Period 2 (Per2) is a core circadian factor responsible for its own negative regulation. It operates in the circadian clock, which affects multiple biological functions such as metabolic rate, hormone release, and core body temperature. The Per2 protein functions directly with factors in other biological functions such as tumor suppression, immune system, and metabolism. In many cases, the Per2 deficiency caused by disrupted expression is sufficient to create severe abnormalities in many of the mentioned functions. The sequence contains several domains and motifs in Per2 that are traditionally involved in protein interactions which suggests that Per2 serving a regulatory role by effecting downstream biological roles dependent on Per2 stability. In this work, we perform a two-hybrid screening assay using the C-terminal region of human Per2 and identified an extensive number of interactors. Utilizing a genetic ontology program, we assorted the list of clones into groups of proteins that are biologically relevant or operated in similar function. Through this program, we validated the two-hybrid screening by the clusters of biological function already attributed to hPer2 and identified new putative biological functions. We use the new putative interactors to gain further insight on the regulatory roles that hPer2 performs, in conjunction with operating as a core factor in circadian rhythmicity. We also show that Translationally Controlled Tumor Protein (TCTP) is capable of binding to hPer2 and is a novel interaction. When a sufficient amount of TCTP (1:1 molar stoichiometric ratio) is present in a system, a cleavage of hPer2 is observed in vitro. This cleavage occurs in reactions independent of ATP, ubiquitin, and the proteasome. The data points towards a method of cleavage similar to that of the archael lon-tk (Thermococcus kodakaraensis) that preferentially cleaved unstructured substrates in ATP-independent reactions. / Master of Science

Transfer ribonucleic acids (tRNAs) are extensively modified, especially their anticodon loops. Modifications at position 34 (wobble base) and 37 in these loops affect the tRNAs’ decoding ability, while modifications outside the anticodon loops, e.g. m1A58 of tRNAMeti, may be crucial for tRNA structure or stability. A number of gene products are required for the formation of modified nucleosides, e.g. at least 26 proteins (including Elongator complex) are needed for U34 modifications in yeast, and methyl transferase activity of the Trm6/61p complex is needed to form m1A58. The aim of the studies which this thesis is based upon was to investigate the functional aspects of tRNA modifications and regulation of the modifying enzymes’ activity. First, the hypothesis that ncm5U34, mcm5U34, or mcm5s2U34 modifications may be essential for reading frame maintenance was investigated. The results show that mcm5 and s2 group of mcm5s2U play a vital role in reading frame maintenance. Subsequent experiments showed that the +1 frameshifting event at Lys AAA codon occurs via peptidyl-tRNA slippage due to a slow entry of the hypomodified tRNA-Lys. Moreover, the hypothesis that Elp1p N-terminal truncation may regulate Elongator activity was investigated. Cleavage of Elp1p was found to occur between residue 203 (Lys) and 204 (Ala) and to depend on the vacuolar protease Prb1p. However, including trichloroacetic acid (TCA) during protein extraction abolished the appearance of truncated Elp1p, showing that its truncation is a preparation artifact. Finally, in glioma cell line C6, PKCα was found to interact with TRM61. RNA silencing of TRM6/61 causes a growth defect that can be partially suppressed by tRNAMeti overexpression. PKCα overexpression reduces the nuclear level of TRM61, likely resulting in reduced level of TRM6/61 complex in the nucleus. Furthermore, lower expression of PKCα in the highly aggressive GBM (relative to its expression in less aggressive Grade II/III glioblastomas) is accompanied by increased expression of TRM6/61 mRNAs and tRNAMeti, highlighting the clinical relevance of the studies.

