DNA-Protein interaction: a response regulatory protein associated with Mo homeostasis in Desulfovibrio alaskensis G20.

Dissertation for a degree in Doctor in Sustainable Chemistry

Efetividade das vacinas anti-VHB (DNA-recombinante) em doadores de sangue de uma região endêmica para hepatite B no sul do Brasil

O objetivo deste estudo foi de estimar a efetividade das vacinas anti-VHB em um estudo longitudinal, retrospectivo composto por 1.012 doadores de sangue que completaram o esquema padrão de vacinação (três doses, incluindo doses de reforço nos doadores com títulos de anti-HBs <10UI/L) durante o período entre 1998 e 2002. Os resultados mostram que a taxa de soroconversão foi significativamente menor nos doadores cujo título de anti-HBs foi mensurado após seis meses decorridos do término do esquema de vacinação e nos doadores com mais de 50 anos. A efetividade média correspondeu a 88,7%, variando de 80,6% nos com maior idade (50 anos ou mais) a 91,4% nos doadores mais jovens (18 a 30 anos). O regime de dose de reforço foi efetivo, principalmente por reduzir o percentual de não-respondedores. Conclui-se que a efetividade da vacina foi significativamente maior nos doadores mais jovens e que o tempo decorrido entre a vacinação e a testagem interferiu na taxa de soroconversão.

Estudo da ação danificadora do 2,2'-Bipyridyl no DNA na presença de radiação UV

Em 2008, foram estimados no mundo, cerca de 12.7 milhões de novos casos de cancro e 7,6 milhões de mortes por cancro, sendo que 56% dos casos e 64% das mortes ocorreram nos países em desenvolvimento. Estes números indicam que é necessário se encontrar novos meios de combater este flagelo. Uma das possibilidades é encontrar moléculas intercalantes do ácido desoxirribonucleico (DNA) que se degradem na presença de radiação ou partículas carregadas de baixa energia que criem condições de induzir lesões no DNA, inibidoras da replicação e que portanto contribuam para a não proliferação de células cancerígenas. Assim, nesta dissertação analisou-se a influência do agente intercalante 2,2'-Bipyridyl do DNA em presença de radiação UV, 254 nm, na degradação do DNA. Os danos criados no DNA foram analisados pelas técnicas de espectroscopia de ultravioleta-visível e de infravermelho. Os resultados obtidos permitiram inferir que a cinética de degradação do DNA é mais eficiente na presença do composto 2,2'-Bipyridyl e que o ataque pelos produtos da decomposição do 2,2'-Bipyridyl é feito a todas as bases, embora com constantes características diferentes, sendo a ataque à guanina com uma constante de tempo maior. Estes resultados permitem concluir que este composto pode ser um possível candidato a indutor de lesões no DNA.

Predictive analytical and numerical modeling for orthogonal cutting

In this thesis, a predictive analytical and numerical modeling approach for the orthogonal cutting process is proposed to calculate temperature distributions and subsequently, forces and stress distributions. The models proposed include a constitutive model for the material being cut based on the work of Weber, a model for the shear plane based on Merchants model, a model describing the contribution of friction based on Zorev’s approach, a model for the effect of wear on the tool based on the work of Waldorf, and a thermal model based on the works of Komanduri and Hou, with a fraction heat partition for a non-uniform distribution of the heat in the interfaces, but extended to encompass a set of contributions to the global temperature rise of chip, tool and work piece. The models proposed in this work, try to avoid from experimental based values or expressions, and simplifying assumptions or suppositions, as much as possible. On a thermo-physical point of view, the results were affected not only by the mechanical or cutting parameters chosen, but also by their coupling effects, instead of the simplifying way of modeling which is to contemplate only the direct effect of the variation of a parameter. The implementation of these models was performed using the MATLAB environment. Since it was possible to find in the literature all the parameters for AISI 1045 and AISI O2, these materials were used to run the simulations in order to avoid arbitrary assumption.

