Experimental Model of Gene Transfection in Healthy Canine Myocardium: Perspectives of Gene Therapy for Ischemic Heart Disease

OBJECTIVE: To assess the transfection of the gene that encodes green fluorescent protein (GFP) through direct intramyocardial injection. METHODS: The pREGFP plasmid vector was used. The EGFP gene was inserted downstream from the constitutive promoter of the Rous sarcoma virus. Five male dogs were used (mean weight 13.5 kg), in which 0.5 mL of saline solution (n=1) or 0.5 mL of plasmid solution containing 0.5 µg of pREGFP/dog (n=4) were injected into the myocardium of the left ventricular lateral wall. The dogs were euthanized 1 week later, and cardiac biopsies were obtained. RESULTS: Fluorescence microscopy showed differences between the cells transfected and not transfected with pREGFP plasmid. Mild fluorescence was observed in the cardiac fibers that received saline solution; however, the myocardial cells transfected with pREGFP had overt EGFP expression. CONCLUSION: Transfection with the EGFP gene in healthy canine myocardium was effective. The reproduction of this efficacy using vascular endothelial growth factor (VEGF) instead of EGFP aims at developing gene therapy for ischemic heart disease.

Estrategias estadístico-computacionales avanzadas para análisis de expresión de proteínas/genes en cáncer. Advanced statistical and computational strategies for protein/gene expression analysis in cancer.

El objetivo de este proyecto, enmarcado en el área de metodología de análisis en bioingeniería-biotecnología aplicadas al estudio del cancer, es el análisis y caracterización a través modelos estadísticos con efectos mixtos y técnicas de aprendizaje automático, de perfiles de expresión de proteínas y genes de las vías metabolicas asociadas a progresión tumoral. Dicho estudio se llevará a cabo mediante la utilización de tecnologías de alto rendimiento. Las mismas permiten evaluar miles de genes/proteínas en forma simultánea, generando así una gran cantidad de datos de expresión. Se hipotetiza que para un análisis e interpretación de la información subyacente, caracterizada por su abundancia y complejidad, podría realizarse mediante técnicas estadístico-computacionales eficientes en el contexto de modelos mixtos y técnias de aprendizaje automático. Para que el análisis sea efectivo es necesario contemplar los efectos ocasionados por los diferentes factores experimentales ajenos al fenómeno biológico bajo estudio. Estos efectos pueden enmascarar la información subycente y así perder informacion relavante en el contexto de progresión tumoral. La identificación de estos efectos permitirá obtener, eficientemente, los perfiles de expresión molecular que podrían permitir el desarrollo de métodos de diagnóstico basados en ellos. Con este trabajo se espera poner a disposición de investigadores de nuestro medio, herramientas y procedimientos de análisis que maximicen la eficiencia en el uso de los recursos asignados a la masiva captura de datos genómicos/proteómicos que permitan extraer información biológica relevante pertinente al análisis, clasificación o predicción de cáncer, el diseño de tratamientos y terapias específicos y el mejoramiento de los métodos de detección como así tambien aportar al entendimieto de la progresión tumoral mediante análisis computacional intensivo.

O polimorfismo VNTR no gene codificador do antagonista do receptor da interleucina-1 está associado com a doença arterial coronariana

FUNDAMENTO: A Doença Arterial Coronariana (DAC) é a aterosclerose das artérias coronárias que transportam o sangue para o coração. A aterosclerose é uma doença inflamatória. As variações gênicas das citocinas - como as associadas à família IL1 - fazem parte da patogênese da aterosclerose. OBJETIVO: O objetivo deste estudo foi determinar a relação entre os polimorfismos da família IL1 (VNTR do IL1RN, posições -511 e +3953 do IL1B) e a DAC na população turca. MÉTODOS: Um total de 427 indivíduos foram submetidos à angiografia coronariana e em seguida divididos da seguinte forma: 170 no grupo controle e 257 no grupo de pacientes com DAC. Os sujeitos com DAC foram divididos em dois subgrupos: 91 no grupo de Doença Coronariana em um único vaso (Single Vessel Disease - SVD) e 166 no grupo Doença Coronariana em múltiplos vasos (Multiple Vessel Disease - MVD). Os genótipos de IL1RN e IL1B (-511, +3953) foram determinados por reação em cadeia da polimerase (RCP), seguida de análise da digestão por enzima de restrição. RESULTADOS: Não foram observadas diferenças significantes nas distribuições de genótipos de IL1RN e IL1B (-511 e +3953) entre os sujeitos com DAC e os controles, ou entre sujeitos com MVD e controles. No entanto, observou-se uma relação significante no genótipo IL1RN 2/2 entre sujeitos portadores de SVD e controles (P= 0,016, x2: 10,289, OR: 2,94IC 95% 1,183 - 7,229). Tampouco foi observada diferença estatisticamente significante nas freqüências dos alelos de IL1RN e IL1B (-511 e +3953) entre os sujeitos com DAC e controles, os sujeitos com MVD e controles, ou ainda os sujeitos SVD e controles. CONCLUSÃO: Não foi observada nenhuma relação na freqüência alélica e nem na distribuição genotípica dos polimorfismos de IL1RN e IL1B entre sujeitos com DAC e grupos controle. No entanto, o genótipo IL1RN 2/2 pode representar um fator de risco para sujeitos com SVD na população turca.

