The E3 ubiquitin ligase \(CRL4^{Cdt2}\) targets proteins for destruction during DNA replication and following DNA damage (Havens and Walter, 2011). Its substrates contain "PIP degrons" that mediate substrate binding to the processivity factor PCNA at replication forks and damage sites. The resulting PCNA-PIP degron complex forms a docking site for \(CRL4^{Cdt2}\), which ubiquitylates the substrate on chromatin. Several \(CRL4^{Cdt2}\) substrates are known, including Cdt1, multiple CDK inhibitors, Drosophila E2f1, human Set8, S. pombe Spd1, and C. elegans \(Pol\eta\) (Havens and Walter, 2011). An emerging theme is that \(CRL4^{Cdt2}\) targets proteins whose presence in S phase is toxic. Here, I used Xenopus egg extract to characterize a new \(CRL4^{Cdt2}\) substrate, thymine DNA glycosylase (TDG). TDG is a base excision repair protein that targets G-U and G-T mispairs, which arise from cytosine and 5-methylcytosine deamination (Cortazar et al., 2007). Thus, TDG may function in epigenetic gene regulation via DNA demethylation, in addition to its canonical DNA repair function. A yet unknown E3 ubiquitin ligase triggers TDG destruction during S phase (Hardeland et al., 2007). Understanding TDG proteolysis in S phase is relevant to the regulation of DNA replication, DNA repair, and epigenetic control of gene expression. I discovered that TDG contains a variant of the "PIP degron" consensus and that TDG is ubiquitylated and destroyed in a PCNA-, Cdt2-, and degron-specific manner during DNA repair and DNA replication in Xenopus egg extract. I further characterized what features of TDG contribute to its proteolysis. Interestingly, I could not identify any defects during DNA replication or during Xenopus embryonic development in response to a non-degradable form of TDG. Additionally, I examined how interactions between \(CRL4^{Cdt2}\) and multiple subunits of the PCNA homotrimer contribute to \(CRL4^{Cdt2}\) function. In a popular model, PCNA functions as a "tool belt" on DNA, binding three separate proteins through its individual subunits to facilitate rapid exchange of DNA replication and repair proteins as they are needed on DNA. To address this model, I generated a single chain polypeptide with three PCNA subunits connected through flexible linker sequences. I used this tool to determine how multiple PCNA subunits contribute to \(CRL4^{Cdt2}\) function. I found that a single wildtype subunit is sufficient for modest destruction of the \(CRL4^{Cdt2}\) substrate Cdt1, but complete Cdt1 destruction requires two separate wildtype subunits. Additionally, a single subunit was sufficient for leading strand elongation, challenging the "tool belt" model during DNA replication. I also discuss implications and future use of the single-chain PCNA.

Reovirus is internalized after interaction of the outer proteins μ1, σ1 and σ3 with the host cell. Proteolysis of σ3 and cleavage of μ1 (into δ and φ) eventually leads to the formation of a more infectious subviral particle named “ISVP”. The infectious entry of viruses, but not of ISVPs, can be blocked using various entry inhibitors and therefore, suggests that there is a threshold of σ3 digestion required to allow particle to bypass entry blockers. By combining protease and detergent to the digestion of virions, data from this work showed distinct particles generated along the transition pathway. In addition, studies involving flow cytometry and specific antibodies (anti-μ1) showed that between virus and ISVP there is a gradual yet heterogeneous particle proteolysis that is directly related to the virus infectivity. The findings and approaches taken for this thesis work can possibly be extended for studying other non-enveloped viruses. Moreover, it may help to shed some light on the development of safe and effective oncolytic agents.

Reovirus is internalized after interaction of the outer proteins μ1, σ1 and σ3 with the host cell. Proteolysis of σ3 and cleavage of μ1 (into δ and φ) eventually leads to the formation of a more infectious subviral particle named “ISVP”. The infectious entry of viruses, but not of ISVPs, can be blocked using various entry inhibitors and therefore, suggests that there is a threshold of σ3 digestion required to allow particle to bypass entry blockers. By combining protease and detergent to the digestion of virions, data from this work showed distinct particles generated along the transition pathway. In addition, studies involving flow cytometry and specific antibodies (anti-μ1) showed that between virus and ISVP there is a gradual yet heterogeneous particle proteolysis that is directly related to the virus infectivity. The findings and approaches taken for this thesis work can possibly be extended for studying other non-enveloped viruses. Moreover, it may help to shed some light on the development of safe and effective oncolytic agents.

