p53 Regulates the Formation of Lamellipodia and Circular Dorsal Ruffles Through Caldesmon and PTEN

Vascular smooth muscle cell migration is a significant contributor to many aspects of heart disease, and specifically atherosclerosis. Tissue damage in the arteries can result in the formation of a fatty streak. Smooth muscle cells (SMC) can then migrate to this site to form a fibrous cap, stabilizing the fatty plaque. Since cardiovascular disease is the leading cause of death in developed countries, this function of SMC is an essential area of study. The formation of lamellipodia and circular dorsal ruffles were studied in this project as indicators that cell migration is occurring. The roles of the proteins p53, Rac, caldesmon and PTEN were investigated with regards to these actin-based structures. The tumour suppressor p53 is often reported to cause apoptosis, senescence or cell cycle arrest when stress is placed on a cell, but has recently been shown to regulate cell migration as well. It was determined in this project that p53 could inhibit the formation of both lamellipodia and circular dorsal ruffles. It was also shown that this could occur directly through an inhibition of the GTPase Rac. Previous studies have shown that p53 can upregulate caldesmon, a protein which is known to bind to and stabilize actin filaments while inhibiting Arp2/3-mediated branching. It was confirmed that p53 could upregulate caldesmon, and that caldesmon could inhibit the formation of lamellipodia and circular dorsal ruffles. The phosphorylation of caldesmon by p21-associated kinase (PAK) or extracellular signal-related kinase (Erk) was shown to effectively reverse the ability of caldesmon to inhibit these structures. The role of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) was also studied with regards to this signalling pathway. PTEN was shown to inhibit lamellipodia and circular dorsal ruffles through its lipid phosphatase activity. It was concluded that p53 can inhibit the formation of lamellipodia and circular dorsal ruffles in vascular SMC, and that this occurs through Rac, caldesmon and PTEN.

Identification of protease and ADP-ribose 1''-monophosphatase activities associated with transmissible gastroenteritis virus non-structural protein 3.

The replicase polyproteins, pp1a and pp1ab, of porcine Transmissible gastroenteritis virus (TGEV) have been predicted to be cleaved by viral proteases into 16 non-structural proteins (nsp). Here, enzymic activities residing in the amino-proximal region of nsp3, the largest TGEV replicase processing product, were characterized. It was shown, by in vitro translation experiments and protein sequencing, that the papain-like protease 1, PL1pro, but not a mutant derivative containing a substitution of the presumed active-site nucleophile, Cys1093, cleaves the nsp2|nsp3 site at 879Gly|Gly880. By using an antiserum raised against the pp1a/pp1ab residues 526–713, the upstream processing product, nsp2, was identified as an 85 kDa protein in TGEV-infected cells. Furthermore, PL1pro was confirmed to be flanked at its C terminus by a domain (called X) that mediates ADP-ribose 1''-phosphatase activity. Expression and characterization of a range of bacterially expressed forms of this enzyme suggest that the active X domain comprises pp1a/pp1ab residues Asp1320–Ser1486.

Impact of oncogenic driver mutations on feedback between the PI3K and MEK pathways in cancer cells

Inhibition of the PI3K (phosphoinositide 3-kinase)/Akt/mTORC1 (mammalian target of rapamycin complex 1) and Ras/MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase]/ERK pathways for cancer therapy has been pursued for over a decade with limited success. Emerging data have indicated that only discrete subsets of cancer patients have favourable responses to these inhibitors. This is due to genetic mutations that confer drug insensitivity and compensatory mechanisms. Therefore understanding of the feedback mechanisms that occur with respect to specific genetic mutations may aid identification of novel biomarkers that predict patient response. In the present paper, we show that feedback between the PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways is cell-line-specific and highly dependent on the activating mutation of K-Ras or overexpression c-Met. We found that cell lines exhibited differential signalling and apoptotic responses to PD184352, a specific MEK inhibitor, and PI103, a second-generation class I PI3K inhibitor. We reveal that feedback from the PI3K/Akt/mTORC1 to the Ras/MEK/ERK pathway is present in cancer cells harbouring either K-Ras activating mutations or amplification of c-Met but not the wild-type counterparts. Moreover, we demonstrate that inhibition of protein phosphatase activity by OA (okadaic acid) restored PI103-mediated feedback in wild-type cells. Together, our results demonstrate a novel mechanism for feedback between the PI3K/Akt/mTORC1 and the Ras/MEK/ERK pathways that only occurs in K-Ras mutant and c-Met amplified cells but not the isogenic wild-type cells through a mechanism that may involve inhibition of a specific endogenous phosphatase(s) activity. We conclude that monitoring K-Ras and c-Met status are important biomarkers for determining the efficacy of PI103 and other PI3K/Akt inhibitors in cancer therapy.