<p>Protein biomarkers can facilitate the diagnosis of many diseases such as cancer and they can be important for the development of effective therapeutic interventions. Current large-scale biomarker discovery and disease state characterization studies have largely focused on the global analysis of gene and protein expression levels, which are not directly tied to function. Moreover, functionally significant proteins with similar expression levels go undetected in the current paradigm of using gene and protein expression level analyses for protein biomarker discovery. Protein-ligand interactions play an important role in biological processes. A number of diseases such as cancer are reported to have altered protein interaction networks. Current understanding of biophysical properties and consequences of altered protein interaction network in disease state is limited due to the lack of reproducible and high-throughput methods to make such measurements. Thermodynamic stability measurements can report on a wide range of biologically significant phenomena (e.g., point mutations, post-translational modifications, and new or altered binding interactions with cellular ligands) associated with proteins in different disease states. Investigated here is the use of thermodynamic stability measurements to probe the altered interaction networks and functions of proteins in disease states. This thesis outlines the development and application of mass spectrometry based methods for making proteome-wide thermodynamic measurements of protein stability in multifactorial complex diseases such as cancer. Initial work involved the development of SILAC-SPROX and SILAC-PP approaches for thermodynamic stability measurements in proof-of-concept studies with two test ligands, CsA and a non-hydrolyzable adenosine triphosphate (ATP) analogue, adenylyl imidodiphosphate (AMP-PNP). In these proof-of-principle studies, known direct binding target of CsA, cyclophilin A, was successfully identified and quantified. Similarly a number of known and previously unknown ATP binding proteins were also detected and quantified using these SILAC-based energetics approaches. </p><p>Subsequent studies in this thesis involved thermodynamic stability measurements of proteins in the breast cancer cell line models to differentiate disease states. Using the SILAC-SPROX, ~800 proteins were assayed for changes in their protein folding behavior in three different cell line models of breast cancer including the MCF-10A, MCF-7, and MDA-MB-231 cell lines. Approximately, 10-12% of the assayed proteins in the comparative analyses performed here exhibited differential stability in cell lysates prepared from the different cell lines. Thermodynamic profiling differences of 28 proteins identified with SILAC-SPROX strategy in MCF-10A versus MCF-7 cell line comparison were also confirmed with SILAC-PP technique. The thermodynamic analyses performed here enabled the non-tumorigenic MCF-10A breast cell line to be differentiated from the MCF-7 and MDA-MB-231 breast cancer cell lines. Differentiation of the less invasive MCF-7 breast cancer cell line from the more highly invasive MDA-MB-231 breast cancer cell line was also possible using thermodynamic stability measurements. The differentially stabilized protein hits in these studies encompassed those with a wide range of functions and protein expression levels, and they included a significant fraction (~45%) with similar expression levels in the cell line comparisons. These proteins created novel molecular signatures to differentiate the cancer cell lines studied here. Our results suggest that protein folding and stability measurements complement the current paradigm of expression level analyses for biomarker discovery and help elucidate the molecular basis of disease.</p> / Dissertation

