Automatic learning for the classification of chemical reactions and in statistical thermodynamics

This Thesis describes the application of automatic learning methods for a) the classification of organic and metabolic reactions, and b) the mapping of Potential Energy Surfaces(PES). The classification of reactions was approached with two distinct methodologies: a representation of chemical reactions based on NMR data, and a representation of chemical reactions from the reaction equation based on the physico-chemical and topological features of chemical bonds. NMR-based classification of photochemical and enzymatic reactions. Photochemical and metabolic reactions were classified by Kohonen Self-Organizing Maps (Kohonen SOMs) and Random Forests (RFs) taking as input the difference between the 1H NMR spectra of the products and the reactants. The development of such a representation can be applied in automatic analysis of changes in the 1H NMR spectrum of a mixture and their interpretation in terms of the chemical reactions taking place. Examples of possible applications are the monitoring of reaction processes, evaluation of the stability of chemicals, or even the interpretation of metabonomic data. A Kohonen SOM trained with a data set of metabolic reactions catalysed by transferases was able to correctly classify 75% of an independent test set in terms of the EC number subclass. Random Forests improved the correct predictions to 79%. With photochemical reactions classified into 7 groups, an independent test set was classified with 86-93% accuracy. The data set of photochemical reactions was also used to simulate mixtures with two reactions occurring simultaneously. Kohonen SOMs and Feed-Forward Neural Networks (FFNNs) were trained to classify the reactions occurring in a mixture based on the 1H NMR spectra of the products and reactants. Kohonen SOMs allowed the correct assignment of 53-63% of the mixtures (in a test set). Counter-Propagation Neural Networks (CPNNs) gave origin to similar results. The use of supervised learning techniques allowed an improvement in the results. They were improved to 77% of correct assignments when an ensemble of ten FFNNs were used and to 80% when Random Forests were used. This study was performed with NMR data simulated from the molecular structure by the SPINUS program. In the design of one test set, simulated data was combined with experimental data. The results support the proposal of linking databases of chemical reactions to experimental or simulated NMR data for automatic classification of reactions and mixtures of reactions. Genome-scale classification of enzymatic reactions from their reaction equation. The MOLMAP descriptor relies on a Kohonen SOM that defines types of bonds on the basis of their physico-chemical and topological properties. The MOLMAP descriptor of a molecule represents the types of bonds available in that molecule. The MOLMAP descriptor of a reaction is defined as the difference between the MOLMAPs of the products and the reactants, and numerically encodes the pattern of bonds that are broken, changed, and made during a chemical reaction. The automatic perception of chemical similarities between metabolic reactions is required for a variety of applications ranging from the computer validation of classification systems, genome-scale reconstruction (or comparison) of metabolic pathways, to the classification of enzymatic mechanisms. Catalytic functions of proteins are generally described by the EC numbers that are simultaneously employed as identifiers of reactions, enzymes, and enzyme genes, thus linking metabolic and genomic information. Different methods should be available to automatically compare metabolic reactions and for the automatic assignment of EC numbers to reactions still not officially classified. In this study, the genome-scale data set of enzymatic reactions available in the KEGG database was encoded by the MOLMAP descriptors, and was submitted to Kohonen SOMs to compare the resulting map with the official EC number classification, to explore the possibility of predicting EC numbers from the reaction equation, and to assess the internal consistency