Biocompatibility of Calcium Phosphate Bone Cement with Optimised Mechanical Properties

The broad aim of this work was to investigate and optimise the properties of calcium phosphate bone cements (CPCs) for use in vertebroplasty to achieve effective primary fixation of spinal fractures. The incorporation of collagen, both bovine and from a marine sponge (Chondrosia reniformis), into a CPC was investigated. The biological properties of the CPC and collagen-CPC composites were assessed in vitro through the use of human bone marrow stromal cells. Cytotoxicity, proliferation and osteoblastic differentiation were evaluated using lactate dehydrogenase, PicoGreen and alkaline phosphatase activity assays respectively. The addition of both types of collagen resulted in an increase in cytotoxicity, albeit not to a clinically relevant level. Cellular proliferation after 1, 7 and 14 days was unchanged. The osteogenic potential of the CPC was reduced through the addition of bovine collagen but remained unchanged in the case of the marine collagen. These findings, coupled with previous work showing that incorporation of marine collagen in this way can improve the physical properties of CPCs, suggest that such a composite may offer an alternative to CPCs in applications where low setting times and higher mechanical stability are important.

Tyrosine phosphorylation and dephosphorylation in Burkholderia cenocepacia affect biofilm formation, growth under nutritional deprivation, and pathogenicity

Burkholderia cenocepacia, a member of the B. cepacia complex (Bcc), is an opportunistic pathogen causing serious chronic infections in patients with cystic fibrosis. Tyrosine phosphorylation has emerged as an important post-translational modification modulating the physiology and pathogenicity of Bcc bacteria. Here, we investigated the predicted bacterial tyrosine kinases BCAM1331 and BceF, and the low molecular weight protein tyrosine phosphatases BCAM0208, BceD and BCAL2200 of B. cenocepacia K56-2. We show that BCAM1331, BceF, BCAM0208 and BceD contributed to biofilm formation, while BCAL2200 was required for growth in nutrient-limited conditions. Multiple deletions of either tyrosine kinase or low molecular weight protein tyrosine phosphatases genes resulted in attenuation of B. cenocepacia intramacrophage survival and reduced pathogenicity in the Galleria mellonella larvae infection model. Experimental evidence indicates that BCAM1331 displays a reduced
tyrosine autophosphorylation activity compared to BceF. Using the artificial substrate p-nitrophenyl phosphate, the phosphatase activity of the three low molecular weight protein tyrosine phosphatases demonstrated similar kinetic parameters. However, only BCAM0208 and BceD could dephosphorylate BceF. Further, BCAL2200 becomes tyrosine phosphorylated in vivo and catalyzes its auto-dephosphorylation. Together, our data suggest that despite having similar biochemical activities low molecular weight protein tyrosine phosphatases and tyrosine kinases have both overlapping and specific roles in the physiology of B. cenocepacia.

Nutritional modulation of innate immune parameters in the epidermal mucus of Senegalese sole (Solea senegalensis)=Influência da dieta nos parâmetros da resposta imunitária inata do muco de linguado (Solea senegalensis)

Dissertação mest., Biologia Marinha, Universidade do Algarve, 2008

Role of insulin and insulin-like peptides in bone formation: identification of bone-specific target genes and regulatory mechanisms, and characterization of the insulin-mimetic effect of vanadium

Tese de Doutoramento em Biologia, Especialidade em Biologia Molecular, Universidade do Algarve, 2008

Efeito da combinação de antibióticos e sinvastatina sobre microrganismos de interesse endodôntico e na expressão de marcadores odontoblásticos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Characterization of the Toxicological Effects of Aminocarb on Rats: Hematological, Biochemical, and Histological Analyses

