High-resolution correlation of coastal cliff sections in the Lagos- Portimao Formation (Lower - Middle Miocene, central Algarve, Portugal)

This paper describes a high-resolution stratigraphic correlation scheme for the early to middle Miocene Lagos-Portimão Formation of central Algarve, southern Portugal. The Lagos Portimão-Formation of central Algarve is a 60 m thick package of horizontally bedded siliciclastics and carbonates. The bryozoan and mollusc dominated biofacies is typical of a shallow marine, warm-temperate climatic environment. We define four stratigraphic marker beds based on biofacies, lithology, and gamma-ray signatures. Marker bed 1 is a reddish shell bed composed predominantly of bivalve shells in various stages of fragmentation. Marker bed 2 is a fossiliferous sandstone / sandy rudstone characterized by bryozoan masses. Marker bed 3 is also a fossiliferous sandstone with abundant larger foraminifers and foliate bryozoans. Marker bed 4 is composed of three distinct layers; two fossiliferous sandstones with an intercalated shell bed. The upper sandstone unit displays thickets of the bryozoan Celleporaria palmate associated with the coral Culizia parasitica. This stratigraphic framework allows to correlate isolated outcrops within the stratigraphic context of the Lagos-Portimão Formation and to establish high resolution chronostratigraphic Sr-isotopic dating.

Kinematic parameters of throwing performance in patients with schizophrenia using a markerless motion capture system

Motor dysfunction is consistently reported but understudied in schizophrenia. It has been hypothesized that this abnormality may reflect a neuro-developmental disorder underlying this illness. The main goal of this study was to analyze movement patterns used by participants with schizophrenia and healthy controls during overarm throwing performance, using a markerless motion capture system. Thirteen schizophrenia patients and 16 healthy control patients performed the overarm throwing task in a markerless motion capture system. Participants were also examined for the presence of motor neurological soft signs (mNSS) using the Brief Motor Scale. Schizophrenia patients demonstrated a less developed movement pattern with low individualization of components compared to healthy controls. The schizophrenia group also displayed a higher incidence of mNSS. The presence of a less mature movement pattern can be an indicator of neuro-immaturity and a marker for atypical neurological development in schizophrenia. Our findings support the understanding of motor dysfunction as an intrinsic part of the disorder of schizophrenia.

Antimicrobial resistance and plasmid detection in strains of the Bacteroides fragilis group

Resistant populations of the Bacteroides fragilis group bacteria (two reference ones and two isolated from human and Callithrix penicillata marmoset) were obtained by the gradient plate technique, to clindamycin, penicillin G, metronidazole and mercuric chloride. All the four tested strains were originaly susceptible to the four antimicrobial drugs at the breakpoint used in this study. MICs determination for the four cultures gave constant values for each antimicrobial, on the several steps by the gradient plate technique. The intestinal human B. fragilis strains showed three DNA bands, that could be representative of only two plasmids in the closed covalently circular (CCC) form with molecular weights of approximately 25 and 2.5 Md. The results do not permit an association between the presence of plasmid in the human strain with the susceptibility to the studied drugs. The four strains were ß-lactamase negative in the two methods used, and no particular chromosomal genetic resistance marker was demonstred. The resistance (MIC) observed, after contact with penicillin G and mercuric chloride, were two-fold in the four tested strains

Acute schistosomiasis: clinical, diagnostic and therapeutic features

Three distinct syndromes caused by schistosomiasis have been described: cercarial dermatitis or swimmer's itch, acute schistosomiasis or Katayama fever, and chronic schistosomiasis. Complications of acute schistosomiasis have also been reported. The absence of a serological marker for the acute stage has hindered early diagnosis and treatment. Recently, an ELISA test using KLH (keyhole limpet haemocyanin) as antigen, has proved useful in differentiating acute from chronic schistosomiasis mansoni. Clinical and experimental evidence indicate that steroids act synergistically with schistosomicides in the treatment of Katayama syndrome. In this paper, clinical, diagnostic and therapeutic features of acute schistosomiasis are updated.