of the EC classification at the class level. A general agreement with the EC classification was observed, i.e. a relationship between the similarity of MOLMAPs and the similarity of EC numbers. At the same time, MOLMAPs were able to discriminate between EC sub-subclasses. EC numbers could be assigned at the class, subclass, and sub-subclass levels with accuracies up to 92%, 80%, and 70% for independent test sets. The correspondence between chemical similarity of metabolic reactions and their MOLMAP descriptors was applied to the identification of a number of reactions mapped into the same neuron but belonging to different EC classes, which demonstrated the ability of the MOLMAP/SOM approach to verify the internal consistency of classifications in databases of metabolic reactions. RFs were also used to assign the four levels of the EC hierarchy from the reaction equation. EC numbers were correctly assigned in 95%, 90%, 85% and 86% of the cases (for independent test sets) at the class, subclass, sub-subclass and full EC number level,respectively. Experiments for the classification of reactions from the main reactants and products were performed with RFs - EC numbers were assigned at the class, subclass and sub-subclass level with accuracies of 78%, 74% and 63%, respectively. In the course of the experiments with metabolic reactions we suggested that the MOLMAP / SOM concept could be extended to the representation of other levels of metabolic information such as metabolic pathways. Following the MOLMAP idea, the pattern of neurons activated by the reactions of a metabolic pathway is a representation of the reactions involved in that pathway - a descriptor of the metabolic pathway. This reasoning enabled the comparison of different pathways, the automatic classification of pathways, and a classification of organisms based on their biochemical machinery. The three levels of classification (from bonds to metabolic pathways) allowed to map and perceive chemical similarities between metabolic pathways even for pathways of different types of metabolism and pathways that do not share similarities in terms of EC numbers. Mapping of PES by neural networks (NNs). In a first series of experiments, ensembles of Feed-Forward NNs (EnsFFNNs) and Associative Neural Networks (ASNNs) were trained to reproduce PES represented by the Lennard-Jones (LJ) analytical potential function. The accuracy of the method was assessed by comparing the results of molecular dynamics simulations (thermal, structural, and dynamic properties) obtained from the NNs-PES and from the LJ function. The results indicated that for LJ-type potentials, NNs can be trained to generate accurate PES to be used in molecular simulations. EnsFFNNs and ASNNs gave better results than single FFNNs. A remarkable ability of the NNs models to interpolate between distant curves and accurately reproduce potentials to be used in molecular simulations is shown. The purpose of the first study was to systematically analyse the accuracy of different NNs. Our main motivation, however, is reflected in the next study: the mapping of multidimensional PES by NNs to simulate, by Molecular Dynamics or Monte Carlo, the adsorption and self-assembly of solvated organic molecules on noble-metal electrodes. Indeed, for such complex and heterogeneous systems the development of suitable analytical functions that fit quantum mechanical interaction energies is a non-trivial or even impossible task. The data consisted of energy values, from Density Functional Theory (DFT) calculations, at different distances, for several molecular orientations and three electrode adsorption sites. The results indicate that NNs require a data set large enough to cover well the diversity of possible interaction sites, distances, and orientations. NNs trained with such data sets can perform equally well or even better than analytical functions. Therefore, they can be used in molecular simulations, particularly for the ethanol/Au (111) interface which is the case studied in the present Thesis. Once properly trained, the networks are able to produce, as output, any required number of energy points for accurate interpolations.

