cAMP is a second messenger in a variety of chemosensory receptors, including taste buds and glucose-sensitive pancreatic beta-cells. cAMP is modulated during taste transduction, yet the significance of cAMP changes and the taste cell types in which they occur (Type I glial-like; Type II Receptor; Type III Presynaptic) remain unclear. I developed techniques to image real-time changes in intracellular cAMP in taste cells using genetically-encoded cAMP reporters. This FRET-based reporter permits one to measure single-cell cAMP levels with excellent spatial and temporal resolution (Zaccolo & Pozzan 2002, Science 295:1711). Using a biolistic approach I have transfected rat fungiform taste buds with cAMP reporter plasmids. Focal application of bitter tastant to living fungiform tastebuds in situ produced a decrease in [cAMP]i within individual taste receptor cells. These results are qualitatively similar to previous biochemical measurements from homogenized taste tissue (Yan et al. 2001, Am J Physiol Cell Physiol 280:C742) but are now allowing us to examine the cAMP response in individual, identified cells. I next explored the effect of elevating cAMP on calcium levels, using Fura-2 imaging of isolated mouse vallate taste buds. Elevating [cAMP]i in taste buds evoked calcium responses in presynaptic/Type III taste cells, which do not express GAD1. cAMP induced responses were generated by calcium influx. Using pharmacological antagonists, I determined that the calcium influx triggered by cAMP is through L-type calcium channels, whereas influx following depolarization is primarily through P/Q-type calcium channels. Consistent with these data, single cell RT-PCR showed that the L-type subunit (alpha 1C) was expressed primarily in GAD-negative Presynaptic cells, while the P/Q-type (alpha 1A) was expressed in all Presynaptic cells. Thus, cAMP may modulate the function of synapses in some taste cells. Finally, we have developed a mouse strain expressing a cAMP reporter in a tissue-specific and tetracycline-inducible manner. We crossed this mouse with another strain expressing tet-activator in beta cells of the pancreas. Such islets responded to increasing concentrations of glucose (5.5 to 35mM) with an increase in cAMP levels. The half maximum of 9mM glucose for the cAMP response corresponds well with reported glucose concentrations that elicit insulin release from whole islets. Stimulating pancreatic islets with glucose is known to drive calcium influx into beta-cells. When we simultaneously imaged both second messengers, we found that cAMP changes precede and are independent of calcium changes. In conclusion, these studies have outlined novel potential functions for cAMP signaling in the transduction of both primary tastant and plasma glucose information. In addition, the flexibility of the tet-system will enable cAMP reporter expression in numerous cell types, including those which mediate gustatory transduction.

The phenolamines octopamine and tyramine control, regulate, and modulate many physiological and behavioral processes in invertebrates. Vertebrates possess only small amounts of both substances, and thus, octopamine and tyramine, together with other biogenic amines, are referred to as “trace amines.” Biogenic amines evoke cellular responses by activating G-protein-coupled receptors. We have isolated a complementary DNA (cDNA) that encodes a biogenic amine receptor from the American cockroach Periplaneta americana, viz., Peatyr1, which shares high sequence similarity to members of the invertebrate tyramine-receptor family. The PeaTYR1 receptor was stably expressed in human embryonic kidney (HEK) 293 cells, and its ligand response has been examined. Receptor activation with tyramine reduces adenylyl cyclase activity in a dose-dependent manner (EC50 350 nM). The inhibitory effect of tyramine is abolished by co-incubation with either yohimbine or chlorpromazine. Receptor expression has been investigated by reverse transcription polymerase chain reaction and immunocytochemistry. The mRNA is present in various tissues including brain, salivary glands, midgut, Malpighian tubules, and leg muscles. The effect of tyramine on salivary gland acinar cells has been investigated by intracellular recordings, which have revealed excitatory presynaptic actions of tyramine. This study marks the first comprehensive molecular, pharmacological, and functional characterization of a tyramine receptor in the cockroach.