Polimorfismo K121Q do gene ENPP1 e cardiopatia isquêmica em pacientes com diabete melito

FUNDAMENTO: O gene ecto-nucleotídeo pirofosfatase/fosfodiesterase 1 (ENPP1) é um gene candidato à resistência insulínica. A resistência à insulina é um componente importante da síndrome metabólica e tem sido implicada no desenvolvimento de doença cardíaca isquêmica (DCI). OBJETIVO: Avaliar a associação entre o polimorfismo K121Q do gene ENPP1 e a presença da DCI em pacientes caucasianos com diabete melito (DM) tipo 2. MÉTODOS: Estudo transversal foi realizado em pacientes com DM tipo 2 (n=573; 50,6% homens; idade 59,5±10,4 anos). DCI foi definida pela presença de angina ou infarto agudo do miocárdio pelo questionário cardiovascular da Organização Mundial da Saúde e/ou alterações compatíveis no ECG (código Minnesota) ou cintilografia miocárdica. O polimorfismo K121Q foi genotipado através da técnica de PCR e digestão enzimática. RESULTADOS: DCI esteve presente em 209 (36,5%) pacientes. A frequência dos genótipos KK, KQ e QQ entre os pacientes com DCI foi 60,8%, 34,4% e 4,8%, semelhante à distribuição dos genótipos entre os pacientes sem DCI (64,0%, 32,7% e 3,3%, P = 0,574). Não se observou diferença nas características clínicas ou laboratoriais entre os três genótipos, nem em relação à presença de síndrome metabólica. CONCLUSÃO: Nenhuma associação foi encontrada entre o polimorfismo K121A do gene ENPP1 e a presença de DCI ou características fenotípicas de resistência insulínica.

Efeitos da atorvastatina e do polimorfismo T-786C do gene eNOS sobre parâmetros do metabolismo lipídico plasmático

FUNDAMENTO: A atividade do óxido nítrico sintase endotelial (eNOS) pode ser modulada pelo colesterol da lipoproteína de alta densidade (HDL-C), estatinas ou polimorfismos, como o T-786C de eNOS. OBJETIVO: Este estudo teve como objetivo avaliar se o polimorfismo T-786C está associado a alterações nos efeitos da atorvastatina no perfil lipídico, nas concentrações de metabólitos de óxido nítrico (NO) e da proteína C reativa de alta sensibilidade (PCR-as). MÉTODOS: Trinta voluntários do sexo masculino, assintomáticos, com idade entre 18-56 anos foram genotipados e classificados de acordo com a ausência (TT, n = 15) ou presença (CC, n = 15) do polimorfismo. Eles foram selecionados aleatoriamente para a utilização de placebo e atorvastatina (10 mg/dia por 14 dias). Após cada tratamento foram medidos lípides, lipoproteínas, frações HDL2 e HDL3, atividade da proteína de transferência de colesteril éster (CETP), metabólitos de NO e PCR-as. RESULTADOS: As comparações entre genótipos após a administração de placebo mostraram aumento da atividade da CETP polimorfismo-dependente (TT, 12 ± 7; CC, 22 ± 12, p < 0,05). As análises da interação entre os tratamentos indicaram que a atorvastatina tem efeito sobre colesterol, LDL, nitrito e razões lípides/proteínas (HDL2 e HDL3) (p < 0,001) em ambos os genótipos. É interessante notar as interações genótipo/droga sobre a CETP (p < 0,07) e a lipoproteína (a) [Lp(a)] (p < 0,056), levando a uma diminuição limítrofe da CETP, embora sem afetar a Lp(a). A PRC-as não mostrou alterações. CONCLUSÃO: Os resultados sugerem que o tratamento com estatinas pode ser relevante para a prevenção primária da aterosclerose em pacientes com o polimorfismo T-786C do eNOS, considerando os efeitos no metabolismo lipídico.

