Ubiquitylation of p53 by the APC/C inhibitor Trim39.

Tripartite motif 39 (Trim39) is a RING domain-containing E3 ubiquitin ligase able to inhibit the anaphase-promoting complex (APC/C) directly. Through analysis of Trim39 function in p53-positive and p53-negative cells, we have found, surprisingly, that p53-positive cells lacking Trim39 could not traverse the G1/S transition. This effect did not result from disinhibition of the APC/C. Moreover, although Trim39 loss inhibited etoposide-induced apoptosis in p53-negative cells, apoptosis was enhanced by Trim39 knockdown in p53-positive cells. Furthermore, we show here that the Trim39 can directly bind and ubiquitylate p53 in vitro and in vivo, leading to p53 degradation. Depletion of Trim39 significantly increased p53 protein levels and cell growth retardation in multiple cell lines. We found that the relative importance of Trim39 and the well-characterized p53-directed E3 ligase, murine double minute 2 (MDM2), varied between cell types. In cells that were relatively insensitive to the MDM2 inhibitor, nutlin-3a, apoptosis could be markedly enhanced by siRNA directed against Trim39. As such, Trim39 may serve as a potential therapeutic target in tumors with WT p53 when MDM2 inhibition is insufficient to elevate p53 levels and apoptosis.

Elucidating the Molecular Composition of Cartilage by Proteomics.

Articular cartilage consists of chondrocytes and two major components, a collagen-rich framework and highly abundant proteoglycans. Most prior studies defining the zonal distribution of cartilage have extracted proteins with guanidine-HCl. However, an unextracted collagen-rich residual is left after extraction. In addition, the high abundance of anionic polysaccharide molecules extracted from cartilage adversely affects the chromatographic separation. In this study, we established a method for removing chondrocytes from cartilage sections with minimal extracellular matrix protein loss. The addition of surfactant to guanidine-HCl extraction buffer improved protein solubility. Ultrafiltration removed interference from polysaccharides and salts. Almost four-times more collagen peptides were extracted by the in situ trypsin digestion method. However, as expected, proteoglycans were more abundant within the guanidine-HCl extraction. These different methods were used to extract cartilage sections from different cartilage layers (superficial, intermediate, and deep), joint types (knee and hip), and disease states (healthy and osteoarthritic), and the extractions were evaluated by quantitative and qualitative proteomic analyses. The results of this study led to the identifications of the potential biomarkers of osteoarthritis (OA), OA progression, and the joint specific biomarkers.

Sorption behavior of cationic and anionic dyes from aqueous solution on different types of activated carbons

The effect of dye molecular charges on their adsorption from solution was investigated by using different types of activated carbon adsorbents. Two types of model systems were used representing cationic and anionic dyes. Screening investigations using single point tests were used throughout the study. Cationic dyes, of which Methylene Blue is an example, showed a higher adsorption tendency towards activated carbon over anionic dyes represented by an ate-type reactive compound. Of the number of activated carbons tested, only one of the adsorbents showed an exception to this behavior, and a good relation was observed between Methylene Blue capacity and activated carbon performance. The high capacity of cationic dyes in comparison to anionic dyes was also evident in the results obtained by a preliminary kinetic study carried out on the selected systems. Surface net charge of activated carbon and the nature of attractions between the molecules were suggested to be one of the reasons attributed for this behavior.

Immune reactions to Bacteroides fragilis populations with three different types of capsule in a model of infection

