Vinte e seis pacientes com paralisia facial unilateral de longa duração foram submetidos à reanimação facial em único estágio com transplante do músculo grácil inervado pelo ramo massetérico do nervo trigêmeo. Foram divididos em 2 grupos não-randomizados de acordo com a técnica de fixação: Grupo I (19 pacientes), fixação do músculo com uso do tendão palmar longo inserido no músculo orbicular nos lábios superior e inferior do lado não paralisado (além da linha média); Grupo II (7 pacientes), fixação do retalho apenas com pontos separados no músculo orbicular dos lábios superior e inferior no lado paralisado. A avaliação qualitativa demonstrou melhores resultados no Grupo I (94,1% vs 66,6%). Na comparação do posicionamento do arco de cúpido em repouso, no pré e pós-operatório, observamos melhora estatisticamente significante (p<0,05) em ambos os grupos. Durante o sorriso, entretanto, houve melhora significativamente maior na centralização do arco de cúpido nos pacientes submetidos à fixação com tendão palmar longo (Grupo I) / Twenty-six patients with unilateral long-stading facial palsy underwent 1-stage reanimation with free gracilis muscle transplant innervated by the masseteric branch of the trigeminal nerve. They were divided into 2 nonrandomized groups according to insertion technique: group I (19 patients), palmaris longus tendon graft placed between the gracilis free flap and the orbicularis oris of the upper and lower lip on the nonparalyzed side; group II (7 patients), interrupted suture between the free flap and the orbicularis oris of the upper and lower lip on the paralyzed side. Qualitative evaluation of the smile demonstrated better results in patients from group I (94,1% vs 66,6%). Comparing the position of the Cupid`s bow at rest, pre- and postoperatively in each patient, we observed significant improvement of facial symmetry in both groups. During smile, however, there was significantly higher rate of centralization of the Cupid`s bow in patients submitted to reanimation with the use of the palmaris longus tendon (group I)

Five chemicals indigenous to the dental office environment that may cause toxic effects are formaldehyde, phenol, acrylic, mercury, and nitrous oxide. These chemicals create abnormal stress on physiological and psychological systems of the body resulting in symptomatology and pathology when the body defenses can no longer maintain homeostasis by adaptation. This study demonstrated serious behavioral consequences of chemical and heavy metal exposure. This study provided evidence that a significant percentage of dental office personnel who are exposed to the dental office chemicals show psycho neurological dysfunction. It was concluded that these individuals suffer adverse reactions to the chemicals in their work environment. The problem areas included perceptual motor difficulty in cognitive functioning, concern with bodily functions, despondency, and interpersonal problems.

I proposed the study of two distinct aspects of Ten-Eleven Translocation 2 (TET2) protein for understanding specific functions in different body systems. In Part I, I characterized the molecular mechanisms of Tet2 in the hematological system. As the second member of Ten-Eleven Translocation protein family, TET2 is frequently mutated in leukemic patients. Previous studies have shown that the TET2 mutations frequently occur in 20% myelodysplastic syndrome/myeloproliferative neoplasm (MDS/MPN), 10% T-cell lymphoma leukemia and 2% B-cell lymphoma leukemia. Genetic mouse models also display distinct phenotypes of various types of hematological malignancies. I performed 5-hydroxymethylcytosine (5hmC) chromatin immunoprecipitation sequencing (ChIP-Seq) and RNA sequencing (RNA-Seq) of hematopoietic stem/progenitor cells to determine whether the deletion of Tet2 can affect the abundance of 5hmC at myeloid, T-cell and B-cell specific gene transcription start sites, which ultimately result in various hematological malignancies. Subsequent Exome sequencing (Exome-Seq) showed that disease-specific genes are mutated in different types of tumors, which suggests that TET2 may protect the genome from being mutated. The direct interaction between TET2 and Mutator S Homolog 6 (MSH6) protein suggests TET2 is involved in DNA mismatch repair. Finally, in vivo mismatch repair studies show that the loss of Tet2 causes a mutator phenotype. Taken together, my data indicate that TET2 binds to MSH6 to protect genome integrity. In Part II, I intended to better understand the role of Tet2 in the nervous system. 5-hydroxymethylcytosine regulates epigenetic modification during neurodevelopment and aging. Thus, Tet2 may play a critical role in regulating adult neurogenesis. To examine the physiological significance of Tet2 in the nervous system, I first showed that the deletion of Tet2 reduces the 5hmC levels in neural stem cells. Mice lacking Tet2 show abnormal hippocampal neurogenesis along with 5hmC alternations at different gene promoters and corresponding gene expression downregulation. Through the luciferase reporter assay, two neural factors Neurogenic differentiation 1 (NeuroD1) and Glial fibrillary acidic protein (Gfap) were down-regulated in Tet2 knockout cells. My results suggest that Tet2 regulates neural stem/progenitor cell proliferation and differentiation in adult brain.