O polimorfismo do gene AKL7 está associado ao risco de síndrome metabólica e à remodelação cardiovascular

FUNDAMENTO: Quinase Tipo Receptor de Ativina 7 (ALK7) é um tipo de receptor I para a superfamília TGF-β e recentemente apresentou ter uma função importante na manutenção de homeostase metabólica. OBJETIVO: Investigar a associação do polimorfismo do gene ALK7 à síndrome metabólica (SMet) e remodelação cardiovascular em pacientes com SMet. MÉTODOS: O polimorfismo de nucleotídeo único rs13010956 no gene ALK7 foi genotipado em 351 indivíduos chineses submetidos à ultrassonografia cardíaca e das carótidas. As associações do polimorfismo do gene ALK7 ao fenótipo e aos parâmetros da síndrome metabólica e características ultrassônicas cardiovasculares foram analisadas. RESULTADOS: O polimorfismo de rs13010956 no gene ALK7 foi considerado significativamente relacionado ao fenótipo de SMet em mulheres (p < 0,05) e significativamente associado à pressão sanguínea em populações totais (p < 0,05) e femininas (p < 0,01). Outras análises revelaram que rs13010956 estava associado à média da espessura íntima-média de artérias carótidas em mulheres (p < 0,05). Após o controle do índice de massa corporal, pressão arterial, glicemia em jejum e triglicérides, o rs13010956 também foi considerado significativamente associado ao índice de massa do ventrículo esquerdo em populações totais (p < 0,05) e femininas (p < 0,05). CONCLUSÃO: Nossos achados sugeriram que o polimorfismo de rs13010956 do gene ALK7 estava significativamente vinculado ao risco de SMet em mulheres e pode estar envolvido na remodelação cardiovascular em pacientes com SMet.

Analyse der Informationsverarbeitung in CHL1-defizienten Mäusen mittels metabolischer Markierung, Expressionsstudien der Immediate-Early-Gene c-fos und arg3.1/arc sowie verhaltensbiologischer Tests

Close homolog of L1, neural cell recognition molecules, c-fos, arg3.1, arc, immediat early genes, novelty, information processing, behavioral tests

Effects of neural nitric oxide synthase gene inactivation on the neurodocrine stress response in mice

Neural nitric oxide synthase, neuroendocrine stress response, forced swimming, nNOS KO mice, hypothalamus, adrenal gland

APOE and LDLR Gene Polymorphisms and Dyslipidemia Tracking. Rio de Janeiro Study

Background:Studies show an association between changes in apolipoprotein E (ApoE) and LDLR receptor with the occurrence of dyslipidemia.Objectives:To investigate the association between polymorphisms of the APOE (ε2, ε3, ε4) and LDLR (A370T) genes with the persistence of abnormal serum lipid levels in young individuals followed up for 17 years in the Rio de Janeiro Study.Methods:The study included 56 individuals (35 males) who underwent three assessments at different ages: A1 (mean age 13.30 ± 1.53 years), A2 (22.09 ± 1.91 years) and A3 (31.23 ± 1.99 years). Clinical evaluation with measurement of blood pressure (BP) and body mass index (BMI) was conducted at all three assessments. Measurement of waist circumference (WC) and serum lipids, and analysis of genetic polymorphisms by PCR-RFLP were performed at A2 and A3. Based on dyslipidemia tracking, three groups were established: 0 (no abnormal lipid value at A2 and A3), 1 (up to one abnormal lipid value at A2 or A3) and 2 (one or more abnormal lipid values at A2 and A3).Results:Compared with groups 0 and 1, group 2 presented higher mean values of BP, BMI, WC, LDL-c and TG (p < 0.01) and lower mean values of HDL-c (p = 0.001). Across the assessments, all individuals with APOE genotypes ε2/ε4 and ε4/ε4 maintained at least one abnormal lipid variable, whereas those with genotype ε2/ε3 did not show abnormal values (χ2 = 16.848, p = 0.032). For the LDLR genotypes, there was no significant difference among the groups.Conclusions:APOE gene polymorphisms were associated with dyslipidemia in young individuals followed up longitudinally from childhood.