The survival and growth of populations of the obligately anaerobic pathogenic bacterium Bacteroides fragilis enriched for large capsules (LCs), small capsules (SCs) or an electron-dense layer (EDL; non-capsulate by light microscopy) were examined in a mouse model of infection over a minimum period of 20 d. Chambers which allowed the influx of leukocytes, but not the efflux of bacteria, were implanted in the mouse peritoneal cavity. The LC and EDL populations consistently attained viable cell densities of the order of 10(8)-10(9) c.f.u. ml-1 within 24 h, whereas the SC population did not. However, after 3 d, all three bacterial populations maintained total viable numbers of 10(8)-10(9) c.f.u. ml-1 within the chambers. LC expression was selected against within 24 h in the model, the populations becoming non-capsulate by light microscopy, whereas in the SC population expression of the SC was retained by approximately 90% of the population. The EDL population remained non-capsulate by light microscopy throughout. Lymphocytes infiltrated the chambers to an equal extent for all three B. fragilis populations and at approximately 1000 times higher concentration than chambers which contained only quarter-strength Ringer's solution. The presence of neutrophils within the chambers did not cause a decrease in the total viable bacterial count. Each population elicited antibodies specific for outer-membrane proteins and polysaccharide, as detected by immunoblotting, which cross-reacted with the other populations. Differences were observed in the immunogenicity of the outer-membrane proteins within the three populations. Neutrophils were initially the predominant cell type in the chambers, but as the total leukocyte count increased with incubation time, neutrophils were outnumbered by other leukocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Agreement between clinical and ultrasound biomicroscopy findings in different types of glaucoma

Purpose. To evaluate agreement between clinical examination (slit lamp examination and gonioscopy) and ultrasound biomicroscopy (UBM) in characterizing various features of the anterior chamber angle and adjacent structures. Methods. Twenty-eight patients (51 eyes) with open angle glaucoma (14), closed angle glaucoma (4), narrow angle and/or plateau iris (10), who had undergone UBM between March 94 and September 95 were studied. Evaluated parameters included angle width, iris configuration and presence of angle closure. The UBMs were reviewed in a masked fashion. Results. In 87.8% of cases there was agreement (within 10 degrees) between gonioscopic and UBM estimates of angle width. In all cases with greater than 10 degrees difference (12.2%) the angle size estimated by UBM was less than that estimated clinically. Iris configuration was graded identically in 51% of cases; the majority of disagreements (76.1%) occurred in cases graded as "regular" by gonioscopy and as "steep" by UBM. Angle closure was judged to be present more frequently by UBM. Conclusions. UBM and gonioscopy do not necessarily yield identical values for angle width, iris configuration, and presence of angle closure.

Activation of MAPK by modified low-density lipoproteins in vascular smooth muscle cells

A high concentration of circulating low-density lipoproteins (LDL) is a major risk factor for atherosclerosis. Native LDL and LDL modified by glycation and/or oxidation are increased in diabetic individuals. LDL directly stimulate vascular smooth muscle cell (VSMC) proliferation; however, the mechanisms remain undefined. The extracellular signal-regulated kinase (ERK) pathway mediates changes in cell function and growth. Therefore, we examined the cellular effects of native and modified LDL on ERK phosphorylation in VSMC. Addition of native, mildly modified (oxidized, glycated, glycoxidized) and highly modified (highly oxidized, highly glycoxidized) LDL at 25 microg/ml to rat VSMC for 5 min induced a fivefold increase in ERK phosphorylation. To elucidate the signal transduction pathway by which LDL phosphorylate ERK, we examined the roles of the Ca(2+)/calmodulin pathway, protein kinase C (PKC), src kinase, and mitogen-activated protein kinase kinase (MEK). Treatment of VSMC with the intracellular Ca(2+) chelator EGTA-AM (50 micromol/l) significantly increased ERK phosphorylation induced by native and mildly modified LDL, whereas chelation of extracellular Ca(2+) by EGTA (3 mmol/l) significantly reduced LDL-induced ERK phosphorylation. The calmodulin inhibitor N-(6-aminohexyl)-1-naphthalenesulfonamide (40 micromol/l) significantly decreased ERK phosphorylation induced by all types of LDL. Downregulation of PKC with phorbol myristate acetate (5 micromol/l) markedly reduced LDL-induced ERK phosphorylation. Pretreatment of VSMC with a cell-permeable MEK inhibitor (PD-98059, 40 micromol/l) significantly decreased ERK phosphorylation in response to native and modified LDL. These findings indicate that native and mildly and highly modified LDL utilize similar signaling pathways to phosphorylate ERK and implicate a role for Ca(2+)/calmodulin, PKC, and MEK. These results suggest a potential link between modified LDL, vascular function, and the development of atherosclerosis in diabetes.

