Methodology for single nucleotide polymorphism selection in promoter regions for clinical use. An example of its applicability

Genetic variability in humans can explain many differences in disease risk factors. Polymorphism-related studies focus mainly on the single nucleotide polymorphisms (SNPs) of coding regions of the genes. SNPs on DNA binding motifs of the promoter region have been less explored. On a recent study of SNPs in patients with non-Hodgkin lymphomas we faced the problem of SNP selection from promoter regions and developed a practical methodology for clinical studies. The process consists in identifying SNPs in the coding and promoter regions of the antigen-processing system using the 'dbSNP' database. With the 'HapMap' program, we select SNPs with frequencies >20% in Caucasian populations. For coding regions, we sought biologically and clinically relevant SNPs described in the literature. For the promoter regions, we determined their chromosomal location on 'QiagenSABioscience' site database. The nucleotide sequence of ancestral and variant alleles is available in the 'dbSNP'. These sequences were used in ` Promoter TESS' to determine binding differences of transcription factors. Each sequence may have affinity to different TFs. Thus, SNP selection on the promoter regions was based in the differences on TF binding pattern between the old and the new allele. The potential clinical relevance of the new TFs was also evaluated before the final selection. With this approach, we found that almost half of the relevant SNP fall within the promoter region. In conclusion, we were able to develop a methodology of oriented selection of promoter regions of human genes, comparing the TF with affinity to the ancestral allele with the TF to a variant allele. We selected those SNPs that change the TF's affinity to a pattern with functional significance.(undefined)info:eu-repo/semantics/publishedVersio

Performance Analysis of a Small-Scale Biogas-Based Trigeneration Plant: An Absorption Refrigeration System Integrated to an Externally Fired Microturbine

Trigeneration or combined cooling, heat and power (CCHP) systems fueled by raw biogas can be an interesting alternative for supplying electricity and thermal services in remote rural areas where biogas can be produced without requiring sophisticated equipment. In this sense, this study considers a performance analysis of a novel small-scale CCHP system where a biogas-fired, 5 kWel externally fired microturbine (EFMT), an absorption refrigeration system (ARS) and heat exchangers are integrated for supplying electricity, refrigeration and hot water demanded by Bolivian small dairy farms. The CCHP solution presents two cases, current and nominal states, in which experimental and design data of the EFMT performance were considered, respectively. The primary energy/exergy rate was used as a performance indicator. The proposed cases show better energy performances than those of reference fossil fuel-based energy solutions (where energy services are produced separately) allowing savings in primary energy utilization of up to 31%. Furthermore, improvements in electric efficiency of the EFMT and coefficient of performance (COP) of the ARS, identified as key variables of the system, allow primary energy savings of up to 37%. However, to achieve these values in real conditions, more research and development of the technologies involved is required, especially for the EFM

SUBDIVIDE AND CONQUER RESOLUTION

This contribution will be freewheeling in the domain of signal, image and surface processing and touch briefly upon some topics that have been close to the heart of people in our research group. A lot of the research of the last 20 years in this domain that has been carried out world wide is dealing with multiresolution. Multiresolution allows to represent a function (in the broadest sense) at different levels of detail. This was not only applied in signals and images but also when solving all kinds of complex numerical problems. Since wavelets came into play in the 1980's, this idea was applied and generalized by many researchers. Therefore we use this as the central idea throughout this text. Wavelets, subdivision and hierarchical bases are the appropriate tools to obtain these multiresolution effects. We shall introduce some of the concepts in a rather informal way and show that the same concepts will work in one, two and three dimensions. The applications in the three cases are however quite different, and thus one wants to achieve very different goals when dealing with signals, images or surfaces. Because completeness in our treatment is impossible, we have chosen to describe two case studies after introducing some concepts in signal processing. These case studies are still the subject of current research. The first one attempts to solve a problem in image processing: how to approximate an edge in an image efficiently by subdivision. The method is based on normal offsets. The second case is the use of Powell-Sabin splines to give a smooth multiresolution representation of a surface. In this context we also illustrate the general method of construction of a spline wavelet basis using a lifting scheme

Metabolic coupling in urothelial bladder cancer compartments and its correlation to tumour aggressiveness

