CityCabin itsekantavan hiilikuitukorin valmistus

Tässä insinöörityössä käydään läpi Stadiassa syksyllä 2006 valmistuneen konsepti-auto CityCabin itsekantavan hiilikuitukorin suunnittelu- ja valmistusprosessi. CityCabin itsekantava hiilikuitukori koostuu viidestä osasta. Eri osat on valmistettu Sprint CBS hiiilikuidusta ja niiden liitoksissa on käytetty liima- ja pulttiliitoksia sekä laminointia. CityCabin itsekantava hiilikuitukori on suunniteltu käyttämällä Catia-mallinnusohjelmistoa ja Algor-lujuuslaskentaohjelmistoa. Itsekantava hiilikuitukori on suunniteltu kolariturvalliseksi nykystandardien mukaan.Työn tavoitteena oli hiilikuitukorin valmistuksen valmistuskustannuksien pienentäminen, käsinlaminoinnin vähentäminen, uuteen valmistusmenetelmään tutustuminen, valmistettavien kappaleiden tasainen laatu sekä aikataulussa pysyminen. Tässä opinnäytetyössä painopiste on enemmän hiilikuituosien valmistuksessa ja korin kasaamisessa. Eri työvaiheet on käyty hyvin tarkasti läpi. Työn tuloksena syntyi CityCabin itsekantava hiilikuitukori, joka on valmistettu komposiittialan uusimpia valmistusmenetelmiä hyödyntäen. Hiilikuitukori pysyi annetuissa toleransseissa ja se täytti sille asetetut vaatimukset. Sprint-hiilikuidun valmistus tekniikoista saatiin paljon uutta hyödyllistä tietoa, mutta on tehtävä vielä paljon kokeita ennen kuin tästä uudesta materiaalista saadaan kaikki hyöty irti.

A química medicinal de N-acilidrazonas: novos compostos-protótipos de fármacos analgésicos, antiinflamatórios e anti-trombóticos

In this article are described new bioactive N-acylhydrazone (NAH) derivatives, structurally designed as optimization of aryl hydrazones precursors planned by molecular hybridization of two 5-lipoxigenase inhibitors, e.g. CBS-1108 and BW-755c. The analgesic, antiedematogenic and anti-platelet aggregating profile of several isosteric compounds was investigated by using classic pharmacological assays in vivo and ex-vivo, allowing to identify new potent peripheric analgesic lead, a new anti-inflammatory and an antithrombotic agent. During this study was discovered dozen of active NAH compounds clarifying the structure-activity relationship for this series of NAH derivatives, indicating the pharmacophore character of the N-acylhydrazone functionality.

Pré-tratamentos de melaço de cana-de-açúcar e água de maceração de milho para a bioprodução de carotenóides

This work studied the pretreatment of sugarcane molasses (CM) and corn steep liquor (CS) for the production of carotenoids by Sporidiobolus salmonicolor (CBS 2636). The acid pretreatment removed less micronutrients than that with activated carbon and led to high removals of Cu and Mn. Reduction in optical density of the prepared medium and removal of glucose from it were 22% and 7% for CM and 95% and 38% for CS, respectively. Total carotenoids obtained with substrates pretreated with acids (541 mg/L) were higher than the results obtained when the medium was treated with activated carbon (208 mg/L).

Ondas ultrassônicas e pérolas de vidro: um novo método de extração de beta-galactosidase para uso em laboratório

The optimization of a traditional technique of cellular disruption by abrasion was carried out and a process using ultrasonic waves associated with glass pearls to extract beta-galactosidase from Kluyveromyces marxianus proposed. In the first case, the effects of the diameter and weight of the pearls in relation to the volume of cellular suspension and amount of time for cellular disruption were evaluated. The efficiency of the new process of cellular disruption was evaluated by varying the length of time of sonification and comparing with the method of abrasion under the same conditions. The proposed method can be efficiently applied to obtain beta-galactosidase at laboratory scale.

