19 resultados para Specifity


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To ensure signalling fidelity, kinases must act only on a defined subset of cellular targets. Appreciating the basis for this substrate specificity is essential for understanding the role of an individual protein kinase in a particular cellular process. The specificity in the cell is determined by a combination of peptide specificity of the kinase (the molecular recognition of the sequence surrounding the phosphorylation site), substrate recruitment and phosphatase activity. Peptide specificity plays a crucial role and depends on the complementarity between the kinase and the substrate and therefore on their three-dimensional structures. Methods for experimental identification of kinase substrates and characterization of specificity are expensive and laborious, therefore, computational approaches are being developed to reduce the amount of experimental work required in substrate identification. We discuss the structural basis of substrate specificity of protein kinases and review the experimental and computational methods used to obtain specificity information. (c) 2005 Elsevier B.V. All rights reserved.

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It is important to detect and treat malnutrition in hospital patients so as to improve clinical outcome and reduce hospital stay. The aim of this study was to develop and validate a nutrition screening tool with a simple and quick scoring system for acute hospital patients in Singapore. In this study, 818 newly admitted patients aged above 18 years old were screened using five parameters that contribute to the risk of malnutrition. A dietitian blinded to the nutrition screening score assessed the same patients using the reference standard, Subjective Global Assessment (SGA) within 48 hours. The sensitivity and specificity were established using the Receiver Operator Characteristics (ROC) curve and the best cutoff scores determined. The nutrition parameter with the largest Area Under the ROC Curve (AUC) was chosen as the final screening tool, which was named 3-Minute Nutrition Screening (3-MinNS). The combination of the parameters weight loss, intake and muscle wastage (3-MinNS), gave the largest AUC when compared with SGA. Using 3-MinNS, the best cutoff point to identify malnourished patients is three (sensitivity 86%, specificity 83%). The cutoff score to identify subjects at risk of severe malnutrition is five (sensitivity 93%, specificity 86%). 3-Minute Nutrition Screening is a valid, simple and rapid tool to identify patients at risk of malnutrition in Singapore acute hospital patients. It is able to differentiate patients at risk of moderate malnutrition and severe malnutrition for prioritization and management purposes.

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Microbial pollution in water periodically affects human health in Australia, particularly in times of drought and flood. There is an increasing need for the control of waterborn microbial pathogens. Methods, allowing the determination of the origin of faecal contamination in water, are generally referred to as Microbial Source Tracking (MST). Various approaches have been evaluated as indicatorsof microbial pathogens in water samples, including detection of different microorganisms and various host-specific markers. However, until today there have been no universal MST methods that could reliably determine the source (human or animal) of faecal contamination. Therefore, the use of multiple approaches is frequently advised. MST is currently recognised as a research tool, rather than something to be included in routine practices. The main focus of this research was to develop novel and universally applicable methods to meet the demands for MST methods in routine testing of water samples. Escherichia coli was chosen initially as the object organism for our studies as, historically and globally, it is the standard indicator of microbial contamination in water. In this thesis, three approaches are described: single nucleotide polymorphism (SNP) genotyping, clustered regularly interspaced short palindromic repeats (CRISPR) screening using high resolution melt analysis (HRMA) methods and phage detection development based on CRISPR types. The advantage of the combination SNP genotyping and CRISPR genes has been discussed in this study. For the first time, a highly discriminatory single nucleotide polymorphism interrogation of E. coli population was applied to identify the host-specific cluster. Six human and one animal-specific SNP profile were revealed. SNP genotyping was successfully applied in the field investigations of the Coomera watershed, South-East Queensland, Australia. Four human profiles [11], [29], [32] and [45] and animal specific SNP profile [7] were detected in water. Two human-specific profiles [29] and [11] were found to be prevalent in the samples over a time period of years. The rainfall (24 and 72 hours), tide height and time, general land use (rural, suburban), seasons, distance from the river mouth and salinity show a lack of relashionship with the diversity of SNP profiles present in the Coomera watershed (p values > 0.05). Nevertheless, SNP genotyping method is able to identify and distinquish between human- and non-human specific E. coli isolates in water sources within one day. In some samples, only mixed profiles were detected. To further investigate host-specificity in these mixed profiles CRISPR screening protocol was developed, to be used on the set of E. coli, previously analysed for SNP profiles. CRISPR loci, which are the pattern of previous DNA coliphages attacks, were considered to be a promising tool for detecting host-specific markers in E. coli. Spacers in CRISPR loci could also reveal the dynamics of virulence in E. coli as well in other pathogens in water. Despite the fact that host-specificity was not observed in the set of E. coli analysed, CRISPR alleles were shown to be useful in detection of the geographical site of sources. HRMA allows determination of ‘different’ and ‘same’ CRISPR alleles and can be introduced in water monitoring as a cost-effective and rapid method. Overall, we show that the identified human specific SNP profiles [11], [29], [32] and [45] can be useful as marker genotypes globally for identification of human faecal contamination in water. Developed in the current study, the SNP typing approach can be used in water monitoring laboratories as an inexpensive, high-throughput and easy adapted protocol. The unique approach based on E. coli spacers for the search for unknown phage was developed to examine the host-specifity in phage sequences. Preliminary experiments on the recombinant plasmids showed the possibility of using this method for recovering phage sequences. Future studies will determine the host-specificity of DNA phage genotyping as soon as first reliable sequences can be acquired. No doubt, only implication of multiple approaches in MST will allow identification of the character of microbial contamination with higher confidence and readability.

