Pluripotency and genetic stability of human pluripotent stem cells

Pluripotent cells have the potential to differentiate into all somatic cell types. As the adult human body is unable to regenerate various tissues, pluripotent cells provide an attractive source for regenerative medicine. Human embryonic stem cells (hESCs) can be isolated from blastocyst stage embryos and cultured in the laboratory environment. However, their use in regenerative medicine is restricted due to problems with immunosuppression by the host and ethical legislation. Recently, a new source of pluripotent cells was established via the direct reprogramming of somatic cells. These human induced pluripotent stem cells (hiPSCs) enable the production of patient specific cell types. However, numerous challenges, such as efficient reprogramming, optimal culture, directed differentiation, genetic stability and tumor risk need to be solved before the launch of therapeutic applications. The main objective of this thesis was to understand the unique properties of human pluripotent stem cells. The specific aims were to identify novel factors involved in maintaining pluripotency, characterize the effects of low oxygen culture on hESCs, and determine the high resolution changes in hESCs and hiPSCs during culture and reprogramming. As a result, the previously uncharacterized protein L1TD1 was determined to be specific for pluripotent cells and essential for the maintenance of pluripotency. The low oxygen culture supported undifferentiated growth and affected expression of stem cell associated transcripts. High resolution screening of hESCs identified a number of culture induced copy number variations and loss of heterozygosity changes. Further, screening of hiPSCs revealed that reprogramming induces high resolution alterations. The results obtained in this thesis have important implications for stem cell and cancer biology and the therapeutic potential of pluripotent cells.

Insights on bovine genetic engineering and cloning

Transgenic technology has become an essential tool for the development of animal biotechnologies, and animal cloning through somatic cell nuclear transfer (SCNT) enabled the generation of genetically modified animals utilizing previously modified and selected cell lineages as nuclei donors, assuring therefore the generation of homogeneous herds expressing the desired modification. The present study aimed to discuss the use of SCNT as an important methodology for the production of transgenic herds, and also some recent insights on genetic modification of nuclei donors and possible effects of gene induction of pluripotency on SCNT.

Thermal radiation in combustion systems

A numerical procedure for solving the nongray radiative transfer equation (RTE) in two-dimensional cylindrical participating media is presented. Nongray effects are treated by using a narrow-band approach. Radiative emission from CO, CO2, H2O, CH4 and soot is considered. The solution procedure is applied to study radiative heat transfer in a premixed CH4-O2, laminar, flame. Temperature, soot and IR-active species molar fraction distributions are allowed to vary in the axial direction of the flame. From the obtained results it is possible to quantify the radiative loss in the flame, as well as the importance of soot radiation as compared to gaseous radiation. Since the solution procedure is developed for a two-dimensional cylindrical geometry, it can be applied to other combustion systems such as furnaces, internal combustion engines, liquid and solid propellant combustion.

Methods of genetic diversity creation and functional display for directed evolution experiments

Protein engineering aims to improve the properties of enzymes and affinity reagents by genetic changes. Typical engineered properties are affinity, specificity, stability, expression, and solubility. Because proteins are complex biomolecules, the effects of specific genetic changes are seldom predictable. Consequently, a popular strategy in protein engineering is to create a library of genetic variants of the target molecule, and render the population in a selection process to sort the variants by the desired property. This technique, called directed evolution, is a central tool for trimming protein-based products used in a wide range of applications from laundry detergents to anti-cancer drugs. New methods are continuously needed to generate larger gene repertoires and compatible selection platforms to shorten the development timeline for new biochemicals. In the first study of this thesis, primer extension mutagenesis was revisited to establish higher quality gene variant libraries in Escherichia coli cells. In the second study, recombination was explored as a method to expand the number of screenable enzyme variants. A selection platform was developed to improve antigen binding fragment (Fab) display on filamentous phages in the third article and, in the fourth study, novel design concepts were tested by two differentially randomized recombinant antibody libraries. Finally, in the last study, the performance of the same antibody repertoire was compared in phage display selections as a genetic fusion to different phage capsid proteins and in different antibody formats, Fab vs. single chain variable fragment (ScFv), in order to find out the most suitable display platform for the library at hand. As a result of the studies, a novel gene library construction method, termed selective rolling circle amplification (sRCA), was developed. The method increases mutagenesis frequency close to 100% in the final library and the number of transformants over 100-fold compared to traditional primer extension mutagenesis. In the second study, Cre/loxP recombination was found to be an appropriate tool to resolve the DNA concatemer resulting from error-prone RCA (epRCA) mutagenesis into monomeric circular DNA units for higher efficiency transformation into E. coli. Library selections against antigens of various size in the fourth study demonstrated that diversity placed closer to the antigen binding site of antibodies supports generation of antibodies against haptens and peptides, whereas diversity at more peripheral locations is better suited for targeting proteins. The conclusion from a comparison of the display formats was that truncated capsid protein three (p3Δ) of filamentous phage was superior to the full-length p3 and protein nine (p9) in obtaining a high number of uniquely specific clones. Especially for digoxigenin, a difficult hapten target, the antibody repertoire as ScFv-p3Δ provided the clones with the highest affinity for binding. This thesis on the construction, design, and selection of gene variant libraries contributes to the practical know-how in directed evolution and contains useful information for scientists in the field to support their undertakings.

