Expression of tumor-related Rac1b antagonizes B-Raf-induced senescence in colorectal cells

Mutations in the BRAF oncogene have been identified as a tumor-initiating genetic event in mainly melanoma, thyroid and colon cancer, resulting in an initial proliferative stimulus that is followed by a growth arrest period known as oncogene-induced senescence (OIS). It remains unknown what triggers subsequent escape from OIS to allow further tumor progression. A previous analysis revealed that overexpression of splice variant Rac1b occurs in around 80% of colorectal tumors carrying a mutation in BRAF. Using both BRaf-V600E-directed RNAi and overexpression we demonstrate that this mutation does not directly lead to Rac1b overexpression, indicating the latter as an independent event during tumor progression. Nonetheless, we observed that expression of oncogenic BRaf-V600E in non-transformed colonocytes (NCM460 cell line) increased both the transcript and protein levels of p14ARF, p15INK4b and p21CIP1 and led to increased expression of β-galactosidase, all indicators of OIS induction. Interestingly, whereas the protein levels of these markers were reduced upon Rac1b overexpression, the levels of their respective transcripts remained unchanged. Importantly, the co-expression of Rac1b with B-Raf-V600E reverted the OIS phenotype, reducing the expression levels of the cell-cycle inhibitors and β-galactosidase to those of control cells. These data identify increased Rac1b expression as one potential mechanism by which colorectal tumor cells can escape from B-Raf-induced OIS.

Obtenção e caracterização de queratinase de bacillus sp. P45 a partir de coprodutos e aplicação na produção de queijos cremosos enriquecidos com chia e quinoa

As proteases constituem 60-65% do mercado global das enzimas industriais e são utilizadas na indústria de alimentos no processo de amaciamento de carne, na síntese de peptídeos, preparo de fórmulas infantis, panificação, cervejarias, produtos farmacêuticos, diagnósticos médicos, como aditivos na indústria de detergentes e na indústria têxtil no processo de depilação e transformação do couro. Proteases específicas produzidas por micro-organismos queratinolíticos são chamadas de queratinases e distinguem-se de outras proteases pela maior capacidade de degradação de substratos compactos e insolúveis como a queratina. Atualmente, processos que apontem o uso total das matérias-primas e que não resultem em impactos negativos ao meio ambiente tem ganhado destaque. Dentro desta temática, destacam-se a reutilização da farinha de penas residual durante o cultivo do Bacillus sp. P45 para produção de proteases e a biomassa residual de levedura, ambas com elevados teores de proteínas, podendo ser utilizadas no cultivo do Bacillus sp. P45 para obtenção de proteases. O objetivo deste trabalho foi obter a enzima queratinase purificada em grandes quantidades, sua caracterização, bem como a sua aplicação em processos de coagulação enzimática do leite para o desenvolvimento de um queijo cremoso enriquecido com farinha de chia e quinoa. Além disso, aplicar diferentes coprodutos para produção de enzimas proteolíticas e queratinolíticas. A presente tese foi dividida em quatro artigos: no primeiro foi realizado a obtenção da queratinase purificada em maiores quantidades e a determinação dos parâmetros de estabilidade térmica e a influência de componentes químicos na atividade enzimática. A obtenção da enzima em maiores quantidades alcançou fatores de purificação de 2,6, 6,7 e 4,0 vezes, paras 1º SAB, 2º SAB e diafiltração, respectivamente. A recuperação enzimática alcançou valores de 75,3% para o 1º SAB, 75,1% no 2º sistema e 84,3% na diafiltração. A temperatura de 55ºC e o pH 7,5 foram determinados como ótimos para atividade da enzima queratinase. O valor da energia de desativação (Ed) médio foi de 118,0 kJ/mol e os valores de z e D variaram de 13,6 a 18,8ºC, e 6,9 a 237,3 min, respectivamente. Além disso a adição de sais (CaCl2, CaO, C8H5KO4 e MgSO4) elevou a atividade da enzima na presença destes compostos. O segundo artigo apresenta a aplicação da queratinase como coagulante de leite bovino e sua aplicação na obtenção de queijo cremoso enriquecido com chia e quinoa. A enzima mostrou atividade de coagulação semelhante ao coagulante comercial, na concentração de 30mg/mL. A enzima purificada foi empregada de forma eficiente na fabricação do queijo cremoso, que apresentou valores de pH de 5,3 e acidez de 0,06 a 0,1 mol/L, com elevação durante os 25 dias de armazenamento. O terceiro artigo apresenta o perfil do queijo cremoso enriquecido com farinha de chia e quinoa, o qual apresentou alto índice de retenção de água (>99,0%) e baixos valores de sinérese (<0,72%). Elevados teores de fibras foi verificado (3,0 a 5,0%), sugerindo seu consumo como fonte de fibras. As análises microbiológicas foram de acordo com a legislação vigente. Na análise sensorial foi verificado altos valores de suavidade ao paladar e verificado maiores valores de consistência e untabilidade nas amostras com maiores concentrações de nata e quinoa. O quarto artigo traz a extração de β-galactosidase por ultrassom e o uso da biomassa residual da levedura, bem como o uso de farinha de penas residuais como substrato para obtenção de proteases. O ultrassom foi eficiente para ruptura celular e extração de β-galactosidase, apresentando alta atividade (35,0 U/mL) e rendimento (876,0 U/g de biomassa). A maior atividade proteolítica (1300 U/mL em 32 h) e queratinolítica (89,2 U/mL) verificadas ocorreram utilizando-se a biomassa e a farinha de penas residuais, respectivamente. Maior produtividade proteolítica (40,8 U/mL/h) foi verificado no meio utilizando biomassa residual como substrato. Já a maior produtividade queratinolítica (2,8 U/mL/h) foi alcançada utilizando farinha de penas reutilizada.

