Síntese de derivados de adenina e guanina

Tese de Doutoramento em Ciências (Especialidade em Química)

Neuroendocrine factors regulate retinoic acid receptors in normal and hypoplastic lung development

Congenital diaphragmatic hernia (CDH) is characterised by a spectrum of lung hypoplasia and consequent pulmonary hypertension, leading to high morbidity and mortality rates. Moreover, CDH has been associated with an increase in the levels of pulmonary neuroendocrine factors, such as bombesin and ghrelin, and a decrease in the action of retinoic acid (RA). The present study aimed to elucidate the interaction between neuroendocrine factors and RA. In vitro analyses were performed on Sprague-Dawley rat embryos. Normal lung explants were treated with bombesin, ghrelin, a bombesin antagonist, a ghrelin antagonist, dimethylsulfoxide (DMSO), RA dissolved in DMSO, bombesin plus RA and ghrelin plus RA. Hypoplastic lung explants (nitrofen model) were cultured with bombesin, ghrelin, bombesin antagonist or ghrelin antagonist. The lung explants were analysed morphometrically, and retinoic acid receptor (RAR) α, β and γ expression levels were assessed via Western blotting. Immunohistochemistry analysis of RAR was performed in normal and hypoplastic lungs 17.5 days post-conception (dpc). Compared with the controls, hypoplastic lungs exhibited significantly higher RARα/γ expression levels. Furthermore considering hypoplastic lungs, bombesin and ghrelin antagonists decreased RARα/γ expression. Normal lung explants (13.5 dpc) treated with RA, bombesin plus RA, ghrelin plus RA, bombesin or ghrelin exhibited increased lung growth. Moreover, bombesin and ghrelin increased RARα/γ expression levels, whereas the bombesin and ghrelin antagonists decreased RARα/γ expression. This study demonstrates for the first time that neuroendocrine factors function as lung growth regulators, sensitising the lung to the action of RA through up-regulation of RARα and RARγ.

Bio-detoxification of mycotoxins by lactic acid bacteria from different food matrices

Lactic acid bacteria (LAB) play a key role in the biopreservation of a wide range of fermented food products, such as yogurt, cheese, fermented milks, meat, fish, vegetables (sauerkraut, olives and pickles), certain beer brands, wines and silage, allowing their safe consumption, which gave to these bacteria a GRAS (Generally Recognised as Safe) status. Besides that, the use of LAB in food and feed is a promising strategy to reduce the exposure to dietary mycotoxins, improving their shelf life and reducing health risks, given the unique mycotoxin decontaminating characteristic of some LAB. Mycotoxins present carcinogenic, mutagenic, teratogenic, neurotoxic and immunosuppressive effects over animals and Humans, being the most important ochratoxin A (OTA), aflatoxins (AFB1), trichothecenes, zearalenone (ZEA), fumonisin (FUM) and patulin. In a previous work of our group it was observed OTA biodegradation by some strains of Pediococcus parvulus isolated from Douro wines. So, the aim of this study was to enlarge the screening of the biodetoxification over more mycotoxins besides OTA, including AFB1, and ZEA. This ability was checked in a collection of LAB isolated from vegetable (wine, olives, fruits and silage) and animal (milk and dairy products, sausages) sources. All LAB strains were characterized phenotypically (Gram, catalase) and genotypically. Molecular characterisation of all LAB strains was performed using genomic fingerprinting by MSP- PCR with (GTG)5 and csM13 primers. The identification of the isolates was confirmed by 16S rDNA sequencing. To study the ability of LAB strains to degrade OTA, AFB1 and ZEA, a MRS broth medium was supplemented with 2.0 g/mL of each mycotoxin. For each strain, 2 mL of MRS supplemented with the mycotoxins was inoculated in triplicate with 109 CFU/mL. The culture media and bacterial cells were extracted by the addition of an equal volume of acetonitrile/methanol/acetic acid (78:20:2 v/v/v) to the culture tubes. A 2 mL sample was then collected and filtered into a clean 2 mL vial using PP filters with 0.45 m pores. The samples were preserved at 4 °C until HPLC analysis. Among LAB tested, 10 strains isolated from milk were able to eliminate AFB1, belonging to Lactobacillus casei (7), Lb. paracasei (1), Lb. plantarum (1) and 1 to Leuconostoc mesenteroides. Two strains of Enterococcus faecium and one of Ec. faecalis from sausage eliminated ZEA. Concerning to strains of vegetal origin, one Lb. plantarum isolated from elderberry fruit, one Lb. buchnerii and one Lb. parafarraginis both isolated from silage eliminated ZEA. Other 2 strains of Lb. plantarum from silage were able to degrade both ZEA and OTA, and 1 Lb. buchnerii showed activity over AFB1. These enzymatic activities were also verified genotypically through specific gene PCR and posteriorly confirmed by sequencing analysis. In conclusion, due the ability of some strains of LAB isolated from different sources to eliminate OTA, AFB1 and ZEA one can recognize their potential biotechnological application to reduce the health hazards associated with these mycotoxins. They may be suitable as silage inoculants or as feed additives or even in food industry.