Glycation, glycoxidation, and cross-linking of collagen by glucose. Kinetics, mechanisms, and inhibition of late stages of the Maillard reaction

The Maillard or browning reaction between sugar and protein contributes to the increased chemical modification and cross-linking of long-lived tissue proteins in diabetes. To evaluate the role of glycation and oxidation in these reactions, we have studied the effects of oxidative and antioxidative conditions and various types of inhibitors on the reaction of glucose with rat tail tendon collagen in phosphate buffer at physiological pH and temperature. The chemical modifications of collagen that were measured included fructoselysine, the glycoxidation products N epsilon-(carboxymethyl)lysine and pentosidine and fluorescence. Collagen cross-linking was evaluated by analysis of cyanogen bromide peptides using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by changes in collagen solubilization on treatment with pepsin or sodium dodecylsulfate. Although glycation was unaffected, formation of glycoxidation products and cross-linking of collagen were inhibited by antioxidative conditions. The kinetics of formation of glycoxidation products proceeded with a short lag phase and were independent of the amount of Amadori adduct on the protein, suggesting that autoxidative degradation of glucose was a major contributor to glycoxidation and cross-linking reactions. Chelators, sulfhydryl compounds, antioxidants, and aminoguanidine also inhibited formation of glycoxidation products, generation of fluorescence, and cross-linking of collagen without significant effect on the extent of glycation of the protein. We conclude that autoxidation of glucose or Amadori compounds on protein plays a major role in the formation of glycoxidation products and cross-liking of collagen by glucose in vitro and that chelators, sulfhydryl compounds, antioxidants, and aminoguanidine act as uncouplers of glycation from subsequent glycoxidation and cross-linking reactions.

Three Types of Temporal Perspective: Characterizing Developmental Changes in Temporal Thought

This paper provides an outline of the development of temporal thinking that is underpinned by the idea that temporal cognition shifts from being event dependent to event independent over the preschool period. I distinguish between three different ways in which it may be possible to have a perspective on time: (1) a perspective that is grounded in script-like representations of repeated events; (2) a more sophisticated perspective that brings in an fundamental categorical distinction between events that have already happened and events that are yet to come; and (3) a mature temporal perspective that involves orienting oneself in time using a linear temporal framework, with a grasp of the distinctions between past, present, and future. I propose that, with development, children possess each of these types of perspective in turn, and that only the last of these involves being able to represent time in an event-independent way.

Prevalence of the different types of age-related cataract in an African population.

PURPOSE: To describe the prevalence of different types of cataract and their association with visual acuity in a Tanzanian population aged 40 years and older. METHODS: A prevalence survey for lens opacity, glaucoma, and visual impairment was carried out on all residents age 40 and older of six villages in Kongwa, Tanzania. One examiner graded the lens for presence of nuclear (NSC), posterior subcapsular (PSC), and cortical cataract (CC), using the new WHO Simplified Cataract Grading System. Visual acuity was measured in each eye, both presenting and best corrected, using an illiterate E chart. RESULTS: The proportion of eligible subjects participating was 90% (3268/3641). The prevalence of cataract was as follows: NSC, 15.6%; CC, 8.8%; and PSC, 1.9%. All types of cataract increased with age, from NSC, 1.7%; CC, 2.4%; and PSC, 0.4% for those aged 40 to 49 years to NSC, 59.2%; CC, 23.5%; and PSC, 5.9% for those aged 70 years and older (P < 0.0001 for all cataract types, chi(2) test for trend). Cataract prevalence was higher among women than men for NSC (P = 0.0001), but not for CC (P = 0.15) or PSC (P = 0.25), after adjusting for age. Prevalence rates of visual impairment (BCVA < 6/12), US blindness (< or = 6/60) and WHO blindness (< 6/120) for this population were 13.3%, 2.1%, and 1.3%, respectively. Older age and each of the major types of pure and mixed cataract were independently associated with worse vision in regression modeling. CONCLUSIONS: Unlike African-derived populations in Salisbury and Barbados, NSC rather than CC was most prevalent in this African population. The seeming lower prevalence of CC may to some extent be explained by different grading schemes, differential availability of cataract surgery, the younger mean age of the Tanzanian subjects, and a higher prevalence of NSC in this population.

