Genotyping of group A rotavirus samples from Brazilian children by probe hybridization

The G genotyping of 74 group A rotavirus samples was done by RNA-DNA hybridization (dot-blot) using oligonucleotide probes for the VP7 gene region of the human rotavirus serotypes/genotypes 1, 2, 3 and 4. Thirty-one samples could be genotyped by dot-blot showing the following results: G1 = 16, G4 = 6, G3 = 5, and G2 = 4. The data show circulation of genotypes G1-G4 and the predominance of G1. The knowledge of genotypes provides important information concerning rotavirus circulation in Central Brazil.

The Brazilian investment in science and technology

An analysis of Brazilian federal expenditures in science and technology is presented is this study. The 1990-1999 data were compiled from records provided by two federal agencies (MCT and CNPq) responsible for managing most of the national budget related to these activities. The results indicate that the federal investments in Brazilian science and technology stagnated during the last decade (US$ 2.32 billion in 1990, US$ 2.39 billion in 1996, and US$ 2.36 billion in 1999). In contrast, a great increase in private investments in research was acknowledged both by industry and by the government during the same period, from US$ 2.12 to US$ 4.64 billion. However, this investment did not result in an increase in invention patents granted to residents (492 in 1990 and only 232 in 1997) or in a reduction of patent costs. Despite this unfavorable scenario, the number of graduate programs in the country has increased two-fold in the last decade and the contribution of Brazilians to the database of the Institute for Scientific Information has increased 4.7-fold from 1990 (2,725 scientific publications) to 2000 (12,686 scientific publications). Unstable federal resources for science, together with the poor returns of private resources in terms of developing new technologies, may jeopardize the future of Brazilian technological development.

A Template for Cloud Technology Deployment in an Energy Trading Environment

Nykyaikaiset pilvipalvelut tarjoavat suurille yrityksille mahdollisuuden tehostaa laskennallista tietojenkäsittelyä. Pilvipalveluiden käyttöönotto tuo mukanaan kuitenkin esimerkiksi useita tietoturvakysymyksiä, joiden vuoksi käyttöönoton tulee olla tarkasti suunniteltua. Tämä tutkimus esittelee kirjallisuuskatsaukseen perustuvan, asteittaisen suunnitelman pilvipalveluiden käyttöönotolle energialiiketoimintaympäristössä. Kohdeyrityksen sisäiset haastattelut ja katsaus nykyisiin energiateollisuuden pilviratkaisuihin muodostavat kokonaiskuvan käyttöönoton haasteista ja mahdollisuuksista. Tutkimuksen päätavoitteena on esittää ratkaisut tyypillisiin pilvipalvelun käyttöönotossa esiintyviin ongelmiin käyttöönottomallin avulla. Tutkimuksessa rakennettu käyttöönottomalli testattiin esimerkkitapauksen avulla ja malli todettiin toimivaksi. Ulkoisten palveluiden herättämien tietoturvakysymysten takia käyttöönoton ensimmäiset osiot, kuten lopputuotteen määrittely ja huolellinen suunnittelu, ovat koko käyttöönottoprosessin ydin. Lisäksi pilvipalveluiden käyttöönotto vaatii nykyiseltä käyttöympäristöltä uusia teknisiä ja hallinnollisia taitoja. Tutkimuksen tulokset osoittavat pilvipalveluiden monipuolisen hyödyn erityisesti laskentatehon tarpeen vaihdellessa. Käyttöönottomallin rinnalle luotu kustannusvertailu tukee kirjallisuuskatsauksessa esille tuotuja hyötyjä ja tarjoaa kohdeyritykselle perusteen tutkimuksen eteenpäin viemiselle.

Utilization of AMI-Technology in a Multilayered Demand Response Service Environment

This thesis studied the performance of Advanced metering infrastructure systems in a challenging Demand Response environment. The aim was to find out what kind of challenges and bottlenecks could be met when utilizing AMI-systems in challenging Demand Response tasks. To find out the challenges and bottlenecks, a multilayered demand response service concept was formed. The service consists of seven different market layers which consist of Nordic electricity market and the reserve markets of Fingrid. In the simulations the AMI-systems were benchmarked against these seven market layers. It was found out, that the current generation AMI-systems were capable of delivering Demand Response on the most challenging market layers, when observed from time critical viewpoint. Additionally, it was found out, that to enable wide scale Demand Response there are three major challenges to be acknowledged. The challenges hindering the utilization of wide scale Demand Response were related to poor standardization of the systems in use, possible problems in data connectivity solutions and the current electricity market regulation model.

