Modelagem molecular na caracterização eletrônica de oligopeptídeos e na descrição quântica da interação fármaco-receptor

In this dissertation, the theoretical principles governing the molecular modeling were applied for electronic characterization of oligopeptide α3 and its variants (5Q, 7Q)-α3, as well as in the quantum description of the interaction of the aminoglycoside hygromycin B and the 30S subunit of bacterial ribosome. In the first study, the linear and neutral dipeptides which make up the mentioned oligopeptides were modeled and then optimized for a structure of lower potential energy and appropriate dihedral angles. In this case, three subsequent geometric optimization processes, based on classical Newtonian theory, the semi-empirical and density functional theory (DFT), explore the energy landscape of each dipeptide during the search of ideal minimum energy structures. Finally, great conformers were described about its electrostatic potential, ionization energy (amino acids), and frontier molecular orbitals and hopping term. From the hopping terms described in this study, it was possible in subsequent studies to characterize the charge transport propertie of these peptides models. It envisioned a new biosensor technology capable of diagnosing amyloid diseases, related to an accumulation of misshapen proteins, based on the conductivity displayed by proteins of the patient. In a second step of this dissertation, a study carried out by quantum molecular modeling of the interaction energy of an antibiotic ribosomal aminoglicosídico on your receiver. It is known that the hygromycin B (hygB) is an aminoglycoside antibiotic that affects ribosomal translocation by direct interaction with the small subunit of the bacterial ribosome (30S), specifically with nucleotides in helix 44 of the 16S ribosomal RNA (16S rRNA). Due to strong electrostatic character of this connection, it was proposed an energetic investigation of the binding mechanism of this complex using different values of dielectric constants (ε = 0, 4, 10, 20 and 40), which have been widely used to study the electrostatic properties of biomolecules. For this, increasing radii centered on the hygB centroid were measured from the 30S-hygB crystal structure (1HNZ.pdb), and only the individual interaction energy of each enclosed nucleotide was determined for quantum calculations using molecular fractionation with conjugate caps (MFCC) strategy. It was noticed that the dielectric constants underestimated the energies of individual interactions, allowing the convergence state is achieved quickly. But only for ε = 40, the total binding energy of drug-receptor interaction is stabilized at r = 18A, which provided an appropriate binding pocket because it encompassed the main residues that interact more strongly with the hygB - C1403, C1404, G1405, A1493, G1494, U1495, U1498 and C1496. Thus, the dielectric constant ≈ 40 is ideal for the treatment of systems with many electrical charges. By comparing the individual binding energies of 16S rRNA nucleotides with the experimental tests that determine the minimum inhibitory concentration (MIC) of hygB, it is believed that those residues with high binding values generated bacterial resistance to the drug when mutated. With the same reasoning, since those with low interaction energy do not influence effectively the affinity of the hygB in its binding site, there is no loss of effectiveness if they were replaced.

Mutagênese induzida por flavonóides presentes do decocto das cascas da aroeira (Schinus terebinthifolius, Raddi)

The decoction of Brazilian pepper tree barks (Schinus terebinthifolius, Raddi), is used in medicine as wound healing and antiinflamatory. Once extracts from this plant are used for acceleration of scar s process, it is important to study their mutagenic and genotoxic potential. In previous works in our laboratory, it was observed mutagenicity caused by the decoction when in high concentrations. Among the chemical compounds of this plant that could be able to induce mutation, the flavonoids were the only group that was referred to have either an oxidant or antioxidant potential. The flavonoids were isolated, purified and quantified by adsorptive column chromatography under silica gel, bacterial and in vitro genotoxic tests were realized to determine if the flavonoids were the responsible agents for this mutagenicity found. The tests realized with plasmidial DNA were indicative that the flavonoids are probably genotoxic, due to the presence of correlation between increase of the flavonoid concentration and in plasmidial DNA double strand breakage visualized in agarose gel, as well as they were capable to generated abasic sites shown by the in vitro treatment with exonuclease III. The same tests with plasmidial DNA in the presence of copper [10 µM] and of a Tris-HCl pH 7.5 [10 µM] buffer were realized with the isolated flavonoids to determine if there would be or not participation of reactive oxygen species (ROS). The transformation of plasmidial DNA in different bacterial strains proficient and deficient in DNA repair enzymes in the presence or not of a Tris-HCl buffer, suggests that the enzymes that repair oxidative lesions are necessary to repair the lesions generated by the flavonoids and that ROS are generated and are necessary to promote the lesions. Bacterial tests with Escherichia coli strains of the CC collection (deficient or not for DNA repair enzymes), showed that the flavonoids are able to increase the frequency of mutations, mainly in strains mutated in repair enzymes (MutM, MutY-glicosylases and double mutant), suggesting that these agents are responsible for the enhancement in the mutation rate. In order to determine the mutation spectrum caused by the flavonoids of the Brazilian pepper tree stem bark, plasmidial DNA previously treated with the flavonoids were transformed in bacterial strains deficient and proficient in the DNA repair enzymes, followed by a blue-white selection with X-gal, DNA amplification by PCR and sequencing the positive mutant clones. Analysis of the mutants obtained from strains CC104, CC104mutM, CC104mutY, CC104mutMmutY, BW9101, BW9109 indicated a predominance of some mutations like G:C to C:G that can be correlated with the origin of 8-oxoG, due to oxidative lesions caused by the flavonoids. So it can concluded that the flavonoid isolated or in fractions enriched on them are genotoxic and mutagenic, and their mutations are predominantly oxidative, mediated by ROS, and the lesions are recognized by the BER system. In this way it is proposed that the flavonoids can act in two different ways to generate the DNA lesion: 1. in a Fenton-like reaction, when the flavonoid are in the presence of metal ions and that together with the water generate ROS that promotes the DNA lesions; 2. in another way the lesions can be generated by the formation of ROS due to the internal chemical structure of the flavonoid molecule due to the quantity and location of hydroxyl groups, and so producing the DNA lesions, those lesions can be directly (suggested by the in vitro experiments) or indirectly done (supported by the experiments using the CC bacterial strains)