Orientador: Lucia Figueiredo Mourão / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-26T23:25:47Z (GMT). No. of bitstreams: 1 Lima_DaniellaPriscilade_M.pdf: 1711012 bytes, checksum: 5823b535276013fc9abfaf369781e7a7 (MD5) Previous issue date: 2015 / Resumo: No envelhecimento normal, alterações fisiológicas tendem a interferir na deglutição, mas o indivíduo idoso pode ser capaz de manter uma alimentação segura. Todavia, em associação com um quadro neurológico, eleva-se o risco de desenvolvimento de disfagia nessa população. Sabe-se que a funcionalidade da deglutição pode estar associada também à interação com fatores contextuais, o que permite descrever seus múltiplos impactos na vida do indivíduo. Essa concepção vai ao encontro do que preconiza a Classificação Internacional de Funcionalidade, Incapacidade e Saúde (CIF). Objetivo: Este trabalho tem como objetivo caracterizar a deglutição de idosos com e sem doença neurológica com base na videofluoroscopia (VFC) e na aplicação da Classificação Internacional de Funcionalidade, Incapacidade e Saúde (CIF). Pretende-se, ainda, verificar se as categorias da CIF, o exame de videofluoroscopia e as escalas de deglutição padronizadas discriminam ou não os grupos de idosos com e sem doença neurológica e se há categorias da CIF ou da VFC que se correlacionam com a presença de penetração e aspiração. Métodos: Compuseram a amostra 63 idosos, organizados em indivíduos sem doença neurológica e com doença neurológica (Esclerose Lateral Amiotrófica, Síndromes Parkinsonianas e Acidente Vascular Cerebral). A avaliação da deglutição foi composta por anamnese, avaliação clínica (indireta e direta) e exame de videofluoroscopia (no qual foram quantificados 17 parâmetros). Aplicou-se as escalas Functional Oral Intake Scale (FOIS), Escala de Penetração e Aspiração e Escala de Severidade da Disfagia. Posteriormente, os participantes foram classificados em relação a 39 categorias da CIF pertencentes a todos os agrupamentos - Funções do corpo (b); Estruturas do Corpo (s); Atividades e Participação (d) e Fatores Ambientais (e). Os dados foram submetidos a análise estatística, com aplicação do Teste de Mann-Whitney e teste de correlação de Spearman. Resultados: No grupo de idosos sem doença neurológica, a maioria dos participantes apresentou graus 0 (ausência de alterações) e 1 (alteração leve) em todas as categorias da CIF pertencentes às Funções do Corpo (b), Estruturas do Corpo (s) e Atividades e Participação (d) e graus 0 e 1 nos parâmetros do exame de videofluoroscopia. Houve diferenças significativas (p < 0,05) entre os grupos neurológicos e o grupo de idosos em relação aos qualificadores da CIF, distribuídas em todos os agrupamentos, sendo: grupo Esclerose Lateral Amiotrófica- 23 categorias (59%); grupo Síndromes Parkinsonianas - 25 categorias (64%) e grupo Acidente Vascular Cerebral - 20 categorias (51,3%). Os parâmetros da videofluoroscopia também se diferenciaram entre os idosos com doença neurológica e o grupo de idosos sem doença neurológica no grupo Esclerose Lateral Amiotrófica em 9 parâmetros (52,9%); no grupo Síndromes Parkinsonianas em 8 parâmetros (47,05%) e no grupo Acidente Vascular Cerebral em 7 parâmetros (41,2%). Identificaram-se diferenças significativas (p < 0,05) em relação ao grupo de idosos sem doença neurológica envolvendo as escalas Functional Oral Intake Scale (FOIS) e Escala de Severidade da Disfagia. Houve significância no teste de correlação entre os itens da CIF e a ocorrência de penetração/aspiração, sendo as categorias "Deglutição oral" e "Deglutição faríngea" as mais predominantes. Houve correlação entre os parâmetros da videofluoroscopia e a ocorrência de penetração/aspiração, sendo o item "Fechamento do vestíbulo laríngeo" o mais predominante. A análise descritiva sugeriu que os grupos apresentaram diferenças na gravidade das alterações em relação a diversas categorias da CIF. Conclusão: Os resultados indicaram que idosos sem doença neurológica apresentam ausência de alterações ou alterações consideradas leves em comparação aos grupos com doença neurológica. O uso de avaliações padronizadas associadas à CIF pode