Competição entre megaspórios em milho

The experiments reported were started as early as 1933, when indications were found in class material that the factor for small pollen, spl, causes not only differences in the size of pollen grains and in the growth of pollen tubes, but also a competition between megaspores, as first observed by RENNER (1921) in Oenothera. Dr. P. C. MANGELSDORF, who had kindly furnished the original seeds, was informed and the final publication delayed untill his publication in 1940. A further delay was caused by other circunstances. The main reason for the differences of the results obtained by SINGLETON and MANGELSDORF (1940) and those reported here, seems to be the way the material was analysed. I applied methods of a detailed statistical analysis, while MANGELSDORF and SINGLETON analysed pooled data. 1) The data obtained on pollen tube competition indicate .that there is about 3-4% of crossing-over between the su and sp factors in chromosome IV. The elimination is not always complete, but from 0 to 10% of the sp pollen tubes may function, instead of the 50% expected without elimination. These results are, as a whole, in accordance with SINGLETON and MANGELSDORF's data. 2) Female elimination is weaker and transmission determined as between 16 to 49,5%, instead of 50% without competition, the values being calculated by a special formula. 3) The variability of female elimination is partially genotypical, partially phenotypical. The former was shown by the difference in the behavior of the two progenies tested, while the latter was very evident when comparing the upper and lower halves of ears. For some unknown physiological reason, the elimination is generally stronger in the upper than in the lower half of the ear. 4) The female elimination of the sp gene may be caused theoretically, by either of two processes: a simple lethal effect in the female gametophyte or a competition between megaspores. The former would lead not only to the abortion of the individual megaspores, but of the whole uniovulate ovary. In the case of the latter, the abortive megaspore carrying the gene sp will be substituted in each ovule by one of the Sp megaspores and no abortion of ovaries may be observed. My observations are completely in favor of the second explication: a) The ears were as a whole very well filled except for a few incomplete ears which always appear in artificial pollinations. b) Row arrangement was always very regular. c) The number of kernels on ears with elimination is not smaller than in normal ears, but is incidentally higher : with elimnation, in back-crosses 354 kernels and in selfed ears 390 kernels, without elimination 310 kernels per ear. d) There is no correlation between the intensity of elimination and the number of grains in individual ears; the coefficient; of linear correlation, equal to 0,24, is small and insignificant. e) Our results are in complete disagreement whit those reported by SINGLETON and MANGELSDORF (1940). Since these authors present only pooled date, a complete and detailed analysis which may explain the cause of these divergences is impossible.

Bases para o estudo da genética de populações dos Hymenoptera em geral e dos Apinae sociais em particular