<p>Nearly thirty percent of all newly synthesized polypeptides are targeted for rapid proteasome-mediated degradation. These rapidly degraded polypeptides (RDPs) are the primary source of antigenic substrates for the major histocompatibility complex (MHC) class I presentation pathway, allowing for the immunosurveillance of newly synthesized proteins by cytotoxic T lymphocytes. Despite the recognized role of RDPs in MHC class I presentation, it remains unclear what molecular characteristics distinguish RDPs from their more stable counterparts. It has been proposed that premature translational termination products may constitute a form of RDP; indeed, in prokaryotes translational drop-off products are normal by-products of protein synthesis and are subsequently rapidly degraded. </p><p>To study the cellular fate of premature termination products, the antibiotic puromycin was used to modulate prematurely terminated polypeptide production in human cells. At low concentrations, puromycin doubled the fraction of rapidly degraded polypeptides, with enhanced degradation predominantly affecting small polypeptides, consistent with rapid degradation of truncated translation products. Immunoprecipitation experiments using anti-puromycin antisera demonstrated that the majority of peptidyl-puromycins are rapidly degraded in a proteasome-dependent manner. Low concentrations of puromycin increased the recovery of cell surface MHC class I-peptide complexes, indicating that prematurely terminated polypeptides can be processed for presentation via the MHC I pathway. In the continued presence of puromycin, MHC I export to the cell surface was inhibited, coincident with the accumulation of polyubiquitinated proteins. The time- and dose-dependent effects of puromycin suggest that the pool of peptidyl-puromycin adducts differ in their targeting to various proteolytic pathways which, in turn, differ in the efficiency with which they access the MHC class I presentation machinery. These studies highlight the diversity of cellular proteolytic pathways necessary for the metabolism and immunosurveillance of prematurely terminated polypeptides which are, by their nature, highly heterogeneous.</p> / Dissertation

Rhomboids are unique membrane proteins that use a serine protease hydrolysis mechanism to cleave a transmembrane substrate within the lipid bilayer. This remarkable proteolytic activity is achieved by a core domain comprised of 6 transmembrane segments that form a hydrophilic cavity submerged in the membrane. In addition to this core domain, many rhomboids also possess aqueous domains of varying sizes at the N- and/or C-terminus, the sequences of which tend to be rhomboid-type specific. The functional role of these extramembranous domains is generally not well understood, although it is thought that they may be involved in regulation of rhomboid activity and specificity. While extramembranous domains may be important for rhomboid activity, they are absent in all x-ray crystal structures available. For this reason, we have focused on uncovering the structural and functional relationship between the rhomboid cytoplasmic domain and its catalytic transmembrane core. To investigate the structure and function of the bacterial rhomboid cytoplasmic domain, full-length rhomboids from Escherichia coli and Pseudomonas aeruginosa were studied using solution nuclear magnetic resonance (NMR) spectroscopy, mutation and activity assays. The P. aeruginosa rhomboid was purified in a range of membrane-mimetic media, evaluated for its functional status in vitro and investigated for its NMR spectroscopic properties. Results from this study suggested that an activity-modulating interaction might occur between the catalytic core transmembrane domain and the cytoplasmic domain. Further investigation of this hypothesis with the E. coli rhomboid revealed that protease activity relies on a short but critical sequence N-terminal to the first transmembrane segment. This sequence was found to have a direct impact on the rhomboid active site, and should be included in future structural studies of this catalytic domain. The structure of the cytoplasmic domain from the E. coli rhomboid was also determined by solution NMR. We found that it forms slowly-exchanging dimers through an exchange of secondary structure elements between subunits, commonly known as three-dimensional domain swapping. Beyond this rare example of domain swapping in a membrane protein extramembranous domain, we found that the rate of exchange between monomeric and dimeric states could be accelerated by transient interactions with large detergent micelles with a phosphocholine headgroup, but not by exposure to other weakly denaturing conditions. This novel example of micelle-catalyzed domain swapping interactions raises the possibility that domain swapping interactions might be induced by similar interactions in vivo. Overall, the results of this thesis have identified detergent conditions that preserve the highest level of activity for bacterial rhomboids, defined the minimal functional unit beyond what had been identified in available x-ray crystal structures, and characterized a novel micelle-catalyzed domain-swapping interaction by the cytoplasmic domain.