Comparação entre métodos compostos no cálculo de afinidades por próton e elétron em sistemas moleculares

The CBS-4M, CBS-QB3, G2, G2(MP2), G3 and G3(MP2) model chemistry methods have been used to calculate proton and electron affinities for a set of molecular and atomic systems. Agreement with the experimental value for these electronic properties is quite good considering the uncertainty in the experimental data. A comparison among the six theories using statistical analysis (average value, standard deviation and root-mean-square) showed a better performance of CBS-QB3 to obtain these properties.

Susceptibility of fruits of the 'Valência' and 'Natal' sweet orange varieties to Guignardia citricarpa and the influence of the coexistence of healthy and symptomatic fruits

The importance of the presence of symptomatic fruits with citrus black symptoms (CBS) on the disease severity level in subsequent crop production was assessed in an orchard planted with Valência and Natal sweet orange varieties. Additionally, the period of susceptibility of the fruits of these varieties was evaluated. Fruits were covered with paper bags at the stage of 75% fallen petals and were then exposed to natural infection at weekly intervals, from October 2000 to April 2001. This process was carried out in plants where the fruits from the previous harvest had been picked as well as in plants where fruits remained until natural drop. The evaluation of disease severity used a scale that varied from 0 (absence of symptoms) to 6 (severe symptoms). It was observed that, for the Valência and Natal varieties, conidia of Phyllosticta citricarpa that had formed on the lesions of fruits from the previous harvest did not significantly increase the severity of disease on the fruits of the subsequent harvest period. In this study, the protection of the fruits until 10 weeks after petal drop did not affect the number of lesions, indicating that ascospore discharges after that date were, probably, responsible for disease severity. Fruits exposed between the 20th and 24th week after 75% of the petals had fallen were symptomatic, indicating that, at this stage, the fruits were still susceptible to the pathogen.

L'Univers

1885/11/16 (Numéro 6556).

The Structural Basis for inorganic Pyrophosphatase Catalysis and Regulation

Inorganic pyrophosphatases (PPases) are essential enzymes for every living cell. PPases provide the necessary thermodynamic pull for many biosynthetic reactions by hydrolyzing pyrophosphate. There are two types of PPases: integral membrane-bound and soluble enzymes. The latter type is divided into two non-homologous protein families, I and II. Family I PPases are present in all kingdoms of life, whereas family II PPases are only found in prokaryotes, including archae. Family I PPases, particularly that from Saccharomyces cerevisiae, are among the most extensively characterized phosphoryl transfer enzymes. In the present study, we have solved the structures of wild-type and seven active site variants of S. cerevisiae PPase bound to its natural metal cofactor, magnesium ion. These structures have facilitated derivation of the complete enzyme reaction scheme for PPase, fulfilling structures of all the reaction intermediates. The main focus in this study was on a novel subfamily of family II PPases (CBSPPase) containing a large insert formed by two CBS domains and a DRTGG domain within the catalytic domain. The CBS domain (named after cystathionine beta-synthase in which it was initially identified) usually occurs as tandem pairs with two or four copies in many proteins in all kingdoms of life. The structure formed by a pair of CBS domains is also known as a Bateman domain. CBS domains function as regulatory units, with adenylate ligands as the main effectors. The DRTGG domain (designated based on its most conserved residues) occurs less frequently and only in prokaryotes. Often, the domain co-exists with CBS domains, but its function remains unknown. The key objective of the current study was to explore the structural rearrangements in the CBS domains induced by regulatory adenylate ligands and their functional consequences. Two CBS-PPases were investigated, one from Clostridium perfringens (cpCBS-PPase) containing both CBS and DRTGG domains in its regulatory region and the other from Moorella thermoacetica (mt CBS-PPase) lacking the DRTGG domain. We additionally constructed a separate regulatory region of cpCBS-PPase (cpCBS). Both full-length enzymes and cpCBS formed homodimers. Two structures of the regulatory region of cpCBS-PPase complexed with the inhibitor, AMP, and activator, diadenosine tetraphosphate, were solved. The structures were significantly different, providing information on the structural pathway from bound adenylates to the interface between the regulatory and catalytic parts. To our knowledge, these are the first reported structures of a regulated CBS enzyme, which reveal large conformational changes upon regulator binding. The activator-bound structure was more open, consistent with the different thermostabilities of the activator- and inhibitor-bound forms of cpCBS-PPase. The results of the functional studies on wild-type and variant CBS-PPases provide support for inferences made on the basis of structural analyses. Moreover, these findings indicate that CBS-PPase activity is highly sensitive to adenine nucleotide distribution between AMP, ADP and ATP, and hence to the energy level of the cell. CBS-PPase activity is markedly inhibited at low energy levels, allowing PPi energy to be used for cell survival instead of being converted into heat.