Bcl-xL (S49) and (S62) sequential phosphorylation/dephosphorylation during mitosis prevents chromosome instability and aneuploidy

Une caractéristique intéressante de la protéine Bcl-xL est la présence d'un domaine en boucle non-structurée entre les hélices α1 and α2 de la protéine. Ce domaine protéique n'est pas essentiel pour sa fonction anti-apoptotique et absent chez CED-9, la protéine orthologue chez Caenorhabditis elegans. A l'intérieur de ce domaine, Bcl-xL subit une phosphorylation et déphosphorylation dynamique sur les résidus Ser49 et Ser62 en phase G2 du cycle cellulaire et lors de la mitose. Lorsque ces résidus sont mutés et les protéines exprimées dans des cellules cancéreuses, les cellules démontrent plusieurs défauts mitotiques liés à l'instabilité chromosomique. Pour analyser les effets de Bcl-xL Ser49 et Ser62 dans les cellules normales, les présentes études ont été réalisées dans des cellules diploïdes humaines normales, et in vivo chez Caenorhabditis elegans. Dans une première étude, nous avons utilisé la lignée cellulaire de cellules fibroblastiques diploïdes humaines normales BJ, exprimant Bcl-xL (type sauvage), (S49A), (S49D), (S62A), (S62D) et les double (S49/62A) et (S49/62D) mutants. Les cellules exprimant les mutants de phosphorylation ont montré des cinétiques de doublement de la population cellulaire réduites. Ces effets sur la cinétique de doublement de la population cellulaire corrèle avec l'apparition de la sénescence cellulaire, sans impact sur les taux de mort cellulaire. Ces cellules sénescentes affichent des phénotypes typiques de sénescence associés notamment à haut niveau de l'activité β-galactosidase associée à la sénescence, la sécrétion d' interleukine-6, l'activation de p53 et de p21WAF1/ Cip1, un inhibiteur des complexes kinase cycline-dépendant, ainsi que la formation de foyers de chromatine nucléaire associés à γH2A.X. Les analyses de fluorescence par hybridation in situ et des caryotypes par coloration au Giemsa ont révélé que l'expression des mutants de phosphorylation de Bcl-xL provoquent de l'instabilité chromosomique et l'aneuploïdie. Ces résultats suggèrent que les cycles de phosphorylation et déphosphorylation dynamiques de Bcl-xL Ser49 et Ser62 sont importants dans le maintien de l'intégrité des chromosomes lors de la mitose dans les cellules normales. Dans une deuxième étude, nous avons entrepris des expériences chez Caenorhabditis elegans pour comprendre l'importance des résidus Ser49 et Ser62 de Bcl-xL in vivo. Les vers transgéniques portant les mutations de Bcl-xL (S49A, S62A, S49D, S62D et S49/62A) ont été générés et leurs effets ont été analysés sur les cellules germinales des jeunes vers adultes. Les vers portant les mutations de Bcl-xL ont montré une diminution de ponte et d'éclosion des oeufs, des variations de la longueur de leurs régions mitotiques et des zones de transition, des anomalies chromosomiques à leur stade de diplotène, et une augmentation de l'apoptose des cellules germinales. Certaines de ces souches transgéniques, en particulier les variants Ser/Ala, ont également montré des variations de durée de vie par rapport aux vers témoins. Ces observations in vivo ont confirmé l'importance de Ser49 et Ser62 à l'intérieur du domaine à boucle de Bcl-xL pour le maintien de la stabilité chromosomique. Ces études auront une incidence sur les futures stratégies visant à développer et à identifier des composés qui pourraient cibler non seulement le domaine anti-apoptotique de la protéine Bcl-xL, mais aussi son domaine mitotique pour la thérapie du cancer.