Κατά τη διάρκεια του κυτταρικού κύκλου ένας από τους σημαντικότερους μηχανισμούς ρύθμισης της ομοιόστασης των πρωτεϊνικών μορίων είναι η ελεγχόμενη στο χώρο και στο χρόνο αποδόμηση τους. Οποιαδήποτε ανωμαλία στη ρύθμιση των μηχανισμών αποδόμησης των πρωτεϊνών μπορεί να προκαλέσει σημαντική βλάβη στη λειτουργία του κυττάρου και να οδηγήσει σε κακοήθη εξαλλαγή του. Η αποδόμηση των κύκλινο-εξατώμενων αναστολέων του κυτταρικού κύκλου, p27 και p21, έχει δειχθεί να αυξάνει κατά την καρκινογένεση. Αν και η πρωτεόλυση των p27 και p21 έχει δειχθεί να μεσολαβείται από διαφορετικά μονοπάτια ωστόσο το περισσότερο γνωστό και καλύτερα τεκμηριωμένο είναι το μονοπάτι μέσω του συμπλόκου SCF (Skp2-cul1-Skp1) Ε3 λιγάσης της ουβικουιτίνης. Αρχικά οι πρωτεΐνες p27 και p21 φωσφορυλιώνονται από το σύμπλκο κυκλίνης Ε/Α-CDK2 στη θρεονίνη 187 (Thr187) και στη σερίνη 130 (Ser130) αντίστοιχα, και εν συνεχεία αναγνωρίζονται από το σύμπλοκο SCFSkp2, το οποίο διευκολύνει την πολύ-ουβικουιτινυλίωση και την αποδόμηση των πρωτεϊνών στο πρωτεόσωμα. Στη παρούσα εργασία μελετήθηκε η έκφραση των πρωτεϊνών: p27, p21, Skp2, cul1, pThr187-p27, κυκλίνη Α, κυκλίνη Ε και CDK2 σε 135 περιπτώσεις Β-λεμφωμάτων με Επιθετική [66 ΔΛΜΒΚ (35 λεμφαδενικά και 31 εξωλεμφαδενικά), 13 ΛΚΜ (8 κλασσικά και 5 βλαστικά) και 5 με ΛΛ βαθμού 3α/β)] και Ήπια βιολογική συμπεριφορά [9 ΛΛ (3 βαθμού 1, 6 βαθμού 2), 20 ΛΟΖ (12 λεμφαδενικά 6 εξωλεμφαδενικά, 2 σπληνικά) και 22 ΛΜΚ]. Η έκφραση της p27 παρατηρήθηκε μέγιστη στα ήπια λεμφώματα. Ωστόσο σε αρκετές περιπτώσεις επιθετικού λεμφώματος, κυρίως ΔΛΜΒΚ (~40%, >30%), η έκφραση της p27 βρέθηκε αυξημένη. Επιπλέον, σε όλες τις περιπτώσεις λεμφωμάτων εκτός του λεμφοζιδιακού και της οριακής ζώνης, παρουσιάστηκε αδυναμία αρνητικής συσχέτισης μεταξύ της p27 και της Skp2. Η μέγιστη έκφραση της p21, αντίθετα με την p27, παρατηρήθηκε στα ΔΛΜΒΚ, γεγονός που συνδέει την p21 με επιθετικότερη νόσο. Η πρωτεΐνη p21 δεν έδειξε να συσχετίζεται με τις πρωτεΐνες που είναι υπεύθυνες για την αποδόμηση της, γεγονός που πιθανόν να υποδεικνύει ότι στα Β-λεμφώματα που μελετήθηκαν η p21 δεν πρωτεολύεται από το σύμπλοκο SCFSkp2. Η υπερέκφραση της Skp2 στα ΔΛΜΒΚ, ΛΚΜ (κυρίως βλαστικά) και ΛΛ (βαθμού 3) καθώς και η ισχυρή θετική συσχέτιση με το δείκτη πολλαπλασιασμού, την κυκλίνη Α και τη CDK2 προσδίδουν στη πρωτεΐνη χαρακτηριστικά δείκτη επιθετικότητας. H cul1 έδειξε τα υψηλότερα επίπεδα έκφρασης και μία ισχυρή θετική συσχέτιση με την Skp2 στα ΔΛΜΒΚ, υποδεικνύοντας την παρουσία ενεργού SCF συμπλόκου, ωστόσο δεν παρατηρήθηκε παρόμοια συσχέτιση στους άλλους τύπους λεμφώματος. Υπερέκφραση της φωσφορυλιωμένης μορφής της p27 (pThr187-p27), δείχθηκε στα επιθετικότερα λεμφώματα. Επιπλέον, ισχυρή θετική συσχέτιση με το δείκτη πολλαπλασιασμού έδειξε η pThr187-p27, σε όλους τους τύπους λεμφώματος, δείχνοντας μία πιθανή σύνδεση της pThr187-p27 με την επιθετικότητα της νόσου. Συσχέτιση των παραπάνω πρωτεϊνών με τα κλινικά και εργαστηριακά ευρήματα των των ασθενών δείχνουν ότι στα επιθετικά λεμφώματα η υπερέκφραση των Skp2 και pThr187-p27 συνδέεται με βραχύτερο διάστημα ελεύθερο νόσο, ενώ η υπερέκφραση της pThr187-p27 έδειξε να συνδέεται και με πτωχότερη ολική επιβίωση. Επίσης κατά την πολυπαραγοντική ανάλυση, δείχθηκε για πρώτη φορά ότι η έκφραση της pThr187-p27 αποτελεί ανεξάρτητο προγνωστικό παράγοντα για τη συνολική