Reductionist biology has yielded tremendous insight into the basis of biochemistry and genetic disease. However, the remarkable failure of reductionist biology to explain complex problems, especially cancer, has led to the development of systems biology. The vast complexity of biological systems remains the most difficult problem in biology today. In order to understand this complexity, we need tools to massively multiplex measurements of a signaling network. Therefore, we developed lysate microarray technology to fill this need. In this work, we discuss three ways in which lysate microarrays were applied to human disease. In the first work, we discuss a key stage in malaria development. The liver-stage malaria parasite represents a promising target for intervention, and we present the first use of lysate microarray technology as a screening tool for host-parasite interactions in an infectious disease. We identified three cancer-related pathways that are modified in malaria infection, and studied the p53 pathway in depth. Our finding that the parasite downregulates p53 and that treatment with Nutlin-3 strongly decreases parasite load may lead to the development of a prophylactic malaria vaccine. In the second work, we began by screening drug combinations and varying dosing schedule in triple-negative breast cancers (TNBCs). We systematically explored stimulation space and collected a large lysate microarray dataset, which was used for statistical analysis. We identified a sensitization effect when a growth factor signaling inhibitor was presented before a genotoxic agent. This sensitization was generalizable among a subset of TNBCs and may generally be important for cancers driven by growth factor signaling, as we found the effect extends to nonTNBC cancers. We hope this data will be useful in guiding cancer treatment strategies in patients. In the third work, we study the changing role of the DNA Damage Response (DDR) as a cell line evolves towards cancer. We used the MCF10A progression series and studied how these cell lines respond to genotoxic agents. We identified differences in cell fates after treatment, and collected a large lysate microarray dataset for statistical analysis. Early analysis of the data indicates gross rewiring within the DDR between the MCF10A cell lines.

In the absence of ligand, certain growth factor receptors can be activated via G protein-coupled receptor (GPCR) activation in a process termed transactivation. Serotonin (5-HT) receptors can transactivate the receptor tyrosine kinase (RTK) platelet-derived growth factor (PDGF) ?? receptors in smooth muscle cells, but it is not known if similar pathways occur in neuronal cells. Here, it is shown that 5-HT can transiently increase the phosphorylation of PDGF?? receptors in a time- and concentration-dependent manner in SH-SY5Y neuroblastoma cells. This transactivation pathway was pertussis-toxin sensitive, and was dependent on phospholipase C activity, intracellular calcium signaling and subsequent protein kinase C activation. Exogenous application of non-lethal concentrations of H2O2 induced the phosphorylation of PDGF?? receptors in a concentration-dependent fashion, similar to that observed with 5-HT. Further investigation revealed reactive oxygen species (ROS) production as a necessary component in the transactivation pathway, as scavenging ROS eliminated PDGF?? receptor phosphorylation. NADPH oxidase was determined to be the likely source of ROS given that the NADPH oxidase inhibitors diphenyleneiodonium chloride and apocynin abrogated PDGF?? receptor transactivation. The role of Src tyrosine kinase was also investigated, and its location in this signaling cascade was determined to be downstream of calcium signaling, but upstream of NADPH oxidase activity. In addition, the activation of ERK1/2 in this system was elucidated to be independent of PDGF?? receptor transactivation. Interestingly, 5-HT also transactivated TrkB receptors, another RTK whose function is implicated in clinical depression. Expectedly, the enzymes in this mechanism were consistent with those revealed in 5-HT-to-PDGF?? receptor signaling. This cross-talk between 5-HT and RTKs such as TrkB and PDGF?? receptors identifies a potentially important signaling link between the serotonergic system and neurotrophic factor signaling in neurons that could have implications in mental health disorders including depression. Furthermore, although transactivation pathways are commonly initiated by a GPCR, recent reports have demonstrated that selective serotonin reuptake inhibitors (SSRIs) were able to block 5-HT-induced transactivation of PDGF?? receptors, suggesting that in addition to GPCRs, monoamine transporters may also be involved in RTK transactivation. SH-SY5Y cells pretreated with the SSRI fluoxetine blocked 5-HT-induced transactivation of the PDGF?? receptors, but not PDGF-induced PDGF?? receptor activation. Upon further examination, it was discovered that during the pretreatment period, fluoxetine itself was transiently transactivating the PDGF?? receptor via 5-HT2 receptors. By the end of the pretreatment period, the effects of fluoxetine on PDGF?? receptor phosphorylation had returned to baseline, and a subsequent transactivating stimulus (5-HT) failed to ???re-transactivate??? the PDGF?? receptor. Additional investigations demonstrated that 5-HT pretreatment can block dopamine-induced PDGF?? receptor transactivation, but not PDGF-induced PDGF?? receptor activation. This is the first demonstration of the heterologous desensitization of an RTK via a transactivation pathway, and this phenomenon is specific for transactivation pathways because in all cases the PDGF?? receptor ligand PDGF-BB was able to directly stimulate receptor activity in spite of GPCR agonist pretreatment. Heterologous desensitization in transactivation signaling reveals a previously unknown short-term ???blackout??? period wherein no further transactivation signaling can occur to potentially exploit the mitogenic effects of RTK activation.