Monocarboxylate transporters (MCTs) are vital for intracellular pH homeostasis by extruding lactate from highly glycolytic cells. These molecules are key players of the metabolic reprogramming of cancer cells, and evidence indicates a potential contribution in urothelial bladder cancer (UBC) aggressiveness and chemoresistance. However, the specific role of MCTs in the metabolic compartmentalization within bladder tumors, namely their preponderance on the tumor stroma, remains to be elucidated. Thus, we evaluated the immunoexpression of MCTs in the different compartments of UBC tissue samples (n = 111), assessing the correlations among them and with the clinical and prognostic parameters. A significant decrease in positivity for MCT1 and MCT4 occurred from normoxic toward hypoxic regions. Significant associations were found between the expression of MCT4 in hypoxic tumor cells and in the tumor stroma. MCT1 staining in normoxic tumor areas, and MCT4 staining in hypoxic regions, in the tumor stroma and in the blood vessels were significantly associated with UBC aggressiveness. MCT4 concomitant positivity in hypoxic tumor cells and in the tumor stroma, as well as positivity in each of these regions concomitant with MCT1 positivity in normoxic tumor cells, was significantly associated with an unfavourable clinicopathological profile, and predicted lower overall survival rates among patients receiving platinum-based chemotherapy. Our results point to the existence of a multi-compartment metabolic model in UBC, providing evidence of a metabolic coupling between catabolic stromal and cancer cells' compartments, and the anabolic cancer cells. It is urgent to further explore the involvement of this metabolic coupling in UBC progression and chemoresistance.This study was supported by the Life and Health Sciences Research Institute (ICVS) from the School of Health Sciences of the University of Minho. JA received a postdoctoral fellowship from ICVS (reference ICVS-SSRL: ON.2 SR&TD Integrated Program, NORTE-07-0124-FEDER-000017).info:eu-repo/semantics/publishedVersio

Association between age at first sexual intercourse and subsequent human papillomavirus infection: results of a Brazilian screening program

Objetivo: descrever a idade de início da atividade sexual (sexarca) e a sua associação com a idade das mulheres com a infecção por papilomavírus humano (HPV) e com as alterações citológicas no exame de papanicolaou. Métodos: mulheres da população geral foram recrutadas para participar de um estudo de rastreamento de câncer cervical e lesões pré-malignas. Após a aplicação de questionário comportamental, foram submetidas ao rastreamento com gia cervical e teste para DNA de HPV de alto risco, por meio de Captura Híbrida 2. Este projeto faz parte do Latin American Screening Study, que envolve mulheres do Brasil e da Argentina, e os dados aqui apresentados referem-se aos centros brasileiros nas cidades de Porto Alegre, São Paulo e Campinas. Resultados: de 8.649 mulheres entrevistadas, 8.641 relataram atividade sexual prévia e foram incluídas na análise. A média de idade no momento da entrevista foi de 38,1±11,04 anos, com início da atividade sexual em média aos 18,5±4,0 anos. Identificamos que a idade do início da atividade sexual aumenta de acordo com o aumento da faixa etária no momento da entrevista, isto é, mulheres mais novas relataram sexarca mais precoce que mulheres mais velhas (p<0,001). Em relação à infecção por HPV de alto risco, do total de mulheres que haviam iniciado as relações sexuais, 3.463 foram testadas, com 17,3% de positividade para HPV. Notadamente, em todos os centros, as mulheres com idade ao início da atividade sexual abaixo da média da população entrevistada apresentaram positividade maior para HPV (20,2%) do que as mulheres com sexarca em idade acima da média (12,5%) – Odds Ratio (OR)=1,8 (IC95%=1,5-2,2; p<0,001). Em relação à citologia, mulheres com sexarca abaixo da média de idade apresentaram também maior percentual de citologia alterada ≥ ASC-US (6,7%) do que mulheres com sexarca em idade maior que a média (4,3%) – OR=1,6 (IC95%=1,3-2,0; p<0,001). Conclusões: a infecção por HPV e a presença de alterações citológicas identificadas no rastreio de lesões cervicais em uma população assintomática estiveram significativamente associadas à idade mais precoce no início das relações sexuais. Ademais, identificamos também que as mulheres desta amostra apresentaram diminuição da idade ao início da atividade sexual, nas últimas décadas, sugerindo importante causa para o acréscimo da prevalência de HPV e as lesões decorrentes desta infecção.Purpose: to investigate women’s age at their first sexual intercourse and its correlation with their present age, human papillomavirus (HPV) infection and cytological abnormalities at Pap smear. Methods: women from the general population were invited to be screened for cervical cancer and pre-malignant lesions. After answering a behavior questionnaire, they were submitted to screening with cervical cytology and high-risk HPV testing with Hybrid Capture 2 (HC2). This report is part of the Latin American Screening (LAMS) study, that comprises centers from Brazil and Argentina, and the data presented herein refer to the Brazilian women evaluated at the cities of Porto Alegre, São Paulo and Campinas. Results: from 8,649 women that answered the questionnaire, 8,641 reported previous sexual activity and were included in this analysis. The mean age at the interview was 38.1±11.0 years and the mean age at the first sexual intercourse was 18.5±4.0 years. The age at the first sexual intercourse increased along with the age at the interview, i.e., younger women reported they had begun their sexual life earlier than older women (p<0.001). From the total of women who had already begun having sexual intercourse, 3,643 patients were tested for high-risk HPV infection and 17.3% of them had positive results. In all the centers, it became clear that the women with the first sexual intercourse at ages below the mean age of all the population interviewed presented higher rates of HPV infection (20.2%) than the women with the first sexual intercourse at ages above the mean (12.5%) – Odds Ratio (OR) 1.8 (IC95% 1.5-2.2;p<0,001). According to the cytology, the women with first sexual intercourse at ages under the mean, presented higher percentage of abnormal cytology ≥ ASC-US (6.7%) than the women with the first sexual intercourse at ages above the mean (4.3%) – OR 1.6 (IC95% 1.3-2.;p<0.001). ConclusionS: the high-risk HPV infection and cytological abnormalities identified during the asymptomatic population screening were significantly associated to the women’s age at the first sexual intercourse. Additionally, we have also identified that the women’s age at the first sexual intercourse has decreased during the last decades, suggesting an important contribution to the increase of HPV infection and the subsequent cervical lesions.Comissão Europeia - programa INCO-DEV (International Cooperation Development) Contrato #ICA4-2001-10013