επιβίωση όχι μόνο ανάμεσα στις υπό μελέτη πρωτεΐνες αλλά και σε συνάρτηση με τις κλινικές παραμέτρους στα επιθετικά Β-λεμφώματα / Cell cycle is tightly regulated by a functionally conserved group of proteins which together constitute the basic cell division machinery that controls cell cycle progression. Altered expression of these proteins are almost always detected in human cancer cells. However, aberrant expression of these proteins can be the cause of malignant transformation but also in some cases, can be the consequence of cancer progression. The cell cycle regulators p27 and p21 play a central role in the suppression of tumorigenesis in a variety of human cancers. Of particular importance for the development of human cancers is the ubiquitin dependent degradation of p27 and p21 by the proteasome. This pathway is controlled by many complexes, however the most well studied is the SCFSkp2 complex. p27 and p21 proteins are phosphorylated at a conserved Threonine (T187) and Serine (Ser130) residue by cyclin E/A-cdk2 complexes, respectively, and the Skp2 protein facilitates the polyubiquitylation of p27 and p21 by the SCF complex. In the present study was examined the immunoexpression of p27, p21, Skp2, cul1, pThr187-p27, cyclin Α, cyclin Ε and CDK2 in 135 cases with B-cell Lymphoma with aggressive [66 DLBCL (35 nodal and 31 extranodal), 13 MCL (8 classical and 5 blastic) and 5 FL grade 3α/β)] and indolent biological behaviour [9 FL (3 G1, 6 G2), 20 MZL (12 nodal, 6 extranodal, 2 splenic) and 22 SLL]. P27 was overexpressed in indolent B-cell lymphomas. However, many cases with aggressive B-cell lymphoma, mainly DLBCLs (~40%, >30%),, showed increased expression of p27. In addition, all lymphoma cases except the FLs and MZLs failed to show an inverse correlation between p27 and Skp2. The highest expression of p21 was observed in DLBCL, indicating that p21 expression is associated with more aggressive neoplasias. The levels of p21 expression did not correlate with Skp2 and cul1, indicating that SCFSkp2 complex might not be capable for p21 degradation in B-cell lymphomas. Overexpression of Skp2 in DLBCLs, MCLs (mainly blastic type) and FL (Grade 3), as well as the strong positive correlation with cyclin A and CDK2, indicate that Skp2 may be a putative biomarker of tumor aggressiveness. The expression of cul1 was higher in DLBCLs, and correlate well with the expression of Skp2, indicating the presence of active SCFSkp2 complex. However, a similar correlation was not observed in other lymphoma groups. The phosphorylated form of p27 (pThr187-p27) was overexpressed in aggressive cases. Furthermore, a strong positive correlation between pThr187-p27 and the proliferation index, was observed in all lymphoma cases. This correlation may indicate that pThr187-p27 could be used as a marker of tumor aggressiveness. The correlation of the studied proteins with the clinical and laboratory data showed that in the aggressive lymphomas the expression of Skp2 and pThr187-p27 is associated with poor disease free survival rate, and pThr187-p27 is also associated with shorter overall survival. In the present study, the multivariant cox analysis showed that the expression of pThr187-p27 is an independent prosgnostic factor for the overall survival among other clinical parameters.