Estrogen receptor dependent genetic and epigenetic factors of tamoxifen resistance

Resumo: A decisão da terapêutica hormonal no tratamento do cancro da mama baseiase na determinação do receptor de estrogénio alfa por imunohistoquímica (IHC). Contudo, a presença deste receptor não prediz a resposta em todas as situações, em parte devido a limitações do método IHC. Investigámos se a expressão dos genes ESR1 e ESR2, bem como a metilação dos respectivos promotores, pode estar relacionada com a evolução desfavorável de uma proporção de doentes tratados com tamoxifeno assim como com a perda dos receptores de estrogénio alfa (ERα) e beta (ERß). Amostras de 211 doentes com cancro da mama diagnosticado entre 1988 e 2004, fixadas em formalina e preservadas em parafina, foram utilizadas para a determinação por IHC da presença dos receptores ERα e ERß. O mRNA total do gene ESR1 e os níveis específicos do transcrito derivado do promotor C (ESR1_C), bem como dos transcritos ESR2_ß1, ESR2_ß2/cx, and ESR2_ß5 foram avaliados por Real-time PCR. Os promotores A e C do gene ESR1 e os promotores 0K e 0N do gene ESR2 foram investigados por análise de metilação dos dinucleotidos CpG usando bisulfite-PCR para análise com enzimas de restrição, ou para methylation specific PCR. Atendendo aos resultados promissores relacionados com a metilação do promotor do gene ESR1, complementamos o estudo com um método quantitativo por matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) suportado pelo software Epityper para a medição da metilação nos promotores A e C. Fez-se a avaliação da estabilidade do mRNA nas linhas celulares de cancro da mama MCF-7 e MDA-MB-231 tratadas com actinomicina D. Baixos níveis do transcrito ESR1_C associaram-se a uma melhor sobrevivência global (p = 0.017). Níveis elevados do transcrito ESR1_C associaram-se a uma resposta inferior ao tamoxifeno (HR = 2.48; CI 95% 1.24-4.99), um efeito mais pronunciado em doentes com tumores de fenótipo ERα/PgR duplamente positivo (HR = 3.41; CI 95% 1.45-8.04). A isoforma ESR1_C mostrou ter uma semi-vida prolongada, bem como uma estrutura secundária da região 5’UTR muito mais relaxada em comparação com a isoforma ESR1_A. A análise por Western-blot mostrou que ao nível da 21 proteína, a selectividade de promotores é indistinguivel. Não se detectou qualquer correlação entre os níveis das isoformas do gene ESR2 ou entre a metilação dos promotores do gene ESR2, e a detecção da proteína ERß. A metilação do promotor C do gene ESR1, e não do promotor A, foi responsável pela perda do receptor ERα. Estes resultados sugerem que os níveis do transcrito ESR1_C sejam usados como um novo potencial marcador para o prognóstico e predição de resposta ao tratamento com tamoxifeno em doentes com cancro da mama. Abstract: The decision of endocrine breast cancer treatment relies on ERα IHC-based assessment. However, ER positivity does not predict response in all cases in part due to IHC methodological limitations. We investigated whether ESR1 and ESR2 gene expression and respective promoter methylation may be related to non-favorable outcome of a proportion of tamoxifen treated patients as well as to ERα and ERß loss. Formalin-fixed paraffin-embedded breast cancer samples from 211 patients diagnosed between 1988 and 2004 were submitted to IHC-based ERα and ERß protein determination. ESR1 whole mRNA and promoter C specific transcript levels, as well as ESR2_ß1, ESR2_ß2/cx, and ESR2_ß5 transcripts were assessed by real-time PCR. ESR1 promoters A and C, and ESR2 promoters 0N and 0K were investigated by CpG methylation analysis using bisulfite-PCR for restriction analysis, or methylation specific PCR. Due to the promising results related to ESR1 promoter methylation, we have used a quantification method by matrixassisted laser desorption/ionization time-of-flight mass spectrometry (MALDITOF MS) together with Epityper software to measure methylation at promoters A and C. mRNA stability was assessed in actinomycin D treated MCF-7 and MDA-MB-231 cells. ERα protein was quantified using transiently transfected breast cancer cells. Low ESR1_C transcript levels were associated with better overall survival (p = 0.017). High levels of ESR1_C transcript were associated with non-favorable response in tamoxifen treated patients (HR = 2.48; CI 95% 1.24-4.99), an effect that was more pronounced in patients with ERα/PgR double-positive tumors (HR = 3.41; CI 95% 1.45-8.04). The ESR1_C isoform had a prolonged mRNA half-life and a more relaxed 5’UTR structure compared to ESR1_A isoform. Western-blot analysis showed that at protein level, the promoter selectivity is undistinguishable. There was no correlation between levels of ESR2 isoforms or ESR2 promoter methylation and ERß protein staining. ESR1 promoter C CpG methylation and not promoter A was responsible for ERα loss. We propose ESR1_C levels as a putative novel marker for breast cancer prognosis and prediction of tamoxifen response.

