A ausência de terapias eficazes para a caquexia permanece como um problema central para o tratamento do câncer no mundo. Em contrapartida, o treinamento de força (i.e. também conhecido como treinamento resistido) tem sido amplamente utilizado como uma estratégia não farmacológica anticatabólica, prevenindo a perda da massa e da função da musculatura esquelética. Entretanto, o papel terapêutico do treinamento de força na caquexia do câncer permanece apenas especulativo. Portanto, nesse estudo avaliamos se o treinamento de força poderia atenuar a perda da massa e da função da musculatura esquelética em um severo modelo de caquexia do câncer em ratos. Para isso, ratos machos da linhagem Wistar foram randomizados em quatro grupos experimentais: 1) ratos sedentários injetados com solução salina na medula óssea (Controle); 2) ratos injetados com solução salina na medula óssea e submetidos ao treinamento de força (Controle + T); 3) ratos sedentários injetados com células do tumor Walker 256 na medula óssea (Tumor); e 4) ratos injetados com células do tumor Walker 256 na medula óssea e submetidos ao treinamento de força (Tumor + T). Foram avaliados a massa e a área de secção transversa da musculatura esquelética, marcadores de disfunção metabólica e do turnover proteico, a função da musculatura esquelética in vivo e ex vivo, o consumo alimentar, o crescimento tumoral e a sobrevida dos grupos experimentais com tumor. O grupo Tumor apresentou atrofia muscular após quinze dias da injeção das células tumorais como pode ser observado pela redução na massa dos músculos Plantaris (- 20,5%) e EDL (-20%). A atrofia no músculo EDL foi confirmada por análises histológicas, demonstrando uma redução de 43,8% na área de secção transversa. Embora o treinamento de força tenha aumentado o conteúdo proteico da lactato desidrogenase e revertido totalmente o conteúdo da forma fosforilada de 4EBP-1 (i.e. repressor da transcrição de mRNA), ele não atuou na morfologia da musculatura esquelética nos animais com tumor. Além disso, o treinamento de força não atenuou a perda de função da musculatura esquelética, a anorexia, o crescimento tumoral ou a taxa de mortalidade. Contudo, a força muscular, avaliada pelo teste de 1RM, apresentou uma correlação negativa com a sobrevida dos animais (p = 0,02), sugerindo que a perda de força prediz a mortalidade nesse modelo experimental de caquexia do câncer. Em suma, a injeção de células do tumor Walker 256 na medula óssea induz caquexia do câncer em ratos. O treinamento de força não foi eficaz em atenuar a perda de massa e função da musculatura esquelética nesse modelo. Entretanto, a força muscular prediz a sobrevida dos animais, sugerindo que novos estudos são necessários para elucidar o possível efeito terapêutico do treinamento de força para atenuar a caquexia do câncer e a progressão tumoral / The lack of therapies for cachexia is a key problem in cancer treatment. In contrast, resistance exercise training (RET) has been adopted as nonpharmacological anti-catabolic strategy, preventing muscle wasting and muscle dysfunction. However, the role of RET to counteract cancer cachexia is still speculative. Presently, we test whether RET would counteract skeletal muscle wasting in a severe cancer cachexia rat model. Methods: Male Wistar rats were randomly assigned into four experimental groups; 1) untrained control rats injected with saline solution in the bone marrow (control), 2) rats injected with saline solution in the bone marrow and submitted to RET (control + RET), 3) untrained rats injected with Walker 256 tumor cells in the bone marrow (tumor) and 4) rats injected with Walker 256 tumor cells in the bone marrow and submitted to RET (tumor + RET). Skeletal muscle mass and fiber cross sectional area, markers of metabolic and protein turnover impairment, in vivo and ex vivo skeletal muscle function, food intake, tumor growth and mortality rate were assessed. Results: Tumor group displayed skeletal muscle atrophy fifteen days post tumor cells injection as assessed by Plantaris (-20.5%) and EDL (-20.0%) muscle mass. EDL atrophy was confirmed by histological analysis, showing 43.8% decline in the fiber cross sectional area. Even though RET increased the lactate dehydrogenase protein content and fully restored phosphorylated form of 4EBP-1 (i.e. a repressor of mRNA translation) to the control levels in skeletal muscle, it failed to rescue muscle morphology in tumorbearing rats. Indeed, RET has not mitigated loss of muscle function, anorexia, tumor growth or mortality rate. However, loss of strength capacity (assessed by 1-RM test performance) demonstrated a negative correlation with rats´ survival (p = 0.02), suggesting that loss of strength capacity predicts cancer mortality. Conclusions: Bone marrow injection of Walker 256 tumor cells in rats induces cancer cachexia. RET is ineffective to mitigate cancer-induced skeletal muscle wasting in this rat model. However, strength capacity predicts cancer survival, suggesting that new studies are needed to elucidate the putative therapeutic role of different exercise training regimens in counteracting cancer cachexia and tumor progression