Identification and differentiation of Candida species from pediatric patients by random amplified polymorphic DNA

Thirty-four Candida isolates were analyzed by random amplified polymorphic DNA using the primer OPG-10:24 Candida albicans; 4 Candida tropicalis; 2 Candida parapsilosis; 2 Candida dubliniensis; 1 Candida glabrata and 1 Candida krusei. The UPGMA-Pearson correlation coefficient was used to calculate the genetic distance between the different Candida groupings. Samples were classified as identical (correlation of 100%); highly related samples (90%); moderately related samples (80%) and unrelated samples (< 70%). The results showed that the RAPD proposed was capable of classifying the isolates coherently (such that same species were in the same dendrogram), except for two isolates of Candida parapsilosis and the positive control (Netherlands, 1973), probably because they are now recognized as three different species. Concerning the only fluconazole-resistant Candida tropicalis isolate with a genotype that was different to the others, the data were insufficient to affirm that the only difference was the sensitivity to fluconazole. We concluded that the Random Amplified Polymorphic DNA proposed might be used to confirm Candida species identified by microbiological methods.

Alteração da metilação do DNA após exposição prolongada a fitoquímicos

Parte do trabalho efetuado durante este projeto de dissertação foi publicado na revista Chemico-Biological Interactions. E apresentado no “50th Congress of the European Societies of Toxicology 7th - 10th September 2014 Edinburgh International Conference Centre, Edinburgh, Scotland” em forma de poster.

Gold nanoparticles for the detection of DNA adducts as biomarkers of exposure to acrylamide

The main objective of this thesis was the development of a gold nanoparticle-based methodology for detection of DNA adducts as biomarkers, to try and overcome existing drawbacks in currently employed techniques. For this objective to be achieved, the experimental work was divided in three components: sample preparation, method of detection and development of a model for exposure to acrylamide. Different techniques were employed and combined for de-complexation and purification of DNA samples (including ultrasonic energy, nuclease digestion and chromatography), resulting in a complete protocol for sample treatment, prior to detection. The detection of alkylated nucleotides using gold nanoparticles was performed by two distinct methodologies: mass spectrometry and colorimetric detection. In mass spectrometry, gold nanoparticles were employed for laser desorption/ionisation instead of the organic matrix. Identification of nucleotides was possible by fingerprint, however no specific mass signals were denoted when using gold nanoparticles to analyse biological samples. An alternate method using the colorimetric properties of gold nanoparticles was employed for detection. This method inspired in the non-cross-linking assay allowed the identification of glycidamide-guanine adducts and DNA adducts generated in vitro. For the development of a model of exposure, two different aquatic organisms were studies: a goldfish and a mussel. Organisms were exposed to waterborne acrylamide, after which mortality was recorded and effect concentrations were estimated. In goldfish, both genotoxicity and metabolic alterations were assessed and revealed dose-effect relationships of acrylamide. Histopathological alterations were verified primarily in pancreatic cells, but also in hepatocytes. Mussels showed higher effect concentrations than goldfish. Biomarkers of oxidative stress, biotransformation and neurotoxicity were analysed after prolonged exposure, showing mild oxidative stress in mussel cells, and induction of enzymes involved in detoxification of oxygen radicals. A qualitative histopathological screening revealed gonadotoxicity in female mussels, which may present some risk to population equilibrium.

Extração de DNA a partir de sangue humano coagulado para aplicação nas técnicas de genotipagem de antígenos leucocitários humanos e de receptores semelhantes à imunoglobulina

O objetivo deste estudo foi padronizar uma metodologia de extração de DNA de alta qualidade a partir de amostras de sangue coagulado. Quarenta e oito amostras de sangue humano coagulado foram utilizadas para a extração de DNA pelo kit comercial EZ-DNA® (Biological Industries, Beit Haemek, Israel), pelo kit de coluna Neoscience® (One Lambda Inc., San Diego, CA) e pelo método modificado de salting out. Apenas o método de salting out foi capaz de extrair altas concentrações de DNA (média, 180ng/µL), as quais foram medidas pelo detector de fluorescência Qubit® (Invitrogen, USA). Este método permitiu a amplificação dos genes HLA (human leukocyte antigens) pela tecnologia PCR-SSO (polymerase chain reaction - specific sequence of oligonucleotides) Luminex, a qual exige DNA de boa qualidade, e de genes KIR (killer cell immunoglobulin-like receptors) pela técnica made in house PCR-SSP (polymerase chain reaction-sequence specific of primers), a qual demanda uma concentração específica de DNA (10ng/µL). Concluímos que a técnica de salting out modificada foi muito eficiente, simples e rápida para a extração de DNA de amostras de sangue humano coagulado, com o objetivo de realizar a genotipagem de genes HLA e KIR.