Catalyzed by protein kinases, phosphorylation is the most important post-translational modification in eukaryotes and is involved in the regulation of almost all cellular processes. Investigating phosphorylation events and how they change in response to different biological conditions is integral to understanding cellular signaling processes in general, as well as to defining the role of phosphorylation in health and disease. A recently-developed technology for studying phosphorylation events is the kinome microarray, which consists of several hundred "spots" arranged in a grid-like pattern on a glass slide. Each spot contains many peptides of a particular amino acid sequence chemically fixed to the slide, with different spots containing peptides with different sequences. Each peptide is a subsequence of a full protein, containing an amino acid residue that is known or suspected to undergo phosphorylation in vivo, as well as several surrounding residues. When a kinome microarray is exposed to cell lysate, the protein kinases in the lysate catalyze the phosphorylation of the peptides on the array. By measuring the degree to which the peptides comprising each spot are phosphorylated, insight can be gained into the upregulation or downregulation of signaling pathways in response to different biological treatments or conditions. There are two main computational challenges associated with kinome microarrays. The first is array design, which involves selecting the peptides to be included on a given array. The level of difficulty of this task depends largely on the number of phosphorylation sites that have been experimentally identified in the proteome of the organism being studied. For instance, thousands of phosphorylation sites are known for human and mouse, allowing considerable freedom to select peptides that are relevant to the problem being examined. In contrast, few sites are known for, say, honeybee and soybean. For such organisms, it is useful to expand the set of possible peptides by using computational techniques to predict probable phosphorylation sites. In this thesis, existing techniques for the computational prediction of phosphorylation sites are reviewed. In addition, two novel methods are described for predicting phosphorylation events in organisms with few known sites, with each method using a fundamentally different approach. The first technique, called PHOSFER, uses a random forest-based machine-learning strategy, while the second, called DAPPLE, takes advantage of sequence homology between known sites and the proteome of interest. Both methods are shown to allow quicker or more accurate predictions in organisms with few known sites than comparable previous techniques. Therefore, the use of kinome microarrays is no longer limited to the study of organisms having many known phosphorylation sites; rather, this technology can potentially be applied to any organism having a sequenced genome. It is shown that PHOSFER and DAPPLE are suitable for identifying phosphorylation sites in a wide variety of organisms, including cow, honeybee, and soybean. The second computational challenge is data analysis, which involves the normalization, clustering, statistical analysis, and visualization of data resulting from the arrays. While software designed for the analysis of DNA microarrays has also been used for kinome arrays, differences between the two technologies prompted the development of PIIKA, a software package specifically designed for the analysis of kinome microarray data. By comparing with methods used for DNA microarrays, it is shown that PIIKA improves the ability to identify biological pathways that are differentially regulated in a treatment condition compared to a control condition. Also described is an updated version, PIIKA 2, which contains improvements and new features in the areas of clustering, statistical analysis, and data visualization. Given the previous absence of dedicated tools for analyzing kinome microarray data, as well as their wealth of features, PIIKA and PIIKA 2 represent an important step in maximizing the scientific value of this technology. In addition to the above techniques, this thesis presents three studies involving biological applications of kinome microarray analysis. The first study demonstrates the existence of "kinotypes" - species- or individual-specific kinome profiles - which has implications for personalized medicine and for the use of model organisms in the study of human disease. The second study uses kinome analysis to characterize how the calf immune system responds to infection by the bacterium Mycobacterium avium subsp. paratuberculosis. Finally, the third study uses kinome arrays to study parasitism of honeybees by the mite Varroa destructor, which is thought to be a major cause of colony collapse disorder. In order to make the methods described above readily available, a website called the SAskatchewan PHosphorylation Internet REsource (SAPHIRE) has been developed. Located at the URL http://saphire.usask.ca, SAPHIRE allows researchers to easily make use of PHOSFER, DAPPLE, and PIIKA 2. These resources facilitate both the design and data analysis of kinome microarrays, making them an even more effective technique for studying cellular signaling.