contribuir para a discriminação dos grupos de idosos bem como ampliar a compreensão de diversos aspectos associados à deglutição / Abstract: In normal aging, physiological changes tend to interfere with swallowing, but elderly individuals may be able to keep feeding safely. However, in association with a neurological condition, the risk of dysphagia arises in this population. The swallowing function can be related to contextual factors, which describe multiple impacts on an individual's life. This view is consistent with the International Classification of Functioning, Disability and Health (ICF). Purpose: This work aims to characterize the swallowing in elderly with and without neurological disease based on videofluoroscopy (FSS) and the International Classification of Functioning, Disability and Health (ICF). Also, we want to verify if ICF categories, FSS and swallowing scales are able to discriminate elderly with and without neurological disease. Besides, we want to know if there is any correlation between ICF categories and the presence of penetration and aspiration. Methods: The sample is composed of 63 elderly men and women, organized in individuals with and without neurological disease and neurological disease (Amyotrophic Lateral Sclerosis-ALS, Parkinsonian Syndromes and Cerebral Vascular Accident-Stroke). The evaluation of swallowing was done through anamnesis, clinical evaluation (direct and indirect) and videofluoroscopy (in which 17 parameters were quantified). We also applied the Functional Oral Intake Scale (FOIS), Penetration Scale and Aspiration and Severity of Dysphagia Scale. The subjects were classified according to 39 ICF categories belonging to Body Functions (b), Body Structures (s), Activities and Participation (d) and Environmental factors (e). We used the Mann-Whitney test to compare each disease group with the group without neurological disease regarding ICF categories. We also compared groups regarding the 17 parameters of videofluoroscopy and the three swallowing scales. We used the Spearman correlation test to identify ICF categories and videofluoroscopy parameters associated with the occurrence of penetration or aspiration. ICF qualifiers assigned to individuals were also analyzed descriptively. Results: In the elderly group without neurological disease, most participants had degrees 0 (no change) and 1 (low change) in all ICF categories belonging to Body Functions (b), Body Structures (s) and Activities and Participation (d) and grades 0 and 1 in the parameters of videofluoroscopy. There were significant differences (p < 0.05) between neurological groups and the control group in relation to ICF qualifiers, distributed in categories in all groups, as follows: ALS group, 23 categories (59%); Parkinsonian Syndromes group - 25 categories (64%) and Stroke group - 20 categories (51.3%). Videofluoroscopy parameters also differed significantly (p < 0.05) between neurological disease and control groups: ALS group with 9 parameters (52.9%); Parkinsonian Syndromes group with 8 parameters (47.05%) and Stroke group with 7 parameters (41.2%). Furthermore, we identified significant differences (p < 0.05) in the neurological group versus the control group involving the scales Functional Oral Intake Scale (FOIS) and Severity of Dysphagia Scale. We also found a correlation between ICF categories and the occurrence of penetration / aspiration: categories "Oral Swallowing" and "Pharyngeal Swallowing" were the most prevalent. There was also a correlation between videofluoroscopy parameters and the occurrence of penetration / aspiration: the item "Laryngeal Vestibular Closure" was predominant. Descriptive analysis suggested that the normal and neurological disease groups had differences in relation to various ICF categories. Conclusion: Elderly without neurological disease have essentially no changes or low changes compared to neurological disease groups. The use of standardized assessments associated with the ICF can contribute to discriminate elderly groups and improve the understanding of many aspects of swallowing / Mestrado / Gerontologia / Mestra em Gerontologia