This paper deals with problems on population genetics in Hymenoptera and particularly in social Apidae. 1) The studies on populations of Hymenoptera were made according to the two basic types of reproduction: endogamy and panmixia. The populations of social Apinae have a mixed method of reproduction with higher percentage of panmixia and a lower of endogamy. This is shown by the following a) males can enter any hive in swarming time; b) males of Meliponini are expelled from hives which does not need them, and thus, are forced to look for some other place; c) Meliponini males were seen powdering themselves with pollen, thus becoming more acceptable in any other hive. The panmixia is not complete owing to the fact that the density of the breeding population as very low, even in the more frequent species as low as about 2 females and 160 males per reproductive area. We adopted as selection values (or survival indices) the expressions according to Brieger (1948,1950) which may be summarised as follows; a population: p2AA + ²pq Aa + q2aa became after selection: x p2AA + 2pq Aa + z q²aa. For alge-braics facilities Brieger divided the three selective values by y giving thus: x/y p2 AA + y/y 2 pq Aa + z/y q²aa. He called x/y of RA and z/y of Ra, that are survival or selective index, calculated in relation to the heterozygote. In our case all index were calculated in relation to the heterozygote, including the ones for haploid males; thus we have: RA surveval index of genotype AA Ra surveval index of genotype aa R'A surveval index of genotype A R'a surveval index of genotype a 1 surveval index of genotype Aa The index R'A ande R'a were equalized to RA and Ra, respectively, for facilities in the conclusions. 2) Panmitic populations of Hymenoptera, barring mutations, migrations and selection, should follow the Hardy-Weinberg law, thus all gens will be present in the population in the inicial frequency (see Graphifc 1). 3) Heterotic genes: If mutation for heterotic gene ( 1 > RA > Ra) occurs, an equilibrium will be reached in a population when: P = R A + Ra - 2R²a _____________ (9) 2(R A + Ra - R²A - R²a q = R A + Ra - 2R²A _____________ (10) 2(R A + Ra - R²A - R²a A heterotic gene in an hymenopteran population may be maintained without the aid of new mutation only if the survival index of the most viable mutant (RA) does not exced the limiting value given by the formula: R A = 1 + √1+Ra _________ 4 If RA has a value higher thah the one permitted by the formula, then only the more viable gene will remain present in the population (see Graphic 10). The only direct proof for heterotic genes in Hymenoptera was given by Mackensen and Roberts, who obtained offspring from Apis mellefera L. queens fertilized by their own sons. Such inbreeding resulted in a rapid loss of vigor the colony; inbred lines intercrossed gave a high hybrid vigor. Other fats correlated with the "heterosis" problem are; a) In a colony M. quadrifasciata Lep., which suffered severely from heat, the percentage of deths omong males was greater .than among females; b) Casteel and Phillips had shown that in their samples (Apis melifera L). the males had 7 times more abnormalities tian the workers (see Quadros IV to VIII); c) just after emerging the males have great variation, but the older ones show a variation equal to that of workers; d) The tongue lenght of males of Apis mellifera L., of Bombus rubicundus Smith (Quadro X), of Melipona marginata Lep. (Quadro XI), and of Melipona quadrifasciata Lep. Quadro IX, show greater variationthan that of workers of the respective species. If such variation were only caused by subviables genes a rapid increasse of homozigoty for the most viable alleles should be expected; then, these .wild populations, supposed to be in equilibrium, could .not show such variability among males. Thus we conclude that heterotic genes have a grat importance in these cases. 4) By means of mathematical models, we came to the conclusion tht isolating genes (Ra ^ Ra > 1), even in the case of mutations with more adaptability, have only the opor-tunity of survival when the population number is very low (thus the frequency of the gene in the breeding population will be large just after its appearence). A pair of such alleles can only remain present in a population when in border regions of two races or subspecies. For more details see Graphics 5 to 8. 5) Sex-limited genes affecting only females, are of great importance toHymenoptera, being subject to the same limits and formulas as diploid panmitic populations (see formulas 12 and 13). The following examples of these genes were given: a) caste-determining genes in the genus Melipona; b) genes permiting an easy response of females to differences in feeding in almost all social Hymenoptera; c) two genes, found in wild populations, one in Trigona (Plebéia) mosquito F. SMITH (quadro XII) and other in Melipona marginata marginata LEP. (Quadro XIII, colonies 76 and 56) showing sex-limited effects. Sex-limited genes affecting only males do not contribute to the plasticity or genie reserve in hymenopteran populations (see formula 14). 6) The factor time (life span) in Hymenoptera has a particular importance for heterotic genes. Supposing one year to be the time unit and a pair of heterotic genes with respective survival indice equal to RA = 0, 90 and Ra = 0,70 to be present; then if the life time of a population is either one or two years, only the more viable gene will remain present (see formula 11). If the species has a life time of three years, then both alleles will be maintained. Thus we conclude that in specis with long lif-time, the heterotic genes have more importance, and should be found more easily. 7) The colonies of social Hymenoptera behave as units in competition, thus in the studies of populations one must determine the survival index, of these units which may be subdivided in indice for egg-laying, for adaptive value of the queen, for working capacity of workers, etc. 8) A study of endogamic hymenopteran populations, reproduced by sister x brother mating (fig. 2), lead us to the following conclusions: a) without selection, a population, heterozygous for one pair of alleles, will consist after some generations (theoretically after an infinite number of generation) of females AA fecundated with males A and females aa fecundated with males a (see Quadro I). b) Even in endogamic population there is the theoretical possibility of the presence of heterotic genes, at equilibrium without the aid of new mutations (see Graphics 11 and 12), but the following! conditions must be satisfied: I - surveval index of both homozygotes (RA e Ra) should be below 0,75 (see Graphic 13); II - The most viable allele must riot exced the less viable one by more than is permited by the following formula (Pimentel Gomes 1950) (see Gra-fic 14) : 4 R5A + 8 Ra R4A - 4 Ra R³A (Ra - 1) R²A - - R²a (4 R²a + 4 Ra - 1) R A + 2 R³a < o Considering these two conditions, the existance of heterotic genes in endogamic populations of Hymenoptera \>ecames very improbable though not - impossible. 