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.

Búsqueda e identificación de polimorfismos en genes asociados con una efectividad farmacológica disminuida. Caracterización molecular, hallazgos bioquímicos y clínicos y su respectiva correlación con la respuesta farmacológica en el tratamiento de enfermedades crónicas e infecciosas.

En medicina, es frecuente encontrar diferencias en la respuesta de una misma droga en distintos individuos. Algunos factores que contribuyen con esta respuesta diferencial incluyen variables como edad, biodisponilidad y absorción gastro-intestinal de los medicamentos, interacción entre fármacos, hábitos alimentarios y factores genéticos. Dentro de los factores genéticos, encontramos polimorfismos genéticos que afectan la absorción, el metabolismo y el transporte de fármacos, como así también receptores de los mismos y/o, la interacción con otros genes. Algunos polimorfismos genéticos que contribuyen a una respuesta farmacológica disminuida han sido descriptos en patologías como, la hipercolesterolemia, artritis reumatoidea, cáncer, diabetes, hipertensión arterial, esquizofrenia, asma, hepatitis C y SIDA, entre otras. Nuestro estudio pretende: I) Identificar polimorfismos en genes que codifican para enzimas metabolizadoras de fármacos, para canales iónicos y, para receptores de fármacos (como por ejemplo polimorfismos en el receptor beta 2 adrenérgico en pacientes tratados con salbutamol que presentan bronquiolitis). II) Identificar la presencia de un polimorfismo en el gen CES 1 que codifica para la enzima carboxilesterasa 1 (en una población hospitalaria), que participa en la activación de la prodroga oseltamivir utilizada en el tratamiento de la Gripe A (H1N1). Los resultados obtenidos podrán ser de gran utilidad en el tratamiento médico, ya que permitirá optimizar el uso de fármacos, disminuir los efectos secundarios causados por los mismos, y proponer el empleo de otros fármacos.

Homocisteína e polimorfismos dos genes MTHFR e VEGF: impacto na doença arterial coronariana

FUNDAMENTO: Polimorfismos em genes relacionados ao desenvolvimento da aterosclerose, angiogênese e metabolismo da homocisteína (Hcy) podem ser fatores de risco para a doença arterial coronariana (DAC). OBJETIVO: Avaliar o efeito dos polimorfismos VEGF C-2578A e MTHFR C677T na DAC e a associação desses polimorfismos com a gravidade e a extensão das lesões ateroscleróticas e concentrações de Hcy. MÉTODOS: 244 indivíduos foram avaliados através de angiografia coronariana e incluídos no estudo (145 com DAC e 99 indivíduos-controle). Os polimorfismos VEGF C-2578A e MTHFR C677T foram investigados através das técnicas de PCR-SSCP e PCR-RFLP, respectivamente. Os níveis de homocisteína plasmática foram mensurados através de cromatografia líquida/espectrometria de massa seqüencial (CL/EMS). RESULTADOS: Não houve diferença significante em relação à distribuição de alelos e genótipos entre os grupos, para ambos os polimorfismos. A análise univariada mostrou uma freqüência maior do genótipo VEGF -2578AA no grupo com doença em três vasos (p=0,044). Além disso, o genótipo VEGF -2578CA foi observado mais freqüentemente entre indivíduos com <95% de estenose (p=0,010). Após ajuste para outros fatores de risco para DAC em um modelo multivariado, observou-se que o polimorfismo VEGF C-2578A não era um correlato independente da DAC (p=0,688). O polimorfismo MTHFR não mostrou qualquer relação com a extensão e/ou gravidade da DAC. O polimorfismo MTHFR C677T não mostrou uma associação direta com hiperhomocisteinemia ou aumento das concentrações médias de Hcy no plasma. CONCLUSÃO: Embora haja uma aparente associação entre o polimorfismo VEGF C-2578A e o desenvolvimento de aterosclerose coronariana, essa associação não é independente dos fatores de risco cardiovasculares convencionais.