Aminocarb is a widely applied carbamate insecticide with action of controlling pests such as Lepidoptera and Coleoptera. In this study, subchronic effects on Wistar rats were investigated using hematological, biochemical, and histological techniques. Rats were exposed orally at sublethal levels of 10, 20, or 40 mg/kg body weight (groups A, B, and C, respectively) for 14 d. Hematological results revealed no statistical differences after 1 d of exposure but significant reduction in white blood cells detected after 7 d of exposure in group C, as well as, in all treated groups after 14 d of exposure. Biochemical data showed a decrease of acetylcholinesterase activity in all groups after 1 d of exposure with a return to normal after 7 and 14 d. Significant increase in alkaline phosphatase activity of rats exposed to aminocarb was noted after 7 d of treatment. The levels of triglycerides were also significantly decreased. The present investigation also showed a significant increase in content of serum urea and creatinine in animals from group A (14 d), and from groups B and C (7 and 14 d). Histological results demonstrated hemorrhagic focus on hepatic and renal parenchyma in all exposed groups. Taken together, the attained results were dose dependent and indicated adverse effects of aminocarb on hepatic and renal functions, as well as on immune responsiveness at sublethal tested doses.

A biochemical predictor of performance during mesophilic anaerobic fermentation of starch wastewater

The aim of this study was to determine the potential of biochemical parameters, such as enzyme activity and adenosine triphosphate (ATP) levels, as monitors of process performance in the Upflow Anaerobic Sludge Blanket (UASB) reactor utilizing a starch wastewater. The acid and alkaline phosphatase activity and the ATP content of the UASB sludge were measured in response to changes in flow rate and nutrient loading. Conventional parameters of process performance, such as gas production, acetic acid production, COD, phosphorus, nitrogen and suspended solids loadings and % COD removal were also monitored. The response of both biochemical and conventional parameters to changing process conditions was then compared. Alkaline phosphatase activity exhibited the highest activity over the entire study perioda A high suspended solids loading was observed to upset the system in terms of gas production, acetic acid production and % COD removala The initial rate of increase in alkaline phosphatase activity following an increase in loading was four times as great during process upset than under conditions of good performance. The change in enzyme actiVity was also more sensitive to process upset than changes in acetic acid production. The change in ATP content of the sludge with time suggested that enzyme actiVity was changing independently of the actual viable biomass present. The bacterial composition of the anaerobic sludge granules was similar to that of other sludge bed systems, at the light and scanning electron microscope level. Isolated serum bottle cultures produced several acids involved in anaerobic carbohydrate metabolism. The overall performance of the UASB system indicated that higher loadings of soluble nutrients could have been tolerated by the system.

Réparation par excision de nucléotides des dommages induits par rayons ultraviolets dans les mélanomes humains