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The purpose of this work was to identify some of the genes of the catabolic route of L-rhamnose in the yeast Pichia stipitis. There are at least two distinctly different pathways for L-rhamnose catabolism. The one described in bacteria has phosphorylated intermediates and the enzymes and the genes of this route have been described. The pathway described in yeast does not have phosphorylated intermediates. The intermediates and the enzymes of this pathway are known but none of the genes have been identified. The work was started by purifying the L-rhamnose dehydrogenase, which oxidates L-rhamnose to rhamnonic acid-gamma-lactone. NAD is used as a cofactor in this reaction. A DEAE ion exchange column was used for purification. The active fraction was further purified using a non-denaturing PAGE and the active protein identified by zymogram staining. In the last step the protein was separated in a SDS-PAGE, the protein band trypsinated and analysed by MALDI-TOF MS. This resulted in the identification of the corresponding gene, RHA1, which was then, after a codon change, expressed in Saccharomyces cerevisiae. Also C- or N-terminal histidine tags were added but as the activity of the enzyme was lost or strongly reduced these were not used. The kinetic properties of the protein were analysed in the cell extract. Substrate specifity was tested with different sugars; L-rhamnose, L-lyxose and L-mannose were oxidated by the enzyme. Vmax values were 180 nkat/mg, 160 nkat/mg and 72 nkat/mg, respectively. The highest affinity was towards L-rhamnose, the Km value being 0.9 mM. Lower affinities were obtained with L-lyxose, Km 4.3 mM, and L-mannose Km 25 mM. Northern analysis was done to study the transcription of RHA1 with different carbon sources. Transcription was observed only on L-rhamnose suggesting that RHA1 expression is L-rhamnose induced. A RHA1 deletion cassette for P. stipitis was constructed but the cassette had integrated randomly and not targeted to delete the RHA1 gene. Enzyme assays for L-lactaldehyde dehydrogenase were done similarly to L-rhamnose dehydrogenase assays. NAD is used as a cofactor also in this reaction where L-lactaldehyde is oxidised to L-lactate. The observed enzyme activities were very low and the activity was lost during the purification procedures.