Base Teórica: A artrite reumatoide (AR) é uma doença crônica e inflamatória que além de sintomas articulares pode levar à perda de massa muscular com peso estável ou aumentado, condição denominada caquexia reumatoide (CR). A CR está associada com pior prognóstico, mas ainda é negligenciada na prática clínica. Objetivo: Avaliar a prevalência de CR em um hospital público terciário de Porto Alegre e determinar sua correlação com características da AR, com níveis de atividade física e com as medicações em uso. Métodos: Estudo transversal com 91 pacientes com AR que foram submetidos à densitometria corporal total (DEXA) para medida total e regional de índice de massa gorda (IMG; Kg/m2), índice de massa magra (IMM; Kg/m2), conteúdo mineral ósseo (CMO) e índice de massa livre de gordura (IMLG; Kg/m2) para avaliar a prevalência de CR pelas duas definições mais recentemene utilizadas na literatura: IMLG < percentil 10 com IMG > percentil 25 e IMLG < percentil 25 com IMG > percentil 50. Foram exploradas as medidas de associação dos parâmetros de composição corporal com características da AR – idade, duração da doença, atividade de doença (através do DAS 28), capacidade funcional (através do HAQ), atividade inflamatória (através da proteína C reativa – PCR – e velocidade de hemossedimentação – VHS), nível de atividade física (através do questionário IPAQ) e medicações em uso. Resultados: A idade média dos participantes foi 56,8 ± 7,3, a duração de doença foi 9 anos (3-18), o DAS 28 3,65 ± 1,32, o HAQ 1,12 (0,25 – 1,87) e o tempo de uso entre os que usaram biológico foi de 25 meses (17,8 – 52,5). A CR foi evidenciada em 17,6% dos pacientes com AR de acordo com a definição mais rigorosa e em 33% de acordo com a classificação mais abrangente. O IMLG teve correlação negativa com idade (r= -0,219; p=0,037) e duração da doença (rs= -0,214; p=0,042). O IMG teve correlação positiva com PCR (rs=0,229; p=0,029), VHS (rs=0,235; p=0,025), DAS 28 (rs=0,273; p=0,009) e HAQ (rs=0,297; p=0,004). Na comparação de pacientes com e sem CR, de acordo com a definição mais rigorosa, dos 26 pacientes usando biológico apenas 1 tinha CR (3,8%), enquanto dos que não usavam, 15 (23%) tinham CR (p=0,033). Conclusão: A prevalência de CR foi considerável e, portanto, merece estudos adicionais. A composição corporal neste estudo, especialmente o IMLG, teve associação inversa com idade e tempo de diagnóstico. Além disso, pacientes em uso de biológico tiveram diferença significativa na prevalência de CR, sugerindo papel protetor do uso de biológico na CR. / Background: Rheumatoid arthritis (RA) is a chronic and inflammatory disease that besides articular symptoms leads to loss of muscle mass in presence of stable or increased fat mass (FM), condition defined as rheumatoid cachexia (RC). RC is associated with a worse prognosis, but it is still overlooked in clinical practice. Objective: To evaluate the prevalence of rheumatoid cachexia (RC) in patients with rheumatoid arthritis (RA) and determine its correlation with the features of RA, the level of physical activity and with the current therapy. Methods: Ninety one RA patients in a cross-sectional study underwent total body dual-energy x-ray absorptiometry (DXA) for measurement of total and regional fat mass index (FMI; Kg/m2), lean mass index (LMI; Kg/m2), bone mineral content (BMC; Kg/m2) and fat free mass index (FFMI; Kg/m2) to assess the prevalence of RC. The associations of measures of body composition with RA features - age, diagnosis time, Health Assessment Questionnaire (HAQ), Disease Activity Score in 28 joints (DAS 28), C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) -, level of physical activity (measured by International Physical Activity Questionnaire – IPAQ) and current therapy were explored. Results: Mean age was 56,8 ± 7,3 , disease duration 9 years (3 – 18), DAS28 3,65 ± 1,32, HAQ 1,12 (0,25 – 1,87) and use duration of biological agents was 25 months (17,8 – 52,5). Seventeen per cent of the patients had FFMI below the 10th percentile and FMI above the 25th percentile of a reference population and 33% of the patients had FFMI below the 25th percentile and FMI above the 50th percentile, condition known as RC, according to the more recently used definitions. FFMI correlated negatively only with age (r=-0,219; p=0,037) and disease duration (rs=-0,214; p=0,042). FMI correlated positively with CRP (rs=0,229; p=0,029), ESR (rs=0,235; p=0,025), DAS 28 (rs=0,273; p=0,009) and HAQ (rs=0,297; p=0,004). Of the 26 patients using biological therapy, 25 were non cachetic (p=0,033) according to the stricter definition of RC. In another words, 3,8% (n=1) and 23% (n=15) of the patients receiving and not receiving biological agents had RC, respectively (p=0,033). Conclusion: The prevalence of RC was considerable and deserves additional research. Body composition, in this study, particularly FFMI is inversely associated with age and disease duration. Besides that, patients under biological therapy had lower prevalence of RC, suggesting a protective effect of biological agents.