PCR-RFLP of 16S ribosomal DNA to confirm the identification of Enterococcus gallinarum and Enterococcus casseliflavus isolated from clinical and food samples

INTRODUCTION: This study aimed to confirm the identification of Enterococcus gallinarum and Enterococcus casseliflavus isolated from clinical and food samples by PCR-RFLP. METHODS: Fifty-two strains identified by conventional biochemical exams were submitted to PCR amplification and digested with HinfI. Only 20 (38.5%) of the 52 strains showed a DNA pattern expected for E. gallinarum and E. casseliflavus. RESULTS: Analysis of the results of this study showed that E. gallinarum and E. casseliflavus are occasionally erroneously identified and confirmed the potential application of 16S rDNA analysis for accurate identification of these species. CONCLUSIONS: A correct identification is important to distinguish between intrinsic and acquired vancomycin resistance.

PCR detection of multiple human herpesvirus DNA in saliva from HIV-infected individuals in Teresina, State of Piauí, Brazil

INTRODUCTION: Human herpesviruses are frequently associated with orofacial diseases in humans (HSV-1, EBV, CMV and HHV-8), some can also cause systemic disease (CMV and HHV-8). The transmission of these viruses occurs by contact with infected secretions, especially saliva. Human immunodeficiency virus infection is associated with an increased risk of HHVs and related diseases. METHODS: This work aimed to detect HSV-1, EBV, CMV and HHV-8 DNA in saliva of HIV-infected patients from Teresina, northeast Brazil, by PCR and compare these findings with age and sex matched HIV-seronegative individuals. RESULTS: No difference in prevalence was verified between HHV detection in the saliva of HIV-seropositive individuals and controls. The individual frequencies of these viruses in these two populations were different. HIV seropositivity correlated positively with the presence of CMV (OR: 18.2, p= 0.00032) and EBV (OR: 3.44, p= 0.0081). No association between CD4 counts and the prevalence of HHVs in the saliva was observed; however, a strong association was determined between seropositivity and the presence of multiple HHV DNAs in saliva (OR: 4.83, p = 0.0028). CONCLUSIONS: These findings suggest the asymptomatic salivary shedding of HHVs is a common event between HIV-seropositive and seronegative individuals from Teresina, Piauí, Brazil, and, especially for HIV-seropositive patients, saliva is a risk factor for the acquisition/transmission of multiple HHVs.

Land cover mapping analysis and urban growth modeling using remote sensing techniques

The rapid growth of big cities has been noticed since 1950s when the majority of world population turned to live in urban areas rather than villages, seeking better job opportunities and higher quality of services and lifestyle circumstances. This demographic transition from rural to urban is expected to have a continuous increase. Governments, especially in less developed countries, are going to face more challenges in different sectors, raising the essence of understanding the spatial pattern of the growth for an effective urban planning. The study aimed to detect, analyse and model the urban growth in Greater Cairo Region (GCR) as one of the fast growing mega cities in the world using remote sensing data. Knowing the current and estimated urbanization situation in GCR will help decision makers in Egypt to adjust their plans and develop new ones. These plans should focus on resources reallocation to overcome the problems arising in the future and to achieve a sustainable development of urban areas, especially after the high percentage of illegal settlements which took place in the last decades. The study focused on a period of 30 years; from 1984 to 2014, and the major transitions to urban were modelled to predict the future scenarios in 2025. Three satellite images of different time stamps (1984, 2003 and 2014) were classified using Support Vector Machines (SVM) classifier, then the land cover changes were detected by applying a high level mapping technique. Later the results were analyzed for higher accurate estimations of the urban growth in the future in 2025 using Land Change Modeler (LCM) embedded in IDRISI software. Moreover, the spatial and temporal urban growth patterns were analyzed using statistical metrics developed in FRAGSTATS software. The study resulted in an overall classification accuracy of 96%, 97.3% and 96.3% for 1984, 2003 and 2014’s map, respectively. Between 1984 and 2003, 19 179 hectares of vegetation and 21 417 hectares of desert changed to urban, while from 2003 to 2014, the transitions to urban from both land cover classes were found to be 16 486 and 31 045 hectares, respectively. The model results indicated that 14% of the vegetation and 4% of the desert in 2014 will turn into urban in 2025, representing 16 512 and 24 687 hectares, respectively.