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Prolyl oligopeptidase (POP, prolyl endopeptidase, EC 3.4.21.26) is a serine-type peptidase (family S9 of clan SC) hydrolyzing peptides shorter than 30 amino acids. POP has been found in various mammalian and bacterial sources and it is widely distributed throughout different organisms. In human and rat, POP enzyme activity has been detected in most tissues, with the highest activity found mostly in the brain. POP has gained scientific interest as being involved in the hydrolyzis of many bioactive peptides connected with learning and memory functions, and also with neurodegenerative disorders. In drug or lesion induced amnesia models and in aged rodents, POP inhibitors have been able to revert memory loss. POP may have a fuction in IP3 signaling and it may be a possible target of mood stabilizing substances. POP may also have a role in protein trafficking, sorting and secretion. The role of POP during ontogeny has not yet been resolved. POP enzyme activity and expression have shown fluctuation during development. Specially high enzyme activities have been measured in the brain during early development. Reduced neuronal proliferation and differentation in presence of POP inhibitor have been reported. Nuclear POP has been observed in proliferating peripheral tissues and in cell cultures at the early stage of development. Also, POP coding mRNA is abundantly expressed during brain ontogeny and the highest levels of expression are associated with proliferative germinal matrices. This observation indicates a special role for POP in the regulation of neurogenesis during development. For the experimental part, the study was undertaken to investigate the expression and distribution of POP protein and enzymatic activity of POP in developing rat brain (from embryonic day 14 to post natal day 7) using immunohistochemistry, POP enzyme activity measurements and western blot-analysis. The aim was also to find in vivo confirmation of the nuclear colocalization of POP during early brain ontogeny. For immunohistochemistry, cryosections from the brains of the fetuses/rats were made and stained using specific antibody for POP and fluorescent markers for POP and nuclei. The enzyme activity assay was based on the fluorescence of 7- amino-4-methylcoumarin (AMC) generated from the fluorogenic substrate succinyl-glycyl-prolyl-7-amino-4-methylcoumarin (Suc-Gly-Pro-AMC) by POP. The amounts of POP protein and the specifity of POP antibody in rat embryos was confirmed by western blot analysis. We observed that enzymatic activity of POP is highest at embryonic day 18 while the protein amounts reach their peak at birth. POP was widely present throughout the developmental stages from embryonic day 14 to parturition day, although the POP-immunoreactivity varied abundantly. At embryonic days 14 and 18 notably amounts of POP was distributed at proliferative germinal zones. Furthermore, POP was located in the nucleus early in the development but is transferred to cytosol before birth. At P0 and P7 the POP-immunoreactivity was also widely observed, but the amount of POP was notably reduced at P7. POP was present in cytosol and in intercellular space, but no nuclear POP was observed. These findings support the idea of POP being involved in specific brain functions, such as neuronal proliferation and differentation. Our results in vivo confirm the previous cell culture results supporting the role of POP in neurogenesis. Moreover, an inconsistency of POP protein amounts and enzymatic activity late in the development suggests a strong regulation of POP activity and a possible non-hydrolytic role at that stage.