Selective neuronal loss in response to loss or dysfunction of a ubiquitously expressed protein is a hallmark of neurodegenerative disease. Proximal spinal muscular atrophy (SMA) is caused by homozygous loss of the ubiquitously expressed survival motor neuron 1 (SMN1) gene, resulting in progressive neuromuscular weakness that eventually leads to flaccid paralysis and death from respiratory failure by two years of age in the most severely affected patients. Despite widespread motor neuron loss, certain motor pools are clinically spared. Type I SMA patients exhibit intercostal recession in conjunction with diaphragmatic sparing that produces a characteristic “bell-shaped chest.” Additionally, patients retain extraocular and external sphincter function, even in late disease stages. In order to fully define this differential vulnerability, I performed an extensive characterization of neuromuscular autopsies from Type I SMA patients and age-matched control patients. I found highly divergent degrees of motor unit degeneration, even within individual cranial nerves or a select anatomical region such as the neck. Remarkably, the diaphragm in a Type I SMA patient kept alive on life support for 17 years was still relatively preserved, despite virtually complete fibro-fatty infiltration in other muscles. Extraocular functions were also normal in this patient. These findings suggest that the molecular determinants of SMA-resistance provide indefinite protection against low SMN protein. Thus, identification and modulation of these genes and pathways represents a promising potential therapeutic strategy. Remarkably, this exquisite pattern of selectivity was preserved in the SMNΔ7 mouse, a widely used SMA mouse model. This suggests that the molecular determinants of differential vulnerability are conserved between mouse and human. Given the high degree of diversity between motor pools, I performed a comparative transcriptional microarray between multiple SMA-vulnerable and –resistant motor pools in healthy mice. This analysis revealed a small number of candidate therapeutic genes that segregate closely with vulnerability. I present a series of preliminary studies evaluating these targets in the SMNΔ7 mouse. Ongoing and future studies combine pharmacological, viral, and genetic approaches to modulate these candidate targets in the SMNΔ7 mouse and assess for improvements in neuromuscular pathology. Given the remarkable preservation of select motor pools in SMA patients, changing expression levels of the candidate targets I have identified may provide substantial clinical benefit.

Neuronal circuit perturbations are emerging as important determinants in the pathogenesis of neurodegenerative diseases, including Alzheimer’s disease, Huntington’s disease, and spinal muscular atrophy (SMA). SMA is a motor neuron disease caused by deficiency in the ubiquitously expressed survival motor neuron (SMN) protein. The hallmarks of SMA include loss of motor neurons, muscle atrophy, and abnormal postural reflexes. Although cell-autonomous mechanisms of motor neuron death have received much attention, recent studies in animal models of SMA revealed that motor circuit deficits resulting from early impairment of synaptic function and sensory-motor connectivity precede motor neuron death. It remains to be established whether motor circuit dysfunction is a consequence of SMN-deficiency in the motor neuron or SMN-dependent alterations in the activity of premotor neurons. Here I sought to address these outstanding issues through the development and characterization of a simplified in vitro model of the motor circuit based on the use of embryonic stem cell-derived motor neurons and interneurons. I found that SMN deficiency caused death of motor neurons in co-culture with other neurons as well as in isolation, demonstrating the cell autonomous origin of this defect. SMN requirement for motor neuron function was investigated using intracellular patch clamp recordings to measure both passive and active membrane properties. Remarkably, SMN deficiency induced hyperexcitability of motor neurons only when they are cultured in the presence of interneurons but not in isolation, providing initial evidence that SMN deficiency induces motor neuron hyperexcitability in a non-cell autonomous manner and that dysfunction and death of motor neurons are uncoupled. To determine the role of SMN-dependent interneuron dysfunction on motor neuron hyperexcitability, the effect of selective SMN depletion in either motor neurons or interneurons was investigated. Importantly, I found that SMN-deficient motor neurons cultured in the presence of wild type interneurons are not hyperexcitable, while the presence of SMN-deficient interneurons is necessary and sufficient to elicit hyperexcitability of wild type motor neurons. Therefore, in the context of SMN deficiency, increased excitability of motor neurons is a homeostatic response to interneuron dysfunction. Although the exact mechanism is currently unknown, reduced glutamatergic drive appears to play a role since glutamatergic receptor blockers phenocopied SMN deficiency in inducing motor neuron hyperexcitability but not neuronal death. Moreover, SMN deficiency caused reduction of excitatory VGluT2 synapses on motor neurons. In addition to changes in intrinsic membrane properties, SMN deficiency caused severe reduction in the spontaneous activity and firing pattern of motor neurons. However, in contrast to death and hyperexcitability, SMN-dependent deficits in both motor neurons and interneurons appear to underlie this complex phenotype. The findings presented in this study validate the use of in vitro models to study SMA disease mechanisms and shed new light on the cellular basis of motor circuit dysfunction induced by SMN deficiency that can have predictive value in vivo.