From Unspecific Quenching to Specific Signaling: Functional GTPase Assays Utilizing Quenching Resonance Energy Transfer (QRET) Technology

The aim of the work presented in this study was to demonstrate the wide applicability of a single-label quenching resonance energy transfer (QRET) assay based on time-resolved lanthanide luminescence. QRET technology is proximity dependent method utilizing weak and unspecific interaction between soluble quencher molecule and lanthanide chelate. The interaction between quencher and chelate is lost when the ligand binds to its target molecule. The properties of QRET technology are especially useful in high throughput screening (HTS) assays. At the beginning of this study, only end-point type QRET technology was available. To enable efficient study of enzymatic reactions, the QRET technology was further developed to enable measurement of reaction kinetics. This was performed using proteindeoxyribonuclei acid (DNA) interaction as a first tool to monitor reaction kinetics. Later, the QRET was used to study nucleotide exchange reaction kinetics and mutation induced effects to the small GTPase activity. Small GTPases act as a molecular switch shifting between active GTP bound and inactive GDP bound conformation. The possibility of monitoring reaction kinetics using the QRET technology was evaluated using two homogeneous assays: a direct growth factor detection assay and a nucleotide exchange monitoring assay with small GTPases. To complete the list, a heterogeneous assay for monitoring GTP hydrolysis using small GTPases, was developed. All these small GTPase assays could be performed using nanomolar protein concentrations without GTPase pretreatment. The results from these studies demonstrated that QRET technology can be used to monitor reaction kinetics and further enable the possibility to use the same method for screening.

A conceptual and practical overview of cDNA microarray technology: implications for basic and clinical sciences

cDNA microarray is an innovative technology that facilitates the analysis of the expression of thousands of genes simultaneously. The utilization of this methodology, which is rapidly evolving, requires a combination of expertise from the biological, mathematical and statistical sciences. In this review, we attempt to provide an overview of the principles of cDNA microarray technology, the practical concerns of the analytical processing of the data obtained, the correlation of this methodology with other data analysis methods such as immunohistochemistry in tissue microarrays, and the cDNA microarray application in distinct areas of the basic and clinical sciences.

Rapid, reliable and inexpensive quality assessment of biotinylated cRNA

The interpretation of oligonucleotide array experiments depends on the quality of the target cRNA used. cRNA target quality is assessed by quantitative analysis of the representation of 5' and 3' sequences of control genes using commercially available Test arrays. The Test array provides an economically priced means of determining the quality of labeled target prior to analysis on whole genome expression arrays. This manuscript validates the use of a duplex RT-PCR assay as a faster (6 h) and less expensive (6 were chosen and classified as degraded cRNAs, and 31 samples with a ß-actin 3'/5' ratio <6 were selected as good quality cRNAs. Blinded samples were then used for the RT-PCR assay. After gel electrophoresis, optical densities of the amplified 3' and 5' fragments of ß-actin were measured and the 3'/5' ratio was calculated. There was a strong correlation (r² = 0.6802) between the array and the RT-PCR ß-actin 3'/5' ratios. Moreover, the RT-PCR 3'/5' ratio was significantly different (P < 0.0001) between undegraded (mean ± SD, 0.34 ± 0.09) and degraded (1.71 ± 0.83) samples. None of the other parameters analyzed, such as i) the starting amount of RNA, ii) RNA quality assessed using the Bioanalyzer Chip technology, or iii) the concentration and OD260/OD280 ratio of the purified biotinylated cRNA, correlated with cRNA quality.

Genotyping hepatitis C virus from hemodialysis patients in Central Brazil by line probe assay and sequence analysis

The present study examined the distribution of hepatitis C virus (HCV) genotypes and subtypes in a hemodialysis population in Goiás State, Central Brazil, and evaluated the efficiency of two genotyping methods: line probe assay (LiPA) based on the 5' noncoding region and nucleotide sequencing of the nonstructural 5B (NS5B) region of the genome. A total of 1095 sera were tested for HCV RNA by RT-nested PCR of the 5' noncoding region. The LiPA assay was able to genotype all 131 HCV RNA-positive samples. Genotypes 1 (92.4%) and 3 (7.6%) were found. Subtype 1a (65.7%) was the most prevalent, followed by subtypes 1b (26.7%) and 3a (7.6%). Direct nucleotide sequencing of 340 bp from the NS5B region was performed in 106 samples. The phylogenetic tree showed that 98 sequences (92.4%) were classified as genotype 1, subtypes 1a (72.6%) and 1b (19.8%), and 8 sequences (7.6%) as subtype 3a. The two genotyping methods gave concordant results within HCV genotypes and subtypes in 100 and 96.2% of cases, respectively. Only four samples presented discrepant results, with LiPA not distinguishing subtypes 1a and 1b. Therefore, HCV genotype 1 (subtype 1a) is predominant in hemodialysis patients in Central Brazil. By using sequence analysis of the NS5B region as a reference standard method for HCV genotyping, we found that LiPA was efficient at the genotype level, although some discrepant results were observed at the subtype level (sensitivity of 96.1% for subtype 1a and 95.2% for subtype 1b). Thus, analysis of the NS5B region permitted better discrimination between HCV subtypes, as required in epidemiological investigations.