Amplificação dos genes que codificam a endotelina-1 e seus receptores em valvas mitrais reumáticas

FUNDAMENTO: As cardiopatias são doenças de alta prevalência, sendo a cardite reumática uma doença de grande relevância em países em desenvolvimento. As alterações em câmaras cardíacas esquerdas se associam à disfunção endotelial, com aumento dos níveis de endotelina-1 (ET-1) e consequências sobre a circulação pulmonar, muitas vezes determinando a hipertensão pulmonar (HP). No entanto, a presença de ET-1 e seus receptores na própria valva mitral, promovendo alterações vasculares pulmonares e aumentando a deformação valvar reumática, ainda é um assunto não abordado na literatura. OBJETIVO: Determinar, mediante técnicas moleculares, a expressão dos genes da endotelina e dos seus receptores em valvas mitrais reumáticas. MÉTODOS: 27 pacientes submetidos à troca valvar mitral tiveram seu tecido valvar analisado, a fim de determinar a presença de genes de ET-1 e seus receptores A e B. Foram feitas análises histológica e molecular das valvas (divididas em fragmentos M1, M2 e M3) e colhidos dados clínicos e epidemiológicos dos pacientes. Foram divididos em três grupos: valvopatia mitral, mitroaórtica e pacientes reoperados. RESULTADOS: O estudo mostrou a manifestação do gene da ET-1 em 40,7% dos espécimes e de seu receptor A em todas as amostras, com manifestação minoritária do gene do receptor B (22,2%). CONCLUSÃO: Todos os pacientes expressaram a presença do gene do receptor A. Não houve diferença estatística quanto à gravidade da doença, expressa em classe funcional, e aos subgrupos estudados (valvopatas mitrais, mitroaórticos e pacientes reoperados), ou quanto à expressão dos genes da ET-1 e seus receptores entre os subgrupos estudados (valvopatas mitrais, mitroaórticos e pacientes reoperados).

Survival and Predictive Factors of Lethality in Hemodyalisis: D/I Polymorphism of The Angiotensin I-Converting Enzyme and of the Angiotensinogen M235T Genes

Background: End-stage kidney disease patients continue to have markedly increased cardiovascular disease morbidity and mortality. Analysis of genetic factors connected with the renin-angiotensin system that influences the survival of the patients with end-stage kidney disease supports the ongoing search for improved outcomes. Objective: To assess survival and its association with the polymorphism of renin-angiotensin system genes: angiotensin I-converting enzyme insertion/deletion and angiotensinogen M235T in patients undergoing hemodialysis. Methods: Our study was designed to examine the role of renin-angiotensin system genes. It was an observational study. We analyzed 473 chronic hemodialysis patients in four dialysis units in the state of Rio de Janeiro. Survival rates were calculated by the Kaplan-Meier method and the differences between the curves were evaluated by Tarone-Ware, Peto-Prentice, and log rank tests. We also used logistic regression analysis and the multinomial model. A p value ≤ 0.05 was considered to be statistically significant. The local medical ethics committee gave their approval to this study. Results: The mean age of patients was 45.8 years old. The overall survival rate was 48% at 11 years. The major causes of death were cardiovascular diseases (34%) and infections (15%). Logistic regression analysis found statistical significance for the following variables: age (p = 0.000038), TT angiotensinogen (p = 0.08261), and family income greater than five times the minimum wage (p = 0.03089), the latter being a protective factor. Conclusions: The survival of hemodialysis patients is likely to be influenced by the TT of the angiotensinogen M235T gene.