Neste trabalho, avaliamos os efeitos da suplementação crônica de leucina e do treinamento de força sobre a miopatia diabética. 40 ratos machos da linhagem Wistar Hannover foram distribuídos em 5 grupos: controle, não diabético (C), diabético não tratado (D), diabético treinado (DT), diabético suplementado com leucina e treinado (DLT). O início das intervenções ocorreu na 4ª semana de vida dos animais, e perdurou por 8 semanas. Foram avaliados: massa corporal, consumo de ração e água, concentrações sanguíneas de glicose, insulina e perfil lipídico; capacidade funcional muscular voluntária através de testes de força de preensão e de ambulação; conteúdo intracelular de proteínas relacionadas à via anabólica mTOR e p70S6K, totais e fosforiladas, no músculo extensor longo dos dedos. Os ratos diabéticos não tratados (grupo D) apresentaram hiperglicemia e hipoinsulinemia moderada, menor massa corporal, maior consumo de ração e água, menor peso absoluto dos músculos extensor longo dos dedos e gastrocnêmio, menor força de preensão, menor capacidade de ambulação e menor atividade das proteínas mTOR e p70S6K comparado ao grupo C, o que caracteriza o quadro de miopatia diabética. O peso relativo do músculo gastrocnêmico (peso absoluto/100g de peso do animal) foi maior nos grupos DT e DLT comparado com o grupo D, e maior no grupo DLT comparado com o grupo DL (p < 0,05). Não houve diferença estatística entre os grupos DL e D sobre os pesos relativos dos músculos, ou seja, a suplementação crônica de leucina não afetou este parâmetro nos ratos diabéticos. Interessantemente, houve diferença estatística entre os grupos DL e D sobre a força muscular (p < 0,05), sem haver diferença entre grupos DL e C quanto à glicemia; ou seja, a dieta suplementada com leucina foi capaz de controlar a glicemia e atenuar a perda de força muscular. O treinamento de força também controlou a glicemia, recuperou a força muscular e melhorou a capacidade de ambulação, bem como a regulação da via mTOR-p70S6K. A fosforilação da via mTOR-p70S6K foi maior nos grupos DT e DLT comparado com o grupo D (p < 0,05), e sem diferença entre estes grupos treinados e o grupo C, sugerindo que o treinamento de força combinado com a suplementação de leucina recuperou a atividade da via do mTOR-p70S6K nos animais diabéticos, que pode refletir em maior síntese proteica muscular. O colesterol total do grupo D foi maior comparado com o do grupo C; e nos grupos diabéticos treinados (DT e DLT), este parâmetro foi menor do que no grupo D (p < 0,05). Adicionalmente, o HDL-c aumentou nos grupos treinados (DT e DLT) quando comparado com o grupo D, mas não alterou no grupo que recebeu apenas a suplementação de leucina (grupo DL). Portanto, neste estudo, a suplementação crônica de leucina por si só normalizou a glicemia e melhorou a força muscular dos animais diabéticos. Além disso, o treinamento de força foi responsável pelo maior aumento de força e da massa muscular, bem como pela normalização da glicemia, pela elevação da concentração de HDL-c e pela redução do colesterol total dos animais diabéticos e ambas foram capaz de recuperar a via mTOR-p70S6K. / In this study, we evaluated the effects of chronic supplementation with leucine and resistance training on diabetic myopathy. 40 Wistar Hannover rats were divided into 5 groups: control, non-diabetic (C), untreated diabetic (D), trained diabetic (DT), diabetic supplemented with leucine and trained (DLT). The beginning of the interventions occurred in the 4th week of life of the animals, and lasted for 8 weeks. Were evaluated: body weight, food and water intake, blood concentrations of glucose, insulin and lipid profile; voluntary muscle functional capacity through grip strength and ambulation test; intracellular content of proteins related to the anabolic mTOR and p70S6K pathway, total and phosphorylated in the extensor digitorum longus muscle. Diabetics untreated mice (group D) had hyperglycemia and moderate hypoinsulinemia, lower body mass, food and water intake, reduced absolute weights of the muscles of the long extensor digitorum, and gastrocnemius, the lower grip strength, lower ambulation capacity and lower activity of mTOR and p70S6K protein compared the C group, featuring diabetic myopathy. The relative weight of the gastrocnemius muscle (absolute weight / 100g of body weight) was greater in DT and DLT groups compared with group D, and higher in the DLT group compared to the DL group (P < 0.05). No statistical difference between the DL and D groups on the relative weights of the muscles, that is, chronic supplementation of leucine did not affect this parameter in diabetic rats. Interestingly, there was statistical difference between the DL and D groups on muscle strength (P < 0.05), with no difference between groups DL and C on the blood glucose; that is, the diet supplemented with leucine was able to control glycemia and avoid loss of muscle strength of diabetic animals. Resistance training also controlled glycemia, recovered muscle strength and improved the capacity of ambulation of diabetic animals and the regulation of the mTOR-p70S6K pathway. The phosphorylation of mTOR-p70S6K pathway was higher only in the DT and DLT groups compared with the D group (P < 0.05), and no difference between the DT and C groups, suggesting that the training recovered muscle mass in diabetic animals. Total cholesterol was greater in Group D compared to the group C; and trained diabetic groups (DLT and DT), this parameter was lower than that of the D group (P < 0.05). In addition, HDL-C increased in trained groups (DT and DLT) as compared to group D, but had no effect the group that received only leucine supplementation (DL group). Therefore, in this study, chronic supplementation of leucine alone normalized glucose and improved muscle strength of diabetic animals. In addition, resistance training was responsible for the largest increase in strength and muscle mass, as well as the normalization of glucose, elevated concentrations of HDL-C and reduction in total cholesterol of animals diabetics and both were able to recover mTOR- p70S6K pathway.