DNA tests probe the genomic ancestry of Brazilians

We review studies from our laboratories using different molecular tools to characterize the ancestry of Brazilians in reference to their Amerindian, European and African roots. Initially we used uniparental DNA markers to investigate the contribution of distinct Y chromosome and mitochondrial DNA lineages to present-day populations. High levels of genetic admixture and strong directional mating between European males and Amerindian and African females were unraveled. We next analyzed different types of biparental autosomal polymorphisms. Especially useful was a set of 40 insertion-deletion polymorphisms (indels) that when studied worldwide proved exquisitely sensitive in discriminating between Amerindians, Europeans and Sub-Saharan Africans. When applied to the study of Brazilians these markers confirmed extensive genomic admixture, but also demonstrated a strong imprint of the massive European immigration wave in the 19th and 20th centuries. The high individual ancestral variability observed suggests that each Brazilian has a singular proportion of Amerindian, European and African ancestries in his mosaic genome. In Brazil, one cannot predict the color of persons from their genomic ancestry nor the opposite. Brazilians should be assessed on a personal basis, as 190 million human beings, and not as members of color groups.

Characterization of alpha thalassemic genotypes by multiplex ligation-dependent probe amplification in the Brazilian population

Alpha-thalassemia is the most common inherited disorder of hemoglobin synthesis. Genomic deletions involving the alpha-globin gene cluster on chromosome 16p13.3 are the most frequent molecular causes of the disease. Although common deletions can be detected by a single multiplex gap-PCR, the rare and novel deletions depend on more laborious techniques for their identification. The multiplex ligation-dependent probe amplification (MLPA) technique has recently been used for this purpose and was successfully used in the present study to detect the molecular alterations responsible for the alpha-thalassemic phenotypes in 8 unrelated individuals (3 males and 5 females; age, 4 months to 30 years) in whom the molecular basis of the disease could not be determined by conventional methods. A total of 44 probe pairs were used for MLPA, covering approximately 800 kb from the telomere to the MSLN gene in the 16p13.3 region. Eight deletions were detected. Four of these varied in size from 240 to 720 kb and affected a large region including the entire alpha-globin gene cluster and its upstream regulatory element (alpha-MRE), while the other four varied in size from 0.4 to 100 kb and were limited to a region containing this element. This study is the first in Brazil to use the MLPA method to determine the molecular basis of alpha-thalassemia. The variety of rearrangements identified highlights the need to investigate all cases presenting microcytosis and hypochromia, but without iron deficiency or elevated hemoglobin A2 levels and suggests that these rearrangements may be more frequent in our population than previously estimated.

Role of a GenoType MTBDRplus line probe assay in early detection of multidrug-resistant tuberculosis at a Brazilian reference center

Resistance to Mycobacterium tuberculosis is a reality worldwide, and its diagnosis continues to be difficult and time consuming. To face this challenge, the World Health Organization has recommended the use of rapid molecular tests. We evaluated the routine use (once a week) of a line probe assay (Genotype MTBDRplus) for early diagnosis of resistance and for assessment of the main related risk factors over 2 years. A total of 170 samples were tested: 15 (8.8%) were resistant, and multidrug resistance was detected in 10 (5.9%). The sensitivity profile took 3 weeks (2 weeks for culture and 1 week for rapid testing). Previous treatment for tuberculosis and the persistence of positive acid-fast smears after 4 months of supervised treatment were the major risk factors observed. The use of molecular tests enabled early diagnosis of drug-resistant bacilli and led to appropriate treatment of the disease. This information has the potential to interrupt the transmission chain of resistant M. tuberculosis.

Determinação de substitutos da manteiga de cacau em coberturas de chocolate através da análise de triacilgliceróis

A legislação brasileira proíbe a adição de substitutos da manteiga de cacau ao chocolate. O presente trabalho teve como objetivo verificar o padrão de qualidade de coberturas comercializadas na região de Campinas. Para isso, foram analisadas cinco marcas de cobertura de chocolate ao leite e quatro de cobertura de chocolate amargo. Para verificar a possível adição de substitutos determinou-se, por cromatografia gasosa a alta temperatura (CGAT), a composição em triacilgliceróis da gordura extraída e os resultados foram analisados pelo método matemático de Padley & Timms. Não foi detectada a presença de substitutos da manteiga de cacau nas amostras de cobertura de chocolate analisadas.

Invoice process analysis and development. Case: Power and automation technology company

This master’s thesis studies the case company’s current purchase invoice process and the challenges that are related to it. Like most of other master’s thesis this study consists of both theoretical- and empirical parts. The purpose of this work is to combine theoretical and empirical parts together so that the theoretical part brings value to the empirical case study. The case company’s main business is frequency converters for both low voltage AC & DC drives and medium voltage AC Drives which are used across all industries and applications. The main focus of this study is on the current invoice process modelling. When modelling the existing process with discipline and care, current challenges can be understood better. Empirical study relays heavily on interviews and existing, yet fragmented, data. This, along with own calculations and analysis, creates the foundation for the empirical part of this master’s thesis.