RKIP Inhibition in Cervical Cancer Is Associated with Higher Tumor Aggressive Behavior and Resistance to Cisplatin Therapy

Cervical cancer is one of the most common cancers in women worldwide, being high-risk group the HPV infected, the leading etiological factor. The raf kinase inhibitory protein (RKIP) has been associated with tumor progression and metastasis in several human neoplasms, however its role on cervical cancer is unclear. In the present study, 259 uterine cervix tissues, including cervicitis, cervical intraepithelial lesions and carcinomas, were analyzed for RKIP expression by immunohistochemistry. We found that RKIP expression was significantly decreased during malignant progression, being highly expressed in non-neoplastic tissues (54% of the samples; 73/135), and expressed at low levels in the cervix invasive carcinomas (∼15% (19/124). Following in vitro downregulation of RKIP, we observed a viability and proliferative advantage of RKIP-inhibited cells over time, which was associated with an altered cell cycle distribution and higher colony number in a colony formation assay. An in vitro wound healing assay showed that RKIP abrogation is associated with increased migratory capability. RKIP downregulation was also associated with an increased vascularization of the tumors in vivo using a CAM assay. Furthermore, RKIP inhibition induced cervical cancer cells apoptotic resistance to cisplatin treatment. In conclusion, we described that RKIP protein is significantly depleted during the malignant progression of cervical tumors. Despite the lack of association with patient clinical outcome, we demonstrate, in vitro and in vivo, that loss of RKIP expression can be one of the factors that are behind the aggressiveness, malignant progression and chemotherapy resistance of cervical cancer.</p

Rifabutin encapsulated in liposomes exhibits increased therapeutic activity in a model of disseminated tuberculosis