Orientador: Athanase Billis / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-11-07T16:10:17Z (GMT). No. of bitstreams: 1 Worschech_Adriana_M.pdf: 2136837 bytes, checksum: e169d43b81c649e3dde44c36f7a96278 (MD5) Previous issue date: 2008 / Resumo: A atrofia parcial (AP) é uma lesão benigna que mais freqüentemente imita adenocarcinoma, particularmente a variante parcial. AP ocorre com maior freqüência no lobo posterior ou zona periférica e ganhou importância maior com o uso das biópsias por agulha na detecção do carcinoma prostático. A atrofia parcial e a hiperplasia pós-atrófica (atrofia hiperplásica) são as lesões benignas que mais freqüentemente são confundidas com adenocarcinoma. Uma das razões que contribuem para dificultar o diagnóstico da atrofia parcial está relacionada com a ausência de células basais em alguns ácinos. Mais recentemente a aplicação da molécula de AMACR (alfa-metilacil Co-enzima A racemase) como marcador de células neoplásicas através de imunoistoquímica tem auxiliado no diagnóstico diferencial com o adenocarcinoma. Entretanto, sua aplicação na rotina diagnóstica ainda não está estabelecida. A imunoexpressão da AMACR pode causar algumas dúvidas em sua interpretação. Na literatura existem poucos estudos que relatam a expressão da AMACR em atrofia parcial. Avaliamos através da imunoistoquímica a expressão da AMACR e do 34ßE12 (citoqueratina de alto peso molecular) através do coquetel P504S+34ßE12 em material proveniente de 74 biópsias por agulha de próstata correspondendo a 61 pacientes. Foram analisados um total de 1198 ácinos prostáticos (324 ácinos com adenocarcinoma, 213 ácinos normais, 190 ácinos com atrofia parcial, 298 ácinos com hiperplasia pós-atrófica, 139 ácinos com atrofia simples e 34 ácinos com atrofia esclerosante). Nos ácinos com adenocarcinoma a intensidade da marcação da AMACR foi forte em 251/324 (77.5%) e fraca 73/324 (22.5%). Não houveram casos negativos. Nos ácinos normais observou-se marcação para a AMACR forte em 13/213 (6.1%), fraca em 33/213 (15.5%) e negativa em 167/213 (78.4%). A atrofia parcial apresentou marcação para a AMACR fraca em 47/190 (24.7%) e negativa em 143/190 (75.3%). Não houve marcação forte em nenhum dos casos de atrofia parcial. Os ácinos normais mostraram expressão para AMACR negativo, fraco e forte onde os valores foram respectivamente 167/213 (78,4%), 33/213 (15,5%) e 13/213 (6,1%). A atrofia parcial mostrou-se negativa, e fraca para imunoexpressão da AMACR em 143/190 (75,3%) e 47/190 (24,7%) respectivamente. Não foi observada forte positividade em atrofia parcial, no entanto, a fraca positividade observada em cerca de 25% dos ácinos pode causar dificuldade para a interpretação correta no diagnóstico diferencial de câncer e atrofia parcial. A AMACR foi negativa em todos os ácinos da atrofia simples, hiperplásica (ou hiperplasia pós-atrófica) e esclerosante, por conseguinte, sem qualquer ajuda no diagnóstico diferencial de adenocar-cinoma. A distribuição das células basais, observadas na atrofia simples, hiperplásica e esclerosante foram descontínuas e as células do compartimento secretor mostraram imunoexpressão aberrante de 34ßE12 sugerindo um fenótipo intermédio. Analisando-se os estes resultados conclui-se que o diagnóstico diferencial do adenocarcinoma com atrofia parcial deve ser feito com cautela considerando-se que a expressão da AMACR, apesar de fraca em nosso estudo, pode ocorrer em cerca de 25% dos ácinos. Soma-se a este achado o fato de que em 23.2% dos ácinos de atrofia parcial as células basais estão ausentes. Estes dados impõem cautela no difícil diagnóstico diferencial de pequenos focos "suspeitos, mas não diagnósticos de adenocarcinoma da próstata", sendo que, em alguns casos, os critérios puramente morfológicos poderão ser os únicos na identificação da lesão. / Abstract: Prostatic atrophy (PA) is the benign lesion that most frequently mimicks adenocarcinoma particularly the partial variant. PA occurs more frequently in the peripheral zone and gained greater importance with the use of needle biopsies in detecting cancer of the prostate. Partial atrophy and post-atrophic hyperplasia (hyperplastic atrophy) are the benign lesions that most often are confused with adenocarcinoma. One of the reasons that contribute to make the diagnosis of partial atrophy difficult is related to the absence of basal cell in some acini. More recently the application of AMACR (alpha-metilacil Co-enzyme A racemase) as a marker of malignant cells through immunohistochemistry has helped in the differential diagnosis with prostate cancer. However, its application