Intestinal microsporidiosis in HIV-positive patients with chronic unexplained diarrhea in Rio de Janeiro, Brazil: diagnosis, clinical presentation and follow-up

After the diagnosis of two cases of microsporidial intestinal infection in 1992, in Rio de Janeiro, we have started looking for this parasite in HIV-infected patients with chronic unexplained diarrhea. We have studied 13 patients from Hospital Evandro Chagas, IOC-FIOCRUZ. Fecal specimens from these patients were examined for the presence of Cryptosporidia and Microsporidia, in addition to routine examination. Spores of Microsporidia were found in the stools of 6 (46.1%) of the 13 patients studied, with 2 histological jejunal confirmations. The Microsporidia-infected patients presented chronic diarrhea with about 6 loose to watery bowel movements a day. Five infected patients were treated with Metronidazole (1.5 g/day). They initially showed a good clinical response, but they never stopped eliminating spores. After about the 4th week of therapy, their diarrhea returned. Two patients utilized Albendazole (400 mg/day-4 weeks) with a similar initial improvement and recurrence of the diarrhea. Intestinal Microsporidiosis seems to be a marker of advanced stages of AIDS, since 5 of our 6 infected patients were dead after a 6 month period of follow-up. The present study indicates that intestinal microsporidiosis may be a burgeoning problem in HIV-infected patients with chronic diarrhea in Brazil, which deserves further investigation.

Human cutaneous leishmaniasis caused by Leishmania (Viannia) braziliensis in Santiago del Estero, Argentina: identification of parasites by monoclonal antibodies and isoenzymes

Diagnostic and parasite characterization and identification studies were carried out in human patients with cutaneous leishmaniasis lesions in Santiago del Estero, Northern Province of Argentina. Diagnostic procedures were biopsies of lesions for smears and inoculations in hamster, needle aspirations of material from ulcers for "in vitro" cultures. Immunodiagnostic techniques applied were IFAT-IgG and Montenegro skin test. Primary isolation of eight stocks of leishmanial parasites was achieved from patients with active lesions. All stocks were biologically characterized by their behaviour in hamster, measurements of amastigote and promastigotes and growth "in vitro". Eight stocks were characterized and identified at species level by their reactivity to a cross-panel of sub-genus and specie-specific Monoclonal Antibodies through an Indirect Immunofluorescence technique and a Dot-ELISA. We conclude from the serodeme analysis of Argentina stocks that: stocks MHOM/AR/92/SE-1; SE-2; SE-4; SE-8; SE-8-I; SE-30; SE-34 and SE-36 are Leishmania (Viannia) braziliensis. Three Leishmania stocks (SE-1; SE-2 and SE-30) did not react with one highly specie-specific Monoclonal Antibody (Clone: B-18, Leishmania (Viannia) braziliensis marker) disclosing two serodeme group patterns. Five out of eight soluble extracts of leishmanial promastigotes were electrophoresed on thin-layer starch gels and examined for the enzyme MPI, Mannose Phosphate Isomerase; MDH, Malate Dehydrogenase; 6PGD, 6 Phosphogluconate Dehydrogenase; NH, Nucleoside Hydrolase, 2-deoxyinosinc as substrate; SOD, Superoxide Dismutase; GPI, Glucose Phosphate Isomerase and ES, Esterase. From the isoenzyme studies we concluded that stocks: MHOM/AR/92/SE-1; SE-2; SE-4; SE-8 and SE-8-I are isoenzymatically Leishmania (Viannia) braziliensis. We need to analyze more enzymes before assigning them to a braziliensis zymodeme.