Study of the retinoic acid regulated genes in the rodent hypothalamus controlling growth and appetite and the potential for the epigenetic control

Dissertação de mestrado em Bioquímica (área de especialização em Biomedicina)

Development of a poly(L-Lactic acid) based drug release system

Dissertação de mestrado em Biofísica e Bionanossistemas

Reactivity studies on chromene derivatives

Dissertação de mestrado em Medicinal Chemistry

Síntese e testes in vitro de novas pirimido[5,4-d] pirimidinas em células resistentes do cancro coloretal

Dissertação de mestrado em Química Medicinal

Fatty Acid and Cholesterol Concentrations in Usually Consumed Fish in Brazil

Background: Several studies have demonstrated clinical benefits of fish consumption for the cardiovascular system. These effects are attributed to the increased amounts of polyunsaturated fatty acids in these foods. However, the concentrations of fatty acids may vary according to region. Objective: The goal of this study was to determine the amount of,cholesterol and fatty acids in 10 Brazilian fishes and in a non-native farmed salmon usually consumed in Brazil. Methods: The concentrations of cholesterol and fatty acids, especially omega-3, were determined in grilled fishes. Each fish sample was divided in 3 sub-samples (chops) and each one was extracted from the fish to minimize possible differences in muscle and fat contents. Results: The largest cholesterol amount was found in white grouper (107.6 mg/100 g of fish) and the smallest in badejo (70 mg/100 g). Omega-3 amount varied from 0.01 g/100 g in badejo to 0.900 g/100 g in weakfish. Saturated fat varied from 0.687 g/100 g in seabass to 4.530 g/100 g in filhote. The salmon had the greatest concentration of polyunsaturated fats (3.29 g/100 g) and the highest content of monounsaturated was found in pescadinha (5.98 g/100 g). Whiting and boyfriend had the best omega-6/omega 3 ratios respectively 2.22 and 1.19, however these species showed very little amounts of omega-3. Conclusion: All studied Brazilian fishes and imported salmon have low amounts of saturated fat and most of them also have low amounts of omega-3.

Tratamento eclético da lepra: 20 anos de experiência

The author gives the clinical records of 20 patients, 14 of which were treated during 17 months, and the rest much less. The method used for treatment was the ecclectical — preferred by the A. since many years —, in which he associates electricity with minor surgery, chaulmestrol (pure or with 0.5 p. c. iodine or 4 p. c. creosote) and other auxilliary curative agents. The chaulmoogra derivatives were used daily as nostrils tamponage (2 p.c. mentholated or thymolated ester), periodically by injections inside the enlarged lymph nodes and nerve abscesses, and twice weekly by subcutaneous infiltrations (MUIR method) 5 c.c. each, and chaulmoogra soap tablets per os. The galvanocauterisation session was once per week, on active leprotic lesions, followed by painting with 30 p. c. trichloracetic acid solution. All 20 patients were bacilliferous before treatment, and became stronger positive after some months treatment. At the end 14 negativated and 6 remained positive and sometimes bacilli being very scanty. 16 out of 20 gave interesting serological reactions, viz.: Wassermann, Stern, Kahn. Rubino, Witebsky and Gaté (Formol-gel) positive in 5; Stern, Rubino. Witebsky and Gaté positive and Wassermann and Kahn negative in 2; Stern. Kahn, Witebsky and Gaté positive and Wassermann anticomplementary in 1; Wassermann. Stern, Kahn, Rubino and Gaté negative in 1. in the beginning, and a few months later Stern. Witebsky and Gaté becoming positive; Stern. Rubino. Witebsky and Gaté positive and anticomplementary W. in 1; Stern, Witebsky and Gaté positive and Wassermann. Kahn and Rubino negative in 1; Witebsky and Gaté positive and Wassermann, Stern, Kahn and Rubino negative in 1; Witebsky 3 times anticomplemantary and strongly positive Gaté in 1; Wassermann and Gaté positive in 1; Stern and Rubino positive in 1 and Stern test negative in one. In 7 cases high Formol-gel were associated with a high sedimentation index. Many cases had very high S.I. being a false measure of the severity of the disease; others remained very high notwithstanding the great improvement of the disease. All patients with more than 12 months treatment became practically symptom free. Lepra reaction amongst them was rare and always started by embolic rash, being controlled by destruction of such skin lesions by galvanocauterisation. In a few cases the lepromata infiltrated with "Subintrol" (a 3 p. c. special chaulmoogra soap prepared by Dr. ASTROGILDO MACHADO) were completely destroyd and never relapsed. In October 1946 Dr. ERNEST MUIR saw here a few cases treated by the author's method and suggested the combination of Diasone with galvanocauterisation which is being done now with satifactory results. The second part of this paper, reporting many leprosy cases treated by the so-called ecclectical method, which are symptom free and negativated since five to ten years, will be published as soon as the sulfone-therapy be summarised in some reliable scientific report to be compared with.

