Evaluation of different natural ingredients as satiety inductors

Dissertation to obtain a Master’s Degree in Chemical and Biochemical Engineering

Evaluation of three different DNA extraction methods from blood samples collected in dried filter paper in Plasmodium subpatent infections from the Amazon region in Brazil

Asymptomatic Plasmodium infection is a new challenge for public health in the American region. The polymerase chain reaction (PCR) is the best method for diagnosing subpatent parasitemias. In endemic areas, blood collection is hampered by geographical distances and deficient transport and storage conditions of the samples. Because DNA extraction from blood collected on filter paper is an efficient method for molecular studies in high parasitemic individuals, we investigated whether the technique could be an alternative for Plasmodium diagnosis among asymptomatic and pauciparasitemic subjects. In this report we compared three different methods (Chelex®-saponin, methanol and TRIS-EDTA) of DNA extraction from blood collected on filter paper from asymptomatic Plasmodium-infected individuals. Polymerase chain reaction assays for detection of Plasmodium species showed the best results when the Chelex®-saponin method was used. Even though the sensitivity of detection was approximately 66% and 31% for P. falciparum and P. vivax, respectively, this method did not show the effectiveness in DNA extraction required for molecular diagnosis of Plasmodium. The development of better methods for extracting DNA from blood collected on filter paper is important for the diagnosis of subpatent malarial infections in remote areas and would contribute to establishing the epidemiology of this form of infection.

SOIL CONTAMINATION IN PUBLIC SQUARES IN BELO HORIZONTE, MINAS GERAIS, BY CANINE PARASITES IN DIFFERENT DEVELOPMENTAL STAGES

SUMMARY To evaluate soil contamination by parasites in different developmental stages in public squares used as recreation and leisure areas for children in Belo Horizonte (MG, Brazil), 210 soil samples and 141 canine fecal samples were collected from 42 squares in the city. These samples were analyzed by the Caldwell and Caldwell technique and the Hoffman, Pons, and Janer technique. Of the samples analyzed, 89 (42.4%) soil samples and 104 (73.5%) fecal samples were contaminated with Ancylostoma sp., Toxocara sp., Trichuris sp., or Dipylidium sp. eggs; Giardia sp. cysts; or Isospora sp. oocysts. The commonest parasite was Ancylostoma sp., found in 85% soil and 99% fecal samples, followed by Toxocara sp., found in 43.7% soil and 30.7% fecal samples.

Viva@Matemática project: a different way to see math

Viva@Mat is a project developed by four Math teachers from the School of Industrial Studies and Management (ESEIG) that was born with the fundamental objective of engaging ESEIG students with different math backgrounds in Math challenging activities. Some of these activities were transformed into real palpable materials and others into small interactive ones, being the great majority of them proposed by ESEIG’ students themselves. This small project rapidly grew into something we didn’t expect – it did flow over the walls of our institution to the general involving community – specifically to pre-university schools through the Viva@Math Exhibits – Orange, Blue and Green (the fourth, the Purple one is still in development). Nowadays, Viva@Math Exhibits – the public face of the Project – are itinerant and have been travelling between several, and different institutions (pre-university schools, preparatory schools, libraries, among others), around ESEIG and IPP area of influence and having registered visitors/participants of all ages. In this article we will describe the Viva@Math Project, its different activities that are categorized in some “great groups” like Numerical Trivia, Logic Activities and Mental Calculation, Puzzles, Geometric Curiosities, Magic Tricks, among others, designed to challenge students to use the underlying logical-mathematical reasoning to any ordinary and everyday activity. We will give specific and concrete examples of some of the activities developed and, also, reproduce of the general stimulating feedback the Project receives from the enrolled “actors” (teachers, students and their relatives, institutions, among others). We feel that this Project has become a small “bridge” between the pre-university schools and Higher Education Institutions (HEI), in trying to shorten the “gap” between the institutions of different levels of education and bring them to work together.

EVALUATION OF FOUR DIFFERENT DNA EXTRACTION METHODS IN COAGULASE-NEGATIVE STAPHYLOCOCCI CLINICAL ISOLATES

Currently there are several methods to extract bacterial DNA based on different principles. However, the amount and the quality of the DNA obtained by each one of those methods is highly variable and microorganism dependent, as illustrated by coagulase-negative staphylococci (CoNS) which have a thick cell wall that is difficult to lyse. This study was designed to compare the quality and the amount of CoNS DNA, extracted by four different techniques: two in-house protocols and two commercial kits. DNA amount and quality determination was performed through spectrophotometry. The extracted DNA was also analyzed using agarose gel electrophoresis and by PCR. 267 isolates of CoNS were used in this study. The column method and thermal lyses showed better results with regard to DNA quality (mean ratio of A260/280 = 1.95) and average concentration of DNA (), respectively. All four methods tested provided appropriate DNA for PCR amplification, but with different yields. DNA quality is important since it allows the application of a large number of molecular biology techniques, and also it's storage for a longer period of time. In this sense the extraction method based on an extraction column presented the best results for CoNS.