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Table of Contents [pdf, 0.01 Mb] Preface [pdf, 0.01 Mb] Masaaki Aota Long-term tendencies of sea ice concentration and air temperature in the Okhotsk Sea coast of Hokkaido [pdf, 0.05 Mb] Hajime Ito & Miki Yoshioka Geography of the seasonally ice covered seas [pdf, 0.5 Mb] George V. Shevchenko & Victor F. Putov On wind and tide induced sea-ice drift on the northeastern shelf of Sakhalin Island (analysis of radar data) [pdf, 0.96 Mb] Boris S. Dyakov, A.A. Nikitin, L. S. Muktepavel & T.A. Shatilina Variability of the Japan and Okhotsk Seas ice cover depending on geopotential field H500 over the Far-Eastern region [pdf, 0.10 Mb] Aleksandr G. Petrov & Nikolay A. Rykov Intermediate cold layer and ice cover in the Sea of Okhotsk [pdf, 0.37 Mb] Vladimir Ponomarev, Olga Trusenkova, Elena Ustinova & Dmitry Kaplunenko Interannual variations of oceanographic and meteorological characteristics in the Sea of Okhotsk [pdf, 0.16 Mb] George V. Shevchenko & Akie Kato Seasonal and interannual changes of atmospheric pressure, air and water temperature in the area of the Kuril Ridge [pdf, 0.13 Mb] George V. Shevchenko & Vladimir Yu. Saveliev Spatial variability of the wind field in the area of the Kuril Islands [pdf, 0.15 Mb] Alexander L. Figurkin & Igor A. Zhigalov Seasonal variability and specifity of the oceanological conditions in the northern Okhotsk Sea in 1997 [pdf, 1.04 Mb] Igor A. Zhabin Ventilation of the upper portion of the intermediate water in the Okhotsk Sea [pdf, 0.80 Mb] Vladimir A. Luchin & Alexander L. Figurkin Oceanographic conditions over the Kashevarov Bank [pdf, 0.61 Mb] Toshiyuki Awaji, Tomohiro Nakamura, Takaki Hatayama, Kazunori Akimoto & Takatoshi Takizawa Tidal exchange through the Kuril Straits [pdf, 2.01 Mb] Tomohiro Nakamura, Toshiyuki Awaji, Takaki Hatayama, Kazunori Akimoto, Takatoshi Takizawa & Masao Fukasawa Vertical mixing induced by tidally generated internal waves in the Kuril Straits [pdf, 0.83 Mb] Katsuro Katsumata & Ichiro Yasuda Water exchange between the Okhotsk Sea and the North Pacific Ocean estimated by simple models [pdf, 0.97 Mb] Konstantin A. Rogachev Oyashio west path culmination as the consequence of a rapid thermohaline transition in the Pacific Subarctic [pdf, 0.22 Mb] Yasuhiro Kawasaki On the year-to-year change in subarctic water characteristics around the Kuril Islands [pdf, 0.39 Mb] Alexander L. Figurkin & Evgeniy E. Ovsyannikov Influence of oceanological conditions of the West Kamchatka shelf waters on spawning grounds and on pollock egg distribution [pdf, 0.97 Mb] Igor E. Kochergin & Alexander A. Bogdanovsky Transport and turbulence characteristics for the northeastern Sakhalin shelf conditions [pdf, 0.08 Mb] Igor E. Kochergin, Alexander A. Bogdanovsky, Valentina D. Budaeva, Vyacheslav G. Makarov, Vasily F. Mishukov, S.N. Ovsienko, Victor F. Putov, L.A. Reitsema, J.W. Sciallabba, O.O. Sergucheva & P.V. Yarosh Modeling of oil spills for the shelf conditions of northeastern Sakhalin [pdf, 0.32 Mb] Valentina D. Budaeva & Vyacheslav G. Makarov A peculiar water regime of currents in the area of eastern Sakhalin shelf [pdf, 0.66 Mb] Nikolay A. Rykov The oceanographic databases on the Sakhalin shelf [pdf, 0.27 Mb] Akifumi Nakata, Iori Tanaka, Hiroki Yagi, Tomomi Watanabe, Gennady A. Kantakov & Andrew D. Samatov Formation of high-density water (over 26.8 sigma-t) near the La Perouse Strait (the Soya Strait) [pdf, 0.09 Mb] Minoru Odamaki & Kouji Iwamoto Currents and tidal observations by Hydrographic Department of Maritime Safety Agency, off the Okhotsk coast of Hokkaido [pdf, 0.16 Mb] Yasushi Fukamachi, Genta Mizuta, Kay I. Ohshima, Motoyo Itoh, Masaaki Wakatsuchi & Masaaki Aota Mooring measurements off Shiretoko Peninsula, Hokkaido in 1997-1998 [pdf, 0.19 Mb] Mikhail A. Danchenkov, David Aubrey & Stephen C. Riser Oceanographic features of the La Perouse Strait [pdf, 0.91 Mb] Iori Tanaka & Akifumi Nakata Results of direct current measurements in the La Perouse Strait (the Soya Strait), 1995-1998 [pdf, 0.06 Mb] Gennady A. Kantakov & George V. Shevchenko In situ observations of Tsushima and West-Sakhalin currents near La Perouse (Soya) Strait [pdf, 0.79 Mb] Irina Y. Bragina Geographical and biological characteristics of the net zooplankton in the southwestern part of the Sea of Okhotsk during 1987-1996 [pdf, 0.27 Mb] List of corresponding authors [pdf, 0.01 Mb] (Document pdf contains 193 pages)