Les mélanomes malins (MM) constituent le deuxième type de cancer le plus fréquent chez les jeunes adultes canadiens (entre 20 et 44 ans) ainsi qu’un des rares cancers dont l’incidence augmente annuellement. À moins que les MM ne soient excisés à temps par chirurgie, les chances de survie des patients sont pratiquement nulles puisque ce type de tumeur est très réfractaire aux traitements conventionnels. Il est bien connu que l’exposition aux rayons ultraviolets (UV), induisant des photoproduits génotoxiques, est une déterminante majeure dans l’acquisition de MM. À cet effet, la réparation par excision de nucléotides (NER) est la ligne de défense principale contre le développement des mélanomes puisqu’elle est la voie de réparation prépondérante en ce qui a trait aux dits photoproduits. Malgré cela, la contribution potentielle de défauts de la NER au développement des MM dans la population normale n’est toujours pas bien établie. Notre laboratoire a précédemment développé une méthode basée sur la cytométrie de flux qui permet de mesurer la NER en fonction du cycle cellulaire. Cette méthode a déjà mise en évidence qu’une déficience de l’activité de la protéine ATR peut mener à une déficience de la NER exclusive à la phase S dans des fibroblastes humains. Pareillement, nous avons démontré que plusieurs lignées cellulaires cancéreuses modèles comportent une déficience en NER en phase S, suggérant qu’une telle déficience puisse caractériser certains types de cancers. Nous avons voulu savoir si une déficience en NER en phase S pouvait être associée à une proportion significative de mélanomes et si le tout pouvait être attribuable à une diminution de l’activité d’ATR. Nos objectifs ont donc été de : (i) mesurer l’efficacité de la NER en fonction du cycle cellulaire dans les MM en comparaison avec les mélanocytes primaires, (ii) vérifier si le niveau d’activité d’ATR corrèle avec l’efficacité de la NER en phase S dans les lignées de MM et (iii) voir si un gène fréquemment muté dans les mélanomes (tels PTEN et BRAF) pouvait coopérer avec ATR pour réguler la NER en phase S dans les mélanomes. Nous avons démontré que 13 lignées de MM sur 16 ont une capacité grandement diminuée à réparer les photoproduits induits par UV spécifiquement en phase S. De plus, cette déficience corrèle fortement avec une réduction de l’activation d’ATR et, dans plusieurs lignées de MM, avec une phosphorylation d’Akt plus importante. L’utilisation d’ARN interférent ou d’un inhibiteur du suppresseur de tumeurs PTEN, a permis, en plus d’augmenter la phosphorylation d’Akt, de réduire la réparation des photoproduits et l’activation d’ATR dans les cellules en phase S. En addition, (i) l’expression ectopique de la protéine PTEN sauvage dans des lignées déficientes en PTEN (mais pas d’une protéine PTEN sans activité phosphatase) ou (ii) l’inhibition pharmacologique d’Akt a permis d’augmenter la réparation en phase S ainsi que l’activation d’ATR. En somme, cette étude démontre qu’une signalisation d’ATR dépendante de PTEN/Akt amenant à une réparation déficiente des photoproduits génomiques causés par les UV en phase S peut être déterminante dans le développement des mélanomes induits par UV.

Acute salinity stress alters the haemolymph metabolic profile of Penaeus monodon and reduces immunocompetence to white spot syndrome virus infection

Influence of acute salinity stress on the immunological and physiological response of Penaeus monodon to white spot syndrome virus (WSSV) infection was analysed. P. monodon maintained at 15‰ were subjected to acute salinity changes to 0‰ and 35‰ in 7 h and then challenged orally with WSSV. Immune variables viz., total haemocyte count, phenol oxidase activity (PO), nitroblue tetrazolium salt (NBT) reduction, alkaline phosphatase activity (ALP), acid phosphatase activity (ACP) and metabolic variables viz., total protein, total carbohydrates, total free amino acids (TFAA), total lipids, glucose and cholesterol were determined soon after salinity change and on post challenge days 2 (PCD2) and 5 (PCD5). Acute salinity change induced an increase in metabolic variables in shrimps at 35‰ except TFAA. Immune variables reduced significantly (Pb0.05) in shrimps subjected to salinity stress with the exception of ALP and PO at 35‰ and the reduction was found to be more at 0‰. Better performance of metabolic and immune variables in general could be observed in shrimps maintained at 15‰ that showed significantly higher post challenge survival following infection compared to those under salinity stress. Stress was found to be higher in shrimps subjected to salinity change to lower level (0‰) than to higher level (35‰) as being evidenced by the better immune response and survival at 35‰. THC (Pb0.001), ALP (Pb0.01) and PO (Pb0.05) that together explained a greater percentage of variability in survival rate, could be proposed as the most potential health indicators in shrimp haemolymph. It can be concluded from the study that acute salinity stress induces alterations in the haemolymph metabolic and immune variables of P. monodon affecting the immunocompetence and increasing susceptibility to WSSV, particularly at low salinity stress conditions

Cereal/legume rotations affect chemical properties and biological activities in two West African soils

A more widespread use of cereal/legume rotations has been suggested as a means to sustainably meet increasing food demands in sub-Saharan West Africa. Enhanced cereal yields following legumes have been attributed to chemical and biological factors such as higher levels of mineral nitrogen (Nmin) and arbuscular mycorrhizae (AM) but also to lower amounts of plant parasitic nematodes. This study was conducted under controlled conditions to examine the relative contribution of AM, plant parasitic nematodes and increased nitrogen (N) and phosphorus (P) availability to cereal/legume rotation effects on two West African soils. Sample soils were taken from field experiments at Gaya (Niger) and Fada (Burkina Faso) supporting continuous cereal and cereal/legume rotation systems and analysed for chemical and biological parameters. Average increases in cereal shoot dry matter (DM) of rotation cereals compared with continuous cereals were 490% at Gaya and 550% at Fada. Shoot P concentration of rotation millet was significantly higher than in continuous millet and P uptake in rotation cereals was on average 62.5-fold higher than in continuous cereals. Rotation rhizosphere soils also had higher pH at both sites. For the Fada soil, large increases in Bray1-P and organic P were observed in bulk and rhizosphere soils. Plant parasitic nematodes in roots of continuous cereals were 60–80-fold higher than in those of rotation cereals. In both cropping systems mycorrhizal infection rates were similar at 37 days after sowing (DAS) but at 57 DAS AM infection was 10–15% higher in rotation sorghum than in continuous sorghum. This study provides strong evidence that cereal/legume rotations can enhance P nutrition of cereals through improved soil chemical P availability and microbiologically increased P uptake.

Causes of legume-rotation effects in increasing cereal yields across the Sudanian, Sahelian and Guinean zone of West Africa

On-farm experiments and pot trials were conducted on eight West African soils to explore the mechanisms governing the often reported legume rotation-induced cereal growth increases in this region. Crops comprised pearl millet (Pennisetum glaucum L.), sorghum (Sorghum bicolor Moench), maize (Zea mays L.), cowpea (Vigna unguiculata Walp.) and groundnut (Arachis hypogaea L.). In groundnut trials the observed 26 to 85% increases in total dry matter (TDM) of rotation cereals (RC) compared with continuous cereals (CC) in the 4th year appeared to be triggered by site- and crop-specific early season differences in nematode infestation (up to 6-fold lower in RC than in CC), enhanced Nmin and a 7% increase in mycorrhizal (AM) infection. In cowpea trials yield effects on millet and differences in nematode numbers, Nmin and AM were much smaller. Rhizosphere studies indicated effects on pH and acid phosphatase activity as secondary causes for the observed growth differences between RC and CC. In the study region legume-rotation effects on cereals seemed to depend on the capability of the legume to suppress nematodes and to enhance early N and P availability for the subsequent cereal.

Effects of phthalic acid esters on the liver and thyroid

The effects, over periods from 3 days to 9 months of administration, of diets containing di-2-ethylhexyl phthalate are very similar to those observed in rats administered diets containing hypolipidemic drugs such as clofibrate. Changes occur in a characteristic order commencing with alterations in the distribution of lipid within the liver, quickly followed by proliferation of hepatic peroxisomes and induction of the specialized P-450 isoenzyme(s) catalyzing omega oxidation of fatty acids. There follows a phase of mild liver damage indicated by induction of glucose-6-phosphatase activity and a loss of glycogen, eventually leading to the formation of enlarged lysosomes through autophagy and the accumulation of lipofuscin. Associated changes are found in the kidney and thyroid. The renal changes are limited to the proximal convoluted tubules and are generally similar to changes found in the liver. The effects on the thyroid are more marked. Although the levels of thyroxine in plasma fail to about half normal values, serum triiodothyronine remains close to normal values while the appearance of the thyroid varies, very marked hyperactivity being noted 7 days after commencement of treatment, this is less marked at 14 days, but even after 9 months treatment there is clear cut evidence for hyperactivity with colloid changes which indicate this has persisted for some time. Straight chain analogs of di-2-ethylhexyl phthalate, di-n-hexyl phthalate and di-n-oxtyl phthalate differ entirely in their short-term effects on the liver and kidney but have similar effects on the thyroid. The short-term in vivo hepatic effects of the three phthalate esters can be reproduced in hepatocytes in tissue culture. All three phthalate esters, as well as clofibrate, have early marked effects on the metabolism of fatty acids in isolated hepatocytes. The nature of these changes is such as to increase storage of lipid in the liver. A hypothesis is presented to explain the progress from these initial metabolic effects to the final formation of liver tumors.