FACTORS ASSOCIATED TO ADHERENCE TO DIFFERENT TREATMENT SCHEMES WITH MEGLUMINE ANTIMONIATE IN A CLINICAL TRIAL FOR CUTANEOUS LEISHMANIASIS

The favorable outcome of the treatment of a disease is influenced by the adherence to therapy. Our objective was to assess factors associated with adherence to treatment of patients included in a clinical trial of equivalence between the standard and alternative treatment schemes with meglumine antimoniate (MA) in the treatment of cutaneous leishmaniasis (CL), in the state of Rio de Janeiro. Between 2008 and 2011, 57 patients with CL were interviewed using a questionnaire to collect socioeconomic data. The following methods were used for adherence monitoring: counting of vial surplus, monitoring card, Morisky test and modified Morisky test (without the question regarding the schedule); we observed 82.1% (vial return), 86.0% (monitoring card), 66.7% (Morisky test) and 86.0% (modified Morisky test) adherence. There was a strong correlation between the method of vial counting and the monitoring card and modified Morisky test. A significant association was observed between greater adherence to treatment and low dose of MA, as well as with a lower number of people sleeping in the same room. We recommend the use of the modified Morisky test to assess adherence to treatment of CL with MA, because it is a simple method and with a good performance, when compared to other methods.

LABORATORY EVALUATION OF THE DEVELOPMENT OF Aedes aegypti IN TWO SEASONS: INFLUENCE OF DIFFERENT PLACES AND DIFFERENT DENSITIES

Aedes aegypti is an important vector in Brazil being the main vector of the dengue-fever. This paper employs survival curves to describe the time in days from larvae to adult forms of Aedes aegypti raised, individually and collectively, and compares it during winter and spring when positioned inside and outside a laboratory. The study was conducted in São Vicente, a coastal city in Southeastern Brazil. The lowest water temperature in winter and in spring was 20 °C and the highest was 26 °C in spring. Higher and more stable temperatures were measured in the intra compared to the peri in both seasons. Consequently, larvae positioned in the intra resulted in the lowest median time to develop in the individual and collective experiment (nine and ten days, respectively). At least 25% of the larvae positioned in the intra in the individual experiment in the spring took only seven days to reach adulthood. Sex ratios and the median time development by sex did not show significant differences. These results indicate that efforts to control Aedes aegypti must be continuous and directed mainly to prevent the intra-domiciliary sites that can be infested in a week in order to reduce the human-vector contact.

EVALUATION OF METHODS IN DETECTING VANCOMYCIN MIC AMONG MRSA ISOLATES AND THE CHANGES IN ACCURACY RELATED TO DIFFERENT MIC VALUES

INTRODUCTION: Methicillin-Resistant Staphylococcus aureus (MRSA) presenting reduced susceptibility to vancomycin has been associated to therapeutic failure. Some methods used by clinical laboratories may not be sufficiently accurate to detect this phenotype, compromising results and the outcome of the patient. OBJECTIVES: To evaluate the performance of methods in the detection of vancomycin MIC values among clinical isolates of MRSA. MATERIAL AND METHODS: The Vancomycin Minimal Inhibitory Concentration was determined for 75 MRSA isolates from inpatients of Mãe de Deus Hospital, Porto Alegre, Brazil. The broth microdilution (BM) was used as the gold-standard technique, as well as the following methods: E-test® strips (BioMérieux), M.I.C.E® strips (Oxoid), PROBAC® commercial panel and the automated system MicroScan® (Siemens). Besides, the agar screening test was carried out with 3 µg/mL of vancomycin. RESULTS: All isolates presented MIC ≤ 2 µg/mL for BM. E-test® had higher concordance (40%) in terms of global agreement with the gold standard, and there was not statistical difference among E-test® and broth microdilution results. PROBAC® panels presented MICs, in general, lower than the gold-standard panels (58.66% major errors), while M.I.C.E.® MICs were higher (67.99% minor errors). CONCLUSIONS: For the population of MRSA in question, E-test® presented the best performance, although with a heterogeneous accuracy, depending on MIC values.