Functional studies on bacterial nucleotide-regulated inorganic pyrophosphatases

CBS domains are ~60 amino acid tandemly repeated regulatory modules forming a widely distributed domain superfamily. Found in thousands of proteins from all kingdoms of life, CBS domains have adopted a variety of functions during evolution, one of which is regulation of enzyme activity through binding of adenylate-containing compounds in a hydrophobic cavity. Mutations in human CBS domain-containing proteins cause hereditary diseases. Inorganic pyrophosphatases (PPases) are ubiquitous enzymes, which pull pyrophosphate (PPi) producing reactions forward by hydrolyzing PPi into phosphate. Of the two nonhomologous soluble PPases, dimeric family II PPases, belonging to the DHH family of phosphoesterases, require a transition metal and magnesium for maximal activity. A quarter of the almost 500 family II PPases, found in bacteria and archaea, contain a 120-250 amino acid N-terminal insertion, comprised of two CBS domains separated in sequence by a DRTGG domain. These enzymes are thus named CBS-PPases. The function of the DRTGG domain in proteins is unknown. The aim of this PhD thesis was to elucidate the structural and functional differences of CBS-PPases in comparison to family II PPases lacking the regulatory insert. To this end, we expressed, purified and characterized the CBS-PPases from Clostridium perfringens (cpCBS-PPase) and Moorella thermoacetica (mtCBS-PPase), the latter lacking a DRTGG domain. Both enzymes are homodimers in solution and display maximal activity against PPi in the presence of Co2+ and Mg2+. Uniquely, the DRTGG domain was found to enable tripolyphosphate hydrolysis at rates similar to that of PPi. Additionally, we found that AMP and ADP inhibit, while ATP and AP4A activate CBSPPases, thus enabling regulation in response to changes in cellular energy status. We then observed substrate- and nucleotide-induced conformational transitions in mtCBS-PPase and found that the enzyme exists in two differentially active conformations, interconverted through substrate binding and resulting in a 2.5-fold enzyme activation. AMP binding was shown to produce an alternate conformation, which is reached through a different pathway than the substrate-induced conformation. We solved the structure of the regulatory insert from cpCBS-PPase in complex with AMP and AP4A and proposed that conformational changes in the loops connecting the catalytic and regulatory domains enable activity regulation. We examined the effects of mutations in the CBS domains of mtCBS-PPase on catalytic activity, as well as, nucleotide binding and inhibition.

Determination of slurry’s viscosity using case based reasoning approach

Case-based reasoning (CBR) is a recent approach to problem solving and learning that has got a lot of attention over the last years. In this work, the CBR methodology is used to reduce the time and amount of resources spent on carry out experiments to determine the viscosity of the new slurry. The aim of this work is: to develop a CBR system to support the decision making process about the type of slurries behavior, to collect a sufficient volume of qualitative data for case base, and to calculate the viscosity of the Newtonian slurries. Firstly in this paper, the literature review about the types of fluid flow, Newtonian and non-Newtonian slurries is presented. Some physical properties of the suspensions are also considered. The second part of the literature review provides an overview of the case-based reasoning field. Different models and stages of CBR cycles, benefits and disadvantages of this methodology are considered subsequently. Brief review of the CBS tools is also given in this work. Finally, some results of work and opportunities for system modernization are presented. To develop a decision support system for slurry viscosity determination, software application MS Office Excel was used. Designed system consists of three parts: workspace, the case base, and section for calculating the viscosity of Newtonian slurries. First and second sections are supposed to work with Newtonian and Bingham fluids. In the last section, apparent viscosity can be calculated for Newtonian slurries.