Orientador: Marcelo Ganzarolli de Oliveira / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Quimica / Made available in DSpace on 2018-08-15T02:21:46Z (GMT). No. of bitstreams: 1 Sassonia_RogerioCorte_D.pdf: 2947110 bytes, checksum: fc316c83bed9508d2e27063d89528d56 (MD5) Previous issue date: 2009 / Resumo: Este trabalho apresenta os resultados da aplicação da titulação calorimétrica isotérmica na caracterização termodinâmica de reações de S-nitrosação de tióis e de interações proteínaproteína e proteína-íon. Foram estudadas as reações de S-nitrosação da N-acetil-L-cisteína (NAC), L-cisteína (CYS), L-glutationa (GLU) e do ácido mercaptosuccínico. Também foram avaliadas as interações entre a proteína sinalizadora Shc (Src homology collagen-like) e as proteínas glutationa S-transferase (GST) e a ciclofilina A (CypA) e a interação entre a região Cterminal da proteína humana EFHC1 (EFHC1-C) com íons Ca e Mg. Os valores da variação de entalpia revelaram que a S-nitrosação é um fenômeno exotérmico e ocorre com diminuição de entropia. Estes dados termodinâmicos revelam que as reações de S-nitrosação investigadas são entalpicamente dirigidas a 25 °C (1 atm) e possuem valores semelhantes de variações de entalpia, entropia e energia livre, apesar das diferenças entre as estruturas químicas dos tióis. Verificou-se que a proteína EFHC1C liga-se tanto a íons Ca quanto Mg numa estequiometria de 1:1, com afinidades definidas por diferentes contribuições entálpicas e entrópicas. Este dado confirmou a existência de um suposto domínio EF-hand ligante de Ca na porção C-terminal previsto pela seqüência primária da EFHC1C. Por outro lado, a EFHC1C perde sua capacidade de interação com íons Ca e Mg em solução sem 1,4-ditiotreitol (DTT), provavelmente, devido à formação de dímeros. A ausência de sinais térmicos de ITC mostrou que nem a proteína GST, nem a proteína CypA interagem com a proteína Shc nas condições experimentais usadas. / Abstract: This work presents the results of isothermal titration calorimetry application in the thermodynamic characterization of thiol nitrosation reactions, protein-protein and protein-ion interactions. The S-nitrosation reactions of N-acetyl-L-cysteine (NAC), L-cysteine (CYS), Lglutathione (GLU) and acid mercaptosuccinic were studied. The interactions of the signaling protein Shc (Src homology collagen-like) with glutathione S-transferase (GST) and ciclofilina A (CypA) and of the EF-hand motif from human EFHC1C with Caand Mg ions were also evaluated. Enthalpy change values revealed that the S-nitrosation reaction is an exothermic phenomenum associated to a decrease in entropy. These thermodynamic data show that the S-nitrosation reactions investigated are enthalpically driven at 25 °C (1 atm) and have similar enthalpic, entropic and free energy change values, despite the differences among the chemical structures of the thiols. It was verified that the EFHC1C protein binds to both Ca and Mg ions in a 1:1 stoichiometry with affinities defined by different enthalpic and entropic contributions. These data confirmed the presence of a putative EF-hand Ca-binding motif at the C-terminal portion as expected by the primary sequence of EFHC1C. On the other hand, EFHC1C losses its ability to interact with Ca and Mgions in solution without 1,4-ditiotreitol (DTT) likely due to protein dimerization. The absence of ITC thermal signals showed that neither GST nor CypA interact with the Shc protein in the experimental conditions used. / Doutorado / Físico-Química / Doutor em Ciências