WATER FROM DIFFERENT SOURCES USED FOR THE IRRIGATION OF VEGETABLES TO BE MARKETED: RESEARCH ON Cryptosporidium spp., Giardia spp., AND COLIFORMS IN PARANA, BRAZIL

SUMMARY The aim of this work was to compare, from a parasitological ( Cryptosporidiumspp. and Giardia duodenalis), bacteriological (total and thermotolerants coliforms) and physicochemical perspective, water sources used for drinking and irrigation of vegetables intended to be sold for human consumption. From January 2010 to May 2011, samples of different water sources from vegetable producing properties were collected; 100 liters for parasitological analysis, 200 mL for bacteriological analysis, and five liters for physicochemical analysis. Water samples were filtered under vacuum with a kit containing a cellulose acetate membrane filter, 1.2 µm (Millipore(r), Barueri, SP, Brazil). The material retained on the membrane was mechanically extracted and analyzed by direct immunofluorescence (Merifluor(r)kit). From 20 rural properties investigated, 10 had artesian wells (40 samples), 10 had common wells (40 samples), and one had a mine (four samples), the latter contaminated by Cryptosporidiumspp. In samples from artesian wells, 90 to 130 meters depth, 42.5% were positive for total coliforms and 5.0% were identified to have abnormal coloration. From the samples of common wells, 14 to 37 meters depth, 87.5% were contaminated with total coliforms, 82.5% were positive for thermotolerant coliforms, and 12.5% had color abnormalities. We did not detect the presence of Giardiaspp. or Cryptosporidiumspp. in artesian and common wells. The use of artesian or common wells is an important step in the control of the spreading of zoonoses, particularly Cryptosporidiumspp. and Giardiaspp., as well as artesian wells for coliform control in local production of vegetables to be marketed.

IMMUNODIAGNOSIS OF HUMAN STRONGYLOIDIASIS: USE OF SIX DIFFERENT ANTIGENIC FRACTIONS FROM Strongyloides venezuelensis PARASITIC FEMALES

SUMMARY The aim of this study was to evaluate six different antigenic fractions from Strongyloides venezuelensis parasitic females for the immunodiagnosis of human strongyloidiasis. Soluble and membrane fractions from S. venezuelensis parasitic females were prepared in phosphate-buffered saline (SSF and SMF, respectively), Tris-HCl (TSF and TMF, respectively), and an alkaline buffer (ASF and AMF, respectively). Serum samples obtained from patients with strongyloidiasis or, other parasitic diseases, and healthy individuals were analyzed by enzyme-linked immunosorbent assay (ELISA). Soluble fractions SSF, TSF, and ASF showed 85.0%, 75.0%, and 80.0% sensitivity and 93.1%, 93.1%, and 87.5% specificity, respectively. Membrane fractions SMF, TMF, and AMF showed 80.0%, 75.0%, and 85.0% sensitivity, and 95.8%, 90.3%, and 91.7% specificity, respectively. In conclusion, the present results suggest that the fractions obtained from parasitic females, especially the SSF and SMF, could be used as alternative antigen sources in the serodiagnosis of human strongyloidiasis.

ANTHELMINTIC ACTIVITY OF CHLOROPHYLLIN AGAINST DIFFERENT LARVAL STAGES OF Fasciola gigantica

Fasciolosis is a food borne zoonosis, caused by the digenetic trematode Fasciola. Freshwater lymnaeid snails are the intermediate host of the trematodes. Chlorophyllin, a semi-synthetic derivative of chlorophyll and its formulations obtained from freeze dried cow urine (FCU) had their toxicity tested against redia and cercaria larvae of F. gigantica. The larvicidal activity of chlorophyllin and its formulations were found to depend on both, time and concentration used against the larvae. Toxicity of chlorophyllin + FCU (1:1 ratio) in sunlight against redia larva (8 h LC50: 0.03 mg/mL) was more pronounced than using just chlorophyllin (8 h LC50: 0.06 mg/mL). Toxicity of chlorophyllin + FCU in sunlight against redia (8 h LC50: 0.03 mg/mL) was higher than against cercaria (8 h LC50: 0.06 mg/mL). The larvicidal activity of chlorophyllin in sunlight (redia/cercaria larvae: 8 h LC50: 0.06 mg/mL) was more pronounced than under laboratory conditions (redia: 8 h LC50: 22.21 mg/mL/, cercaria 8 h LC50: 96.21 mg/mL). Toxicity of FCU against both larvae was lower than that of chlorophyllin and chlorophyllin + FCU. Chlorophyllin and its formulations + FCU were 357.4 to 1603.5 times more effective against redia/cercaria larvae in sunlight than under laboratory conditions. The present study has shown that chlorophyllin formulations may be used as potent larvicides against fasciolosis.

SURVIVAL CAPACITY OF Arcobacter butzleri INOCULATED IN POULTRY MEAT AT TWO DIFFERENT REFRIGERATION TEMPERATURES

Arcobacter spp. are emerging enteropathogens and potential zoonotic agents that can be transmitted by food and water, being considered a public health risk. The high isolation rate of these bacteria from poultry products suggests that it may be a major source of human infections. One hallmark for differentiating the genus Arcobacter fromCampylobacter includes their growing capacity at low temperatures (15-30 °C) under aerobic conditions. However, little is known about the population density variation of these bacteria at different refrigeration temperatures. The aim of this study was to determine the survival behavior of two different Arcobacter butzleri concentrations (104 CFU/mL and 107 CFU/mL) inoculated on chicken legs and held at two different refrigeration temperatures (4 and 10 °C) throughout storage time. Results have shown that A. butzleri had growing capacity both at 4 and 10 °C. No statistical difference between the survival trends was found for both bacterial concentrations and temperatures tested. This study shows that A. butzleri is a robust species with regard to storage temperature, and represents a potential health risk for poultry meat consumers.

