The decline of the notion of soul and the rise of the concept of self

This thesis examines the ethics of genetic engineering from the frame of reference of the human individual.

Microbial biosynthesis of biopolymers and applications in the biopharmaceutical, biomedical and food industries

Biopolymers can be produced through a variety of mechanisms. They can be derived from microbial systems, extracted from higher organisms such as plants, or synthesized chemically from basic biological building blocks. A wide range of emerging applications rely on all three of these production techniques. In recent years, considerable attention has been given to biopolymers produced by microbes. It is on the microbial level where the tools of genetic engineering can be most readily applied. A number of novel materials are now being developed or introduced into the market. Biopolymers are being developed for use as medical materials, packaging, cosmetics, food additives, clothing fabrics, water treatment chemicals, industrial plastics, absorbents, biosensors, and even data storage elements. This review identifies the possible commercial applications and describes the various methods of production of microbial biopolymers.

Production and characterization of hypoallergenic chicken eggs

This project aimed to develop novel therapeutics and methods to tackle egg allergy. In this study, recombinant variants of egg allergens were produced that may be used in future immunotherapy. Genetic engineering was also used to develop new strategies to produce non-allergenic chicken eggs.

Functionalization of dendrimers for improved gene delivery to mesenchymal stem cell

Disease, injury, and age problems compromise human quality of life and continuously motivate the search for new and more efficacious therapeutic approaches. The field of Tissue Regeneration and Engineering has greatly evolved over the last years, mainly due to the combination of the important advances verified in Biomaterials Science and Engineering with those of Cell and Molecular Biology. In particular, a new and promising area arose – Nanomedicine – that takes advantage of the extremely small size and especial chemical and physical properties of Nanomaterials, offering powerful tools for health improvement. Research on Stem Cells, the self-renewing progenitors of body tissues, is also challenging to the medical and scientific communities, being expectable the appearance of new and exciting stem cell-based therapies in the next years. The control of cell behavior (namely, of cell proliferation and differentiation) is of key importance in devising strategies for Tissue Regeneration and Engineering. Cytokines, growth factors, transcription factors and other signaling molecules, most of them proteins, have been identified and found to regulate and support tissue development and regeneration. However, the application of these molecules in long-term regenerative processes requires their continuous presence at high concentrations as they usually present short half-lives at physiological conditions and may be rapidly cleared from the body. Alternatively, genes encoding such proteins can be introduced inside cells and be expressed using cell’s machinery, allowing an extended and more sustained production of the protein of interest (gene therapy). Genetic engineering of stem cells is particularly attractive because of their self-renewal capability and differentiation potential. For Tissue Regeneration and Engineering purposes, the patient’s own stem cells can be genetically engineered in vitro and, after, introduced in the body (with or without a scaffold) where they will not only modulate the behavior of native cells (stem cell-mediated gene therapy), but also directly participate in tissue repair. Cells can be genetically engineered using viral and non-viral systems. Viruses, as a result of millions of years of evolution, are very effective for the delivery of genes in several types of cells, including cells from primary sources. However, the risks associated with their use (like infection and immunogenic reactions) are driving the search for non-viral systems that will efficiently deliver genetic material into cells. Among them, chemical methods that are promising and being investigated use cationic molecules as carriers for DNA. In this case, gene delivery and gene expression level remain relatively low when primary cells are used. The main goal of this thesis was to develop and assess the in vitro potential of polyamidoamine (PAMAM) dendrimers based carriers to deliver genes to mesenchymal stem cells (MSCs). PAMAM dendrimers are monodispersive, hyperbranched and nanospherical molecules presenting unique characteristics that make them very attractive vehicles for both drug and gene delivery. Although they have been explored for gene delivery in a wide range of cell lines, the interaction and the usefulness of these molecules in the delivery of genes to MSCs remains a field to be explored. Adult MSCs were chosen for the studies due to their potential biomedical applications (they are considered multipotent cells) and because they present several advantages over embryonic stem cells, such as easy accessibility and the inexistence of ethical restrictions to their use. This thesis is divided in 5 interconnected chapters. Chapter I provides an overview of the current literature concerning the various non-viral systems investigated for gene delivery in MSCs. Attention is devoted to physical methods, as well as to chemical methods that make use of polymers (natural and synthetic), liposomes, and inorganic nanoparticles as gene delivery vectors. Also, it summarizes the current applications of genetically engineered mesenchymal stem cells using non-viral systems in regenerative medicine, with special focus on bone tissue regeneration. In Chapter II, the potential of native PAMAM dendrimers with amine termini to transfect MSCs is evaluated. The level of transfection achieved with the dendrimers is, in a first step, studied using a plasmid DNA (pDNA) encoding for the β-galactosidase reporter gene. The effect of dendrimer’s generation, cell passage number, and N:P ratio (where N= number of primary amines in the dendrimer; P= number of phosphate groups in the pDNA backbone) on the level of transfection is evaluated, being the values always very low. In a second step, a pDNA encoding for bone morphogenetic protein-2, a protein that is known for its role in MSCs proliferation and differentiation, is used. The BMP-2 content produced by transfected cells is evaluated by an ELISA assay and its effect on the osteogenic markers is analyzed through several classical assays including alkaline phosphatase activity (an early marker of osteogenesis), osteocalcin production, calcium deposition and mineralized nodules formation (late osteogenesis markers). Results show that a low transfection level is enough to induce in vitro osteogenic differentiation in MSCs. Next, from Chapter III to Chapter V, studies are shown where several strategies are adopted to change the interaction of PAMAM dendrimers with MSCs cell membrane and, as a consequence, to enhance the levels of gene delivery. In Chapter III, generations 5 and 6 of PAMAM dendrimers are surface functionalized with arginine-glycine-aspartic acid (RGD) containing peptides – experiments with dendrimers conjugated to 4, 8 and 16 RGD units were performed. The underlying concept is that by including the RGD integrin-binding motif in the design of the vectors and by forming RGD clusters, the level of transfection will increase as MSCs highly express integrins at their surface. Results show that cellular uptake of functionalized dendrimers and gene expression is enhanced in comparison with the native dendrimers. Furthermore, gene expression is dependent on both the electrostatic interaction established between the dendrimer moiety and the cell surface and the nanocluster RGD density. In Chapter IV, a new family of gene delivery vectors is synthesized consisting of a PAMAM dendrimer (generation 5) core randomly linked at the periphery to alkyl hydrophobic chains that vary in length and number. Herein, the idea is to take advantage of both the cationic nature of the dendrimer and the capacity of lipids to interact with biological membranes. These new vectors show a remarkable capacity for internalizing pDNA, being this effect positively correlated with the –CH2– content present in the hydrophobic corona. Gene expression is also greatly enhanced using the new vectors but, in this case, the higher efficiency is shown by the vectors containing the smallest hydrophobic chains. Finally, chapter V reports the synthesis, characterization and evaluation of novel gene delivery vectors based on PAMAM dendrimers (generation 5) conjugated to peptides with high affinity for MSCs membrane binding - for comparison, experiments are also done with a peptide with low affinity binding properties. These systems present low cytotoxicity and transfection efficiencies superior to those of native dendrimers and partially degraded dendrimers (Superfect®, a commercial product). Furthermore, with this biomimetic approach, the process of gene delivery is shown to be cell surface receptor-mediated. Overall, results show the potential of PAMAM dendrimers to be used, as such or modified, in Tissue Regeneration and Engineering. To our knowledge, this is the first time that PAMAM dendrimers are studied as gene delivery vehicles in this context and using, as target, a cell type with clinical relevancy. It is shown that the cationic nature of PAMAM dendrimers with amine termini can be synergistically combined with surface engineering approaches, which will ultimately result in suitable interactions with the cytoplasmic membrane and enhanced pDNA cellular entry and gene expression. Nevertheless, the quantity of pDNA detected inside cell nucleus is always very small when compared with the bigger amount reaching cytoplasm (accumulation of pDNA is evident in the perinuclear region), suggesting that the main barrier to transfection is the nuclear membrane. Future work can then be envisaged based on the versatility of these systems as biomedical molecular materials, such as the conjugation of PAMAM dendrimers to molecules able to bind nuclear membrane receptors and to promote nuclear translocation.

Estudo do Cultivo de dois clones de Escherichia coli recombinantes (eIF, LACK) para a Expressão de Antígenos da Leishmania chagasi

With advent of the technology of the recombinant DNA, the recombinant protein expression becomes an important tool in the studies of the structure, function and identification of new proteins, mainly with therapeutical purposes. The Escherichia coli has been procarioto predominant in the studies of genetic engineering due to wealth of information regarding its metabolism. Despite the expressivo advance of the studies of molecular biology and the immunology of the infections, it does not exist, currently, no prophylactic drug capable to prevent calazar. Of this form, it exists a great necessity of specific antigen identification for the vaccine development and kits for disgnostic against the visceral Leishmaniose. In this context, this work objectified to study the recombinant antigen expression of the Leishmania chagasi during the culture of Escherichia coli in shaker. A first set of assays was carried through with the objective of if knowing the kinetic behavior of the growth of two clones recombinant proteins (eIF, LACK) in two different compositions of culture medium (2xTY, TB) supplemented by antibiotics, without IPTG addition. In the second stage of the assays, the procedure of induction for IPTG was carried through, in order to verify the influence of the composition of the ways tested in the expression them recombinant proteins. On the basis of the gotten results, can be observed that the high complexity of culture medium favored the kinetic one of growth of clones recombinant (eIF, LACK), however, to if to deal with the assays submitted to the procedure of induction for IPTG, the raised complexity of culture medium did not favor the expression of recombinant proteins. On the other hand, they had been gotten resulted positive for all clones recombinant (eIF, LACK) tested, confirmed through the eletroforético profile

Análise de fluxos metabólicos para otimização da síntese do antibiótico cosmomicina por Streptomyces olindensis ICB20

Metabolic flux analysis (MFA) is a powerful tool for analyzing cellular metabolism. In order to control the growth conditions of a specific organism, it is important to have a complete understanding of its MFA. This would allowed us to improve the processes for obtaining products of interest to human and also to understand how to manipulate the genome of a cell, allowing optimization process for genetic engineering. Streptomyces olindensis ICB20 is a promising producer of the antibiotic cosmomycin, a powerful antitumor drug. Several Brazilian researchers groups have been developing studies in order to optimize cosmomycin production in bioreactors. However, to the best of our knowledge, nothing has been done on metabolic fluxes analysis field. Therefore, the aim of this work is to identify several factors that can affect the metabolism of Streptomyces olindensis ICB20, through the metabolic flux analysis. As a result, the production of the secondary metabolite, cosmomycin, can be increased. To achieve this goal, a metabolic model was developed which simulates a distribution of internal cellular fluxes based on the knowledge of metabolic pathways, its interconnections, as well as the constraints of microorganism under study. The validity of the proposed model was verified by comparing the computational data obtained by the model with the experimental data obtained from the literature. Based on the analysis of intracellular fluxes, obtained by the model, an optimal culture medium was proposed. In addition, some key points of the metabolism of Streptomyces olindensis were identified, aiming to direct its metabolism to a greater cosmomycin production. In this sense it was found that by increasing the concentration of yeast extract, the culture medium could be optimized. Furthermore, the inhibition of the biosynthesis of fatty acids was found to be a interesting strategy for genetic manipulation. Based on the metabolic model, one of the optimized medium conditions was experimentally tested in order to demonstrate in vitro what was obtained in silico. It was found that by increasing the concentration of yeast extract in the culture medium would induce to an increase of the cosmomycin production

Biotechnology and fish culture

Biotechnology can currently be considered of importance in aquaculture. The increase in the production of aquatic organisms over the last two decades through the use of biotechnology indicates that in a few generations biotechnology may overtake conventional techniques, at least for the commercially more valuable species. In the last few years, genetics has contributed greatly to fish culture through the application of the more recent techniques developed in biotechnology and in genetic engineering. At present, the most commonly used methods in fish biotechnology are chromosome manipulation and hormonal treatments, which can be used to produce triploid, tetraploid, haploid, gynogenetic and androgenetic fish. These result in the production of individuals and lineages of sterile, monosex or highly endogamic fish. The use of such strategies in fish culture has as a practical objective the control of precocious sexual maturation in certain species; other uses are the production of larger specimens by control of the reproductive process and the attainment of monosex lines containing only those individuals of greater commercial value. The use of new technologies, such as those involved in gene transfer in many species, can result in modified individuals of great interest to aquaculturists and play important roles in specific programmes of fish production in the near future.

Raciocínio moral na tomada de decisões em relação a questões sociocientíficas: o exemplo do melhoramento genético humano

Nos últimos anos, os avanços nas Ciências Biológicas têm levado a sociedade a discutir diversas questões no campo da moral e da ética. Questões como engenharia genética, clonagem e pesquisas com células-tronco são questões chamadas de sociocientíficas por estarem na interface entre a ciência e a sociedade. Nesse trabalho buscamos entender como estudantes de Ensino Médio percebem e interpretam questões relacionadas à manipulação genética em seres humanos. Houve divisão de opiniões em relação à eugenia negativa, que se destina a remover características desfavoráveis das pessoas; mas a eugenia positiva, que busca melhoramento de características estéticas, foi rejeitada por todos os estudantes. As variações nas opiniões em relação ao assunto tratado podem ser, em grande medida, devidas às representações sociais dos estudantes.

Determinação de parâmetros genéticos em população de gonçalo-alves (Astronium fraxinifolium) através das características fisiológicas da semente

Seeds of Astronium fraxinifolium (Anarcadiaceae) were collected in two years, and submitted to tests of seed germination and vigour (first count, aging and electric conductivity), in order to verify the potencial of conservation and if variations in the seed quality presented on association with genetic parameters. Coefficient of heritability in average (ĥm 2) were 0.91 and 0.97 to germination; 0.90 and 0.96 to first count; 0.93 and 0.97 to aging and 0.95 and 0.99 to electric conductivity, for seeds collected in 1997 and 1998, respectively, evidencing a great heritability to all studied characteristics. Genetic variability was influenced more by environmental conditions of blooming and harvest of seeds than storage time.

Seleção simultânea de caracteres em progênies de Pinus caribaea Morelet var. bahamensis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Parâmetros genéticos para caracteres de crescimento, de produção e tecnológicos da madeira em clones de Eucalyptus spp

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Variabilidade genética intrapopulacional em Myracrodruon urundeuva Fr. All. por marcador AFLP

Levels of genetic variability for in situ and ex situ genetic conservation were estimated in a population of Myracrodruon urundeuva using the PCR (polymerase chain reaction) technique with the AFLP (Amplified fragment-length polymorphism) genetic marker. Seeds for progeny tests were collected from 30 open-pollination trees (matrices) at Paulo de Faria Ecological Station - SP. From this genetic material, three progeny tests were installed on the Teaching and Research Farm of Ilha Solteira Faculty of Engineering - University of São Paulo State (UNESP), which is located in Selvlria - MS, Brazil. The analysis by genetic marker was conducted with three combinations of different starters EcoRl-Msel, resulting in a total number of 137 polymorphic bands, thus forming a table of binary data. These data were used for the analysis of genetic divergence and distance between progenies. High levels of genetic divergence were observed among families. Based on the Analysis of Molecular Variance (AMOVA), it was shown that 16.2% of genetic diversity is found among progenies and 83.8% within progenies, which suggests deviances of random matings. The grouping of progenies, based on genetic distances, suggests that progenies deriving from trees which are close to each other tend to be more similar. This, in turn, indicates that the population originating the seeds may be genetically structured.

Efeito do desbaste seletivo nas estimativas de parâmetros genéticos em progénies de Pinus caribaea Morelet var. hondurensis

The objective of this work was to study the effect of selective thinning on the estimates of genetic parameters in progenies of Pinus caribaea var. hondurensis. The progeny test was installed in June 1986, following a 10 × 10, triple square lattice design, containing ten trees in linear plots in 3.0 × 3.0m spacing. Twelve years after the planting, a selective thinning based on Multi-effect index (selection for DBH) was performed, leaving six trees per plot. The assessments were done in four situations: A (before thinning); B (among thinned trees); C (among remnant trees at 13 years of age) and D (two years after the thinning). The following traits were analyzed: total height (H), diameter at breast height (DBH), volume (VOL), stem form (FOR), foxtail (FT), wood density at 1.3 m (WD1) and wood density at the half height (WD2). The thinning led to increase in the estimates of variability among progenies for VOL and decrease for ALT and DBH. There was a high and positive genetic correlation among the growth traits, mainly between DBH and VOL. The latter remained unchanged after thinning. The narrow-sense heritabilities at the individual level, showed a slight increase while at the family mean level had a reduction with the thinning. The estimates of breeding values increased with the thinning. This may lead to an easier identification of the best progenies in further selection.

Estratégias de seleção dentro de progênies em duas populações de myracrodruon urundeuva Fr. All.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Variação genética, interação genótipo solo e ganhos na seleção em teste de progěnies de Corymbia citriodora Hook em Luiz Antonio, São Paulo

The aim of this study was to investigate the genetic variation, the genotype × soil interaction and the selection among and within Corymbia citriodora progenies in three different kinds of soils (Red Latossol, Quartz Sand and Purple Latossol), which occur in the Luiz Antônio Experimental Station, São Paulo State, Brazil. The progeny test was established 1983, using 56 open-pollinated families of C. citriodora. Twenty fve years after planting the following traits were measured: height, diameter at breast height (Dbh), stem form and survival. Best growth occurred in Purple Latossol. Significant differences among progenies were detected for most traits in all sites, suggesting the possibility of improvement by selection. In the analysis with sites, Significant differences among locals, progenies and genotypes × soil interaction were detected, confiming that the tested material has sufficient genetic variation to be explored by selection in all sites. The analysis of genotype × soil interaction indicates that growth traits present single interaction and the same progenies can be selected in each site. On the order hand, genotype × soil interaction for stem form and survival was complex; and specifc progenies need to be selected for each kind of soil. The population, due to its high genetic variation and strong genetic control of traits, permits to obtain considerable genetic gains by selection among and within progenies.