9) Genie mutation offects more hymenopteran than diploid populations. Thus we have for lethal genes in diploid populations: u = q2, and in Hymenoptera: u = s, being u the mutation ratio and s the frequency of the mutant in the male population. 10) Three factors, important to competition among species of Meliponini were analysed: flying capacity of workers, food gathering capacity of workers, egg-laying of the queen. In this connection we refer to the variability of the tongue lenght observed in colonies from several localites, to the method of transporting the pollen in the stomach, from some pots (Melliponi-ni storage alveolus) to others (e. g. in cases of pillage), and to the observation that the species with the most populous hives are almost always the most frequent ones also. 11) Several defensive ways used for Meliponini to avoid predation are cited, but special references are made upon the camouflage of both hive (fig. 5) and hive entrance (fig. 4) and on the mimetism (see list in page ). Also under the same heading we described the method of Lestrimelitta for pillage. 12) As mechanisms important for promoting genetic plasticity of hymenopteran species we cited: a) cytological variations and b) genie reserve. As to the former, duplications and numerical variations of chromosomes were studied. Diprion simile ATC was cited as example for polyploidy. Apis mellife-ra L. (n •= 16) also sugests polyploid origen since: a) The genus Melipona, which belongs to a" related tribe, presents in all species so far studied n = 9 chromosomes and b) there occurs formation of dyads in the firt spermatocyte division. It is su-gested that the origin of the sex-chromosome of Apis mellifera It. may be related to the possible origin of diplo-tetraploidy in this species. With regards to the genie reserve, several possible types of mutants were discussed. They were classified according to their survival indices; the heterotic and neutral mutants must be considered as more important for the genie reserve. 13) The mean radius from a mother to a daghter colony was estimated as 100 meters. Since the Meliponini hives swarm only once a year we may take 100 meters a year as the average dispersion of female Meliponini in ocordance to data obtained from Trigona (tetragonisca) jaty F. SMITH and Melipona marginata LEP., while other species may give different values. For males the flying distance was roughly estimated to be 10 times that for females. A review of the bibliography on Meliponini swarm was made (pg. 43 to 47) and new facts added. The population desity (breeding population) corresponds in may species of Meliponini to one male and one female per 10.000 square meters. Apparently the males are more frequent than the females, because there are sometimes many thousands, of males in a swarm; but for the genie frequency the individuals which have descendants are the ones computed. In the case of Apini and Meliponini, only one queen per hive and the males represented by. the spermatozoos in its spermateca are computed. In Meliponini only one male mate with the queen, while queens of Apis mellijera L. are fecundated by an average of about 1, 5 males. (Roberts, 1944). From the date cited, one clearly sees that, on the whole, populations of wild social bees (Meliponini) are so small that the Sewall Wright effect may become of great importance. In fact applying the Wright's formula: f = ( 1/aN♂ + 1/aN♀) (1 - 1/aN♂ + 1/aN♀) which measures the fixation and loss of genes per generation, we see that the fixation or loss of genes is of about 7% in the more frequent species, and rarer species about 11%. The variation in size, tergite color, background color, etc, of Melipona marginata Lep. is atributed to this genetic drift. A detail, important to the survival of Meliponini species, is the Constance of their breeding population. This Constance is due to the social organization, i. e., to the care given to the reproductive individuals (the queen with its sperm pack), to the way of swarming, to the food storage intended to control variations of feeding supply, etc. 14) Some species of the Meliponini are adapted to various ecological conditions and inhabit large geographical areas (e. g. T. (Tetragonisca jaty F. SMITH), and Trigona (Nanno-trigona testaceicornis LEP.) while others are limited to narrow regions with special ecological conditions (e. g. M. fuscata me-lanoventer SCHWARZ). Other species still, within the same geographical region, profit different ecological conditions, as do M. marginata LEP. and M. quadrifasciata LEP. The geographical distribution of Melipona quadrifasciata LEP. is different according to the subspecies: a) subsp anthidio-des LEP. (represented in Fig. 7 by black squares) inhabits a region fron the North of the S. Paulo State to Northeastern Brazil, ,b) subspecies quadrifasciata LEP., (marked in Fig. 7 with black triangles) accurs from the South of S. Paulo State to the middle of the State of Rio Grande do Sul (South Brazil). In the margined region between these two areas of distribution, hi-brid colonies were found (Fig. 7, white circles); they are shown with more details in fig. 8, while the zone of hybridization is roughly indicated in fig. 9 (gray zone). The subspecies quadrifasciata LEP., has 4 complete yellow bands on the abdominal tergites while anthidioides LEP. has interrupted ones. This character is determined by one or two genes and gives different adaptative properties to the subspecies. Figs. 10 shows certains meteorological isoclines which have aproximately the same configuration as the limits of the hybrid zone, suggesting different climatic adaptabilities for both genotypes. The exis-tance of a border zone between the areas of both subspecies, where were found a high frequency of hybrids, is explained as follows: being each subspecies adapted to a special climatic zone, we may suppose a poor adaptation of either one in the border region, which is also a region of intermediate climatic conditions. Thus, the hybrids, having a combination of the parent qualities, will be best adapted to the transition zone. Thus, the hybrids will become heterotic and an equilibrium will be reached with all genotypes present in the population in the border region.