A ação dos gens gametofíticos com referência especial ao milho

1) The first part deals with the different processes which may complicate Mendelian segregation and which may be classified into three groups, according to BRIEGER (1937b) : a) Instability of genes, b) Abnormal segregation due to distur- bances during the meiotic divisions, c) obscured segregation, after a perfectly normal meiosis, caused by elimination or during the gonophase (gametophyte in higher plants), or during zygophase (sporophyte). Without entering into detail, it is emphasized that all the above mentioned complications in the segregation of some genes may be caused by the action of other genes. Thus in maize, the instability of the Al factor is observed only when the gene dt is presente in the homozygous conditions (RHOADES 1938). In another case, still under observation in Piracicaba, an instability is observed in Mirabilis with regard to two pairs of alleles both controlling flower color. Several cases are known, especially in corn, where recessive genes, when homozigous, affect the course of meiosis, causing asynapsis (asyndesis) (BEADLE AND MC CLINTOCK 1928, BEADLE 1930), sticky chromosomes (BEADLE 1932), supermunmerary divisions (BEADLE 1931). The most extreme case of an obscured segregatiou is represented by the action of the S factors in self stetrile plants. An additional proof of EAST AND MANGELSDORF (1925) genetic formula of self sterility has been contributed by the studies on Jinked factors in Nicotina (BRIEGER AND MANGELSDORF (1926) and Antirrhinum (BRIEGER 1930, 1935), In cases of a incomplete competition and selection between pollen tubes, studies of linked indicator-genes are indispensable in the genetic analysis, since it is impossible to analyse the factors for gametophyte competition by direct aproach. 2) The flower structure of corn is explained, and stated that the particularites of floral biology make maize an excellent object for the study of gametophyte factors. Since only one pollen tube per ovule may accomplish fertilization, the competition is always extremely strong, as compared with other species possessing multi-ovulate ovaries. The lenght of the silk permitts the study of pollen tube competitions over a varying distance. Finally the genetic analysis of grains characters (endosperm and aleoron) simpliflen the experimental work considerably, by allowing the accumulation of large numbers for statistical treatment. 3) The four methods for analyzing the naturing of pollen tube competition are discussed, following BRIEGER (1930). Of these the first three are: a) polinization with a small number of pollen grains, b) polinization at different times and c) cut- ting the style after the faster tubes have passe dand before the slower tubes have reached the point where the stigma will be cut. d) The fourth method, alteration of the distatice over which competition takes place, has been applied largely in corn. The basic conceptions underlying this process, are illustrated in Fig. 3. While BRINK (1925) and MANGELSDORF (1929) applied pollen at different levels on the silks, the remaining authors (JONES, 1922, MANGELSDORF 1929, BRIEGER, at al. 1938) have used a different process. The pollen was applied as usual, after removing the main part of the silks, but the ears were divided transversally into halves or quarters before counting. The experiments showed generally an increase in the intensity of competition when there was increase of the distance over which they had to travel. Only MANGELSDORF found an interesting exception. When the distance became extreme, the initially slower tubes seemed to become finally the faster ones. 4) Methods of genetic and statistical analysis are discussed, following chiefly BRIEGER (1937a and 1937b). A formula is given to determine the intensity of ellimination in three point experiments. 5) The few facts are cited which give some indication about the physiological mechanism of gametophyte competition. They are four in number a) the growth rate depends-only on the action of gametophyte factors; b) there is an interaction between the conductive tissue of the stigma or style and the pollen tubes, mainly in self-sterile plants; c) after self-pollination necrosis starts in the tissue of the stigma, in some orchids after F. MÜLLER (1867); d) in pollon mixtures there is an inhibitory interaction between two types of pollen and the female tissue; Gossypium according to BALLS (1911), KEARNEY 1923, 1928, KEARNEY AND HARRISON (1924). A more complete discussion is found in BRIEGER 1930). 6) A list of the gametophyte factors so far localized in corn is given. CHROMOSOME IV Ga 1 : MANGELSDORF AND JONES (1925), EMERSON 1934). Ga 4 : BRIEGER (1945b). Sp 1 : MANGELSDORF (1931), SINGLETON AND MANGELSDORF (1940), BRIEGER (1945a). CHROMOSOME V Ga 2 : BRIEGER (1937a). CHROMOSOME VI BRIEGER, TIDBURY AND TSENG (1938) found indications of a gametophyte factor altering the segregation of yellow endosperm y1. CHROMOSOME IX Ga 3 : BRIEGER, TIDBURY AND TSENG (1938). While the competition in these six cases is essentially determined by one pair of factors, the degree of elimination may be variable, as shown for Ga2 (BRIEGER, 1937), for Ga4 (BRIEGER 1945a) and for Spl (SINGLETON AND MANGELSDORF 1940, BRIEGER 1945b). The action of a gametophyte factor altering the segregation of waxy (perhaps Ga3) is increased by the presence of the sul factor which thus acts as a modifier (BRINCK AND BURNHAM 1927). A polyfactorial case of gametophyte competition has been found by JONES (1922) and analysed by DEMEREC (1929) in rice pop corn which rejects the pollen tubes of other types of corn. Preference for selfing or for brothers-sister mating and partial elimination of other pollen tubes has been described by BRIEGER (1936). 7) HARLAND'S (1943) very ingenious idea is discussed to use pollen tube factors in applied genetics in order to build up an obstacle to natural crossing as a consequence of the rapid pollen tube growth after selfing. Unfortunately, HARLAND could not obtain the experimental proof of the praticability of his idea, during his experiments on selection for minor modifiers for pollen tube grouth in cotton. In maize it should be possible to employ gametophyte factors to build up lines with preference for crossing, though the method should hardly be of any practical advantage.