In vitro assessment of three dimensional dense chitosan-based structures to be as bioabsorbable implants

Chitosan biocompatibility and biodegradability properties make this biopolymer promising for the development of advanced internal fixation devices for orthopedic applications. This work presents a detailed study on the production and characterization of three dimensional (3D) dense, non-porous, chitosan-based structures, with the ability to be processed in different shapes, and also with high strength and stiffness. Such features are crucial for the application of such 3D structures as bioabsorbable implantable devices. The influence of chitosan's molecular weight and the addition of one plasticizer (glycerol) on 3D dense chitosan-based products' biomechanical properties were explored. Several specimens were produced and in vitro studies were performed in order to assess the cytotoxicity of these specimens and their physical behavior throughout the enzymatic degradation experiments. The results point out that glycerol does not impact on cytotoxicity and has a high impact in improving mechanical properties, both elasticity and compressive strength. In addition, human mesenchymal stem/stromal cells (MSC) were used as an ex-vivo model to study cell adhesion and proliferation on these structures, showing promising results with fold increase values in total cell number similar to the ones obtained in standard cell culture flasks. (C) 2014 Elsevier Ltd. All rights reserved.

Aspectos clínicos e sorológicos de 47 pacientes com paracoccidioidomicose tratados pela anfotericina B

In the present study, clinic and serologic aspects of 47 paracoccidioidomycosis patients were reviewed. The clinic-radiologie-laboratorial data of each patient were obtained from the medical chart before, during and after therapy with Amphotericin B. The age of the patients ranged between 13 to 50 years; the ratio male: female was 10:1. The most frequently associated diseases were alcoholism and tabagism; tuberculosis was present in 14.2% of the cases. Most patients came from Botucatu of adjacent towns (central area of the State of São Paulo). Cough with sputum production, dyspnea and anorexia were the most frequent clinic manifestations. All 47 patients, but 5 presented pulmonary involvement which showed the radiologic pattern of interstitial exudate nodular type and fibrocicatritial lesions in 85.7%. There was associated emphysema in 37.7% of the patients. Blood urea, creatinin and kaliemia increased during therapy with Amphotericin B. Clinic-laboratorial follow-up showed electrocardiographic changes in 13, hypertension in 9 and hyperreactivity in Thorn test in 1 patient. Complement fixation was the best serologic test for monitoring patients with paracoccidioidomycosis. Incomplete dosage of Amphotericin B led to therapeutic ineffectivenes.

Resistance of mice immunized with killed culture trypomastigotes against infection by insect-derived trypomastigotes of Trypanosoma cruzi

Mice immunized with heat or merthiolate-killed culture trypomastigotes of the non-virulent G strain were resistant to the challenge by insect-derived trypomastigotes of the CL strain of Trypanosoma cruzi. No parasitemia was detected, by direct microscopic examination of blood samples, in 90% of immunized mice while all control animals developed a high parasitemia. Trypsinization before heat-inactivation, or fixation with paraformaldehyde, apparently reduced the immunogenicity of the G strain trypomastigotes. Mice immunized with trypomastigotes treated by either of these procedures were not protected against infection by virulent T. cruzi. Analysis of the 13I-labeled surface proteins of G strain trypomastigotes inactivated by the various methods suggests that these components are involved in eliciting protective immunity against T. cruzi infection.

Molluscum contagiosum: serology and electron microscopy findings in twenty one patients

Twenty one cases of molluscum contagiosum virus disease were collected for electron microscopical and serological tests. Molluscum virus was detected in the crust, inside the vacuoles formed in the keratinocytes cells. The patients developed specific antibodies to the virus detected by complement fixation test.

Predictive value of serologic tests in the diagnosis and follow-up of patients with paracoccidioidomycosis

A serologic study was undertaken in a group of 43 patients with active paracoccidioidomycosis who were treated in the same form (ketoconazole), for identical periods of time (6 months), and folio wed-up for various periods posttherapy. The tests employed were agar gel immunodiffusion (AGID) and complement fixation (FC). Also studied were 50 sera from patients with proven histoplasmosis and pulmonary aspergilloma, 30 patients with culturaly proven tuberculosis as well as 92 specimens from healthy individuals, residents in the endemic area for paracoccidioidomycosis. A single lot of yeast filtrate antigen was used throughout the study. The value of each test was measured according to GALEN and GAMBINO6. Both tests were highly sensitive, 89 and 93% respectively. Regarding their specificity, the AGID was totally specific while the CF exhibited 96.6% and 97% specificity in front of tuberculosis patients and healthy individuals respectively and 82% in comparison with patients with other mycoses. The concept of predictive value, that is, the certainty one has in accepting a positive test as diagnostic of paracoccidioidomycosis, favored the AGID procedure (100%) over the CF test. The latter could sort out with 93% certainty a patient with paracoccidioidomycosis among a group of healthy individuals and with 97.5% in the case of TB patients; when the group in question was composed by individuals with other deep mycoses, such certainty was lower (81%). The above results indicate that both the AGID and the CF tests furnish results of high confidence; one should not relay, however, in the CF alone as a means to establish the specific diagnosis of paracoccidioidomycosis.