mRNA metabolism: nonsense mediated mRNA decay as a tool for gene therapy and the role of human DIS3L2 in transcript degradation

Tese de mestrado em Biologia Humana e Ambiente, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2015

Changes in reproductive roles are associated with changes in gene expression in fire ant queens.

Abstract In species with social hierarchies, the death of dominant individuals typically upheaves the social hierarchy and provides an opportunity for subordinate individuals to become reproductives. Such a phenomenon occurs in the monogyne form of the fire ant, Solenopsis invicta, where colonies typically contain a single wingless reproductive queen, thousands of workers and hundreds of winged nonreproductive virgin queens. Upon the death of the mother queen, many virgin queens shed their wings and initiate reproductive development instead of departing on a mating flight. Workers progressively execute almost all of them over the following weeks. To identify the molecular changes that occur in virgin queens as they perceive the loss of their mother queen and begin to compete for reproductive dominance, we collected virgin queens before the loss of their mother queen, 6 h after orphaning and 24 h after orphaning. Their RNA was extracted and hybridized against microarrays to examine the expression levels of approximately 10 000 genes. We identified 297 genes that were consistently differentially expressed after orphaning. These include genes that are putatively involved in the signalling and onset of reproductive development, as well as genes underlying major physiological changes in the young queens.

Proteomic and Metabolomic Analyses of Mitochondrial Complex I-deficient Mouse Model Generated by Spontaneous B2 Short Interspersed Nuclear Element (SINE) Insertion into NADH Dehydrogenase (Ubiquinone) Fe-S Protein 4 (Ndufs4) Gene.

Eukaryotic cells generate energy in the form of ATP, through a network of mitochondrial complexes and electron carriers known as the oxidative phosphorylation system. In mammals, mitochondrial complex I (CI) is the largest component of this system, comprising 45 different subunits encoded by mitochondrial and nuclear DNA. Humans diagnosed with mutations in the gene NDUFS4, encoding a nuclear DNA-encoded subunit of CI (NADH dehydrogenase ubiquinone Fe-S protein 4), typically suffer from Leigh syndrome, a neurodegenerative disease with onset in infancy or early childhood. Mitochondria from NDUFS4 patients usually lack detectable NDUFS4 protein and show a CI stability/assembly defect. Here, we describe a recessive mouse phenotype caused by the insertion of a transposable element into Ndufs4, identified by a novel combined linkage and expression analysis. Designated Ndufs4(fky), the mutation leads to aberrant transcript splicing and absence of NDUFS4 protein in all tissues tested of homozygous mice. Physical and behavioral symptoms displayed by Ndufs4(fky/fky) mice include temporary fur loss, growth retardation, unsteady gait, and abnormal body posture when suspended by the tail. Analysis of CI in Ndufs4(fky/fky) mice using blue native PAGE revealed the presence of a faster migrating crippled complex. This crippled CI was shown to lack subunits of the "N assembly module", which contains the NADH binding site, but contained two assembly factors not present in intact CI. Metabolomic analysis of the blood by tandem mass spectrometry showed increased hydroxyacylcarnitine species, implying that the CI defect leads to an imbalanced NADH/NAD(+) ratio that inhibits mitochondrial fatty acid β-oxidation.