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Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

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Le syndrome reproducteur et respiratoire porcin (SRRP) est une des maladies les plus dévastatrices économiquement pour l'industrie mondiale du porc. L'agent étiologique du SRRP est le virus du SRRP (VSRRP) lequel est connu pour avoir une spécificité d'hôte très restreinte et pour sa transmission par voie aerosol. Les antigènes et les ARN du VSRRP ont été trouvés dans des cellules épithéliales du tractus respiratoire de porcs infectés par le virus. L’interaction entre les macrophages alvéolaires porcins (PAMs) et le VSRRP a été démontrée comme jouant un rôle important dans l’infection causée par le virus. Malgré cela, l’interaction prenant place entre les cellules épithéliales du tractus respiratoire porcin et le virus ne devrait pas être négligée. Jusqu’à présent, la réplication du VSRRP in vitro dans des cellules épithéliales du tractus respiratoire porcin n’a pas été conduite avec succès et les tentatives pour le faire ont échoué. Une nouvelle lignée de cellules épithéliales de poumon de porc (SJPL) est maintenant disponible et sera utilisée dans cette étude afin de déterminer si elle est permissive à la réplication du VSRRP et si elle peut être un modèle approprié pour l’étude de la pathogénèse virale du VSRRP. L’expérimentation a démontré que cette nouvelle lignée cellulaire était permissive à l’infection et à la réplication du VSRRP. Afin de corroborer ces résultats, la cinétique de réplication du virus à été effectuée avec les cellules MARC-145 et SJPL. Aucune différence significative dans la production virale totale n’a été trouvée entre les deux lignées cellulaires. Les cellules SJPL ont permis la réplication de plusieurs souches Nord-Américaines du VSRRP, quoiqu’elles sont légèrement moins efficaces que les cellules MARC-145 pour l’isolement du virus. De plus, les cellules SJPL sont phénotypiquement différentes des cellules MARC-145. Plus précisément, les cellules SJPL sont plus sensibles à l’activation par le VSRRP des pro-caspases 3/7 et plusieurs inducteurs apoptotiques. Elles ont également montré de 8 à 16 fois plus de sensibilité à l’effet antiviral causé par l’IFN-α sur la réplication du virus contrairement aux cellules MARC-145. Ces résultats démontrent que les cellules SJPL pourraient représenter un substitut intéressant aux cellules MARC-145 pour la production d’antigènes pour un vaccin anti-VSRRP. Également, dû à leurs origines (poumon de l’hôte naturel), elles pourraient s’avérer être un modèle in vitro plus approprié pour l’étude de la pathogénèse du VSRRP.

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We investigate the relationship between consensus measures used in different settings depending on how voters or experts express their preferences. We propose some new models for single-preference voting, which we derive from the evenness concept in ecology, and show that some of these can be placed within the framework of existing consensus measures using the discrete distance. Finally, we suggest some generalizations of the single-preference consensus measures allowing the incorporation of more general notions of distance.