in routine diagnosis is not yet established. The immunoexpression of AMACR may cause some doubt in interpretation. In literature there are few studies that reported the expression of AMACR in partial atrophy. We evaluated by immunohistochemistry the expression of AMACR and 34ßE12 (cytokeratin high-molecular weight) using the cocktail P504S +34ßE12 in 74 needle prostatic biopsies corresponding to 61 patients. We analyzed a total of 1198 prostate acini (324 acini with adenocarcinoma, 213 normal acini, 190 acini with partial atrophy, 298 acini with post-atrophic hyperplasia, 139 acini with simple atrophy and 34 acini with sclerosing atrophy). In adenocarcinoma acini the staining of AMACR was strong in 251/324 (77.5%) and weak in 73/324 (22.5%). There were no negative acini. In normal acini AMACR was strong in 13/213 (6.1%), weak in 33/213 (15.5%) and negative in 167/213 (78.4%). In partial atrophy, acini showed weak AMACR in 47/190 (24.7%) and were negative in 143/190 (75.3%). There was no strong staining in partial atrophy. The immunoexpression of AMACR was negative in all variants of complete atrophy: simple atrophy, hyperplastic atrophy and sclerosing atrophy. Normal acini showed negative, weak, or strong expression in 167/213 (78.4%), 33/213 (15.5%), and 13/213 (6.1%) acini, respectively. Partial atrophy showed negative, and weak expression in 143/190 (75.3%), and 47/190(24.7%) acini, respectively. No strong positivity was seen in partial atrophy, however, the weak positivity seen in approximately 25% of the acini may be a pitfall for the correct interpretation in the differential diagnosis of cancer and partial atrophy. AMACR was negative in all acini of simple, postatrophic hyperplasia and sclerosing atrophy, therefore, with no help in the differential diagnosis of adenocarcinoma. The distribution of basal cells in simple, postatrophic hyperplasia and sclerotic atrophy was discontinuous and the cells of the secretory compartment showed aberrant expression of 34ßE12 suggesting an intermediate phenotype. Analyzing these results it is concluded that the differential diagnosis of prostate cancer with partial atrophy must be done carefully considering that the expression of AMACR, although weak in our study, can occur in about 25% of the acini. Furthermore, in 23.2% acini of partial atrophy the basal cells are absent. In some cases the microscopic identification of partial atrophy will rely only on morphologic criteria. / Mestrado / Ciencias Biomedicas / Mestre em Ciências Médicas

Rotavírus (RV) é um importante patógeno viral que causa diarreia em leitões e indivíduos jovens de várias outras espécies animais. RV dos grupos A, B, C e E foram descritos como causa de diarreia em suínos. Este estudo reúne achados histopatológicos, imunohistoquímicos e de reação em cadeia da polimerase com transcriptase reversa (RT-PCR) presentes em quatro surtos de diarreia causados por RV de um e de múltiplos grupos na região sul do Brasil. Vacinação para RV não era aplicada em nenhuma das granjas estudadas. Necropsia, exames histológicos e imuni-histoquímicos foram realizados em 34 suínos de maternidade que apresentavam diarreia severa, além disso, realizou-se cultivo bacteriano e RT-PCR para RV dos grupos A, B e C, vírus da gastroenterite transmissível (TGEV), vírus da diarreia epidêmica dos suínos (PEDV), sapovírus (SaV), norovírus (NoV) e kobuvírus (Aichi vírus C) em 30 dessas amostras. Desidratação e conteúdo pastoso a líquido no cólon foram observados em todos os suínos. Exame histológico revelou atrofia de vilosidades em 29 casos, vacuolização de enterócitos em 27 casos e debris celulares na lâmina própria em 20 casos. Houve marcação imunohistoquímica positiva em 21 casos. RT-PCR foi positiva para RV em 20 casos e RV do grupo C foi o mais frequentemente detectado, presente em 17 amostras. Cultivo e isolamento de Escherichia coli ocorreu em todos os casos e quatro destes foram de E. coli α-hemolítica. Em 15 amostras houve isolamento de Clostridium sp. Sapovirus foi detectado em oito amostras, duas amostras foram positivas para norovírus e detectou-se kobuvírus em 11 animais. Os achados histológicos foram consistentes com infecção por RV e a imuno-histoquímica revelou dois padrões de marcação para o agente no intestino delgado. Os resultados de RT-PCR mostraram que o RV do grupo C foi o principal agente detectado neste estudo. O isolamento de E. coli e a detecção de SaV os destacou como agentes associados à infecção por RV. A detecção de kobuvírus o enfatiza como um novo candidato em associação com RV. / Rotavirus is an important