S100A6 Amyloid Fibril Formation Is Calcium-modulated and Enhances Superoxide Dismutase-1 (SOD1) Aggregation

S100A6 is a small EF-hand calcium- and zinc-binding protein involved in the regulation of cell proliferation and cytoskeletal dynamics. It is overexpressed in neurodegenerative disorders and a proposed marker for Amyotrophic Lateral Sclerosis (ALS). Following recent reports of amyloid formation by S100 proteins, we investigated the aggregation properties of S100A6. Computational analysis using aggregation predictors Waltz and Zyggregator revealed increased propensity within S100A6 helices HI and HIV. Subsequent analysis of Thioflavin-T binding kinetics under acidic conditions elicited a very fast process with no lag phase and extensive formation of aggregates and stacked fibrils as observed by electron microscopy. Ca2+ exerted an inhibitory effect on the aggregation kinetics, which could be reverted upon chelation. An FT-IR investigation of the early conformational changes occurring under these conditions showed that Ca2+ promotes anti-parallel β-sheet conformations that repress fibrillation. At pH 7, Ca2+ rendered the fibril formation kinetics slower: time-resolved imaging showed that fibril formation is highly suppressed, with aggregates forming instead. In the absence of metals an extensive network of fibrils is formed. S100A6 oligomers, but not fibrils, were found to be cytotoxic, decreasing cell viability by up to 40%. This effect was not observed when the aggregates were formed in the presence of Ca2+. Interestingly, native S1006 seeds SOD1 aggregation, shortening its nucleation process. This suggests a cross-talk between these two proteins involved in ALS. Overall, these results put forward novel roles for S100 proteins, whose metal-modulated aggregation propensity may be a key aspect in their physiology and function.

HEPATITIS B VIRUS INFECTION PROFILE IN CENTRAL BRAZILIAN HEMODIALYSIS POPULATION

Hepatitis B has proved to be a major health hazard in hemodialysis patients. In order to investigate the hepatitis B virus (HBV) infection profile in the hemodialysis population of Goiânia city - Central Brazil, all dialysis patients (N=282) were studied. The prevalence of any HBV marker (HBsAg, anti-HBs, and anti-HBc) was 56.7% (95% CI: 51.1-62.7), ranging from 33.3% to 77.7% depending on dialysis unit. HBV-DNA was detected in 67.6% and 88.2% of the HBsAg-positive serum samples, in 91.3% and 100% of the HBsAg/HBeAg-positive samples, and in 18.2% and 63.6% of the HBsAg/anti-HBe-reactive sera by hybridization and PCR, respectively. The length of time on hemodialysis was significantly associated with HBV seropositivity. Only 10% of the patients reported received hepatitis B vaccination. The findings of a high HBV infection prevalence in this population and the increased risk for HBV infection on long-term hemodialysis suggest the environmental transmission, emphasizing the urgent need to evaluate strategies of control and prevention followed in these units.