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Para a realização deste estudo, partiu-se da definição que hipoglicemia corresponde a uma taxa de glicose menor ou igual a 40mg/dl e hiperglicemia a uma concentração sangüínea de glicose maior que 120mg/dl. Foi realizado um estudo transversal, selecionando RNs com patologias potencialmente modificadoras da concentração de glicose e que deveriam ter suas glicemias monitorizadas e RNs com quadros clínicos os mais variados indicando a necessidade de coleta de sangue para sua assistência. A amostra de escolha para as dosagens de glicose é a venosa, porém há uma série de inconvenientes para se realizar essa determinação, uma vez que há necessidade de punção venosa, o que exige habilidade na execução devido ao diâmetro dos vasos e da própria fragilidade dos RNs, principalmente os prematuros, os quais constituem o grupo de maior risco para hipoglicemia. Outro problema que se observa é a demora em se obter os resultados, devido à estrutura da maioria dos nossos hospitais. Como existe no mercado um aparelho manual eletrônico que utiliza tiras-teste eletroquímicas capaz de dosar a glicemia capilar em 20 segundos, elaborou-se este estudo 19 trabalho para verificar se as determinações da glicemia em sangue capilar coincidiam com a realizada em sangue venoso (padrão-ouro), contribuindo assim para que o diagnóstico e o tratamento possam ser efetivados o mais precocemente possível. Foram estudados 177 exames, encontrando-se o seguinte: como desempenho do teste Precision Plus®, usando o ponto de corte tradicional para hipoglicemia (≤40) e (n=28), sensibilidade de 90,3 (IC 95%: 73,1 a 97,5) e especificidade de 88,4 (IC 95%: 81,7 a 92,9); como desempenho do teste Precision Plus®, usando o ponto de corte tradicional para hiperglicemia (≥120) e (n=17), sensibilidade 77,3 (IC 95%: 54,2 a 91,3) e especificidade 93,5 (IC 95%:88,1 a 96,7). Modificando o corte tradicional para taxas de 50 mg/dl e 100 mg/dl, respectivamente, hipo e hiperglicemia encontrou-se como desempenho do teste Precision Plus® para hipoglicemia (≤ 50), sensibilidade de 96,8 e especificidade de 82,9; como desempenho do teste Precision Plus® para hiperglicemia (≥100), sensibilidade de 95,5 e especificidade de 87,7. O desempenho do aparelho Precision Plus® no teste é adequado para realizar rastreamento de alterações glicêmicas nas populações de risco em UTIs, apesar das oscilações. Este método não deverá ser o indicado para tomadas de condutas terapêuticas. O método bioquímico deverá ser sempre utilizado para a confirmação da glicemia quando esta for realizada por métodos mais simples.

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Pós-graduação em Pediatria - FMB

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

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Pós-graduação em Medicina Veterinária - FCAV

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

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Mit der Zielsetzung der vorliegenden Arbeit wurde die detailierten Analyse von Migrationsdynamiken epithelilaler Monolayer anhand zweier neuartiger in vitro Biosensoren verfolgt, der elektrischen Zell-Substrat Impedanz Spektroskopie (electrical cell-substrate impedance sensing, ECIS) sowie der Quarz Kristall Mikrowaage (quartz crystal microbalance, QCM). Beide Methoden erwiesen sich als sensitiv gegenüber der Zellmotilität und der Nanozytotoxizität.rnInnerhalb des ersten Projektes wurde ein Fingerprinting von Krebszellen anhand ihrer Motilitätsdynamiken und der daraus generierten elektrischen oder akkustischen Fluktuationen auf ECIS oder QCM Basis vorgenommen; diese Echtzeitsensoren wurdene mit Hilfe klassicher in vitro Boyden-Kammer Migrations- und Invasions-assays validiert. Fluktuationssignaturen, also Langzeitkorrelationen oder fraktale Selbstähnlichkeit aufgrund der kollektiven Zellbewegung, wurden über Varianz-, Fourier- sowie trendbereinigende Fluktuationsanalyse quantifiziert. Stochastische Langzeitgedächtnisphänomene erwiesen sich als maßgebliche Beiträge zur Antwort adhärenter Zellen auf den QCM und ECIS-Sensoren. Des weiteren wurde der Einfluss niedermolekularer Toxine auf die Zytoslelettdynamiken verfolgt: die Auswirkungen von Cytochalasin D, Phalloidin und Blebbistatin sowie Taxol, Nocodazol und Colchicin wurden dabei über die QCM und ECIS Fluktuationsanalyse erfasst.rnIn einem zweiten Projektschwerpunkt wurden Adhäsionsprozesse sowie Zell-Zell und Zell-Substrat Degradationsprozesse bei Nanopartikelgabe charackterisiert, um ein Maß für Nanozytotoxizität in Abhangigkeit der Form, Funktionalisierung Stabilität oder Ladung der Partikel zu erhalten.rnAls Schlussfolgerung ist zu nennen, dass die neuartigen Echtzeit-Biosensoren QCM und ECIS eine hohe Zellspezifität besitzen, auf Zytoskelettdynamiken reagieren sowie als sensitive Detektoren für die Zellvitalität fungieren können.