Différents facteurs sont connus pour affecter les paramètres de qualité du foie gras. Parmi ces paramètres, la fonte est considérée comme étant la plus critique du point de vue des producteurs et des consommateurs. Outre les facteurs déjà connus, le but de ce travail est d’étudier si les activités protéolytiques hépatiques pourraient jouer un rôle dans le déterminisme de la qualité du foie gras. Pour atteindre cet objectif, trois expérimentations ont été menées, dans lesquelles nous avons utilisé des approches biochimiques et protéolytiques ainsi que des mesures de qualité. Dans ces trois expériences nous avons évalué : 1) l’effet du gavage chez deux types génétiques de canards, 2) l’effet de la vitesse de refroidissement et du stockage post mortem et 3) l’effet de la variabilité de taux de fonte sur les activités protéolytiques. Nos résultats montrent que les activités protéolytiques restent constantes ou baissent avec le gavage. De plus, malgré des différences connues quant à leur capacité à développer une stéatose hépatique, les canards de Barbarie et les canards Pékin ont réagis de la même façon en ce qui concerne leurs activités protéolytiques. En ce qui concerne l’effet des différentes vitesses de refroidissement, nous avons montré que plus le refroidissement est lent, plus le foie fond et que ceci est –au moins partiellement- due à des activités protéolytiques plus élevées. Finalement, nous avons montré que les foies à fonte élevée présentés également des activités protéolytiques supérieures à celles des foies à fonte faible. Dans cette thèse, nous avons pu mettre en évidence certaines relations existantes entre la protéolyse hépatique et les qualités du foie gras de canard. Cependant, d’autres études seront nécessaires pour mieux comprendre les mécanismes responsables de ces relations dans le but d’exploiter le potentiel des protéases dans la maîtrise de la qualité du foie gras. / Different factors are known to affect the quality parameters of « foie gras ». Among those parameters, the cooking loss is considered to be the most critical one in the eyes of both producers and consumers. Besides already known factors, the objective of this work is to investigate whether proteolytic activities might play a role in the determinism of « foie gras» cooking losses. To achieve this objective three experiments were conducted in which we used biochemical and proteomic approaches as well as quality analysis. In these three different experiments we evaluated: 1) the effect of overfeeding in two breeds of ducks (Muscovy and Pekin ducks) 2) the effect of chilling rate and post mortem storage and 3) the effect of cooking loss variability on proteolytic activities. Our results show that overfeeding either does not affect proteases activities, either causes their decrease. In addition, despite the known differences regarding their ability to develop hepatic steatosis, in our study Muscovy and Pekin ducks’ proteolytic activities responded similarly to overfeeding. Concerning the effect of chilling rates, we showed that the slower the chilling rate the higher the melting after cooking. This result is - at least partially- due to higher proteolytic activities. Finally we showed that livers with a high melting rate presented higher proteolytic activities than those with a low melting rate. In this thesis, we were able to expose certain relations between hepatic proteolysis and fatty liver qualities. However, more studies are required to better understand the mechanisms underlying these relations in the goal of exploiting the potential of proteases in fatty liver quality control.

Ensiling high moisture grain often increases starch and protein digestibilities due proteolysis during storage. However, the length of storage is fundamental to allow great protein matrix break down. The central objective of this work was to evaluate the effect of length of storage of reconstituted sorghum grain silage (RSGS) on dairy cows performance. Simultaneously it was evaluated the effect of sodium benzoate on silage nutritive value and its impact on animal performance. The hypothesis was that sodium benzoate reduces proteolitic activity due its antimicrobial properties. Two sequential experiments with mid-lactation Holstein dairy cows were set. The first experiment evaluated the effect of different length of storages on RSGS treated or not with sodium benzoate (0.2% as fed). Silages treated with additive (Benzoate) and non-treated (Control) were stored for 30 or 90 days prior feeding. Twenty mid-lactation dairy cows with 168 &#177; 87 days in milk (DIM) were used in 5 replicated 4 × 4 Latin square design with a 2 × 2 factorial arrangement. Dry ground sorghum grain was reconstituted to 35% moisture and ensiled in 200-L plastic drums. Treatments were: RSGS stored for 30 days without additive (30 CON), RSGS stored for 30 days with sodium benzoate (30 BEN), RSGS stored for 90 days without additive (90 CON) and RSGS stored for 90 days with sodium benzoate (90 BEN). Lengthening silage storage increased 1,2-propanediol concentration and protein solubility of silages. Milk yield was greater in favor of cows fed silage stored for 90 days compared to 30 days (31.2 vs. 30.0 kg/d). Starch (89.3 vs. 86.9%) and protein (57.1 vs. 54.0%) digestibility was also greater for silages stored for 90 days compared to 30 days. Sodium benzoate reduced silage ethanol concentration (0.20 vs. 0.08% of DM), but did not altered statistically protein solubility (CON = 18.9 vs. BEN = 15.6% of CP) or ammonium-N (CON = 4.38 vs. BEN = 3.94 % of N). The second trial were conducted with 12 mid-lactation dairy cows (170 &#177; 47 DIM) to evaluated the effect of sodium benzoate on nutritive value and dairy cows performance fed RSGS stored for 150 days, treated (Benzoate) or not (Control) with sodium benzoate. Cows received a standard diet containing dry ground sorghum for 14 days. At the end of adaptation period, cows were paired blocked and randomly assigned to one of two treatments (Control or Benzoate) for 28 experimental days. During experimental period cows received the same diet with the exception of dry ground sorghum, which was totally replaced with RSGS. Silages treated with sodium benzoate had low ethanol (0.84 vs. 0.18 % of DM) and ethyl-lactate (388 vs. 157 mg/kg of DM) concentration as a consequence of low yeast population (4.73 vs. 2.52 log cfu/g). Soluble protein was reduced on treated silages (26.2 vs. 20.6 % of CP). Aerobic stability was higher for treated silages (51 vs. 146 h). Dairy cow performance was not altered by treating silages with sodium benzoate. In conclusion, extending the length of storage of RSGS increased dairy cows use feed efficient and nitrogen use efficiency due higher starch and protein digestibility. Sodium benzoate promoted typical response on silage fermentation and did not alter animal performance. / A ensilagem de grãos úmidos geralmente aumenta a digestão do amido e da proteína devido a proteólise durante o armazenamento. Porém, o tempo de armazenamento é fundamental para permitir que a matriz proteica seja degradada. O objetivo central desse trabalho foi avaliar o efeito do tempo de estocagem em silagens de sorgo grão reconstituído (SSGR) no desempenho de vacas leiteiras. De forma simultânea foi avaliado o efeito do benzoato de sódio no valor nutritivo de silagens de sorgo grão reconstituído. A hipótese foi que o uso de benzoato de sódio em SSGR reduz a atividade proteolítica devido suas propriedades antimicrobianas impactando no desempenho animal. Uma sequência de dois experimentos com vacas leiteiras da raça Holandesa foram formatados. O primeiro experimento avaliou o efeito de diferentes tempos de estocagem em SSGR tratadas ou não com benzoato de sódio (0.2 % base na matéria natural). Silagens não tratadas (Controle) e tratadas com aditivo (Benzoato) foram armazenadas por 30 ou 90 dias antes do fornecimento. Vinte vacas leiteiras (168 &#177; 87 dias em lactação) foram usadas em cinco quadrados latinos replicados 4 × 4 em arranjo fatorial 2 × 2. Sorgo grão foi reconstituído para 35 % de umidade em ambos os experimentos e ensilados em tambores plásticos com capacidade de 200-L. Os tratamentos foram: SSGR armazenados por 30 dias sem aditivo (30 CON), SSGR armazenados por 30 dias com benzoato de sódio (30 BEN), SSGR armazenados por 90 dias sem aditivo (90 BEN) ou SSGR armazenados por 90 dias com benzoato de sódio (90 BEN). Prolongando o tempo de armazenamento a concentração de 1,2-propanodiol e proteína solúvel aumentaram. A produção de leite foi maior em favor das vacas alimentadas com silagens armazenadas por 90 dias comparadas à 30 dias (31.2 vs. 30.0 kg/d). A digestibilidade do amido (89.3 vs. 86.9%) e da proteína (57.1 vs. 54.0%) foi maior para silagens armazenadas por 90 dias. O benzoato de sódio reduziu a concentração de etanol (0.20 vs. 0.08% of DM), porém não alterou ao nível de significância estatística adotada nesse trabalho a proteína solúvel (CON = 18.9 vs. BEN = 15.6% of PB) e o N-amoniacal (CON = 4.38 vs. BEN = 3.94 % of N). O segundo experimento foi conduzido com 12 vacas leiteiras (170 &#177; 47 DEL) para avaliar o efeito do benzoato de sódio no valor nutritivo e desempenho de vacas leiteiras alimentadas com SSGR estocadas por 150 dias, tratadas (Benzoato) ou não (Controle) com benzoato de sódio. Uma dieta padrão contendo sorgo grão seco moído foi fornecida por 14 dias. No final do período de adaptação, os animais foram pareados dois a dois e distribuídos aleatoriamente a um de dois tratamentos (Controle ou Benzoato) fornecidos por 28 dias. Durante o período experimental as vacas receberam a mesma dieta sendo o sorgo seco totalmente substituído por SSGR. Silagens tradadas com benzoato de sódio tiveram menor concentração de etanol (0.84 vs. 0.18 % de MS) e etil lactato (388 vs. 157 mg/kg de MS) como consequência de uma menor população de leveduras (4.73 vs. 2.52 log ufc/g). A proteína solúvel foi reduzida nas silagens tratadas (26.2 vs. 20.6 % da PB). A estabilidade aeróbia foi mais alta nas silagens tratadas (51 vs. 146 h). Em conclusão, estender o tempo de estocagem em SSGR aumentou a eficiência alimentar e do uso do nitrogênio devido ao aumento na digestibilidade do amido e da proteína. O benzoato de sódio promoveu respostas típicas na fermentação de silagens, e não alterou o desempenho animal.

Orientador: Elisa Maria Aparecida Giro / Resumo: O objetivo deste trabalho foi avaliar o perfil proteômico e a proteólise da histatina 1 e da histatina 5 na saliva total estimulada de indivíduos com síndrome de Down (SD) e não-sindrômicos (NS) na presença e na ausência da doença periodontal (DP). Foram selecionados 24 indivíduos, os quais foram divididos em 4 grupos experimentais (n=6): SD com DP (SDcDP), SD sem DP (SDsDP), não sindrômicos com DP (NScDP) e não sindrômicos sem DP (NSsDP - controle). Inicialmente, os participantes passaram por exame clínico intra-bucal para avaliação da condição periodontal e determinação do índice CPO-D. Foi realizada a coleta da saliva estimulada até a obtenção de 3,0 mL. Parte da saliva foi utilizada para a análise microbiológica e parte foi centrifugada para obtenção do sobrenadante da saliva total (SST), aliquotada e armazenada em freezer -80ºC para as análises proteômica e de degradação. Os níveis salivares de Aggregatibacter actinomycetemcomitans e Porphyromonas gingivalis foram quantificados pela Reação em Cadeia da Polimerase em Tempo Real (qPCR). A análise espectrométrica foi realizada com os pools de saliva de cada um dos quatro grupos, os quais foram submetidos a nLC-ESI-MS/MS (Cromatografia Líquida por ionização electrospray Tandem Espectrometria de Massas). A degradação proteica foi realizada pela adição de histatina 1 e histatina 5 sintéticas ao SST diluído (1:10) e incubação à 37ºC pelos tempos 0, 0,5, 1,5, 4, 6, 8, 24 e 48 horas. Em seguida, foram realizadas as análises d... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: This study aimed to evaluate the proteomic profile and histatins 1 and 5 proteolysis in stimulated whole saliva of individuals with Down syndrome (DS) and non-syndromics (NS) in the presence and absence of periodontal disease (PD). Twenty-four individuals were selected and divided in the following groups (n=6): DS with PD (DSwPD), DS without PD (DSwtPD), NS with PD (NSwPD) and NS without PD (NSwtPD - control). First, periodontal condition and DMFT index were evaluated and 3.0 mL of stimulated whole saliva was collected. Then, part of whole saliva was used to microbiological analysis and the remaining samples were centrifuged in order to obtain the whole saliva supernatant (WSS), aliquoted and stored at -80ºC to proteomic and degradation assays. Levels of Porphyromonas gingivalis (Pg) and Aggregatibacter actinomycetemcomitans (Aa) were quantified by real-time polymerase chain reaction (qPCR). Spectrophotometric analyses were carried out with saliva pools of each group by using nLC-ESI-MS/MS (Liquid chromatography electrospray ionization tandem mass spectrometry). Protein degradation assay was carried out with synthetic histatins 1 or 5 added to WSS (1:10) followed by samples incubation at 37°C for 0, 0.5, 1.5, 4, 6, 8, 24 and 48 hours. Next, polyacrylamide cationic gel electrophoresis (PAGE) analysis and measurement of bands density (%) were performed. Kruskal-Wallis test complemented by Dunn's test was applied to analyze clinical and microbiological data. Total protein and hi... (Complete abstract click electronic access below) / Doutor

De précédents travaux de recherche ont montré la présence de phénomènes inflammatoires dans les muscles de patients souffrant d’une maladie génétique rare nommée dystrophie musculaire des ceintures de type 2A (en anglais, LGMD2A), et ceci très tôt dans le développement de cette maladie. Il a aussi été observé les mêmes phénomènes chez le modèle animal de la maladie. C’est pourquoi nous avons mené une étude sur l’animal pour tenter d’étudier ces phénomènes afin d’en comprendre leur origine et leur(s) mécanisme(s). En parallèle de ce projet, nous avons développé et testé deux outils de thérapie génique : il s’agit de deux concepts qui vont permettre soit de réparer l’ARN au niveau du transcrit du gène CAPN3 muté responsable de la maladie, soit de permettre aux cellules du muscle de produire une protéine calpaïne-3 fonctionnelle, qui ne peut exercer son rôle correctement à cause des mutations qui la rende non opérationnelle. / Previous research works showed inflammatory phenomena early in muscles of patients affected with a rare genetic disease called Limb Girdle Muscular Dystrophy type 2A (LGMD2A). This very same phenomena have been observed in LGMD2A animal models. For these reasons we conducted an animal-oriented study to apprehend these phenomena from its origin to mechanism(s). In parallel of this project, we designed and evaluated two gene therapy tools: one to repair RNA on CAPN3 gene transcript that is responsible of LGMD2A, another one to help muscle cells to produce a functional calpain-3 protein based on an inter molecular compensation with vector expressing domains of calpain 3

O presente trabalho teve como objetivo geral estudar a qualidade de carne (Longissimus dorsi) de machos inteiros da raça Nelore através de análise proteômica bidimensional. Para tanto, dois objetivos específicos foram propostos: (i) estudar a proteólise das carnes em diferentes tempos de maturação, no caso um, sete e 14 dias, e (ii) estudar o perfil protéico das carnes de bovinos machos inteiros Nelore com genótipos contrastantes para maciez, em diferentes tempos de maturação (um, sete e 14 dias), associados aos marcadores moleculares CAPN4751 (&mu;-calpaína) e UOGCAST1 (calpastatina). Amostras de sangue foram coletadas para realização de análises genotípicas. Para as análises eletroforéticas e de maciez, amostras maturadas por um, sete e 14 dias foram coletadas. A caracterização e determinação dos genótipos polimórficos (SNP) para os marcadores CAPN4751 e UOGCAST1 foram realizadas por meio de PCR em tempo real. Seis pools de amostras de animais foram preparados para a realização de eletroforese bidimensional. As análises estatísticas foram realizadas no pacote Statistical Analysis System(SAS). Foram encontrados 256 spots em comum na análise de proteólise, sendo 25 significativos (P&lt;0,05). Na análise de regressão múltipla, sendo maciez a variável dependente e os spots significativos a variável independente, foi possível verificar que 13 spots explicaram em 73% a variação de maciez durante a maturação. As análises de variância da intensidade para os volumes de expressão normalizados (IVEN) indicaram 21 spots significativos (P&lt;0,05) para os marcadores CAPN4751 e UOGCAST1. Análises de regressão múltipla para os spots significativos mostraram que alguns spots, além dos marcadores estudados, podem predizer a maciez da carne aos 14 dias de maturação. Portanto, as avaliações de spots permitirão identificar as proteínas diferencialmente expressas, auxiliando no melhor entendimento do complexo mecanismo que determina a maciez da carne. / The present work aimed to study beef quality (Longissimus dorsi) from Nellore bull through two-dimensional proteomic analysis. For this purpose, two specific objectives were proposed: (i) to study the proteolysis of meat at different aging times, if 24 hours, seven and 14 days, and (ii) to study the protein profile of meat from Nellore bull with contrasting genotypes for tenderness at different aging times (24 hours, seven and 14 days), associated with molecular markers CAPN4751 (&mu;-calpain) and UOGCAST1 (calpastatin). Blood samples were collected for genomic analyses. For electrophoretic analysis and tenderness, samples aged for 24 hours, seven and 14 days were collected. The characterization and determination of genotypes polymorphic (SNP) markers for CAPN4751 and UOGCAST1 were performed using real time PCR. Six samples of animals pools were set up to carry out two-dimensional electrophoresis. Statistical analyzes were performed in the package Statistical Analysis System (SAS). 256 spots were found in common in the analysis of proteolysis, with 25 spots significant. We found that 13 spots accounted for 73% of the variation in tenderness during aging. Analyses of variance of the intensity of expression for the normalized volumes (IENV), 21 spots indicated significant (P&lt;0,05) for the markers CAPN4751 and UOGCAST1. Multiple regressions analysis to significant spots showed to some spots, in addition to the markers studied may predict meat tenderness at 14 days of aging. Therefore, the assessments will identify the spots differentially expressed proteins, aiding in better understanding the complex mechanism that determines the softness of the meat.

A expressão do PIF/DCD em tumores tem sido relacionada ao crescimento e resistência à morte celular e à indução de caquexia em camundongos. O objetivo deste estudo foi investigar o papel do PIF/DCD nestes processos. Foram construídos três PIF/DCD-RNAi chamados de IBC-I, II, III e pKLO (controle), compreendendo diferentes áreas do mRNA, geramos clones celulares PIF/DCD-RNAi e comparamos a expressão do mRNA por RT-PCR em tempo real. Depois, foi avaliado o crescimento e formação de colônias dos clones RNAi in vitro e a progressão tumoral in vivo em camundongo Nude. Os RNAi IBC-I, II e III reduziram a expressão em 93,25% dos transcritos do PIF/DCD, alem de reduzir em 61,7% a capacidade de formação de colônias em comparação ao controle (p<0.001). Nos experimentos in vivo, os camundongos 2 meses do grupo pKLO tiveram um retardo no desenvolvimento muscular. O grupo pKLO de 8 meses apresentou uma redução de 35% do seu peso. Ambos mostraram diminuição da massa muscular (p<0,05). Estes resultados confirmam o papel do PIF/DCD na progressão tumoral. / The PIF/DCD expression in tumors has been related to the growth and resistance to cellular death and induction of cachexia in mice. The aim of this study was to investigate the role of PIF/DCD in these processes. It was designed three PIF/DCD-RNAi named IBC-I, II and III and one control pKLO, comprising different areas of the mRNA. Next, it was generated PIF/DCD-RNAi cell clones and compared mRNA expression by RT-PCR real time. After that, we evaluated the growth and colony formation ability of RNAi clones in vitro and in vivo tumorogenicity in Nude mice. The IBC-I, II and III RNAi reduced in 93,25% the expression of transcripts for PIF/DCD. There was also a significant reduction (61,7%) in the colony formation compared to the control (p<0.001). In the in vivo experiments, the 2-month-old mice in the pKLO group had muscular development retardation. The 8-month-old pKLO group presented a 35% reduction on its weight. Both groups had shown muscular mass reduction (p<0,05). These results confirm the role of PIF/DCD in the tumoral progression.