A objetivação do controle concreto de constitucionalidade nas decisões do Supremo Tribunal Federal

The independence of the United States and the revolutions that emerged in Europe in the eighteenth century led to the birth of the written constitution, with a mission to limit the power of the State and to ensure fundamental rights to citizens. Thus, the Constitution has become the norm and ultimate founding of the State. Because of this superiority felt the need to protect her, emerging from that constitutional jurisdiction, taking control of constitutionality of provisions his main instrument. In Brazil, the constitutionality control began with the Constitution of 1891, when "imported" the American model, which is named after incidental diffuse model of judicial review. Indeed, allowed that any judge or court could declare the unconstitutionality of the law or normative act in a concrete case. However, the Brazilian Constituent did not bring the U.S. Institute of stare decisis, by which the precedents of higher courts eventually link the below. Because of this lack, each tribunal Brazilian freely decide about the constitutionality of a rule, so that the decision took effect only between the parties to the dispute. This prompted the emergence of conflicting decisions between judicantes organs, which ultimately undermine legal certainty and the image of the judiciary. As a solution to the problem, was incorporated from the 1934 Constitution to rule that the Senate would suspend the law declared unconstitutional by the Supreme Court. With the introduction of abstract control of constitutionality, since 1965, the Supreme Court went on to also have the power to declare the invalidity of the provision unconstitutional, effectively against all without the need for the participation of the Senate. However, it remained the view that in case the Supreme Court declared the unconstitutionality of the fuzzy control law by the Senate would continue with the competence to suspend the law unconstitutional, thus the decision of the Praetorium Exalted restricted parties. The 1988 Constitution strengthened the abstract control expanding legitimized the Declaratory Action of Unconstitutionality and creating new mechanisms of abstract control. Adding to this, the Constitutional Amendment. No. 45/2004 brought the requirement of general repercussion and created the Office of Binding Precedent, both to be applied by the Supreme Court judgments in individual cases, thus causing an approximation between the control abstract and concrete constitutional. Saw themselves so that the Supreme Court, to be the guardian of the Constitution, its action should be directed to the trial of issues of public interest. In this new reality, it becomes more necessary the participation of the Senate to the law declared unconstitutional in fuzzy control by the Supreme Court can reach everyone, because such an interpretation has become obsolete. So, to adapt it to this reality, such a rule must be read in the sense that the Senate give publicity to the law declared unconstitutional by the Supreme Court, since mutated constitutional

Expressão imuno-histoquimica da cicloxigenase-2 e p53 em cancinoma epidermóide oral

Squamous cell carcinoma is the most common malignant neoplasm in the oral cavity, accounting for more than 90% of all malignancies in this location. Cyclooxygenases (COX s) are key enzymes on arachidonic acid metabolism and prostaglandin synthesis, being expressed basically in two forms: the constitutive (COX-1) and the inducible (COX-2). Increased levels on the expression of COX-2 have been implicated in the pathogenesis tumor progression of various forms of human cancer, including oral squamous cell carcinoma, some of what suggesting a possible interaction between COX-2 and the protein expressed by the tumor suppressor gene p53, mutated in more than 50% of all human cancers. The mean of the present research consisted in analyze the correlation between the expression of COX-2 and p53, at the protein level, as well as evaluate the difference on the expression of these two proteins with the histological grading of malignancy. 34 cases of oral squamous cell carcinoma were selected and graded according to the histological grading system proposed by Bryne (1998) and the labeling indexes (LI s) for COX-2 and p53 evaluated using immunohistochemistry method. The results revealed that COX-2 was expressed in increased levels in most of the specimens, although there was no statistic significant correlation between LI s from COX-2 and p53 (p>0.05), and there were no statistical differences on the expression of these proteins between tumors of high and low grade of malignancy (p>0.05). Interestingly, the expression of COX-2 and p53 was detected in fragments of dysplastic oral epithelium adjacent to tumor areas, on basal and suprabasal layers. The absence of statistical correlation between the expression of COX-2 and p53 proteins do not rule ot the existence of a relation between them, were it may reflect the diversity of regulatory pathways between both, different direct and indirect inhibitory effects of COX-2 over p53, as well as the wide range of activation macheenisms for COX-2 and mutational status of the p53 gene Another conclusion point that the increased expression of COX-2 observed in oral squamous cell carcinomas suggest a role for this protein in the processes of pathogenesis and tumoral evolution of this malignant neoplasm