Clinical and diagnostic aspects of intestinal microsporidiosis in HIV-infected patients with chronic diarrhea in Rio de Janeiro, Brazil

The objectives of this study were to determine both the prevalence of microsporidial intestinal infection and the clinical outcome of the disease in a cohort of 40 HIV-infected patients presenting with chronic diarrhea in Rio de Janeiro, Brazil. Each patient, after clinical evaluation, had stools and intestinal fragments examined for viral, bacterial and parasitic pathogens. Microsporidia were found in 11 patients (27.5%) either in stools or in duodenal or ileal biopsies. Microsporidial spores were found more frequently in stools than in biopsy fragments. Samples examined using transmission electron microscopy (n=3) or polymerase chain reaction (n=6) confirmed Enterocytozoon bieneusi as the causative agent. Microsporidia were the only potential enteric pathogens found in 5 of the 11 patients. Other pathogens were also detected in the intestinal tract of 21 patients, but diarrhea remained unexplained in 8. We concluded that microsporidial infection is frequently found in HIV infected persons in Rio de Janeiro, and it seems to be a marker of advanced stage of AIDS.

Hepatitis B and C virus markers among patients with hepatosplenic mansonic schistosomiasis

PURPOSE: To evaluate the frequency and the consequences of the co-infection of hepatitis B and C viruses in patients with hepatosplenic schistosomiasis (HSS). METHODS: B and C serologic markers, exposure to risk factors, biochemical assays, upper gastrointestinal endoscopies, and abdominal ultrasonograms were evaluated in 101 patients with HSS from 1994 to 1997. Whenever possible, PCR was tested and histopathological studies were reviewed. RESULTS: At least one HBV virus marker was found in 15.8%, and anti-HCV was detected in 12.9% of the subjects. The seropositive subjects tended to be older than the seronegative ones. A history of blood transfusion was significantly related to the presence of anti-HCV. Three (18.75%) out of 16 subjects exposed to B virus were HBsAg positive. Eleven (84.6%) out of thirteen patients who were anti-HCV positive demonstrated viral activity. Patients with ongoing viral infection presented a higher average level of liver aminotransferases, a higher frequency of cell decompensation and a higher rate of chronic hepatitis. Portal hypertension parameters were not influenced by viral exposure. CONCLUSIONS: The rate of hepatitis B and C viruses serologic markers observed in the patients with HSS was higher than the control group. The co-infection was responsible for a higher frequency of cell decompensation.