Direct binding of peptides to MHC class I molecules on living cells. Analysis at the single cell level.

To directly assess the binding of exogenous peptides to cell surface-associated MHC class I molecules at the single cell level, we examined the possibility of combining the use of biotinylated peptide derivatives with an immunofluorescence detection system based on flow cytometry. Various biotinylated derivatives of the adenovirus 5 early region 1A peptide 234-243, an antigenic peptide recognized by CTL in the context of H-2Db, were first screened in functional assays for their ability to bind efficiently to Db molecules on living cells. Suitable peptide derivatives were then tested for their ability to generate positive fluorescence signals upon addition of phycoerythrin-labeled streptavidin to peptide derivative-bearing cells. Strong fluorescent staining of Db-expressing cells was achieved after incubation with a peptide derivative containing a biotin group at the C-terminus. Competition experiments using the unmodified parental peptide as well as unrelated peptides known to bind to Kd, Kb, or Db, respectively, established that binding of the biotinylated peptide to living cells was Db-specific. By using Con A blasts derived from different H-2 congenic mouse strains, it could be shown that the biotinylated peptide bound only to Db among > 20 class I alleles tested. Moreover, binding of the biotinylated peptide to cells expressing the Dbm13 and Dbm14 mutant molecules was drastically reduced compared to Db. Binding of the biotinylated peptide to freshly isolated Db+ cells was readily detectable, allowing direct assessment of the relative amount of peptide bound to distinct lymphocyte subpopulations by three-color flow cytometry. While minor differences between peripheral T and B cells could be documented, thymocytes were found to differ widely in their peptide binding activity. In all cases, these differences correlated positively with the differential expression of Db at the cell surface. Finally, kinetic studies at different temperatures strongly suggested that the biotinylated peptide first associated with Db molecules available constitutively at the cell surface and then with newly arrived Db molecules.

Aplicació de la termoquimiolisi - cromatografia de gasos - espectrometria de masses a l'estudi de la matèria orgànica del sòl

Estudi realitzat a partir d’una estada a la Université de Poitiers, França, entre 2007 i 2009. El treball s'ha centrat en dues activitats bàsiques. El treball realitzat s’ha centrat en dues activitats bàsiques. D’una banda, la posada a punt d'un protocol de fraccionament de la matèria orgànica del sòl, per extraccions successives amb solvents alcalins després d'una seqüència de pretractaments al sòl: cap pretractament, atac amb àcid (per destruir els carbonats), atac amb ditionit (per reduir els òxids de Fe i Al i facilitar l'extracció de la matèria orgànica associada a aquests compostos). El protocol dóna una visió de conjunt de la situació de la matèria orgànica del sòl, combinant aspectes físics (protecció, precipitació, oclusió per carbonats) i químics (grau d'humificació). D’altra banda, l'aprenentatge de la tècnica de termoquimiolisi-cromatografia de gasos-espectrometria de masses. Aquest era l'objectiu de l'estada a Poitiers, al qual hem donat prioritat. Ens hem centrat en l'estudi de fraccions físiques (densimètriques) obtingudes en estudis anteriors sobre sòls forestals. Les fraccions considerades són: fracció lleugera (FL), tres fraccions ocluïdes (OC1, OC2 i OC3) i fracció densa (FD). L’aplicació de la termoquimiolisi permet de caracteritzar diversos grups de substàncies, de les quals ens hem centrat en alguns indicadors bioquímics: àcids grasos, alcohols, diàcids, productes fenòlics i altres productes aromàtics, derivats de carbohidrats. L’estudi de conjunt d’aquests productes indica que és a les fraccions ocluïdes (que solen ser minoritàries a tots els horitzons) on la matèria orgànica d’origen microbià és dominant, mentre que a les fraccions lleugera (FL) i densa (FD) la matèria orgànica d’origen vegetal sembla dominant. Es preveu aplicar aquesta tècnica a l’estudi de les fraccions obtingudes a la primera part del treball, actualment congelades i a l’espera de ser processades.

Once-yearly zoledronic acid for treatment of postmenopausal osteoporosis.

BACKGROUND: A single infusion of intravenous zoledronic acid decreases bone turnover and improves bone density at 12 months in postmenopausal women with osteoporosis. We assessed the effects of annual infusions of zoledronic acid on fracture risk during a 3-year period. METHODS: In this double-blind, placebo-controlled trial, 3889 patients (mean age, 73 years) were randomly assigned to receive a single 15-minute infusion of zoledronic acid (5 mg) and 3876 were assigned to receive placebo at baseline, at 12 months, and at 24 months; the patients were followed until 36 months. Primary end points were new vertebral fracture (in patients not taking concomitant osteoporosis medications) and hip fracture (in all patients). Secondary end points included bone mineral density, bone turnover markers, and safety outcomes. RESULTS: Treatment with zoledronic acid reduced the risk of morphometric vertebral fracture by 70% during a 3-year period, as compared with placebo (3.3% in the zoledronic-acid group vs. 10.9% in the placebo group; relative risk, 0.30; 95% confidence interval [CI], 0.24 to 0.38) and reduced the risk of hip fracture by 41% (1.4% in the zoledronic-acid group vs. 2.5% in the placebo group; hazard ratio, 0.59; 95% CI, 0.42 to 0.83). Nonvertebral fractures, clinical fractures, and clinical vertebral fractures were reduced by 25%, 33%, and 77%, respectively (P<0.001 for all comparisons). Zoledronic acid was also associated with a significant improvement in bone mineral density and bone metabolism markers. Adverse events, including change in renal function, were similar in the two study groups. However, serious atrial fibrillation occurred more frequently in the zoledronic acid group (in 50 vs. 20 patients, P<0.001). CONCLUSIONS: A once-yearly infusion of zoledronic acid during a 3-year period significantly reduced the risk of vertebral, hip, and other fractures. (ClinicalTrials.gov number, NCT00049829.)

Valproic acid related idiosyncratic drug induced hepatotoxicity in a glioblastoma patient treated with temozolomide.

Glioblastoma patients undergoing treatment with surgery followed by radiation and temozolomide chemotherapy often develop a state of immunosuppression and are at risk for opportunistic infections and reactivation of hepatitis and herpes viruses. We report the case of a 48-year-old glioblastoma patient who developed acute cholestatic hepatitis with hepatic failure during adjuvant treatment with temozolomide and the integrin inhibitor cilengitide. A viral hepatitis was excluded and valproic acid treatment was stopped. Upon normalisation of the liver tests, temozolomide treatment was resumed without perturbation of the liver tests. Valproic acid related idiosyncratic drug induced hepatotoxicity should be considered as a differential diagnosis in glioblastoma patients undergoing adjuvant therapy.

Quaternary ammonium salt derivatives of allylphenols with peripheral analgesic effect

Ammonium salt derivatives of natural allylphenols were synthesized with the purpose of obtaining potential peripheral analgesics. These drugs, by virtue of their physicochemical properties, would not be able to cross the blood brain barrier. Their inability to enter into the central nervous system (CNS) should prevent several adverse effects observed with classical opiate analgesics (Ferreira et al., 1984). Eugenol (1) O-methyleugenol (5) and safrole (9) were submitted to nitration, reduction and permethylation, leading to the ammonium salts 4, 8 and 12. Another strategy applied to eugenol (1), consisting in its conversion to a glycidic ether (13), opening the epoxide ring with secondary amines and methylation, led to the ammonium salts 16 and 17. All these ammonium salts showed significant peripheral analgesic action, in modified version of the Randall-Sellito test (Ferreira et al. 1978), at non-lethal doses. The ammonium salt 8 showed an activity comparable to that of methylnalorphinium, the prototype of an ideal peripheral analgesic (Ferreira et al., 1984).