GM2 gangliosidoses are a family of lysosomal storage disorders that include both Tay-Sachs and Sandhoff diseases. These disorders result from deficiencies in the lysosomal enzyme β-N-acetylhexosaminidase (HexA). Impairment of HexA leads to accumulation of its substrate, GM2 ganglioside, in cells resulting in cellular dysfunction and death. There is currently no treatment for GM2 gangliosidoses. Patients primarily present with neurological dysfunction and degeneration. Here we developed a central nervous system gene therapy through direct injection that leads to long-term survival in the Sandhoff disease mouse model. We deliver an equal mixture of AAVrh8 vectors that encode for the two subunits (α and β) of HexA into the thalami and lateral ventricle. This strategy has also been shown to be safe and effective in treating the cat model of Sandhoff disease. We tested the feasibility and safety of this therapy in non-human primates, which unexpectedly lead to neurotoxicity in the thalami. We hypothesized that toxicity was due to high overexpression of HexA, which dose reduction of vector could not compensate for. In order to maintain AAV dose, and therefore widespread HexA distribution in the brain, six new vector designs were screened for toxicity in nude mice. The top three vectors that showed reduction of HexA expression with low toxicity were chosen and tested for safety in non-human primates. A final formulation was chosen from the primate screen that showed overexpression of HexA with minimal to no toxicity. Therapeutic efficacy studies were performed in Sandhoff disease mice to define the minimum effective dose.

Analyzing protein tertiary structure is an effective method to understanding protein function. In my thesis study, I aimed to understand how surface features of protein can affect the stability and specificity of enzymes. I focus on 2 proteins that are involved in human disease, Profilin (PFN1) and APOBEC3A (A3A). When these proteins are functioning correctly, PFN1 modulates actin dynamics and A3A inhibits retroviral replication. However, mutations in PFN1 are associated with amyotrophic lateral sclerosis (ALS) while the over expression of A3A are associated with the development of cancer. Currently, the pathological mechanism of PFN1 in this fatal disease is unknown and although it is known that the sequence context for mutating DNA vary among A3s, the mechanism for substrate sequence specificity is not well understood. To understand how the mutations in Profilin could lead to ALS, I solved the structure of WT and 2 ALS-related mutants of PFN1. Our collaborators demonstrated that ALS-linked mutations severely destabilize the native conformation of PFN1 in vitro and cause accelerated turnover of the PFN1 protein in cells. This mutation-induced destabilization can account for the high propensity of ALS-linked variants to aggregate and also provides rationale for their reported loss-of-function phenotypes in cell-based assays. The source of this destabilization was illuminated by my X-ray crystal structures of several PFN1 proteins. I found an expanded cavity near the protein core of the destabilized M114T variant. In contrast, the E117G mutation only modestly perturbs the structure and stability of PFN1, an observation that reconciles the occurrence of this mutation in the control population. These findings suggest that a destabilized form of PFN1 underlies PFN1-mediated ALS pathogenesis. To characterize A3A’s substrate specificity, we solved the structure of apo and bound A3A. I then used a systematic approach to quantify affinity for substrate as a function of sequence context, pH and substrate secondary structure. I found that A3A preferred ssDNA binding motif is T/CTCA/G, and that A3A can bind RNA in a sequence specific manner. The affinity for substrate increased with a decrease in pH. Furthermore, A3A binds tighter to its substrate binding motif when in the loop region of folded nucleic acid compared to a linear sequence. This result suggests that the structure of DNA, and not just its chemical identity, modulates A3 affinity and specificity for substrate.

Amyotrophic lateral sclerosis (ALS) is a terminal neurodegenerative disease that affects motor neurons causing progressive muscle weakness and respiratory failure. In 2011, the presence of a hexanucleotide repeat expansion within chromosome 9 open reading frame 72(C9ORF72) was identified in ALS patient samples, becoming the major known genetic cause for ALS and frontotemporal dementia (FTD). Carriers of this mutation present reduced levels of C9ORF72 mRNA, RNA foci produced by the aggregating expansion and toxic dipeptides generated through repeat-associated non-ATG translation. These findings have led to multiple hypotheses on the pathogenesis of C9ORF72: 1) Haploinsufficiency, 2) RNA gain-of-function, 3) RAN Translation, and 4) Disrupted nucleocytoplasmic trafficking. Due to lack of treatments for this disease, we have pursued an AAV-RNAi dependent gene therapy approach, using an artificial microRNA (amiR) packaged in a recombinant adeno-associated virus (rAAV). After validating our in vitro results, we advanced to in vivo experiments using transgenic mice that recapitulate the major histopathological features seen in human ALS/FTD patients. Adult and neonate mice were injected through clinically relevant routes and our results indicate that AAV9-mediated amiR silencing not only reduced mRNA and protein levels of C9ORF72 but also the expansion derived toxic GP dipeptides. Although our amiR is not targeting the expansion itself but exon 3, we illustrate here that the evident dipeptide decrease is achievable due to the presence of aberrant transcripts in the cytoplasm containing miss-spliced Intron-HRE-C9ORF72 species. These encouraging results have led to the continued testing of this treatment as a therapeutic option for C9ORF72 - ALS patients.

Many steps go into developing a clinical viral gene therapy. The course starts with appropriate disease selection and moves through the many hurdles of in-vitro testing, animal model validation and proof-of-concept studies, all the way through pre-clinical large animal studies. In this thesis, I propose to outline the process of developing a translation pathway for a gene therapy using recombinant adeno-associated virus (rAAV). I will expand on this outline using data that I have generated during the course of my Ph.D. that ranges from animal model validation all the way through pre-clinical vector stability studies. Two disease models will be discussed throughout this thesis, Cockayne Syndrome (CS) and Alpha-1 Antitrypsin Deficiency (AATD). Cockayne Syndrome is a rare autosomal recessive genetic disorder involving mutations in either the CSA or CSB gene, leading to defects in DNA repair. Clinically this presents as progressive degeneration of the central nervous system, retina, cardiovascular system, and cochlea, which leads to mental retardation, post-natal growth defects, ocular abnormalities, and shortened life expectancy. Alpha-1 antitrypsin is a serine protease inhibitor largely produced in the liver that mainly functions to inhibit neutrophil elastase within the lung. AATD leads to an increased risk of emphysema, with shortened life expectancy, and also results in accumulations of mutant AAT polymers in the liver, sometimes leading to liver failure. Using these two disease models I will outline the upstream and downstream pre-clinical work as well as the transition to clinical trials of a rAAV based gene therapy.

Rationale: Delirium is a serious, morbid condition affecting 2.6 million older Americans annually. A major problem plaguing delirium research is difficulty in identification, given a plethora of existing tools. The lack of consensus on key features and approaches has stymied progress in delirium research. The goal of this project was to use advanced measurement methods to improve delirium’s identification. Aims and Findings: (1) Determine the 4 most commonly used and well-validated instruments for delirium identification. Through a rigorous systematic review, I identified the Confusion Assessment Method (CAM), Delirium Observation Screening Scale (DOSS), Delirium Rating Scale-Revised-98 (DRS-R-98), and Memorial Delirium Assessment Scale (MDAS). (2) Harmonize the 4 instruments to generate a delirium item bank (DEL-IB), a dataset containing items and estimates of their population level parameters. In a secondary analysis of 3 datasets, I equated instruments on a common metric and created crosswalks. (3) Explore applications of the harmonized item bank through several approaches. First, identifying different cut-points that will optimize: (a) balanced high accuracy (Youden’s J-Statistic), (b) screening (sensitivity), and (c) confirmation of diagnosis (specificity) in identification of delirium. Second, comparing performance characteristics of example forms developed from the DEL-IB. Impact: The knowledge gained includes harmonization of 4 instruments for identification of delirium, with crosswalks on a common metric. This will pave the way for combining studies, such as meta-analyses of new treatments, essential for developing guidelines and advancing clinical care. Additionally, the DEL-IB will facilitate creating big datasets, such as for omics studies to advance pathophysiologic understanding of delirium.

It is increasingly being recognised that agrichemical exposure may have adverse chronic health effects in humans, particularly on central nervous system function. However, much of this evidence sterns from studies relating to the effects of acute intoxications (i.e. short-term high dose exposures) and little data exist on the chronic effects of long-term low-dose exposures to agrichemicals in the absence of acute poisoning. Such a finding would have substantial public health implications for prevention and control of chronic morbidity and mortality. This is particularly important in South Africa, where a sizeable portion of the rural population are employed in agricultural work, often under extremely unhealthy living and working conditions, and where occupational agrichemical exposures appear to be substantial. To address this question, this study investigated the prevalence of neurological and neurobehavioural abnormalities amongst 247 fruit farm workers in the Kouebokkeveld in the Western Cape, of whom 163 were current agrichemical applicators. Outcomes measured included neurological symptoms, peripheral vibration sense, motor tremor, as well as performance on the World Health Organisation Neurobehavioural Core Test Battery (WHO NCTB) and a set of neurobehavioural tests based on the Information Processing model of cognitive psychology. These latter tests have been developed in South Africa for subjects of low educational levels and aim to by-pass the powerful effects of culture that complicate traditional neuropsychological testing, which may mask the smaller effects due to occupational chemical exposures. Cumulative, and average lifetime intensity of exposure to organophosphates were estimated using a job- exposure matrix based on a combination of secondary industry data, interview reports and farmer records. Confounders measured included age, education, smoking and alcohol habits, non-occupational exposure to agrichemicals and other potential neurotoxins, past medical history and usage of personal protective equipment. The study results confirmed low levels of education and high alcohol consumption amongst the sample of farm workers. Multiple logistic and linear regression were used to identify exposure-effect relationships and to control for confounding. Neurological symptoms were significantly associated with a history of previous pesticide poisoning, although this may have arisen as a result of reporting bias. Vibration sense and the neurobehavioural tests exhibited associations with established covariates, and regression modelling of the WHO NCTB tests was remarkably similar to findings in another study of solvent-exposed factory workers in South Africa. None of the vibration sense, tremor or neurobehavioural outcomes were associated with past agrichemical poisoning in the sample, and only two tests showed significant relationships with long-term occupational exposure. These included the Pursuit Aiming subtest of the WHO NCTB and one of the tests of long-term semantic memory in the Information Processing tests. However, the strength of these the associations were small (partial r²s less than 2%) and these findings may have occurred due to chance arising from multiple comparisons. The neurobehavioural tests based on the Information Processing model appeared to offer little improvement on the WHO NCTB in terms of being less sensitive to cultural effects, although some evidence was present that tests of semantic access were able to detect occupational effects and were less sensitive to education. The absence of a demonstrable and consistent long-term agrichemical exposure-effect relationship appears to suggest that long-term agrichemical exposure is not associated with adverse chronic nervous system effects, although the lack of organophosphate specificity in construction of exposure indices in the job-exposure matrix may partly contribute to this finding. Recommendations to improve the characterisation of agrichemical exposures at farming work place are made, as well as suggestions concerning the role of biological monitoring for agrichemicals, improving working and living conditions on South African farms, and methods of neurological and neurobehavioural assessment in occupational health.