The role of chondrocyte senescence in the pathogenesis of canine osteoarthritis

The aims of this study were to (1) evaluate cellular senescence in chondrocytes from osteoarthritic articular cartilage, (2) investigate the hypothesis that oxidative stress is a feature of canine OA chondrocytes and that oxidative stress contributes to cellular senescence in canine chondrocytes, (3) investigate the hypothesis that osteoarthritic chondrocytes alter the gene expression of adjacent normal chondrocytes in OA joints leading to modulation of genes known to play a role in the pathogenesis of OA and (4) evaluate the presentation of dogs undergoing femoral head excision in veterinary referral practice in the UK as a treatment for osteoarthritis of the coxofemoral joint, and to categorise the distribution and severity of associated pathological lesions. Chondrocytes from osteoarthritic and normal cartilage were examined for levels of senescence. Initially chondrocytes were cultured using an alginate bead culture system, thought to mimic the extracellular matrix of articular cartilage. However, these chondrocytes showed almost no growth as compared to monolayer culture where they grew rapidly. OA chondrocytes entered the senescent state after 1.5 to 4.9 population doublings in monolayer culture, while normal chondrocytes underwent 4.8 to 14.6 population doublings before entering the senescent state. Osteoarthritic chondrocytes had increased levels of markers of cellular senescence (senescence associated beta-galactosidase accumulation and p16 protein accumulation) as compared to normal chondrocytes, suggesting that chondrocyte senescence is a feature of canine osteoarthritis. An experimental model for the induction of oxidative stress in chondrocyte cell culture was developed using tert-Butyl hydroperoxide and total cellular glutathione was measured as an indicator of cellular oxidative stress levels. Experimental induction of oxidative stress in both normal and osteoarthritic chondrocytes in cell culture resulted in increased amounts of cellular senescence, shown by an increase in levels of senescence associated beta-galactosidase accumulation and decreased replicative capacity. Experimental induction of oxidative stress also resulted in altered gene expression of three genes important to the degradation of the extracellular matrix; MMP-13, MMP-3 and Col-3A1, measured by RT-PCR, in normal canine chondrocytes in monolayer cell culture. MMP-3 showed the greatest relative expression change, with a fold-change of between 1.43 and 4.78. MMP-13 had a fold change of 1.16 to 1.38. Col-3A1 was down regulated, with a fold-change of between 0.21 and 0.31. These data demonstrate that experimentally induced oxidative stress in chondrocytes in monolayer culture increases levels of cellular senescence and alters the expression of genes relevant to the pathogenesis of canine OA. Coculture of osteoarthritic chondrocytes with normal canine chondrocytes resulted in gene modulation in the normal chondrocytes. Altered gene expression of ten genes known to play a role in the pathogenesis of osteoarthritis was detected in the normal chondrocytes (fold change shown in brackets); TNF-alpha (11.95), MMP-13 (5.93), MMP-3 (5.48), IL-4 (7.03), IL-6 (5.3), IL-8 (4.92), IL-F3 (4.22), COL-3A1 (4.12), ADAMTS-4 (3.78) and ADAMTS-5 (4.27). In total, 594 genes were significantly modulated suggesting that osteoarthritic chondrocytes contribute to the disease propagation by altering the gene expression of adjacent normal chondrocytes, thus recruiting them into the disease process. Gene expression changes were measured by microarray analysis and validated by RT-PCR and Western blot analysis. An epidemiological study of femoral heads collected from dogs undergoing total hip replacement surgery as a treatment for osteoarthritis of the coxofemoral joint secondary to canine hip dysplasia revealed that there was no characteristic pattern of cartilage lesion for canine hip dysplasia. Severe pathology of the femoral head with cartilage erosion occurred in 63.9% of cases and exposure of subchondral bone in 31.3% of cases. The work presented in this thesis has demonstrated that cellular senescence is a feature of chondrocytes from canine osteoarthritic cartilage and suggests that cellular senescence and oxidative stress play an important role in the pathogenesis of osteoarthritis in dogs.