viral pathogen causing diarrhea in piglets and other animal species worldwide. Groups A, B, C and E have been described causing diarrhea in swine. This study reunites histopathological, immunohistochemical and reverse transcriptase polymerase chain reaction (RT-PCR) findings present in four outbreaks of diarrhea caused by single and multiple groups of rotavirus in the south of Brazil. None of the herds studied applied vaccination against rotavirus. Necropsy, histological examination and immunohistochemistry were performed in 34 nursing piglets that presented severe diarrhea, bacterial culture and reverse transcriptase polymerase chain reaction (RT-PCR) for rotavirus from groups A, B and C, transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), sapovirus (SaV), norovirus (NoV) and kobuvirus (Aichi virus C) were carried out in 30 of the animals necropsied. Additionally, RT-PCR was performed in fecal pools from two outbreaks. Dehydration and fluid to pasty contents were observed in the colon of all 34 swine examined. Histological examination revealed villus atrophy in 29 cases, vacuolation of enterocytes in 27 cases and necrotic debris in the lamina propria of 20 cases. IHC was positive in 21 samples. RT-PCR was positive for rotavirus in 20 samples and group C rotavirus was the most frequently detected, present in 17 samples. Escherichia coli was isolated from all cases, and in four cases, it was α-hemolytic. Clostridium sp. was isolated from 15 samples. Sapovirus was detected in eight samples, samples from two animals were positive for norovirus and kobuvirus was detected from 11 samples. Histological findings were consistent with rotavirus infection and immunohistochemistry revealed two patterns of staining for rotavirus in the small intestine. RT-PCR results have shown group C rotavirus as the main agent detected in this study. The isolation of Escherichia coli and the detection of sapovirus highlighted them as possible agents associated to rotavirus infection. Kobuvirus detection has emphasized it as a new candidate in the association with rotavirus.

Estudos preliminares através de microarray nos mostraram que a E3 ligase Mdm2 foi regulado positivamente no músculo de animais hipertireoideos. Dessa forma, nós inferimos uma possível relação de Mdm2 com a atrofia causada por T3. Para testar nossa hipótese, ratos foram induzidos ao hipertireoidismo para análises subsequentes. Concomitante com a perda de massa muscular foi confirmado um aumento da expressão de Mdm2 tanto no nível gênico (p<0.05) quanto protéico. Interessantemente, Mdm2 foi preferencialmente expresso em fibras tipo I, mostrando maior sensibilidade dessas fibras ao T3. Além disso, foi observado uma diminuição severa na expressão de Pax7/MyoD associado à superexpressão de Mdm2, sugerindo inatividade das células satélites. Surpreendentemente, a inibição de Mdm2 em miotubos cultivados provocou uma diminuição severa no diâmetro destes (~35%, p<0.05), ou seja, tal inibição foi incapaz de minimizar a proteólise muscular causada por T3. Portanto, nós concluímos que a responsividade de Mdm2 ao T3 agiria como um mecanismo compensatório numa tentativa de minimizar a proteólise muscular causada pelo hipertireoidismo. Esta conclusão é reforçada pela atrofia observada em miotubos durante a inibição de Mdm2 sem a presença de T3. / Previous studies in our lab through microarray assay observed Mdm2, an E3 ligase, up regulated in soleus muscle from hyperthyroid rats. In this sense, we inferred that Mdm2 could be related to muscle atrophy caused by T3. To test our hypothesis, rats were induced to experimental hyperthyroidism for subsequent analysis. Along the muscle mass loss, the increase on Mdm2 gene expression was confirmed (p<0.05) as well as protein expression by RT-PCR and Western Blot, respectively. Interestingly, Mdm2 was expressed predominantly in fiber I type during T3 treatment, demonstrating a higher sensibility when compared to type II fiber. Moreover, it was observed a severe decrease in Pax7/MyoD labeling, associated to an increase on Mdm2 labeling, suggesting that T3 could be associated with inactivation of satellite cells. Surprisingly, Mdm2 inhibition in myotubes have induced severe decrease on myotubes diameter (~35%, p<0.05), in other words, Mdm2 inhibition was not able to decrease muscle proteolysis during high levels of T3. Thus, the increase on Mdm2 levels could be a compensatory effect to reduce the muscle mass loss during T3 treatment. This conclusion is highlighted by the myotubes atrophy observed during the Mdm2 inhibition without T3 treatment.