Mastalgias cíclicas: tratamento não-medicamentoso (orientação verbal)

Objetivos: avaliar o tratamento não-medicamentoso (orientação verbal) como primeira opção terapêutica para mulheres com mastalgia cíclica e observar se o tempo de existência da sintomatologia dolorosa altera os resultados. Métodos: conduzimos um estudo do tipo experimental não-controlado com uma amostra de 128 mulheres com história clara de mastalgia cíclica, tratadas com orientação verbal. Uma escala analógica visual da dor foi usada antes e após o tratamento, a fim de avaliar a severidade, e classificamos as mastalgias em graus I (leve), II (moderado) e III (severo), de acordo com a intensidade da dor. Usamos também o "Cardiff Breast Score" (CBS) modificado para avaliar a resposta clínica ao tratamento. A análise dos dados foi feita com o teste do chi² (Epi-Info 6.04). Resultados: verificou-se um índice de sucesso de 59,4% com a orientação verbal, mas não houve resposta com diferença estatisticamente significante entre os grupos (p = 0,16) com diferentes graus de mastalgia. A resposta menos satisfatória ao tratamento não-medicamentoso nas pacientes que apresentavam um período de tempo mais longo de sintomatologia foi apenas aparente, pois não houve diferença estatisticamente significante (p = 0.14). Conclusão: a orientação verbal deve ser tentada sempre como a primeira escolha terapêutica para mulheres com mastalgia cíclica, independentemente do grau de intensidade da dor. O curso prolongado da dor não interferiu nos resultados.

Fatores que Influenciam os Resultados do Tratamento Não-Medicamentoso das Mastalgias Cíclicas

Objetivo: avaliar fatores que possam influenciar os resultados do tratamento não-medicamentoso (orientação verbal) em mulheres com mastalgia cíclica. Métodos: conduzimos um estudo do tipo experimental não controlado com uma amostra de 128 mulheres com uma história clara de mastalgia cíclica, tratadas com orientação verbal. Uma escala analógica visual da dor foi usada antes e após tratamento, a fim de avaliar a sua gravidade, e classificamos as mastalgias em graus I (leve), II (moderado) e III (intensa), de acordo com a intensidade da dor. Usamos também o Cardiff Breast Score (CBS) modificado para avaliar a resposta clínica ao tratamento. A análise dos dados foi feita com o teste do chi² (Epi-Info 6.04). Resultados: verificamos que fatores como paridade, menarca, idade do primeiro parto a termo e amamentação não influenciaram significativamente (p=0,19, p=0,31, p=0,80 e p=0,54, respectivamente) os resultados do tratamento não-medicamentoso (orientação verbal). Por outro lado, quando levamos em consideração a idade, observamos que 26 (78,8%) pacientes com 40 anos ou mais de idade foram mais beneficiadas com a orientação verbal, com diferença estatisticamente significante (p=0,01) quando comparadas aos grupos mais jovens. Conclusão: fatores reprodutivos como paridade, menarca, idade do primeiro parto a termo e amamentação não influenciaram os resultados do tratamento não medicamentoso (orientação verbal). O fator idade, especificamente, influenciou significativamente os resultados.