Trends of T. cruzi infection based on data from blood bank screening

Between October 1988 and April 1989 a cross-sectional survey was carried out in six out of eight blood banks of Goiânia, Central Brazil. Subjects attending for first-time blood donation in the mornings of the study period (n = 1358) were interviewed and screened for T. cruzi infection as a part of a major study among blood donors. Tests to anti-T. cruzi antibodies were performed, simultaneously, by indirect hem agglutination test (IHA) and complement fixation test (CFT). A subject was considered seropositive when any one of the two tests showed a positive result. Information on age, sex, place of birth, migration and socio-economic level was recorded. Results from this survey were compared with seroprevalence rates obtained in previous studies in an attempt to analyse trend of T. cruzi infection in an endemic urban area. The overall seroprevalence of T. cruzi infection among first-time donors was found to be 3.5% (95% confidence interval 2.5%-4.5% ). The seroprevalence rate increased with age up to 45 years and then decreased. Migrants from rural areas had higher seroprevalence rates than subjects from urban counties (1.8%-16.2% vs. 0%-3.6%). A four fold decrease in prevalence rates was observed when these rates were compared with those of fifteen years ago. Two possible hypotheses to explain this difference were suggested: 1. a cohort effect related with the decrease of transmission in rural areas and/or 2. a differential proportion of people of rural origin among blood donors between the two periods. The potential usefulness of blood banks as a source of epidemiological information to monitor trends of T. cruzi infection in an urban adult population was stressed.

The sensitivity, specificity and efficiency values of some serological tests used in the diagnosis of paracoccidioidomycosis

This work reports on the results of double immunodiffusion (ID), counterimmunoelectrophoresis (CIE), complement fixation (CF) and indirect immunofluorescence (IIF) techniques in the serodiagnosis of paracoccidioidomycosis. The study was undertaken on four groups of individuals: 46 patients with untreated paracoccidioidomycosis, 22 patients with other deep mycoses, 30 with other infectious diseases (tuberculosis and cutaneous leishmaniasis) and 47 blood donors as negative controls. Data were obtained using Paracoccidioides brasiliensis antigens, i.e.,a yeast culture filtrate for ID, CIE and CF, and a yeast cell suspension for IIF. The sensitivity, specificity and efficiency values were measured according to GALEN & GAMBINO8.The gel precipitation tests (ID and CIE) showed the greatest sensitivity (91.3 and 95.6%, respectively), maximum specificity (100%) and the highest efficiency values when compared to the CF and IIF tests.

Antibodies against Entamoeba histolytica in individuals with intestinal amoebiasis presenting cysts and/or trophozoites in the feces

Serum samples were obtained from 154 individuals infected with Entamoeba histolytica (78 symptomatic and 76 asymptomatic). Twelve had trophozoites in the feces whereas 142 had only cysts. The sera were used to test the existence of antibodies anti-Entamoeba histolytica employing the Indirect Hemagglutination (IHA), Indirect Immunofluoresccnce (IFAT), Complement Fixation Reaction (CFR) and Counterimmunoelectrophoresis (CIEP). For those individuals with trophozoites in their feces, 75.0 were positive by IHA and IFAT, 83.0 by CFR and 41.7 by CIEP. In individuals who had only cysts, positive results by the same tests were respectively, 5.6%, 12.0%, 19.0% and 5.6%. The difference in relation to the tilers of antibodies detected through IHA, IFAT, CFR and CIEP and in relation to the presence of trophozoites or cysts in the feces was significative for four immunological reactions when X², was employed (P < 0.05).

Isolation and characterization of partially purified leptospiral antigens

The methanol extract of Leptospira interrogans serovar canicola was purified by precipitation with acetone or acetone and chloroform. The antigenicity of the antigen was not altered by heating or treatment with pepsin and pronase. However the antigenicity was lost when the antigen was treated with periodic acid. Chemical analysis revealed the presence of 40% carbohydrate (22% methylpentose, 28%; hexoses),4% protein, 20% lipid and 2,7% phosphate. The complement fixation test with sera from patients with leptospirosis agreed with the microscopic agglutination reaction.