The catecholamines norepinephrine and epinephrine are important regulators of vertebrate physiology. Insects such as honeybees do not synthesize these neuroactive substances. Instead, they use the phenolamines tyramine and octopamine for similar physiological functions. These biogenic amines activate specific members of the large protein family of G protein-coupled receptors (GPCRs). Based on molecular and pharmacological data, insect octopamine receptors were classified as either - or -adrenergic-like octopamine receptors. Currently, one - and four -receptors have been molecularly and pharmacologically characterized in the honeybee. Recently, an 2-adrenergic-like octopamine receptor was identified in Drosophila melanogaster (DmOct2R). This receptor is activated by octopamine and other biogenic amines and causes a decrease in intracellular cAMP ([cAMP]i). Here, we show that the orthologous receptor of the honeybee (AmOct2R), phylogenetically groups in a clade closely related to human 2-adrenergic receptors. When heterologously expressed in an eukaryotic cell line, AmOct2R causes a decrease in [cAMP]i. The receptor displays a pronounced preference for octopamine over tyramine. In contrast to DmOct2R, the honeybee receptor is not activated by serotonin. Its activity can be blocked eciently by 5-carboxamidotryptamine and phentolamine. The functional characterization of AmOct2R now adds a sixth member to this subfamily of monoaminergic receptors in the honeybee and is an important step towards understanding the actions of octopamine in honeybee behavior and physiology.

The tumor suppressor gene HIC1 encodes a BTB/POZ transcription repressor. Its promotor is frequently hypermetylated in large numbers of tumors. HIC1 also functions as a negative modulator of the Wnt signalling pathway, which fundamentally participates in regulation of stem cell renewal of the intestinal epithelium. Thanks to its structural features the intestinal epithelium represents a convenient model tissue to study stem cells and their pathology. To overcome the embryonic lethality of the complete Hic1 "knock-out" the conditional deletion of the gene in adult mouse tissue was chosen to evaluate the Hic1 biological aktivity. By the chip expression analysis of mouse embryonic fibroblasts we discovered a number of new target genes of Hic1, the most interesting of them - in respect to cancer - we considered the Toll-like receptor 2 gene. The expression of Hic1 target genes is likely to be co-regulated by p53 although the direct regulation wasn't proved. Hic1 affects the proportion of the differentiated intestinal epithelial cells types possibly via regulation of Atoh1. After conditional deletion of Hic1 in the intestinal epithelium we observed and quantitatively confirmed a significant increase of the amounts of goblet cells. We concluded that Hic1 affects differentiation pathways in intestinal...

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Alžbeta Horvátová Supervisor: prof. PharmDr. Petr Pávek PhD. Title of diploma thesis: Anti-inflammatory effects of ursodeoxycholyl lysophosphatidylethanolamide on THP-1 human macrophages via Toll-like receptor 4 Nonalcoholic steatohepatitis (NASH) became the most common liver disease in developed countries. It is well-known that the level of protectant phosphatidylcholine (PC) is decreased in NASH. The bile acid-phospholipid conjugate ursodeoxycholyl lysophosphatidylethanolamide (UDCA-LPE) was designed in order to specifically deliver PC to hepatocytes. However, previous studies have proved that UDCA-LPE possesses its proper hepatoprotectant capacity and exhibits anti-apoptotic, anti-inflammatory, anti-fibrotic properties and also improved steatosis and hyperlipidaemia in various models in vivo. These effects may be mediated secondary through modulation of immune system. Therefore, in order to dissect if UDCA-LPE directly influences immune cells in vitro, release of pro-inflammatory cytokines TNFα, IL-6 and IL-1β in LPS-induced THP-1-derived human macrophages was measured by ELISA. Moreover, effects of UDCA-LPE on MAPK signalling pathways and nuclear translocation of NFκB were...