Genes, chromosomes and development

Procura-se mostrar, no presente artigo, que nenhuma teoria micromerista consegue explicar os fenômenos genéticos, por não ser possível conferir a partículas a independência funcional relacionada com o trabalho que devem exercer de maneira específica no organismo. O fato dos gens se encontrarem em tôdas as células do organismo em desenvolvimento, mostra que essas entidades desenvolvem distintas atividades em diferentes tecidos. Mas acontece, que nem a microscopia eletrônica, nem a bioquímica, consegue descobrir nos cormossômios algo que possa corresponder ao conceito de gen-conta-de rosário da genética clássica. Entretanto, o cromossômio considerado como um todo pode com vantagem substituir os gens no seu papel de determinar os caracteres do organismo. Admitindo-se que os cromossômios se determinam com as células de que fazem parte, uns para trabalhar nos esboços de asas, outros nos de olhos, patas ou outras estruturas, compreende-se fàcilmente, que, por intermédio de distintos membros do clone que se inicia com a primeira divisão do ovo, uma dada sorte de cromossômio, funcionando como um todo especializado, pode exercer as atividades específicas que lhe são atribuídas. E isso, de pleno acôrdo com a embriologia experimental.

Early life stress-induced histone acetylations correlate with activation of the synaptic plasticity genes Arc and Egr1 in the mouse brain

Magdeburg, Univ., Fak. für Naturwiss., Diss., 2013

Flippase (FLP) recombinase-mediated marker recycling in the dermatophyte Arthroderma vanbreuseghemii.

Biological processes can be elucidated by investigating complex networks of relevant factors and genes. However, this is not possible in species for which dominant selectable markers for genetic studies are unavailable. To overcome the limitation in selectable markers for the dermatophyte Arthroderma vanbreuseghemii (anamorph: Trichophyton mentagrophytes), we adapted the flippase (FLP) recombinase-recombination target (FRT) site-specific recombination system from the yeast Saccharomyces cerevisiae as a selectable marker recycling system for this fungus. Taking into account practical applicability, we designed FLP/FRT modules carrying two FRT sequences as well as the flp gene adapted to the pathogenic yeast Candida albicans (caflp) or a synthetic codon-optimized flp (avflp) gene with neomycin resistance (nptII) cassette for one-step marker excision. Both flp genes were under control of the Trichophyton rubrum copper-repressible promoter (PCTR4). Molecular analyses of resultant transformants showed that only the avflp-harbouring module was functional in A. vanbreuseghemii. Applying this system, we successfully produced the Ku80 recessive mutant strain devoid of any selectable markers. This strain was subsequently used as the recipient for sequential multiple disruptions of secreted metalloprotease (fungalysin) (MEP) or serine protease (SUB) genes, producing mutant strains with double MEP or triple SUB gene deletions. These results confirmed the feasibility of this system for broad-scale genetic manipulation of dermatophytes, advancing our understanding of functions and networks of individual genes in these fungi.

Effect of single nucleotide polymorphisms in cytochrome P450 isoenzyme and N-acetyltransferase 2 genes on the metabolism of artemisinin-based combination therapies in malaria patients from Cambodia and Tanzania.

The pharmacogenetics of antimalarial agents are poorly known, although the application of pharmacogenetics might be critical in optimizing treatment. This population pharmacokinetic-pharmacogenetic study aimed at assessing the effects of single nucleotide polymorphisms (SNPs) in cytochrome P450 isoenzyme genes (CYP, namely, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, and CYP3A5) and the N-acetyltransferase 2 gene (NAT2) on the pharmacokinetics of artemisinin-based combination therapies in 150 Tanzanian patients treated with artemether-lumefantrine, 64 Cambodian patients treated with artesunate-mefloquine, and 61 Cambodian patients treated with dihydroartemisinin-piperaquine. The frequency of SNPs varied with the enzyme and the population. Higher frequencies of mutant alleles were found in Cambodians than Tanzanians for CYP2C9*3, CYP2D6*10 (100C → T), CYP3A5*3, NAT2*6, and NAT2*7. In contrast, higher frequencies of mutant alleles were found in Tanzanians for CYP2D6*17 (1023C → T and 2850C → T), CYP3A4*1B, NAT2*5, and NAT2*14. For 8 SNPs, no significant differences in frequencies were observed. In the genetic-based population pharmacokinetic analyses, none of the SNPs improved model fit. This suggests that pharmacogenetic data need not be included in appropriate first-line treatments with the current artemisinin derivatives and quinolines for uncomplicated malaria in specific populations. However, it cannot be ruled out that our results represent isolated findings, and therefore more studies in different populations, ideally with the same artemisinin-based combination therapies, are needed to evaluate the influence of pharmacogenetic factors on the clearance of antimalarials.