Metabolism and gene polymorphisms of the folate pathway in Brazilian women with history of recurrent abortion

PURPOSE: To investigate the association between polymorphisms in genes that encode enzymes involved in folate- and vitamin B12-dependent homocysteine metabolism and recurrent spontaneous abortion (RSA).METHODS: We investigated the C677T and A1298C polymorphisms of the methylenetetrahydrofalate reductase gene (MTHFR), the A2756G polymorphism of the methionine synthase gene (MS) and the 844ins68 insertion of the cystathionine beta synthetase gene (CBS). The PCR technique followed by RFLP was used to assess the polymorphisms; the serum levels of homocysteine, vitamin B12 and folate were investigated by chemiluminescence. The EPI Info Software version 6.04 was used for statistical analysis. Parametric variables were compared by Student's t-test and nonparametric variables by the Wilcoxon rank sum test.RESULTS: The frequencies of gene polymorphisms in 89 women with a history of idiopathic recurrent miscarriage and 150 controls were 19.1 and 19.6% for the C677T, insertion, 20.8 and 26% for the A1298C insertion, 14.2 and 21.9% for the A2756G insertion, and 16.4 and 18% for the 844ins68 insertion, respectively. There were no significant differences between case and control groups in any of the gene polymorphisms investigated. However, the frequency of the 844ins68 insertion in the CBS gene was higher among women with a history of loss during the third trimester of pregnancy (p=0.003). Serum homocysteine, vitamin B12 and folate levels id not differ between the polymorphisms studied in the case and control groups. However, linear regression analysis showed a dependence of serum folate levels on the maintenance of tHcy levels.CONCLUSION: The investigated gene polymorphisms and serum homocysteine, vitamin B12 and folate levels were not associated with idiopathic recurrent miscarriage in the present study. Further investigations are needed in order to confirm the role of the CBS 844ins68 insertion in recurrent miscarriage.

Chromatoid body mediated RNA regulation in mouse male germline

Male germ cell differentiation, spermatogenesis is an exceptional developmental process that produces a massive amount of genetically unique spermatozoa. The complexity of this process along with the technical limitations in the germline research has left many aspects of spermatogenesis poorly understood. Post-meiotic haploid round spermatids possess the most complex transcriptomes of the whole body. Correspondingly, efficient and accurate control mechanisms are necessary to deal with the huge diversity of transcribed RNAs in these cells. The high transcriptional activity in round spermatids is accompanied by the presence of an uncommonly large cytoplasmic ribonucleoprotein granule, called the chromatoid body (CB) that is conjectured to participate in the RNA post-transcriptional regulation. However, very little is known about the possible mechanisms of the CB function. The development of a procedure to isolate CBs from mouse testes was this study’s objective. Anti-MVH immunoprecipitation of cross-linked CBs from a fractionated testicular cell lysate was optimized to yield considerable quantities of pure and intact CBs from mice testes. This protocol produced reliable and reproducible data from the subsequent analysis of CB’s protein and RNA components. We found that the majority of the CB’s proteome consists of RNA-binding proteins that associate functionally with different pathways. We also demonstrated notable localization patterns of one of the CB transient components, SAM68 and showed that its ablation does not change the general composition or structure of the CB. CB-associated RNA analysis revealed a strong accumulation of PIWI-interacting RNAs (piRNAs), mRNAs and long non-coding RNAs (lncRNAs) in the CB. When the CB transcriptome and proteome analysis results were combined, the most pronounced molecular functions in the CB were related to piRNA pathway, RNA post-transcriptional processing and CB structural scaffolding. In addition, we demonstrated that the CB is a target for the main RNA flux from the nucleus throughout all steps of round spermatid development. Moreover, we provided preliminary evidence that those isolated CBs slice target RNAs in vitro in an ATPdependent manner. Altogether, these results make a strong suggestion that the CB functions involve RNA-related and RNA-mediated mechanisms. All the existing data supports the hypothesis that the CB coordinates the highly complex haploid transcriptome during the preparation of the male gametes for fertilization. Thereby, this study provides a fundamental basis for the future functional analyses of ribonucleoprotein granules and offers also important insights into the mechanisms governing male fertility.