Nosso objetivo foi investigar o efeito da suplementação de leucina na via intracelular acionada por miostatina, uma proteína reguladora negativa da massa muscular, em um modelo de atrofia gerada por imobilização em ratos. Nossa hipótese inicial contemplava que a expressão de miostatina se elevaria com a imobilização e que a leucina poderia atenuar esse aumento. Nossos resultados, entretanto, mostraram uma regulação gênica no sentido de suprimir a via da miostatina. A suplementação com leucina, associada à imobilização, também provocou queda da expressão gênica de miostatina. Em ambos os grupos a expressão proteica permaneceu inalterada. Em contrapartida, a folistatina, inibidor endógeno da miostatina, apresentou acentuado aumento de expressão no grupo imobilizado e imobilizado/suplementados com leucina. Nossos resultados mostraram que o efeito protetor da leucina no modelo atrófico utilizado não se dá através da sinalização da miostatina. Os dados também sugerem uma resposta biológica coordenada entre o ligante, miostatina, e seu inibidor endógeno, a folistatina. / Our purpose was to investigate the effect of leucine supplementation upon the intracellular pathway triggered by myostatin, a negative regulator of muscle mass, in an atrophic model driven by immobilization in rats. Our initial hypothesis contemplated that myostatin expression would be elevated under immobilization and that leucine supplementation would be able to attenuate this raise. However our results showed a strong downregulation of myostatin gene expression in atrophy induced by cast. The immobilization associated with leucine supplementation also caused mRNA myostatin downregulation. Protein levels were unchanged. On the other hand, follistatin, a myostatin endogenous inhibitor, was markedly upregulated in both immobilized and immobilized/supplemented groups. Our results revealed that leucine protective effect in this atrophic model is not through myostatin signailing. Furthermore, these data suggest a coordinated biological response between ligand, myostatin, and its endogenous inhibitor, follistatin.

Skeletal muscle atrophy is a common, debilitating consequence of muscle disuse, malnutrition, critical illness, musculoskeletal conditions, neurological disease, cancer, and organ failure. Despite its prevalence, little is known about the molecular pathogenesis of this devastating condition due in large part to an incomplete understanding of the molecular mechanisms that drive the atrophy process. In previous studies, we identified the transcription factor ATF4 as a critical mediator of skeletal muscle atrophy. We found that ATF4 is necessary and sufficient for skeletal muscle atrophy during limb immobilization. However, ATF4 mKO mice were only partially protected from skeletal muscle atrophy during limb immobilization, indicating the existence of another pro-atrophy factor that acts independently of the ATF4 pathway. Using mouse models, we identify p53 as this ATF4-independent factor. We show that skeletal muscle atrophy increases p53 expression in skeletal muscle fibers. In addition, overexpression of p53 causes skeletal muscle atrophy. Further, p53 mKO mice are partially resistant to muscle atrophy during limb immobilization. Taken together, these data indicate that like ATF4, p53 is sufficient and required for skeletal muscle atrophy during limb immobilization. Importantly, overexpression of p53 induces muscle atrophy in the absence of ATF4, whereas ATF4-mediated muscle atrophy does not require p53. Furthermore, overexpression of p53 and ATF4 induces greater muscle atrophy than p53 or ATF4 alone. Moreover, skeletal muscle lacking both p53 and ATF4 is more resistant to skeletal muscle atrophy than muscle lacking either p53 or ATF4 alone. Taken together, these data indicate that p53 and ATF4 mediate distinct and additive mechanisms to skeletal muscle atrophy. However, the precise mechanism by which p53 and ATF4 cause skeletal muscle atrophy remained unclear. Using genome-wide expression arrays, we identify p21 as a skeletal muscle mRNA that is highly induced by p53 and ATF4 during limb immobilization. Further, overexpression of p21 causes skeletal muscle atrophy. In addition, p21 is required for muscle atrophy due to limb immobilization, p53, and ATF4. Collectively, these results identify p53 and ATF4 as critical and complementary mediators of skeletal muscle atrophy during limb immobilization, and discover p21 as an essential downstream mediator of the p53 and ATF4 pathways.