Comparison of different techniques for mouse transgenesis

RESUMO: O uso de ratinhos transgénicos em neurociências aumentou consideravelmente nos últimos anos devido ao crescente interesse em compreender o cérebro e a necessidade de solucionar situações clínicas do foro neurológico e psiquiátrico. Para esse efeito, diferentes métodos de produção de animais transgénicos têm sido testados. O objectivo desta tese foi comparar métodos de integração aleatória de um transgene no genoma de ratinhos em termos de eficiência, estabilidade da integração do transgene, número de animais e de horas de trabalho necessárias para cada método. Assim, foi comparado o método mais utilizado - microinjecção pronuclear (PNMI) - com duas outras técnicas cujo desempenho foi considerado promissor – a transferência génica através dos testículos por electroporação e transfecção por lentivírus in vivo. As três técnicas foram realizadas usando um gene repórter sob o controlo de um promotor constitutivo, e depois reproduzidas usando um gene de interesse de modo a permitir obtenção de um animal capaz de ser usado em experimentação laboratorial. O transgene de interesse utilizado codifica uma proteína de fusão correspondendo a uma variante da rodopsina (channelrhodopsin) fundida à proteína enhanced yellow fluorescente protein ((EYFP) resultando num produto designado ChR2-EYFP. Este animal transgénico apresentaria expressão deste canal iónico apenas em células dopaminergicas, o que, com manipulação optogenética, tornaria possivel a activação especifica deste grupo de neurónios e, simultaneamente, a observação do impacto desta manipulação no comportamento num animal em livre movimento. Estas ferramentas são importantes na investigação básica em neurociências pois ajudam a esclarecer o papel de grupos específicos de neurónios e compreender doenças como a doença de Parkinson ou a esquizofrenia onde a função de certos tipos de neurónios de encontra alterada. Quando comparados os três métodos realizados verifica-se que usando um gene repórter PMNI resulta em 31,3% de, a de animais transgénicos obtidos, a electroporação de testículos em 0% e a injecção de lentivírus em 0%. Quando usado o gene de interesse, os resultados obtidos são, respectivamente, 18,8%, 63,9% e 0%.--------------ABSTRACT: The use of transgenic mice is increasing in all fields of research, particularly in neuroscience, due to the widespread need of animal models to solve neurological and psychiatric medical conditions. Different methodologies have been tested in the last decades in order to produce such transgenic animals. The ultimate goal of this thesis is to compare different methods of random integration of a transgene in the genome of mice in terms of efficiency, stability of the transgene integration, number of animals required and the labour intensity of each technique. We compared the most used method – pronuclear microinjection (PNMI) – with two other promising techniques – Testis Mediated Gene Transfer (TMGT) by electroporation and in vivo lentiviral transfection. The three techniques were performed using a reporter gene – green fluorescent protein (GFP), whose transcription was driven by the constitutive cytomegalovirus (CMV) promoter. These three techniques were later reproduced using the tyrosine hydroxylase promoter (TH) and the neuronal manipulator, channelrhodopsin-2 fused to the enhanced yellow fluorescent reporter protein (ChR2-EYFP). The transgenic animal we sough to produce would express the light driven channel only in dopaminergic cells, making possible to specifically activate this group of neurons, while simultaneously observe the behaviour in a freely moving animal. This is a very important tool in basic neuroscience research since it helps to clarify the role of specific groups of neurons, map circuits in the brain, and consequently understand neurological diseases such as Parkinson’s disease or schizophrenia, where the function of certain types of neurons is affected. When comparing the three methods, it was verified that using a reporter gene PNMI resulted in 31.3% of transgenic mice obtained, testis electroporation in 0% and lentiviral injection in 0%. When using the gene of interest, the results obtained were, respectively, 18.8%, 63.9% and 0%.

The avirulence of the cultivated PF strain of Trypanosoma cruzi: V - the evaluation of parasitologic tests after vaccination of different animal species

After vaccination with the live PF strain of Trypanosoma cruzi, 194 blood cultures were performed in 143 mice, 9 dogs, 5 Cebus monkeys and 7 human subjects. Some of these blood cultures were simultaneously done with xenodiagnosis, subinoculation in baby mice and/or culture of viscerae. The trypanosomes isolated from the few positive cases (6,1%) were incapable of infecting baby mice were considered as cases of immunotolerance. All the other tests were negative.