O objetivo deste estudo foi investigar os efeitos do T3 e do seu análogo GC-24, agonista TR<font face=\"symbol\">b seletivo, na proteólise muscular mediada pela via ubiquitina-proteassoma. Avaliamos o efeito do T3 e GC-24 no trofismo radial de fibras musculares, no nível de ubiquitinação e na expressão de genes envolvidos na via ubiquitina-proteassoma. Para tanto foram utilizados, ratos Wistar divididos em 4 grupos (Controle, 12 horas, 1 e 7 dias) e tratados com T3 e GC-24. Determinou-se a área de secção transversa dos cortes histológicos através do programa \"Image Pro-Plus\". O nível de ubiquitinação foi determinado através de Western Blot para proteína ubiquitinada e a expressão gênica por PCR em Tempo real. T3 e GC-24 promoveram redução do diâmetro das fibras musculares e aumentaram o nível de proteínas ubiquitinadas em ambos os músculos. Com relação à expressão gênica, T3 e GC-24 modularam a expressão dos genes analisados de maneira diferenciada, demonstrando que GC-24 é capaz de modular genes pouco ou não responsivos ao T3. / Triiodothyronine (T3) is known to play a key role in the function of several tissues/organs via the thyroid hormone receptor isoforms a/pha (TRa) and beta (TRI3). Abnormalities in skeletal muscle function have been associated with increased leveis of T3, which is a major sarcopenia (Ioss of sarcomeres). Although the phenomenon of sarcopenia induced by T3 has been widely reported, little is known about the molecular mechanisms invo/ved in proteolysis induced by T3. In this study we have investigated the effects of T3 and GC-24, a novel synthetic TRI3¬selective compound, on the ubiquitin proteasome pathway. We analyzed the effect of T3 and GC-24 on the radial trophism, ubiquitination leveis and gene expression of the ubiquitin-proteasome pathway, which are important regulators of muscle proteolysis in the skeletal muscle. We have addressed the ubiquitin ligases (Atrogin¬1, MuRF-1 and E3a) and the deubiquitinating enzymes (UBP45, UBP69 and USP28). Wistar male rats (170-200g) were divided in 4 groups (Control, 12, 1 and 7 days). Rats received T3 (30l-\'g/100g) and GC-24 (16 I-\'g/1 OOg). After decapitation, EDL and soleus muscles were removed for histological ana/ysis, protein expression and gene expression. Cross sectional area was determined in histological sections through the software \"Image-Pro Plus. The ubiquitination leveis was determined by Western Blot and gene expression determined by Real Time PCR analysis. T3 and GC-24 reduced the diameter of the muscle fibers vs control group. Both T3 and GC-24 incresed the ubiquitination leveis, in the soleus and EDL. Regarding gene expression analysis, T3 and GC-24 modulate the gene expression in a differential manner. In the soleus, T3 increased Atrogin-1 and E3 alpha gene expression, while did not alter Murf-1 gene expression. On the other hand, in EDL Atrogin-1 gene expression is not altered, while E3 alpha and Murf-1 are elevated by T3. In the soleus and EDL deubiquitinating gene expression is mostly not altered, exception made for UBP 45, which is reduced by T3 in soleus muscle. GC-24, increased gene expression of E3a and MuRF-1 in the soleus, while did not alter Atrogin-1 gene expression. However, in EDL muscle, GC-24 increased Atrogin-1 and E3a mRNA, while did not alter MuRF-1. Finally, GC-24 decreased UBP 45 gene expression in EDL muscle and USP 28 gene expression was robustly elevated by GC-24 in both muscles analyzed. This data shows that GC-24 is able to strongly modulate genes that are less responsive or even unresponsive to T3, pointing that the GC-24-TRb complex might trans-activate differently target genes. However, both T3 and GC-24 are able to modulate the muscle proteolysis.