issues concerning patients and their families. Participants suggested increasing training and psychology input, and making screening a standardised protocol to all patients may alleviate these barriers. Cognition and behaviour are critically related to advancing disease stage, both cross-sectionally and longitudinally. Declining cognitive and behavioural symptoms has important implications for clinical practice, caregiver impact, and end-of-life decision making. However, clinicians report that cognitive and behavioural screening is not common practice and that significant barriers exist. The newly developed alternate forms of the ECAS provide an accurate, effective, and clinically useful means of monitoring cognitive function over the course of the disease in ALS.

No description available.