Descending pain modulation in a Kaolin-induced hydrocephalus rat model

Pain transmission at the spinal cord is modulated by descending actions that arise from supraspinal areas which collectively form the endogenous pain control system. Two key areas involved of the endogenous pain control system have a circunventricular location, namely the periaqueductal grey (PAG) and the locus coeruleus (LC). The PAG plays a crucial role in descending pain modulation as it conveys the input from higher brain centers to the spinal cord. As to the LC, it is involved in descending pain inhibition by direct noradrenergic projections to the spinal cord. In the context of neurological defects, several diseases may affect the structure and function of the brain. Hydrocephalus is a congenital or acquired disease characterized by an enlargement of the ventricles which leads to a distortion of the adjacent tissues, including the PAG and LC. Usually, patients suffering from hydrocephalus present dysfunctions in learning and memory and also motor deficits. It remains to be evaluated if lesions of the periventricular brain areas involved in pain control during hydrocephalus may affect descending pain control and, herein, affect pain responses. The studies included in the present thesis used an experimental model of hydrocephalus (the rat injected in the cisterna magna with kaolin) to study descending modulation of pain, focusing on the two circumventricular regions referred above (the PAG and the LC). In order to evaluate the effects of kaolin injection into the cisterna magna, we measured the degree of ventricular dilatation in sections encompassing the PAG by standard cytoarquitectonic stanings (thionin staining). For the LC, immunodetection of the noradrenaline-synthetizing enzyme tyrosine hydroxylase (TH) was performed, due to the noradrenergic nature of the LC neurons. In general, rats with kaolin-induced hydrocephalus presented a higher dilatation of the 4th ventricle, along with a tendency to a higher area of the PAG. Due to the validated role of detection the c-fos protooncogene as a marker of neuronal activation, we also studied neuronal activation in the several subnuclei which compose the PAG, namely the dorsomedial, dorsolateral, lateral and ventrolateral (VLPAG) parts. A decrease in the numbers of neurons immunoreactive for Fos protein (the product of activation of the c-fos protooncogene) was detected in rats injected with kaolin, whereas the remaining PAG subnuclei did not present changes in Fos-immunoreactive nuclei. Increases in the levels of TH in the LC, namely at the rostral parts of the nucleus, were detected in hydrocephalic animals. The following pain-related parameters were measured, namely 1) pain behavioural responses in a validated pain inflammatory test (the formalin test) and 2) the nociceptive activation of spinal cord neurons. A decrease in behavioral responses was detected in rats with kaolin-induced hydrocephalus was detected, namely in the second phase of the test (inflammatory phase). This is the phase of the formalin test in which the motor behaviour is less important, which is important since a semi-quantitative analysis of the motor performance of rats injected with kaolin indicates that these animals may present some motor impairments. Collectively, the results of the behavioral studies indicate that rats with kaolin-induced hydrocephalus exhibit hypoalgesia. A decrease in Fos expression was detected at the superficial dorsal layers of the spinal cord in rats with kaolin-induced hydrocephalus, further indicating that hydrocephalus decreases nociceptive responses. It remains to be ascertained if this is due to alterations in the PAG and LC in the rats with kaolin-induced hydrocephalus, which may affect descending pain modulation. It remains to be evaluated what are the mechanisms underlying the increased pain inhibition at the spinal dorsal horn in the hydrocephalus rats. Regarding the VLPAG, the decrease in neuronal activity may impair descending modulation. Since the LC has higher levels of TH in rats with kaolininduced hydrocephalus, which also appears to increase the noradrenergic innervation in the spinal dorsal horn, it is possible that an increase in the release of noradrenaline at the spinal cord accounts for pain inhibition. Our studies also determine the need to study in detail patients with hydrocephalus namely in what concerns their thresholds to pain and to perform imaging studies focused on the structure and function of pain control areas in the brain.

The involvement of CDH1 in cancer angiogenesis

Dissertação para obtenção do Grau de Mestre em Biotecnologia

The biological meaning of anti-HBC positive result in blood donors: relation to HBV-DNA and to other serological markers

In order to assess the potential risk of anti-HBc-positive blood donors for post-transfusional hepatitis and to investigate whether other HBV serological markers are capable of identifying the presence of the virus, 1000 first-time blood donors were enrolled between June and July 1997. These donors were screened using routine Brazilian blood center tests (HIV 1 and 2, HTLV 1 and 2, Chagas disease, Syphilis, HCV, HBsAg, anti-HBc and ALT ). The 120 (12%) found to be anti-HBc-positive underwent further tests: HBe, anti-HBe, anti-HBs and HBV-DNA by PCR. Ten cases were HBsAg positive and all were HBV-DNA positive by PCR. Three HBsAg-negative donors were HBV-DNA-positive. Two HBV-DNA-positive donors were also anti-HBs-positive. All the HBV-positive donors had at least one HBV marker other than anti-HBc. Anti-HBc is an important cause of blood rejection. Testing for HBsAg alone is not fully protective and anti-HBc remains necessary as a screening test. The presence of anti-HBs is not always indicative of absence of the virus. The addition of other HBV serological markers could represent an alternative in predicting the presence of the virus when compared with PCR. It is recommended that other studies should be carried out to confirm this finding.

Basement membrane alterarions in kernicteric brain microvasculature and pericyte response to bilirubin

Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina