Orientador: Fernando Cendes / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-17T23:00:31Z (GMT). No. of bitstreams: 1 Bovi_AnaCarolinaNunes_M.pdf: 869097 bytes, checksum: 13486e80f1bc1dfd4cfb4d480a9031d0 (MD5) Previous issue date: 2011 / Resumo: Epilepsia do Lobo Temporal (ELT) é uma doença que apresenta atrofia das estruturas mesiais temporais, como resultado da esclerose mesial temporal (EMT), presente em 60-70% dos casos em que a cirurgia é a mais recomendada. A ELTM associada EMT possui um alto grau de refratariedade, em que pequena percentagem de indivíduos alcança o controle das crises, uma vez que a causa do EMT é desconhecida. Muitos estudos mostram que a atrofia do hipocampo contralateral ao foco epileptogênico também sofre alterações. A ressonância magnética tem sido uma ferramenta fundamental para o diagnóstico e quantificação dessas mudanças. Assim, o estudo avaliou 60 pacientes, sendo 30 indivíduos em tratamento medicamentoso e 30 em tratamento cirúrgico, com o objetivo de avaliar e quantificar essas alterações morfológicas encontradas nas estruturas envolvidas na ELTM por meio de um software manual que permite o traçado das estruturas e a sua associação com os achados clínicos da doença, a fim de elucidar o impacto dos tratamentos clínico e cirúrgicos. Os resultados mostraram que o grupo controle não apresentou progressão da atrofia tanto o hipocampo considerado menor (p = 0,533), o hipocampo considerado maior (p = 0,494) e nem do corpo caloso (p = 0,260). A análise do grupo clínico identificou uma redução no volume do hipocampo, tanto ipsilateral quanto contralateral nos dois subgrupos, refratários e benignos, e em relação ao hipocampo ipsilateral (atrófico) ocorreu redução volumétrica significativa no subgrupo benigno (p = 0,001) e também no subgrupo refratário (p = 0,003). O hipocampo contralateral ao foco epileptogênico no grupo CLR apresentou um grau significativo de atrofia (p = 0,001) sendo que o mesmo ocorreu com o subgrupo CLB (p = 0,011). No grupo cirúrgico (CX), o hipocampo contralateral (remanescente/saudável) mostrou uma progressão de atrofia que foi pronunciada em ambos os casos, sendo para o subgrupo CX sem controle de crises (CXR) (p = 0,001) e para o subgrupo CX com controle de crises após a cirurgia (CXB) (p = 0,002). A comparação do volume do hipocampo na RM1 entre os dois subgrupos não revelou diferenças significativas tanto para o hipocampo ipsilateral (p = 0,852) e para o hipocampo contralateral (p = 0,290), afirmando que os pacientes foram igualmente selecionados para a cirurgia. A análise pareada entre os grupos revelou uma diminuição significativa no volume do corpo caloso para o 4 subgrupos / Abstract: Temporal Lobe Epilepsy (TLE) is a condition that is associated to atrophy of the mesial temporal structures as a result of mesial temporal sclerosis (MTS), present in 60 to 70% of patients who undergo surgery. The MTLE associated with MTS has a high degree of resistance to antiepileptic drugs, as a small proportion of individuals can achieve seizure control. Many studies show that the atrophy of the hippocampus contralateral to the epileptogenic focus also undergoes changes. The MR imaging has been a fundamental tool for the diagnosis and quantification of these changes. Thus the study investigate 60 patients, being 30 individuals in drug treatment and 30 in surgical treatment, with the objective of assessing and quantifying these morphological changes found in structures involved in MTLE by means of a software which enables the tracing of the structure; and the association of these volumes with the clinical findings, in order to elucidate on the different responses to medical and surgical treatments. The results shown that the control group had no progression of atrophy of either hippocampus (p > 0.05) or corpus callosum (p = 0.260). The analysis of clinical group identified a reduction in the volume of the hippocampi, both the ipsilateral (affected) and the contralateral in both subgroups, resistant and benign. In relation to the hippocampus ipsilateral there was significant volumetric reduction in the benign group (p = 0.001) and also in the refractory group (p = 0.003). The hippocampus contralateral to the epileptogenic focus in the CLR group presented a significant atrophy (p = 0.001) and the same occurred to CLB (p = 0.011). In the surgery group (SG), the contralateral hippocampus (healthy remnant) showed a progression of atrophy that was pronounced in both the subgroup who continued with seizures after surgery (p = 0.001) and the subgroup who became seizure-free after surgery (p = 0.002). The paired analysis between the subgroups revealed a significant decrease in volume of the corpus callosum for the 4 subgroups / Mestrado / Neurociencias / Mestre em Fisiopatologia Médica

Orientadores: Lilian Tereza Lavras Costallat, Fernando Cendes / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-08T06:36:35Z (GMT). No. of bitstreams: 1 Appenzeller_Simone_D.pdf: 2923218 bytes, checksum: ead37b4a881b531bfab95cbff3f95dff (MD5) Previous issue date: 2006 / Resumo: As manifestações do sistema nervoso central (SNC) no Lúpus Eritematoso Sistêmico (LES) são complexas, podendo ser causadas diretamente pela atividade do LES ou serem secundárias a comorbidades. O nosso objetivo foi avaliar as manifestações do SNC no LES e orrelacioná-las às alterações cerebrais estruturais e funcionais à ressonância magnética. Todos os pacientes preenchiam quatro ou mais critérios classificatórios de LES e foram selecionados no ambulatório de Reumatologia da UNICAMP. Observamos que crises epilépticas ocorreram em 11,6% dos pacientes, estando associadas a acidente vascular cerebral e a presença de anticorpos antifosfolípides. A recorrência de crises foi rara, associada somente a presença de anticorpos antifosfolípides. A migrânea ocorreu mais frequentemente no LES que no grupo controle e estava associada a atividade de doença, ao Fenômeno de Raynaud e a presença de anticorpos antifosfolípides. Pacientes com história pregressa de migrânea apresentavam mais dano permanente. Analisando as ressonâncias magnéticas em pacientes com LES, observamos tanto atrofia de substância branca como de substância cinzenta. Embora ambos estivessem associados à presença de manifestações pregressas do SNC e ao maior tempo de doença, somente a atrofia de substância cinzenta esteve associada à dose cumulativa de corticosteróides. Pacientes com distúrbios cognitivos apresentaram mais frequentemente atrofia de corpo caloso e de hipocampo. Observamos também uma disfunção axonal no LES, associada a atividade de doença. De acordo com os nossos resultados, os métodos de neuroimagem estruturais e funcionais são úteis na confirmação do envolvimento do SNC e também na identificação do envolvimento subclínico no LES / Abstract: Central nervous system (CNS) manifestations in systemic lupus erythematosus (SLE) arecomplex. They may be directly caused by SLE disease activity or may be secondary to comorbities. Our objective was to determine CNS manifestations in SLE patients and to determine structural and functional neuroimaging abnormalities associated with its occurrence. Patients with four or more classification criteria for SLE, followed at the Rheumatology Unit of the State University of Campnas were included. We observed 11.6% of epileptic seizures in SLE patients. The occurrence of epileptic seizures was associated with the presence of stroke and antiphospholipid antibodies. Recurrence of seizures was rare and associated only with the presence of antiphospholipid antibodies. Migraine was more frequently observed in SLE patients than controls and was associated with disease activity, Raynaud¿s phenomenon and antiphospholipid antibodies. Pacients with past history of migraine had more frequently organ damage. We observed white and gray matter atrophy in SLE patients. Although both were associated with disease duration and past history of CNS involvement, only gray matter atrophy was associated with the total corticosteroid dose. Patients with cognitive impairment had more frequently corpus callosum and hippocampal atrophy. A transient axonal dysfunction, secondary to disease activity and not to CNS involvement, was observed in SLE. Our results suggest that structural and functional neuroimaging methods are useful in confirming CNS involvement, but also identify subclinical involvement in SLE patients / Doutorado / Clinica Medica / Doutor em Clínica Médica

Submitted by Edineia Teixeira (edineia.teixeira@unioeste.br) on 2017-11-27T18:31:17Z No. of bitstreams: 2 MARCELA LEITE2017.pdf: 2527004 bytes, checksum: 37310bebca45bcc15d56cfda1b505434 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-11-27T18:31:17Z (GMT). No. of bitstreams: 2 MARCELA LEITE2017.pdf: 2527004 bytes, checksum: 37310bebca45bcc15d56cfda1b505434 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-01-25 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Objective: To evaluate whether periodontal disease, through systemic inflammation, can potentiates the deleterious effects of immobilization of the skeletal striated muscle, contributing to the development of muscle atrophy due to disuse. Methods: Forty Wistar rats were divided into four groups: 1) Control Group (CG), 2) Periodontal Disease (GPD) 3) Immobilized (IG) and 4) Immobilized with Periodontal Disease (IPDG). Periodontal disease was induced for 30 days, with ligature method, and the immobilization of the right pelvic limb was performed with cast bandage for 15 days. Prior to euthanasia, the nociceptive threshold and muscular grasping force were evaluated. Afterwards, the soleus muscle was dissected and processed for sarcomere counting and morphological and morphometric analysis. For analysis of the data, the one-way ANOVA followed by the Tukey post test, with p < 0.05. Results: The IG and IPDG groups presented lower muscle weight, lower muscular grip strength and less number of sarcomeres compared to CG. The PDG showed reduction of muscle strength and nociceptive threshold after 15 days of periodontal disease and increase of connective tissue compared to CG. The IPDG presented lower muscle length and nociceptive threshold compared to the other groups. The IG presented a reduction in the cross-sectional area and a smaller diameter, an increase in the number of nuclei and a nucleus/fiber ratio, a decrease in the number of capillaries and a capillary/fiber ratio, with an increase in connective tissue. In the IPDG group, there were significant results for increased nucleus/fiber ratio, decreased capillaries and increased connective tissue when compared to the IG group. The IPDG group presented greater muscle tissue degeneration and increased inflammatory cells when compared to the other groups. Conclusion: Periodontal disease potentiated the deleterious effects of immobilization of the skeletal striated muscle, through intense destruction of muscle tissue, with significant increase of connective tissue, nucleus/fiber ratio and inflammatory infiltrate, significant reduction of vascularization and reduction of muscle length, with consequent reduction of muscle strength and nociceptive threshold. / Objetivo: Avaliar se a doença periodontal, por meio da inflamação sistêmica, potencializa os efeitos deletérios da imobilização do músculo estriado esquelético, colaborando para o desenvolvimento da atrofia muscular por desuso. Metodologia: Foram utilizados 40 ratos Wistar, divididos em quatro grupos: 1) Grupo Controle (GC); 2) Doença Periodontal (GDP); 3) Imobilizado (GI); 4) Doença Periodontal Imobilizado (GDPI). A doença periodontal foi induzida pelo método de ligadura, durante 30 dias e a imobilização do membro pélvico direito foi realizada com atadura gessada, por 15 dias. Antes da eutanásia, foram avaliados o limiar nociceptivo e força muscular de preensão. Após, o músculo sóleo foi dissecado e processado para contagem de sarcômeros e análise morfológica e morfométrica. Para análise dos dados, foi utilizado o teste ANOVA de uma via seguida do post test Tukey, com p<0,05. Resultados: Os grupos GI e GDPI apresentaram menor peso muscular, força muscular de preensão e número de sarcômeros comparados ao GC. O GDP apresentou redução da força muscular e do limiar nociceptivo após 15 dias de doença periodontal e aumento de tecido conjuntivo comparado ao GC. O GDPI apresentou menor comprimento muscular e limiar nociceptivo comparados aos demais grupos. O GI apresentou redução da área de secção transversa e menor diâmetro, aumento no número de núcleos e razão núcleo/fibra, diminuição no número de capilares e razão capilar/fibra, com aumento de tecido conjuntivo. No grupo GDPI, houve resultados significativos para aumento da razão núcleo/fibra, diminuição de capilares e aumento de tecido conjuntivo quando comparado ao grupo GI. O grupo GDPI apresentou maior degeneração do tecido muscular e aumento de células inflamatórias quando comparado aos outros grupos. Conclusão: A doença periodontal potencializou os efeitos deletérios da imobilização do músculo estriado esquelético, por meio da intensa destruição do tecido muscular, com significativo aumento do tecido conjuntivo, da razão núcleo/fibra e infiltrado inflamatório, significativa diminuição da vascularização e redução do comprimento muscular, com consequente redução da força muscular e limiar nociceptivo.

Orientador: Evanisi Teresa Palomari / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-26T00:31:52Z (GMT). No. of bitstreams: 1 Rissi_Renato_M.pdf: 1479367 bytes, checksum: e178a8d14769b59ec03814960ceef145 (MD5) Previous issue date: 2014 / Resumo: No âmbito da ortopedia, a imobilização articular é um recurso terapêutico eficiente e amplamente utilizado na prática clínica. Apesar de seu uso ser indispensável no tratamento de doenças álgicas ou fraturas, a imobilização ocasiona no paciente uma limitação física temporária de suas habilidades, podendo influenciar em sua locomoção e em suas atividades do cotidiano. A obesidade interfere muitas vezes na relação do hormônio insulina com os mecanismos de síntese e degradação proteica nos músculos. Considerando que a insulina exerce papel fundamental facilitando a síntese e inibindo a proteólise, os pacientes obesos, podem apresentar um balanço negativo no que se diz respeito à formação e degradação da massa muscular em virtude das desordens que estes pacientes geralmente apresentam no perfil insulinêmico. O tecido muscular é a reserva mais importante de proteína disponível no organismo, porém, este tecido se encontra consideravelmente reduzido nos casos de desnutrição proteica. Durante o jejum parcial ou total, a proteína corporal é destruída para proporcionar aminoácidos ao organismo, traduzindo-se desta forma em uma perda de massa corporal total. Quando consideramos a obesidade e a desnutrição proteica, associadas a um paciente imobilizado, a interação dessas condições podem vir a potencializar os prejuízos musculoesqueléticos do paciente. O objetivo deste estudo foi verificar experimentalmente se a condição de imobilização articular potencializa as alterações musculoesqueléticas em animais obesos e desnutridos. Para tal, 60 camundongos da linhagem C57BL6 foram utilizados e divididos em quatro grupos: Controle (GC), Controle Imobilizado (GCI), Obeso Imobilizado (GOI), Desnutrido Imobilizado (GDI). A imobilização articular foi realizada utilizando-se um modelo com esparadrapo/gesso adaptado para uso em camundongos. Os animais permaneceram imobilizados durante 14 dias. A obesidade e a desnutrição proteica foram desenvolvidas por meio de ingestão alimentar de dieta específica para cada grupo. Realizou-se análise da atividade locomotora; quantificação sérica da enzima creatina quinase; análise histomorfométrica da tíbia e dos músculos gastrocnêmio e tibial anterior; determinação do conteúdo de glicogênio intramuscular e zimografia das metaloproteinases (2 e 9). Como resultado, verificou-se a redução da atividade locomotora noturna nos animais imobilizados em relação ao GC; aumento dos níveis séricos da enzima CK nos animais imobilizados em relação ao GC; redução na área e no diâmetro das fibras musculares do gastrocnêmio e tibial anterior nos grupos imobilizados em relação ao GC; diminuição do conteúdo de glicogênio intramuscular no GDI em relação ao GCI; aumento da expressão das metaloproteinases 2 e 9 nos grupos imobilizados em relação ao GC. Portanto, podemos concluir que o protocolo de imobilização articular utilizado é capaz de gerar atrofia musculoesquelética nos animais. Já no caso da interação entre as condições de obesidade/imobilização e desnutrição/imobilização, o tecido musculoesquelético apresenta acréscimo na atrofia, revelando elevado prejuízo muscular nessas condições / Abstract: Within orthopedics, joint immobilization is an effective therapeutic tool and widely used in clinical practice. Although their use is essential in the treatment of painful diseases or fractures, the patient immobilization causes a temporary physical limitation of their abilities and may influence its locomotion and in their daily activities. Obesity often interferes to the hormone insulin in relation with the mechanisms of protein synthesis and degradation in muscle. Considering that insulin plays a key role in facilitating the synthesis and inhibiting proteolysis, obese patients may have a negative balance as regards the formation and degradation of muscle mass because of disorders in these patients insulinemic profile. Muscle tissue is the most important reserve available protein in the body, however, this tissue is considerably reduced in cases of malnutrition. During the partial or total fasting, the body protein is destroyed to provide amino acids to the body, thus translating into a loss of total body mass. When we consider obesity and protein malnutrition associated with an immobilized patient, the interaction of clinical conditions can come to enhance the patient's musculoskeletal damage. The aim of this study was to verify experimentally if the condition of joint immobilization enhances musculoskeletal changes in obese and malnourished animals. To this end, 60 mice C57BL6 were used and divided into four groups: Control (CG), Immobilized Control (ICG), Immobilized obese (IOG), Immobilized Malnourished (IMG). The joint immobilization was performed using a model with tape / plaster adapted for use in mice. The animals remained immobilized for 14 days. Obesity and protein malnutrition were developed by means of specific diets food intake for each group. We performed analysis of locomotor activity; quantification of serum CK; histomorphometric analysis of the tibia and the gastrocnemius and tibialis anterior muscles; determining the content of intramuscular glycogen; zymography of the metalloproteinases (2 and 9). As a result, we found reduction in nocturnal locomotor activity in immobilized animals relative to CG; increased serum levels of creatine kinase in the immobilized animals relative to CG; reduction in the area and diameter of the muscle fibers of the gastrocnemius and tibialis anterior in groups immobilized relative to CG; decreased content of intramuscular glycogen in the group IMG relative to ICG; increased expression of metalloproteinases 2 and 9 in groups immobilized relative to CG. Therefore, we conclude that the joint immobilization protocol used is able to generate skeletal muscle atrophy in animals. In the case of the interaction between the conditions of obesity / immobilization and malnutrition / immobilization, tissue musculoskeletal presents increase in atrophy, revealing high muscle injury in these conditions / Mestrado / Anatomia / Mestre em Biologia Celular e Estrutural

Orientador: Aarão Mendes Pinto-Neto / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-23T11:12:13Z (GMT). No. of bitstreams: 1 Fernandes_T._M.pdf: 2208281 bytes, checksum: 1ec9008b41578db5b1f708c5dcb2480b (MD5) Previous issue date: 2013 / Resumo: Introdução: A atrofia vaginal é uma condição crônica frequente em mulheres na pós-menopausa que acarreta alterações em sua sexualidade e consequentemente em sua qualidade de vida. Recentes estudos avaliam novas alternativas de tratamento para essa ascendente queixa da população feminina. Entretanto, atualmente dispomos de poucas opções terapêuticas adequadamente avaliadas. Objetivo: Comparar a função sexual feminina após o uso tópico de estrogênio, testosterona e ácido poliacrílico com o uso de lubrificante vaginal. Métodos: Ensaio clinico randomizado com 80 mulheres na pós-menopausa, entre 40 e 70 anos, em seguimento no Ambulatório de Menopausa do CAISM Unicamp. As mulheres foram randomizadas para o tratamento tópico via vaginal com estrogênio, testosterona, ácido poliacrílico e lubrificante, três vezes na semana, por um período de 12 semanas, entre novembro de 2011 a janeiro 2013. Utilizou-se o Índice de Função Sexual Feminina para avaliar as mudanças da resposta sexual no início e após 6 e 12 semanas. Resultados: O ácido poliacrílico e a testosterona tópica, em comparação com o lubrificante após 12 semanas de tratamento, apresentaram aumento nos domínios: desejo sexual, lubrificação, satisfação, dor na relação sexual e escore total. O tratamento com o estrogênio tópico em comparação com o lubrificante apresentou melhora no domínio desejo. A análise intragrupo ao longo do tempo de tratamento evidenciou melhora nos domínios desejo, lubrificação, dor para as mulheres que utilizaram ácido poliacrílico, testosterona e estrogênio. Além disso, as mulheres que utilizaram testosterona apresentaram melhora ao longo do tempo nos domínios excitação, orgasmo e satisfação. Conclusão: O tratamento por 12 semanas- em mulheres na pós-menopausa com sintomas de atrofia vaginal - realizado com ácido poliacrílico, testosterona e estrogênio demonstrou melhora na função sexual feminina. quando comparado ao uso de lubrificante vaginal / Abstract: Introduction. Female libido is multifactorial and complex. Declining estrogen levels in postmenopausal women affects vaginal function. Aim. To evaluate female sexual function after using topical estrogen, testosterone or polyacrylic acid as vaginal lubricants with K-Y jelly as a placebo lubricant. Methods. This was a randomized controlled clinical trial on 80 postmenopausal women between 40 and 70 years of age with follow-up at the Menopause Clinic of the CAISM / Unicamp. The women were randomized to treatment with topical vaginal estrogen, testosterone, polyacrylic acid or oil lubricant alone, three times a week for a period of 12 weeks from November 2011 to January 2013. Main Outcome Measures. We used the Female Sexual Function Index (FSFI) to assess changes in sexual response at baseline, and after 6 and 12 weeks. Results. After 12 weeks of treatment, polyacrylic acid and topical testosterone produced improvements in the FSFI domains of sexual desire, lubrication, satisfaction, reduced pain during intercourse and total score compared with lubricant alone. Treatment with topical estrogen in comparison with lubricant alone showed an improvement in the FSFI field of desire. The intragroup analysis over the time of the treatment showed improvements in the fields of desire, lubrication, and reduced pain for polyacrylic acid, testosterone and estrogen. Furthermore, women who used testosterone showed improvements over time in the fields of arousal, orgasm and satisfaction. Conclusions. Treatment of postmenopausal women with symptoms of vaginal atrophy with polyacrylic acid, testosterone and estrogen for 12 weeks produced improvements in self-reported female sexual function when compared with a lubricant / Mestrado / Fisiopatologia Ginecológica / Mestra em Ciências da Saúde

Abstract The complete exon-intron organization of the gene coding for the mouse α1(XIII) collagen chain, Col13a1, was characterized from genomic clones and multiple transcription initiation points were determined. Detailed comparison of the human and mouse genes showed that the exon-intron structures are completely conserved between the species, and both genes have their 5' untranslated region preceded by a highly conserved putative promoter region. The chromosomal location of the mouse gene was determined to be at chromosome 10, band B4, between markers D10Mit5 – (2.3 ± 1.6 cM) – Col13a1 – (3.4 ± 1.9 cM) – D10Mit15. The location of the genes for both the catalytically important α-subunit of prolyl 4-hydroxylase (P4HA) and human type XIII collagen (COL13A1) were previously mapped to 10q21.3-23.1. Prolyl-4-hydroxylase catalyzes the formation of 4-hydroxyproline in collagens by the hydroxylation of peptide-bound proline and plays a crucial role in the synthesis of these proteins. The order and transcriptional orientation of the COL13A1 and P4HA was determined. These two genes were found to lie at tail to tail orientation on chromosome 10 and the distance between these genes was determined to be about 550 kbp. To study the function of type XIII collagen we used gene targeting in ES cells to generate a mouse line that carries a mutated type XIII collagen gene. Instead of normal protein, mutant mice express type XIII collagen with an altered amino-terminus in which the cytosolic and the transmembrane domains have been replaced with an unrelated sequence. The homozygous mice are fertile and viable but they show alterations in skeletal muscles, mainly wavy sarcolemma and increased variation in muscle fiber diameter. Ultrastructural studies revealed additional abnormalities such as streaming of z-disks, accumulation and enlargement of mitochondria, and disorganized myofilaments. The basement membranes of the muscle cells showed areas of detachment from the plasma membrane and the fibrillar matrix of the cells was less compact than in control animals. Fibroblasts cultured from mutant mice had normal levels of type XIII collagen but exhibited decreased adhesion to substratum which might be explained by a reduced anchoring strength of the altered protein.

Spinal muscular atrophy is caused by mutation of the SMN1 gene resulting in the selective loss of spinal cord motor neurons. HuD has been shown to interact with SMN and to localize to RNA granules along axons. In conditions where SMN is decreased, like in SMA, HuD’s localization to RNA granules affected. Overexpression of HuD in an SMA cell culture model was shown to rescue SMA-like axonal defects. Here, existence of a signaling pathway downstream of PKC leading to the activation of HuD was investigated in MN-1 cells. Stimulation of this pathway using a pharmacological agonist of PKC increased HuD levels and enhanced its binding to GAP-43 and Tau mRNAs. An scAAV9 viral expression system to overexpress HuD in vivo was established, laying the foundation for the next phase of the study. Overall, modulating HuD expression and activity would be beneficial and could constitute an attractive therapeutic approach for SMA.

Abstract The carbonic anhydrases (CAs) catalyze the reversible hydration of carbon dioxide. In mammals, there are 13 active isoenzymes, which clearly differ in their cell localisation, tissue distributions and functions. CA IX, a unique transmembrane member of the CA gene family, is a tumour-associated protein which is thought to be involved in malignant cell invasion, adhesion and the regulation of cell proliferation. The main focus in the present study was on elucidating the function and expression of CA IX in normal and malignant tissues, especially in the alimentary tract. The functional studies also included CA II, which is regarded as another important CA isoenzyme in the alimentary tract. CA IX immunostaining showed a decrease in the staining intensity of gastric adenomas with increasing dysplasia grade. Well differentiated carcinomas of the intestinal type showed expression comparable to that in the normal mucosa, while expression was decreased in the less differentiated tumours. CA IX deficiency (Car9-/-) genotype and C57/BL6 strain were the main factors which increased the susceptibility of CA IX deficient mice fed on either a normal or high-salt diet to histological abnormalities, including foveolar hyperplasia and glandular atrophy in the gastric body mucosa, while CA II deficiency was associated with only minor histological abnormalities. In a physiological analysis, CA IX played only a minor role in duodenal bicarbonate secretion (DBS), whereas absence of CA II in mice completely abolished the stimulatory effect of E-type prostaglandin 2 (PGE2) on duodenal alkalisation. The results demonstrate that CA IX expression is diminished in most gastric tumours. The variations observed in its expression support the concept that gastric adenomas and carcinomas do not emerge as progressive steps on a single pathway but may instead represent distinct entities with heterogenic genetic backgrounds. In the stomach, CA IX is mainly involved in the regulation of tissue morphogenesis in the body mucosa, while CA II has a major role in maintaining the gastroduodenal acid/base balance.

Polycomb Proteins (PcG) are chromatin proteins that control the maintenance of “transcriptional memory” and cell identity by fixing the repressed state of developmentally regulated genes. This function has been linked to interaction with RNA moieties, in particular long non-coding RNAs (lncRNAs). However, specificity of PcG-RNA interactions has been controversial (Beltran et al., 2016; Chen Davidovich, Leon Zheng, Karen J. Goodrich, & Thomas R. Cech, 2013). In this study we took advantage of recent work published from our lab reporting about a novel and reversible mechanism regulating genome wide Ezh1-PRC2 activation in mouse skeletal muscle cells in response to atrophic stress (Bodega et al., 2017). Using this physiological, in vivo tool we could identify a functional dynamic crosstalk between Malat1 (Metastasis Associated Lung Adenocarcinoma Transcript 1) and PRC2-Ezh1 complex. By combining immuno-fluorescence, biochemistry, epigenomics, ChIRP, DNA and RNA immunoprecipitation we identified a novel pathway in which Malat1 plays a role in compartmentalization, assembly and activity of PRC2 in chromatin, allowing epigenetic plastic response to atrophic stress and recovery. We conclude that Malat1 is an essential partner for PRC2-Ezh1 adaptive function in skeletal muscle cells.

Disruption of RNA homeostasis is a shared feature across multiple neurodegenerative diseases that are associated with mutations in RNA binding proteins or factors involved in RNA processing. One prime example is the neurodegenerative disease spinal muscular atrophy (SMA), which is characterized by the degeneration of spinal motor neurons and atrophy of skeletal muscle through poorly defined mechanisms. SMA is the consequence of ubiquitous deficiency in the survival motor neuron (SMN) protein, which has a well-characterized role in the assembly of small nuclear ribonucleoproteins (snRNPs). SMN-dependent dysfunction of major (U2) and minor (U12) spliceosomal snRNPs as well as U7 snRNP – which functions in histone mRNA processing – along with consequent RNA misprocessing events have been characterized in SMA. Additionally, SMN has been implicated in additional RNA pathways that may also be involved in SMA etiology. With the broad implications of multifaced roles of SMN in RNA regulation, an outstanding challenge in the SMA field has been the identification of key downstream RNA-dependent events and their contributions to pathogenesis. While the selective loss of spinal motor neurons is a key hallmark of SMA pathology, the molecular mechanisms remain incompletely understood. Through my dissertation work, I aimed to characterize the RNA mediated pathways that underlie neurodegeneration in SMA. We previously demonstrated that SMA motor neuron death is driven by converging mechanisms of p53 activation that include upregulation and phosphorylation – the latter of which establishes the vulnerability of specific motor neuron pools – however, the upstream triggers remained unknown. Here, I show that the function of SMN in the assembly of Sm-class snRNPs of the splicing machinery regulates alternative splicing of Mdm2 and Mdm4 – two non-redundant repressors of p53 – and increased skipping of critical exons in these genes is associated with p53 stabilization. Further investigation uncovered that dysfunction of Stasimon – a U12 intron-containing gene regulated by SMN – converges on p53 upregulation to induce phosphorylation of p53 through the activation of p38α MAPK and contributes to the demise of SMA motor neurons. Thus, this work elucidated the upstream RNA mediated mechanisms underlying multiple modes of p53 activation, implicating impairments in both the U2 and U12 snRNP pathways in SMA motor neuron death. It further established Mdm2, Mdm4, and Stasimon as effector genes that are regulated by SMN’s role in snRNP assembly and play key roles in the degeneration of SMA motor neurons. Studies in mouse models of SMA have revealed broader deficits in the motor circuit beyond motor neuron death, which include reduced excitatory drive on motor neurons brought on by a loss of proprioceptive synapses. Restoration in SMA mice revealed that Stasimon dysfunction also contributes to the deafferentation of motor neurons – a cellular defect originating in proprioceptive neurons – revealing Stasimon’s dual contribution to motor circuit pathology in SMA. However, the Stasimon-dependent molecular mechanisms that mediate synaptic loss in proprioceptive neurons, along with the pathway through which Stasimon impairment induces p38α MAPK activation in motor neurons are not well established. Stasimon was initially identified as a novel contributor to motor circuit dysfunction in Drosophila and motor axon outgrowth deficits in zebrafish models of SMN deficiency. However its cellular roles remain poorly understood. In an effort to address this, I identify Stasimon as an endoplasmic reticulum (ER) resident protein that localizes at mitochondria-associated ER membranes (MAM) – specialized contact sites between ER and mitochondria membranes. Additionally, through characterization of novel knockout mice, I show that Stasimon is an essential gene for mouse embryonic development. These findings provide key insight into Stasimon function and set the stage for further investigation of the p53-dependent mechanisms of motor neuron degeneration as well as cellular pathways driving proprioceptive synaptic loss in SMA. This dissertation also expands beyond RNA mediated mechanisms of SMA – which occur as a consequence of SMN-deficiency – to translational efforts aimed to treat the disease by describing an unexpected gain of toxic function associated with SMN overexpression that is accompanied by RNA dysregulation and sensory-motor circuit pathology. I further explore these surprising findings of neuronal toxicity induced by AAV9-mediated SMN overexpression that paradoxically affects sensory-motor function and reveal that they parallel features of SMA pathogenesis. Accordingly, I find that the functional basis of long-term motor toxicity of AAV9-SMN involves motor neuron deafferentation and proprioceptive neurodegeneration. At the cellular level, toxicity is associated with the accumulation of large cytoplasmic aggregates of SMN in motor circuit neurons that sequester Sm proteins, disrupt snRNP biogenesis, and induce widespread transcriptome alterations and splicing deficits. These findings identify a novel deleterious role for SMN when expressed at supraphysiological levels that acts through inhibition of SMN’s normal function in snRNP biogenesis, akin to disease mechanisms of SMA. These observations have important implications regarding the approved use of AAV9-SMN for gene therapy in humans and suggest a need for further, careful consideration of potential detrimental effects when SMN is expressed at supraphysiological levels. Collectively, this dissertation identifies the direct involvement of key SMN-dependent splicing events in select aspects of SMA pathology in a mouse model, in particular those converging on the p53-mediated mechanisms of motor neuron death and the loss of proprioceptive synapses. This work also establishes causal links between impairments in snRNP biology and neuronal dysfunction in SMA, providing mechanistic insight into the process of motor neuron death. Lastly, it uncovers a new, clinically relevant aspect of SMN biology associated with its long-term overexpression which has shared features with the RNA mediated mechanisms of neurodegeneration in SMA.