As vias de sinalização celular por meio de moléculas são um dos principais meios de controle funcional de um organismo. O entendimento das funções de moléculas sinalizadoras facilita a compreensão das vias metabólicas de um organismo, assim possibilitando uma melhor compreensão de vários eventos biológicos e também de várias doenças. A sinalização pelo ácido retinóico (AR), e seus derivados, é responsável pelo controle de várias funções, por exemplo: crescimento celular, diferenciação celular, formação da retina, desenvolvimento cardíaco e também relacionado a várias patologias como diabetes, obesidades, cânceres, e doenças cardiovasculares. A ação do ácido retinóico é controlada em dois níveis: no metabolismo de síntese/degradação e na sua utilização na sinalização para a expressão gênica. A maquinaria que controla o metabolismo inclui as enzimas de síntese do AR (aldeído desidrogenase ALDH) e as enzimas de degradação do AR (Cyp26), que controlam a distribuição espaço-temporal do AR durante a embriogênese. As ALDHs são enzimas NAD(P)+ dependentes, que oxidam uma ampla gama de aldeídos para os seus correspondentes ácidos carboxílicos, sendo ALDH1A2 a principal enzima na transformação de retinal em ácido retinóico. A maquinaria da sinalização celular por AR contém os receptores nucleares controlados por AR (RARs) que estão envolvidos com o controle da transcrição gênica. Os mecanismos de controle de expressão mais comuns são os que ocorrem na fase transcricional. Um desses mecanismos envolve proteínas que se ligam às regiões promotoras de transcrição, representadas por trechos de DNA que geralmente estão localizados próximo à região de início da transcrição, mas que também podem estar a centenas ou até milhares de pares de bases desse início. Essas proteínas modulam a maquinaria transcricional, podendo ativá-la ou inibi-la. A associação de várias técnicas como a biologia molecular, bioinformática, filogenia, análises estruturais de biomoléculas, mecânica molecular e métodos termodinâmicos tem se mostrado uma poderosa abordagem para compreensão de sistemas biológicos simplificando e agilizando o desenvolvimento do conhecimento científico. Nessa direção, esse estudo desenvolveu duas análises: a primeira estudando a evolução das funções das enzimas ALDH, utilizando-se de técnicas de genômica combinatória, filogenia, bioinformática, estrutura de biomoléculas e de biologia do desenvolvimento, tentando compreender o modo como as ALDHs, que apresentam as seqüências de aminoácidos bastante similares, puderam divergir para gerar funções diversas como a destoxificação e a sinalização. Para este estudo foram analisados os genomas de 487 organismos em busca de seqüências de ALDHs e também o genoma do organismo modelo Branchiostoma floridae. Foram obtidas 190 seqüências que foram utilizadas em uma análise filogenética para tentar compreender a função primordial e também para definir grupos de aminoácidos candidatos a marcadores das diferentes famílias de ALDHs. Essas 190 seqüências também foram modeladas estruturalmente e analisada a forma e o volume do canal onde se aloja o aldeído a ser oxidado. A partir dessas informações foi possível prever que as ALDHs passaram das funções ancestrais de controle do padrão corporal para algo mais abrangente como funções protetoras. A segunda análise, utilizando-se das estruturas tridimensionais dos fatores de transcrição ligados ao DNA em diferentes posições e submetendo esses complexos a processos de mecânica molecular, cálculos termodinâmicos e análises das ligações de hidrogênio para tentar prever os mais prováveis sítios de interação entre os receptores e o DNA. O modelo escolhido para essa análise foram os fatores de transcrição regulados por ácido retinóico o RAR e RXR utilizando a região promotora do gene RARE-2 para avaliar as mais prováveis regiões de ligação desses fatores. Para esse estudo foram construídos 71 complexos proteína-DNA que foram submetidos a processos de mecânica molecular e cálculos termodinâmicos. A partir dessas informações foi possível prever uma região de maior afinidade entre o fator de transcrição e o DNA. As análises de ligações de hidrogênio possibilitaram definir exatamente a região de interação entre os fatores de transcrição e o DNA, e também descrever as interações moleculares responsáveis pela especificidade da