Photoinactivation of viruses by porphyrins

A inativação fotodinâmica tem sido usada com sucesso na inativação de microorganismos. Diversos aspetos da inativação fotodinâmica foram já estudados para diferentes microrganismos, contudo, existe ainda pouca informação disponível no que diz respeito à inativação de bacteriófagos por processos fotodinâmicos. Este trabalho pretendeu elucidar e avaliar vários aspetos da fotoinativação de vírus, em particular de bacteriófagos, incluindo (i) o efeito de diversos parâmetros de luz utilizados na fotoinativação de bacteriófagos; (ii) a eficiência da inativação fotodinâmica de diferentes tipos de bacteriófagos (fagos do tipo DNA e RNA); (iii) o principal mecanismo através do qual a inativação fotodinâmica tem lugar; (iv) o efeito da fotoinativação nas proteínas do bacteriófago; e (v) o possível desenvolvimento de resistência e recuperação da viabilidade após vários tratamentos fotodinâmicos consecutivos. Para avaliar o efeito dos diferentes parâmetros de luz, suspensões fágicas com 107 UFP mL-1 foram irradiadas com diferentes fontes e doses de luz, intensidades luminosas e tempos de irradiação (30,90 e 270 min) na presença de 0,5; 1,0 e 5,0 μM dos derivados porfirínicos catiónicos Tri- Py+-Me-PF e Tetra-Py+-Me. A eficiência da fotoinativação de diferentes fagos do tipo DNA e RNA, foi avaliada através da irradiação da suspensão fágica com luz branca (40 W m-2) durante 270 min na presença de 0,5 e 5,0 μM do derivado porfirínico Tri-Py+-Me-PF, respetivamente para os fagos do tipo RNA e DNA. O mecanismo através do qual a fotoinativação de fagos de DNA (fago do tipo T4) e de RNA (fago Qb) tem lugar foi avaliado por exposição da suspensão fágica à luz branca com uma potência de 40 W m-2, na presença de fotossensibilizador (Tri-Py+-Me-PF e Tetra-Py+-Me) e inibidores, quer do oxigénio singuleto (azida de sódio e L-histidina) quer de radicais livres (Dmanitol e L-cisteína). Os danos nas proteínas do fago do tipo T4, induzidos pelas espécies reativas de oxigénio geradas por 5,0 μM Tri-Py+-Me-PF, foram avaliados pelo método convencional de SDS-PAGE e por espectroscopia de infravermelho. O possível desenvolvimento de resistência e recuperação da viabilidade após a inativação fotodinâmica dos bacteriófagos foi avaliado após dez ciclos consecutivos de tratamento fotodinâmico incompletos (120 min sob irradiação de luz branca a uma potência de 40 W m-2) na presença de 5,0 μM do derivado porfirínico Tri-Py+-Me-PF. Os resultados deste trabalho mostraram que (i) quando uma quantidade de energia (dose de luz) determinada foi aplicada numa suspensão fágica, a partir de uma mesma fonte irradiação, a fotoinactivação do fago foi tanto mais eficiente quanto mais baixa foi a potência luminosa aplicada; (ii) os bacteriófagos foram eficientemente inativados até ao limite de deteção (redução de 6-7 log); (ii) os fagos do tipo RNA foram inativados mais facilmente do que os fagos do tipo DNA (tempos de exposição mais curtos e com concentração de fotossensibilizador dez vezes menor do que a usada para inativar os fagos do tipo DNA); (iii) o mecanismo do tipo II (via produção de oxigénio singuleto) foi o principal mecanismo através do qual a fotoinativação dos bacteriófagos teve lugar; (iv) foi possível detectar danos no perfil proteico após tratamento fotodinâmico e a espectroscopia de infravermelho apresentou-se como uma metodologia promissora de screening para avaliação dos danos induzidos pela inativação fotodinâmica em proteínas; e (v) após dez ciclos consecutivos de tratamento fotodinâmico, o fago do tipo T4 não revelou nenhum tipo de resistência ao tratamento fotodinâmico nem recuperou a sua viabilidade. Como conclusão, a inativação fotodinâmica microbiana é uma tecnologia bastante eficaz para a fotoinativação de bacteriófagos do tipo DNA e RNA sem invólucro, a qual pode ser considerada como uma alternativa ao tratamento convencional com agentes antivíricos, mesmo com intensidades luminosas baixas, sem o risco associado de desenvolvimento de mecanismos de resistência.