Made available in DSpace on 2014-06-11T19:32:59Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-07-14Bitstream added on 2014-06-13T19:03:46Z : No. of bitstreams: 1 santos_gb_dr_botfmvz.pdf: 656292 bytes, checksum: de36c7b0fca5c7c6ac79ae4fae22735a (MD5) / Universidade Estadual Paulista (UNESP) / O trabalho objetivou identificar por eletroforese as mudanças nas frações das proteínas musculares, separadas no período post-mortem de bovinos de quatro diferentes grupos genéticos (Nelore, TRC-Brahman, Brangus e TRC-Pardo), submetidos ao modelo biológico superprecoce, assim como determinar os valores da força de cisalhamento destas amostras. Foram utilizadas 12 amostras do músculo Longissimus dorsi por grupo genético. De cada uma delas foram retiradas duas fatias, uma foi refrigerada por 24 horas e a outra foi maturada por 7 dias a 2oC. As amostras submetidas a 7 dias de maturação não diferiram quanto à maciez da carne nos grupos genéticos, mas todas apresentaram maciez aceitável, o que pode ser explicado pela baixa idade ao abate destes animais. Na análise das bandas da eletroforese, notou-se degradação da miosina (MHC) e da Troponina-T (TN-T) e o aparecimento do fragmento de 30 kDa para todos os grupos genéticos durante o período de 7 dias de maturação. / This study aimed to identify by means of eletrophoresis the changes in fractions of the muscular proteins, separated within the post-mortem period from cattle from four different genetic groups ( Nelore, TRC- Brahman, Brangus and TRC- Pardo), submitted to the superprecoce biologic model, as well as to determine the values of those samples shear force. Twelve samples of the muscle Longissimus dorsi were used for each genetic group. From each of those there were taken two slices, one of them was kept cold along 24 hours and the other one was aged during 7 days at 2oC. The samples that came through a seven-day-aging process didn't show any changes concerning the meat tenderness in the genetic groups, even though all of them had acceptable tenderness, which can be explained due to the low age of these animals when slaughtered. In the analysis of the eletrophoresis' bands, it was noticed some degradation of the myosin ( MHC) and of the Troponin-T (TN-T) and the presence of the fragment of 30 kDa for all the genetic groups during the seven-day-ageing period.

Made available in DSpace on 2014-06-11T19:27:43Z (GMT). No. of bitstreams: 0 Previous issue date: 2004-02-19Bitstream added on 2014-06-13T19:15:07Z : No. of bitstreams: 1 hadlich_jc_me_botfmvz.pdf: 277198 bytes, checksum: 032e9607da08d48a7163bb8934baa7d2 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O experimento foi realizado no Setor de Confinamento de Gado de Corte da Faculdade de Medicina Veterinária e Zootecnia e no Laboratório de Bioquímica de Proteínas do Instituto de Biociências. Foram utilizados animais da raça Nelore, mestiços u Aberdeen X Nelore e mestiços u Simental X Nelore, abatidos com idade entre 12 e 15 meses conforme estabelecido pelo modelo biológico superprecoce. O experimento foi conduzido em um delineamento inteiramente casualizado. O objetivo do presente estudo foi análise de componentes da maciez de novilhos superprecoces de grupos genéticos distintos. Não foi verificada diferença estatística (p>0,01) entre os grupos genéticos para a força de cisalhamento, Índice de Fragmentação Miofibrilar (MFI) e frações do colágeno, entretanto houve influência (p<0,01) do período postmortem, exceto para o colágeno. A carne de animais abatidos entre 12 e 15 meses de idade apresenta atributos de qualidade independente do grupo genético utilizado e com sete dias de maturação todos os animais apresentaram carne com grau de maciez desejável. / The experiment was accomplished in the Section of Feedlot of cattle of Faculdade de Medicina Veterinária e Zootecnia and in the Laboratório de Bioquímica de Proteínas do Instituito de Biociências. Nelore breed, u Aberdeen X Nelore crossbreed and u Simental X Nelore crossbreed were used and slaughtered accordingly with the brazilian system called superprecoce. The experiment was accomplished in a completely randomized design. The objective of the present study was the evaluation of tenderness components of superprecoce of different genetic groups. There was no statistics difference (p>0,01) between genetic groups for the shear force values, Myofibrillar Fragmentation Index (MFI) and collagen, however there was influence (p<0,01) of the ageing, except for the collagen. The meat of animals slaughtered between 12 and 15 months of age showed attributes of quality independent of the genetic group and with seven days of ageing all animals had a desirable tenderness.