Flexible multivariate GARCH modeling with an application to international stock markets

This paper offers a new approach to estimating time-varying covariance matrices in the framework of the diagonal-vech version of the multivariate GARCH(1,1) model. Our method is numerically feasible for large-scale problems, produces positive semidefinite conditional covariance matrices, and does not impose unrealistic a priori restrictions. We provide an empirical application in the context of international stock markets, comparing the nev^ estimator with a number of existing ones.

Comparison among three polymerase chain reaction assays on detection of DNA from Leishmania in biological samples from patients with american cutaneous leishmaniasis

INTRODUCTION: The study analyzed positivity of polymerase chain reaction (PCR) on detection of DNA from Leishmania in patients' samples. METHODS: Extracted DNA was submitted to L150/L152, 13Y/13Z, and seminested PCR (snPCR). RESULTS: Results were evidenced by bands of approximately 120, 720, and 670 bp for L150/L152, 13Y/13Z, and snPCR, respectively. L150/L152, 13Y/13Z, and snPCR positivity indexes were 76.9, 56.4, and 9.2 (p>0.05), respectively, for suspected and 93.7, 68.7, and 84.4 (p<0.05), respectively, for confirmed. CONCLUSIONS: Preliminary results showed that these assays, mainly L150/L152 and snPCR, can detect Leishmania DNA and carry potential on laboratory diagnosis of leishmaniasis.

Experimental and modeling studies on reverse electrodialysis for sustainable power generation

A potentially renewable and sustainable source of energy is the chemical energy associated with solvation of salts. Mixing of two aqueous streams with different saline concentrations is spontaneous and releases energy. The global theoretically obtainable power from salinity gradient energy due to World’s rivers discharge into the oceans has been estimated to be within the range of 1.4-2.6 TW. Reverse electrodialysis (RED) is one of the emerging, membrane-based, technologies for harvesting the salinity gradient energy. A common RED stack is composed by alternately-arranged cation- and anion-exchange membranes, stacked between two electrodes. The compartments between the membranes are alternately fed with concentrated (e.g., sea water) and dilute (e.g., river water) saline solutions. Migration of the respective counter-ions through the membranes leads to ionic current between the electrodes, where an appropriate redox pair converts the chemical salinity gradient energy into electrical energy. Given the importance of the need for new sources of energy for power generation, the present study aims at better understanding and solving current challenges, associated with the RED stack design, fluid dynamics, ionic mass transfer and long-term RED stack performance with natural saline solutions as feedwaters. Chronopotentiometry was used to determinate diffusion boundary layer (DBL) thickness from diffusion relaxation data and the flow entrance effects on mass transfer were found to avail a power generation increase in RED stacks. Increasing the linear flow velocity also leads to a decrease of DBL thickness but on the cost of a higher pressure drop. Pressure drop inside RED stacks was successfully simulated by the developed mathematical model, in which contribution of several pressure drops, that until now have not been considered, was included. The effect of each pressure drop on the RED stack performance was identified and rationalized and guidelines for planning and/or optimization of RED stacks were derived. The design of new profiled membranes, with a chevron corrugation structure, was proposed using computational fluid dynamics (CFD) modeling. The performance of the suggested corrugation geometry was compared with the already existing ones, as well as with the use of conductive and non-conductive spacers. According to the estimations, use of chevron structures grants the highest net power density values, at the best compromise between the mass transfer coefficient and the pressure drop values. Finally, long-term experiments with natural waters were performed, during which fouling was experienced. For the first time, 2D fluorescence spectroscopy was used to monitor RED stack performance, with a dedicated focus on following fouling on ion-exchange membrane surfaces. To extract relevant information from fluorescence spectra, parallel factor analysis (PARAFAC) was performed. Moreover, the information obtained was then used to predict net power density, stack electric resistance and pressure drop by multivariate statistical models based on projection to latent structures (PLS) modeling. The use in such models of 2D fluorescence data, containing hidden, but extractable by PARAFAC, information about fouling on membrane surfaces, considerably improved the models fitting to the experimental data.

Modeling and forecasting value-at-risk for the Portuguese stock market

The aim of this work project is to find a model that is able to accurately forecast the daily Value-at-Risk for PSI-20 Index, independently of the market conditions, in order to expand empirical literature for the Portuguese stock market. Hence, two subsamples, representing more and less volatile periods, were modeled through unconditional and conditional volatility models (because it is what drives returns). All models were evaluated through Kupiec’s and Christoffersen’s tests, by comparing forecasts with actual results. Using an out-of-sample of 204 observations, it was found that a GARCH(1,1) is an accurate model for our purposes.