Does this case hold the answer to one of the worse types of pain in medicine--that of loin pain haematuria syndrome (LPHS)

A patient with loin pain haematuria syndrome suffering chronic throbbing pulsing pain overlaid with prolonged periods of incapacitating colic and overnight vomiting was presented 10 months following diagnosis. Ultrasound was normal. No renal or ureteral stones, or filling defects were seen on CT. At cytoscopy, bladder and urethra were normal, and bloody urine effluxed from the left ureteric orifice. The ureters were normal at diagnosis, and developed new abutting non‐penetrating calcifications by 8 months. Pain episodes of complete incapacitating intensity of 2–4 h duration were reduced to 10 min with 5 mg crushed tadalafil administered at onset. If tadalafil was delayed to after onset, the original course of agony resulted. Daily tadalafil reduced loin pain intensity, but not the exacerbations. Tadalafil efficacy may indicate that the pain exacerbations are due to spasm of ureter smooth muscle. 5 mg tadalafil taken at onset alleviated severe loin pain exacerbations in this case of loin pain haematuria syndrome.

How does developmental plasticity differ between diverse types of nutrition regimes?

Tese de mestrado em Biologia Evolutiva e do Desenvolvimento, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2016

Brewer's spent grain from different types of malt: Evaluation of the antioxidant activity and identification of the major phenolic compounds

The antioxidant activity and phenolic composition of brewer's spent grain (BSG) extracts obtained by microwave-assisted extraction from twomalt types (light and darkmalts) were investigated. The total phenolic content (TPC) and antioxidant activity among the light BSG extracts (pilsen, melano, melano 80 and carared)were significantly different (p b 0.05) compared to dark extracts (chocolate and black types), with the pilsen BSG showing higher TPC (20 ± 1 mgGAE/g dry BSG). In addition, the antioxidant activity assessed by 2,2-diphenyl- 1-picrylhydrazyl, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) and deoxyribose assays decreased as a result of increasing kilning temperatures in the following order: pilsen N melano N melano 80 N carared N chocolate N black. HPLC-DAD/ESI-MS/MS analysis indicated the presence of phenolic acids, such as ferulic, p-coumaric and syringic acids, as well as several isomeric ferulate dehydrodimers and one dehydrotrimer. Chocolate and black extracts, obtained frommalts submitted to the highest kilning temperatures, showed the lowest levels of ferulic and p-coumaric acids. These results suggested that BSG extracts from pilsen malt might be used as an inexpensive and good natural source of antioxidants with potential interest for the food, pharmaceutical and/or cosmetic industries after purification.

Assigning real-time tasks on heterogeneous multiprocessors with two unrelated types of processors

A preliminary version of this paper appeared in Proceedings of the 31st IEEE Real-Time Systems Symposium, 2010, pp. 239–248.

Electrodialytic remediation of two types of air pollution control residues and their applicability in construction materials

Dissertação para obtenção do Grau de Mestre em Engenharia do Ambiente Perfil de Engenharia de Sistemas Ambientais