interação. / Cellular signaling paths through molecules are one of the main processes of functional control of an organism. The comprehension of signaling molecules functions enables one to understand the metabolic pathways of an organism, along with related biological events and several diseases. The signaling through retinoic acid (RA) and its secondary products is responsible for controlling several functions, such as cellular growth and differentiation, retinas formation and cardio development, and is also related to several pathologies such as diabetes, obesity, cancers and cardiovascular disorders. There are two levels of control of retinoic acid activity: synthesis/degradation metabolism and its use in gene expression signaling. The machinery that controls the metabolism includes RAs synthesis (aldehyde dehydrogenase ALDH) and degradation (Cyp26) enzymes, which control the space-temporal distribution of RA during the embryogenesis. The ALDHs are NAD(P)+ dependent enzymes that oxidize many types of aldehydes into the related carboxylic acids, being the ALDH1A2 the main enzyme involved in the process of transformation of retinal into retinoic acid. The machinery of cellular signaling through RA contains the nuclear receptors controlled by RA (RARs) that are involved in the control of gene transcription. The most common mechanisms of expression control are the ones that occur during the transcriptional phase. One of these mechanisms involves proteins that bind to the transcription promoter regions, represented by DNA sequences that are usually located close to the region where the transcription starts, but can also be hundreds or thousands of base pairs apart from the starting point. These proteins modulate the transcriptional machinery, being responsible for both its activation and inhibition. The association of several techniques as molecular biology, bioinformatics, phylogeny, structural analysis of biomolecules, molecular mechanics and thermodynamic methods has been shown as a powerful tool for the understanding of biological systems, simplifying and speeding up the production of related scientific knowledge. Facing this direction, the present study developed two analyses. The first one studied the evolution of ALDH enzymes functions, using the techniques of combinatory genomic, phylogeny, bioinformatics, structure of biomolecules and developmental biology, in the attempt of understanding how the ALDHs could diverge and acquire different functions as detoxification and signaling, despite the fact that they have very similar aminoacid sequences. For this study, ALDHs sequences were searched for in the genome of 487 organisms plus the model organisms, Branchiostoma floridae. All 190 sequences obtained were used in a phylogenetic analysis, in the attempt of understanding the primordial function of the enzyme and defining possible groups of conserved aminoacids in the different families of ADLHs. These 190 sequences were also structurally modeled and the shape and volume of the channel where the aldehyde is placed to be oxidized were analyzed. Based on this information, it became possible to predict that the ALDHs moved from ancestral functions of corporal pattern control to a wider spectrum of protection functions. For the second analysis we submitted the complex formed by tridimensional structures of the transcriptional factors bond to DNA in different positions to processes of molecular mechanics, thermodynamic calculi and analysis of the hydrogen bonds, in order to predict the most probable sites of interaction between the receptors and the DNA. The model chosen for this analysis were the transcription factors regulated by retinoic acid, RAR and RXR, using the promoter region of the gene RARE-2 to assay the most probable binding regions of these factors. For this study, 71 protein-DNA complexes were built and submitted to processes of molecular mechanics and thermodynamic calculi. Based on the resulting data, it became possible to predict a region of greater affinity between the transcription factor and the DNA. The analyses of hydrogen bonds enabled us to define the exact region where the interaction between the transcription factor and the DNA takes place and also enabled us to describe the molecular interactions responsible for the specificity of this interaction.