Laboratory acquired infection by the virus SP H 114202 (Arenavirus: Arenaviridae): clinical and laboratory findings

Here in is described the clinical and laboratorial findings of a laboratory-acquired infection caused by the virus SP H 114202 (Arenavirus, family Arenaviridae) a recently discovered agent responsible for a viral hemorrhagic fever. The patient was sick for 13 days. The disease had an abrupt onset characterized by high fever (39ºC.), headache, chills and myalgias for 8 days. In addition, on the 3rd day, the patient developed nauseas and vomiting, and in the 10th, epigastralgia, diarrheia and gengivorrhagia. Leucopenia was seen within the 1 st week of onset, with counts as low as 2,500 white cells per mm³. Counts performed after the 23th day of the onset were within normal limits. With the exception of moderate lymphocitosis, no changes were observed in differential counts. An increase in the liter of antibodies by complement fixation, neutralization and ELISA (IgM) was detected. Suckling mice and baby hamsters were inoculated intracerebrally with 0.02 ml of blood samples collected in the 2nd and 7th days of disease. Attempts to isolate the virus were also made in Vero cells. No virus was isolated. This virus was isolated before in a single occasion in São Paulo State, in 1990, from the blood of a patient with hemorrhagic fever with a fatal outcome. The manipulation of the virus under study, must be done carefully, since the transmission can occur through aerosols.

Jungle yellow fever: clinical and laboratorial sudies emphasizing viremia on a human case

The authors report the clinical, laboratorial and epidemiological aspects of a human case of jungle yellow fever. The patient suffered from fever, chills, sweating, headaches, backaches, myalgia, epigastric pains, nausea, vomiting, diarrhea and prostration. He was unvaccinated and had been working in areas where cases of jungle yellow fever had been confirmed. Investigations concerning the yellow fever virus were performed. Blood samples were collected on several days in the course of the illness. Three of these samples (those obtained on days 5,7 and 10) were inoculated into suckling mice in attempt to isolate virus and to titrate the viremia level. Serological surveys were carried out by using the IgM Antibodies Capture Enzyme Linked Immunosorbent Assay (MAC-ELISA), Complement Fixation (CF), Hemagglulinalion Inhibition (HI) and Neutralization (N) tests. The yellow fever virus, recovered from the two first samples and the virus titration, showed high level of viremia. After that, specific antibodies appeared in all samples. The interval between the end of the viremia and the appearance of the antibodies was associated with the worsening of clinical symptoms, including bleeding of the mucous membrane. One must be aware of the risk of having a urban epidemics in areas where Aedes aegypti is found in high infestation indexes.

Coccidioidomycosis in Brazil. A case report

Coccidioidomycosis is an endemic infection with a relatively limited geographic distribution: Mexico, Guatemala, Honduras, Colombia, Venezuela, Bolivia, Paraguai, Argentina and the southwest of the United States. In these countries, the endemic area is restricted to the semiarid desert like regions which are similar to the northeast of Brazil. Case report: The patient is a 32 year-old male, born in the state of Bahia (Northeast of Brazil) and has been living in São Paulo (Southeast) for 6 years. He was admitted at Hospital das Clínicas, at the Department of Pneumology in October 1996, with a 6 month history of progressive and productive cough, fever, malaise, chills, loss of weight, weakness and arthralgia in the small joints. Chest x-rays and computerized tomography disclosed an interstitial reticulonodular infiltrate with a cavity in the right upper lobe. The standard potassium hydroxide preparation of sputum and broncoalveolar lavage demonstrated the characteristic thickened wall spherules in various stages of development. Sabouraud dextrose agar, at 25° C and 30° C showed growth of white and cottony aerial micelium. The microscopic morphology disclosed branched hyphae characterized by thick walled, barrel shaped arthroconidia alternated with empty cells. The sorological studies with positive double immunodiffusion test, and also positive complement fixation test in 1/128 dilution confirmed the diagnosis. The patient has been treated with ketoconazole and presents a favorable clinical and radiological evolution

Liquid crystal mobility and PDLC memory effects

Dissertation presented at Faculdade de Ciências e Tecnologia of Universidade Nova de Lisboa to obtain the Degree of Master in Chemical and Biochemical Engineering

Light controlled synthesis of nucleic acids

Dissertação apresentada para obtenção do grau de Doutor em Bioquímica - especialidade Biotecnologia, pela Universidade Nova de Lisboa,Faculdade de Ciências e Tecnologia