Made available in DSpace on 2015-03-26T13:51:55Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1046907 bytes, checksum: 3e2156440d7134bf2b8e8f5a387cc61a (MD5) Previous issue date: 2011-07-15 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Among the psychrotrophic micro-organisms that are able to grow during refrigeration, some of them produce thermostable proteases which can cause many technological problems in the dairy industry. The food industries need to high throughput methods for the quality control raw of milk, thus, fast and sensitive methods for psychrotrophic detection, specially Pseudomonas fluorescens, predominant specie, are economically interesting to the dairy industry. In order to establish a protocol for the detection of P. fluorescens by using the polymerase chain reaction (PCR), five DNA extraction methods for milk samples, two target genes for the PCR amplification and the detection limit in raw milk and sterilized milk inoculated with P. fluorescens were evaluated. A commercial DNA extraction kit and a modified filtration method were the most appropriate for successfully bacterial DNA extraction from milk samples. These methods were selected and used for the detection of the target genes. Nevertheless, the modified filtration method was more efficient showing a lower detection limit in raw milk and sterilized milk inoculated with P. fluorescens. By means of PCR amplification of the 16S rRNA target gene, an 850 bp amplicon was observed and it was possible to detect as few as 102 UFC/ml of P. fluorescens from inoculated milk. In raw milk samples, Pseudomonas and psychrotrophic bacteria were detected at 106 UFC/mL and 107 to 109 UFC/mL, respectively. The relationship between the psychrotrophic population and the degradation of milk proteins was evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The results showed the proteolysis degree in raw milk doesn t depend just on the psychrotrophic bacteria count. / Entre os micro-organismos psicrotróficos que são capazes de crescer durante a refrigeração, alguns produzem proteases termorresistentes relacionadas com problemas tecnológicos nos derivados lácteos. Visto que a demanda por métodos que permitem alta taxa de processamento exigida em indústrias de alimentos para controle da qualidade de leite cru é crescente, a avaliação de métodos mais sensíveis e mais rápidos para detecção de psicrotróficos, especialmente Pseudomonas fluorescens, espécie predominante, é de grande importância. Com a finalidade de estabelecer um protocolo para detecção de P. fluorescens pela reação em cadeia da polimerase (PCR), foram avaliados cinco métodos de extração de DNA total do leite, dois genes alvos na PCR e o limite de detecção dos métodos para análise de leite cru e leite esterilizado e inoculado com P. fluorescens. O kit comercial e o método de filtração modificado foram os métodos mais adequados para extração de DNA total de amostras de leite, por isso, foram utilizados nas análises seguintes para detecção dos genes alvos. No entanto, a utilização do método de filtração modificado para análise de leite inoculado com P. fluorescens e leite cru resultou em um menor limite de detecção quando comparado com o kit comercial. Por meio da amplificação do gene alvo rDNA 16S, foi possível observar um amplificado de 850 pb quando a população do leite inoculado com P. fluorescens era da ordem de 102 UFC/ml. Em amostras de leite cru, foi possível detectar o produto quando a contagem de Pseudomonas era da ordem de 106 UFC/ml e a população de psicrotróficos estava entre 107 e 109 UFC/ml. A análise do grau de proteólise das amostras de leite cru por eletroforese em gel de poliacrilamida demonstrou que a relação entre a população de psicrotróficos e a degradação das proteínas do leite depende de outros fatores além da contagem de psicrotróficos.

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.