Tuberculosis (TB) is a leading cause of death amongst infectious diseases. The low permeation of antimycobacterial agents and their difficult access to infected macrophages necessitate long-term use of high drug doses. Liposomes preferentially accumulate in macrophages, increasing the efficacy of antibiotics against intracellular parasites. In the present work, several rifabutin (RFB) liposomal formulations were developed and characterised and their in vivo profile was compared with free RFB following intravenous administration. With the RFB liposomal formulations tested, higher concentrations of the antibiotic were achieved in liver, spleen and lungs 24h post administration compared with free RFB. The concentration of RFB in these organs was dependent on the rigidity of liposomal lipids. The liposomal RFB formulation prepared with dipalmitoyl phosphatidylcholine:dipalmitoyl phosphatidylglycerol (DPPC:DPPG) was the most effective and was selected for biological evaluation in a mouse model of disseminated TB. Compared with mice treated with free RFB, mice treated with the DPPC:DPPG RFB formulation exhibited lower bacterial loads in the spleen (5.53 log(10) vs. 5.18 log(10)) and liver (5.79 log(10) vs. 5.41 log(10)). In the lung, the level of pathology was lower in mice treated with encapsulated RFB. These results suggest that liposomal RFB is a promising approach for the treatment of extrapulmonary TB in human immunodeficiency virus co-infected patients.This work was supported by grants from FCT (POCTI/FCB/36416/99) and from the Health Services of FCG. Fellowships were granted by FCT (SFRH/BD/9624/2002- GABBA Program to A. Cruz and SFRH/BD/15911/2005 to A.G. Fraga

Hormonal contraceptives and the length of their use are not independent risk factors for high-risk HPV infections or high-grade CIN

AIMS: To evaluate the role of hormonal contraceptives as a risk factor of high-risk human papillomavirus (HR-HPV), cervical intraepithelial lesions (CIN) and cervical cancer in our multi-center population-based LAMS (Latin American Screening) study. METHODS: A cohort study with >12,000 women from Brazil and Argentina using logistic regression to analyze the covariates of hormonal contraception (HOC - oral, injections, patches, implants, vaginal ring and progesterone intrauterine system) use followed by multivariate modeling for predictors of HR-HPV and CIN2+. RESULTS: HR-HPV infection was a consistent risk factor of high-grade CIN in all three groups of women. The length of HOC use was not significantly related to high-grade squamous intraepithelial lesions (HSIL)+ Pap (p = 0.069), LSIL+ Pap (p = 0.781) or ASCUS+ (p = 0.231). The same was true with the length of HOC use and histology CIN3+ (p = 0.115) and CIN2+ (p = 0.515). Frequently, HOC users have previously shown more HPV-related lesions, as well as lower HPV prevalence if they were current smokers. But HOC use and time of usage were not independent risk factors of either HR-HPV infection or high-grade CIN using multiple logistic regressions. CONCLUSIONS: No evidence was found for an association between the use of HOC with an increased risk for HR-HPV infection or high-grade CIN in this cohort.This study is a part of the ongoing LAMS (Latin American Screening) study, entitled: Improving Health Systems Towards Equality-Based Control of Cervical Cancer in Latin America, and is supported by the INCO-DEV Program of the European Commission (Project No. ICA4-CT-2001-10013). The generous contribution of Digene Corporation (USA) who donated the HCII tests at our disposal is gratefully acknowledged

Human papillomavirus testing as an optional screening tool in low-resource settings of Latin America: experience from the Latin American Screening study

Hybrid capture II (HC II) test for oncogenic human papillomaviruses (HPV) was carried out in a cohort of 4284 women at their first clinical visit. Overall prevalence of HPV was 17.1%, decreasing with age from 33.9% among women below 20 years to only 11.0% among those older than 41 years. HPV prevalence was significantly higher among current smokers (odds ratio [OR] ¼ 1.31; 95% CI 1.1–1.6), in women with two or more lifetime sexual partners (OR ¼ 1.9; 95% CI 1.6–2.4), and those women with two or more sexual partners during the past 12 months prior to examination (OR ¼ 1.6; 95% CI 1.2–2.2). HPV detection increased in parallel with increasing cytologic abnormality, being highest in women with high-grade squamous intraepithelial lesion (P ¼ 0.001). Specificity of the HPV test in detecting histologically confirmed cervical disease was 85% (95% CI 83.9–86.1). Sensitivity of the HPV test in detecting histologic abnormalities increased in parallel with disease severity, ranging from 51.5% for cervical intraepithelial neoplasia (CIN) 1 to 96.5% for CIN 3 and 100.0% for cancer, with respective decline of positive predictive value. These data suggest that HPV testing with HC II assay might be a viable screening tool among this population with relatively high prevalence of cervical disease