The effects of maternal hyperglycemia on human umbilical vascular gene expression and neonatal rat lung development

Background: Maternal diabetes affects many fetal organ systems, including the vasculature and the lungs. The offspring of diabetic mothers have respiratory adaptation problems after birth. The mechanisms are multifactorial and the effects are prolonged during the postnatal period. An increasing incidence of diabetic pregnancies accentuates the importance of identifying the pathological mechanisms, which cause the metabolic and genetic changes that occur in offspring, born to diabetic mothers. Aims and methods: The aim of this thesis was to determine changes both in human umbilical cord exposed to maternal type 1 diabetes and in neonatal rat lungs after streptozotocin-induced maternal hyperglycemia, during pregnancy. Rat lungs were used as a model for the potential disease mechanisms. Gene expression alterations were determined in human umbilical cords at birth and in rat pup lungs at two week of age. During the first two postnatal weeks, rat lung development was studied morphologically and histologically. Further, the effect of postnatal hyperoxia on hyperglycemia-primed rat lungs was investigated at one week of age to mimic the clinical situation of supplemental oxygen treatment. Results: In the umbilical cord, maternal diabetes had a major negative effect on the expression of genes involved in blood vessel development. The genes regulating vascular tone were also affected. In neonatal rat lungs, intrauterine hyperglycemia had a prolonged effect on gene expression during late alveolarization. The most affected pathway was the upregulation of extracellular matrix proteins. Newborn rat lungs exposed to intrauterine hyperglycemia had thinner saccular walls without changes in airspace size, a smaller relative lung weight and lung total tissue area, and increased cellular apoptosis and proliferation compared to control lungs, possibly reflecting an aberrant maturational adaptation. At one and two weeks of age, cell proliferation and secondary crest formation were accelerated in hyperglycemia-exposed lungs. Postnatal hyperoxic exposure, alone caused arrested alveolarization with thin-walled and enlarged alveoli. In contrast, the dual exposure of intrauterine hyperglycemia and postnatal hyperoxia resulted in the phenotype of thick septa together with arrested alveolarization and decreased number of small pulmonary arteries. Conclusions: Maternal diabetic environment seems to alter the umbilical cord gene expression profile of the regulation of vascular development and function. Fetal hyperglycemia may additionally affect the genetic regulation of the postnatal lung development and may actually induce prolonged structural alterations in neonatal lungs together with a modifying effect on the deleterious pulmonary exposure of postnatal hyperoxia. This, combined with the novel human umbilical cord gene data could serve as stepping stones for future therapies to curb developmental aberrations.

Effects of irrigated rice sowing season and imazapyr + imazapic time of application on rice grain yield and red rice management

Irrigated rice sowing season and red rice competition are among the main factors affecting grain yield. The objective of this work was to evaluate the sowing date of irrigated rice and moments of application of the herbicide imazapyr + imazapic to control red rice management and irrigated rice grain yield. Eight experiments were performed at the following dates (09/30, 10/19, 11/08 and 12/01) for the 2010/2011 harvest season and (09/27, 10/17, 11/08 and 12/05) for the 2011/2012 harvest season. The treatments were: application of the herbicide imazapyr + imazapic at doses of 105+35 g ha-1 in pre-emergence (PRE); 52.5+17.5 g ha‑1 in pre-emergence and 52.5+17.5 g ha-1 in post-emergence (PRE + POST); and 105+35 g ha-1 in post- emergence (POST), and a control without application and no weeding. The cultivar Puitá Inta CL was used and a randomized block design with four replicates. A joint analysis of the experiments was carried out. There was less emergence of red rice and higher grain yield of the irrigated rice at the early periods (09/30/10 and 09/27/11), with 10,578 and 8,653 kg ha-1, respectively. At the end of the season (12/01/10 and 12/05/11), there was greater reduction of the red rice seed bank. Sowing at the beginning of the recommended period provided more irrigated rice grain yield. The application of imazapyr + imazapic at a dose of 52.5+17.5 g ha-1 in PRE + 52.5+17.5 g ha-1 POST, and 105+35 g ha-1 only in PRE and POST was effective in the control of red rice.

Effect of light and temperature on germination and early growth of Vochysia tucanorum Mart., Vochysiaceae, in cerrado and forest soil under different radiation levels

Vochysia tucanorum Mart. (Vochysiaceae) is a very widely distributed species in the cerrado and forests in Brazil, from the State of Bahia to Paraná. Its seed germination was analysed under eight treatments using different conditions of light and temperature. Up to 73 days, at least ca. 93% of the seeds germinated in all treatments, except under alternating temperatures of 35°/10°C (62%). Light enhanced germination, except under constant temperature of 25°C when seeds are not photoblastic. At this temperature, the highest percentage of germination was found. Initial vegetative growth was analysed by comparing the effects of shading, by means of different percentages of full daylight (100%, 45%, 10.6%, 4.8%), and soils from cerrado and forest. During 123 days, no mortality was recorded and cotyledons persisted on the seedlings of all treatments. In general, the seedlings of V. tucanorum were found to be more or less indifferent to the soil from cerrado or forest and shading seems to promote height increment. However, total dry mass was larger under a radiation of 45% full daylight and smaller under extreme conditions (100% or 4.8% full daylight).

Effects of light stress on the growth of the epiphytic orchid Cattleya forbesii Lindl. X Laelia tenebrosa Rolfe

Considering the performance of CAM epiphytes under high levels of radiation or in shaded environments, with growth rate proportional to light intensity, the objective of this work was to evaluate the effects of long-term light stress on the growth of a Brazilian epiphytic orchid, Cattleya forbesii Lindl. X Laelia tenebrosa Rolfe. Two groups of plants were used in the first experiment, one under 90% (@ 1,650 µmol.m-2.s-1) of Photosynthetically Active Radiation (PAR) and the other maintained under 22.5% (@ 400 µmol.m-2.s-1). In the second experiment the diffusive resistance, transpiration rate and fluorescence levels were monitored for plants that were under 22.5% of PAR, under 90% and plants transferred from 22.5 to 90%. Our results show that light intensity interfered with growth and development of this orchid. Data on the changes in pseudobulb volume throughout the time course of growth suggest that water and reserves stored in the back shoots are translocated to the current shoot. Regarding stomatal resistance, plants under 22.5% of PAR reached a largest stomatal aperture during the night, whereas those under 90% only after dawn. After transfer from 22.5% PAR to 90% PAR the ratio of Fv/Fm decreased from approximately 0.8 to 0.7. This suggests the limitation of photoprotection mechanisms in the leaf and the results observed after the transfer of plants from 22.5% to 90% reinforce the possibility that a photoinhibition is reflected in a decrease in growth rate.

The post-fire effects on the outcrossing rate of a Brazilian savannah shrub, Helicteres sacarolha A.St.-Hil

The effect of post-fire, plant density and number of flowers per plant on outcrossing rates was examined in a Brazilian savannah shrub, Helicteres sacarolha. Data on number of flowering plants and flowers per plant were collected during the flowering season in January and February of 1994. In October of 1994, a fire swept through the studied area after 30 years of fire absence. The burnt plants of H. sacarolha resprouted, producing flowers and fruits in 1995. Seeds from several plants in both years were collected. Allozyme markers were used to estimate the multilocus outcrossing rates for 1994 and 1995 progenies. After the fire, outcrossing rates increased. In 1995, plants flowered vigorously, increasing flower density and probably pollinator activity. Helicteres sacarolha seems to be fire resistant, like other plant species of the Brazilian savannah, but several plants tagged in 1994 were not found after the fire, and may have died. Thus, although genetic diversity and outcrossing rates increased following the fire in 1994, repeated events of fire may decrease drastically the population size of H. sacarolha, leading to a diminished genetic diversity and outcrossing rates.

Microsatellite-dense genetic map: towards genome coverage in a tropical maize (Zea mays L.) population

Dense molecular genetic maps are used for an efficient quantitative trait loci (QTL) mapping and in the marker-assisted selection programs. A dense genetic map was generated with 139 microsatellite markers using 256 F2 plants generated by the crossing of two tropical maize inbred lines (L-02-03D and L-20-01F). This map presented 1,858.61 cM in length, where 10 linkage groups were found spanned, with an average interval of 13.47 cM between adjacent markers. Seventy seven percent of the maize genetic mapping bins were covered, which means an increase of 14% coverage in relation to the previous tropical maize maps. The results provide a more detailed and informative genetic map in a tropical maize population representing the first step to make possible the studies of genetic architecture to identify and map QTL and estimate their effects on the variation of quantitative traits, thus allowing the manipulation and use in tropical maize breeding programs.

Multivariate genetic divergence and hybrid performance of cacao (Theobroma cacao L.)

Genetic distances among cacao cultivars were calculated through multivariate analysis, using the D2 statistic, to examine racial group classification and to assess heterotic hybrids. A 5 x 5 complete diallel was evaluated. Over a five-year period (1986-1990), five cultivars of the S1 generation, pertaining to the Lower Amazon Forastero and Trinitario racial groups and 20 crosses between the corresponding S0 parents were analyzed, based upon five yield components - number of healthy and collected fruits per plant (NHFP and NCFP), wet seed weight per plant and per fruit (WSWP and WSWF), and percentage of diseased fruits per plant (PDFP). The diversity analysis suggested a close relationship between the Trinitario and Lower Amazon Forastero groups. A correlation coefficient (r) was calculated to determine the association between genetic diversity and heterosis. Genetic distance of parents by D2 was found to be linearly related to average performance of hybrids for WSWP and WSWF (r = 0.68, P < 0.05 and r = 0.76, P < 0.05, respectively). The heterotic performance for the same components was also correlated with D2, both with r = 0.66 (P < 0.05). A relationship between genetic divergence and combining ability effects was suggested because the most divergent cultivar exhibited a high general combining ability, generating the best performing hybrids. Results indicated that genetic diversity estimates can be useful in selecting parents for crosses and in assessing relationships among cacao racial groups.

Genetic interactions involving major genes at the dwarf locus in egg-type chickens

Three egg-type stocks segregating dwarf (dw) and bantam (dwB) genes in female progeny were produced from the same 18 heterozygous (dwB/dw) sires used to inseminate dams of three different genotypes: normal (dw+), dwarf (dw) and bantam (dwB) dams. The heritability of 8-week body weight estimated from full-sibs of the same phenotype of progeny was 0.40, and that estimated from paternal half-sibs of the same phenotype (dwarf or bantam), and from the same genotype of dam was 0.38. Therefore, maternal and non-additive effects within genotypic classes of dam made little contribution to the genetic variance for 8-week body weight among their progeny. The interaction of sires (S) with genotypes (dw+, dw and dwB) of dam (G) was significant at the 5% level. This indicates that the rankings of the sires within each one of the three genotypes of dam were not the same, probably due to non-additive genetic variation among genotypes of dams. The evidence indicated that in general the genes from individual sires combined differently with each type of dam (G). Those genes which combined well with the genes from normal (dw+) dams combined poorly with both the genes from the dwarf (dw) and the genes from the bantam (dwB) dams. The interaction of sires (S) with phenotypes (dwarf and bantam) of progeny (P) was significant at the 10% level. The results indicated a probable gene x genotype interaction for 8-week weight between genes at the dwarf locus (dw and dwB) and the background genotype (single and/or polygenes). The correlation among paternal half-sibs was influenced more by the S x G than by the S x P interaction, but the effects tended to be cumulative

Calculation of breed direct and maternal genetic fractions and breed specific direct and maternal heterozygosity for crossbreeding data

Teaching, research, and herd breeding applications may require calculation of breed additive contributions for direct and maternal genetic effects and fractions of heterozygosity associated with breed specific direct and maternal heterosis effects. These coefficients can be obtained from the first NB rows of a pseudo numerator relationship matrix where the first NB rows represent fractional contributions by breed to each animal or group representing a specific breed cross. The table begins with an NB x NB identity matrix representing pure breeds. Initial animals or representative crosses must be purebreds or two-breed crosses. Parents of initial purebreds are represented by the corresponding column and initial two-breed cross progeny by the two corresponding columns of the identity matrix. After that, usual rules are used to calculate the NB column entries corresponding to breeds for each animal. The NB entries are fractions of genes expected to be contributed by each of the pure breeds and correspond to the breed additive direct fractions. Entries in the column corresponding to the dam represent breed additive maternal fractions. Breed specific direct heterozygosity coefficients are entries of an NB x NB matrix formed by the outer product of the two NB by 1 columns associated with sire and dam of the animal. One minus sum of the diagonals represents total direct heterozygosity. Similarly, the NB x NB matrix formed by the outer product of columns associated with sire of dam and dam of dam contains breed specific maternal heterozygosity coefficients. These steps can be programmed to create covariates to merge with data. If X represents these coefficients for all unique breed crosses, then the reduced row echelon form function of MATLAB or SAS can be used on X to determine estimable functions of additive breed direct and maternal effects and breed specific direct and maternal heterosis effects

Pollen mother cells of Tradescantia clone 4430 and Tradescantia pallida var. purpurea are equally sensitive to the clastogenic effects of X-rays

The Tradescantia micronucleus test is a sensitive bioassay for mutagenesis that may be employed both under field and laboratory conditions. This test has been standardized mostly on the basis of the results obtained with clone 4430. However, this clone is not well adapted to tropical weather, frequently showing problems with growth and flowering. In addition, it is attacked by parasites and insects, a fact that limits its use in field studies aiming at the biomonitoring of air pollution. In the city of São Paulo, Tradescantia pallida (Rose) Hunt. var. purpurea Boom is widely distributed as an ornamental plant in gardens and along roadsides and streets, mostly because of its natural resistance and its easy propagation. In this report, we present dose-response curves indicating that the sensitivity of T. pallida and clone 4430 to X-radiation (1, 10, 25 and 50 cGy) is similar. The results confirm our previous suggestion that T. pallida represents a good alternative for in situ mutagenesis testing in tropical regions, especially biomonitoring studies in which the exposure conditions may not be fully controllable.

Effects of a PPARG gene variant on obesity characteristics in Brazil

The contribution of genetic factors to the development of obesity has been widely recognized, but the identity of the genes involved has not yet been fully clarified. Variation in genes involved in adipocyte differentiation and energy metabolism is expected to have a role in the etiology of obesity. We assessed the potential association of a polymorphism in one candidate gene, peroxisome proliferator-activated receptor-gamma (PPARGg), involved in these pathways and obesity-related phenotypes in 335 Brazilians of European descent. All individuals included in the sample were adults. Pregnant women, as well as those individuals with secondary hyperlipidemia due to renal, liver or thyroid disease, and diabetes, were not invited to participate in the study; all other individuals were included. The gene variant PPARG Pro12Ala was studied by a PCR-based method and the association between this genetic polymorphism and obesity-related phenotypes was evaluated by analysis of covariance. Variant allele frequency was PPARG Ala12 = 0.09 which is in the same range as described for European and European-derived populations. No statistically significant differences were observed for mean total cholesterol, LDL cholesterol, HDL cholesterol, or triglyceride levels among PPARG genotypes in either gender. In the male sample, an association between the PPARG Pro12Ala variant and body mass index was detected, with male carriers of the Ala variant presenting a higher mean body mass index than wild-type homozygotes (28.3 vs 26.2 kg/m², P = 0.037). No effect of this polymorphism was detected in women. This finding suggests that the PPARG gene has a gender-specific effect and contributes to the susceptibility to obesity in this population.