Changes in enzymatic activity, accumulation of proteins and softening of persimmon (Diospyros kaki Thunb.) flesh as a function of pre-cooling acclimatization.

One of the major causes of ?Fuyu? persimmon loss after cold storage (CS) is the breakdown of its flesh, which results in the production of a translucent fruit (a water-soaked fruit). It is believed that the cause of this disturbance is linked to disorganization of the cytoskelet and endomembrane system, which changes the synthesis and transport of proteins and metabolites, resulting in incomplete ripening. To test this hypothesis, ?Fuyu? persimmon was subjected to three different postharvest treatments (T): Control ? harvested and kept at 23±3 ◦C and relative humidity (RH) of 85±5% (room temperature, RT) for 12 days, T1 ? harvested and kept under cold storage (CS) (1±1 ◦C and RH of 85±5%) for 30 days followed by RT storage for 2 days, T2 ? kept under RT for 2 days (acclimatization) followed by CS for 30 days. Control and T2 resulted in fruit with decreased flesh firmness (FF), and increased soluble solids (SS) and ascorbic acid (AA) contents. In these fruit the activity of endo-1,4-ß-glucanase (endo-1,4-ß-gluc), pectin methylesterase (PME), polygalacturonase (PG) and ß-galactosidase (ß-gal) increased. T1 resulted in translucent fruit with decreased FF, without any enzymatic activity changes, probably due to the physical disruption of the cytoskeleton. Further, there was an increased content of proteins corresponding to expansins in fruit kept under Control and T2 conditions, which suggests that these conditions do contribute to the synthesis and/or transport of proteins involved in the process of solubilization of the cell wall. In these fruit, there was also a major accumulation of gene transcripts corresponding to heat shock proteins (HSPs) of organelles related to endomembrane, which suggests participation of these genes in the prevention of damage caused by cold conditions. These data proved the hypotheses that acclimatization contributes to the expression of HSPs, and synthesis and transportat of proteins involved in the solubilization of the cell wall. The expression of these genes results in the normal ripening of the persimmon, as confirmed by the evolution of ethylene production.