Searching for molecular markers in head and neck squamous cell carcinomas (HNSCC) by statistical and bioinformatic analysis of larynx-derived SAGE libraries

Background: Head and neck squamous cell carcinoma (HNSCC) is one of the most common malignancies in humans. The average 5-year survival rate is one of the lowest among aggressive cancers, showing no significant improvement in recent years. When detected early, HNSCC has a good prognosis, but most patients present metastatic disease at the time of diagnosis, which significantly reduces survival rate. Despite extensive research, no molecular markers are currently available for diagnostic or prognostic purposes. Methods: Aiming to identify differentially-expressed genes involved in laryngeal squamous cell carcinoma (LSCC) development and progression, we generated individual Serial Analysis of Gene Expression (SAGE) libraries from a metastatic and non-metastatic larynx carcinoma, as well as from a normal larynx mucosa sample. Approximately 54,000 unique tags were sequenced in three libraries. Results: Statistical data analysis identified a subset of 1,216 differentially expressed tags between tumor and normal libraries, and 894 differentially expressed tags between metastatic and non-metastatic carcinomas. Three genes displaying differential regulation, one down-regulated (KRT31) and two up-regulated (BST2, MFAP2), as well as one with a non-significant differential expression pattern (GNA15) in our SAGE data were selected for real-time polymerase chain reaction (PCR) in a set of HNSCC samples. Consistent with our statistical analysis, quantitative PCR confirmed the upregulation of BST2 and MFAP2 and the downregulation of KRT31 when samples of HNSCC were compared to tumor-free surgical margins. As expected, GNA15 presented a non-significant differential expression pattern when tumor samples were compared to normal tissues. Conclusion: To the best of our knowledge, this is the first study reporting SAGE data in head and neck squamous cell tumors. Statistical analysis was effective in identifying differentially expressed genes reportedly involved in cancer development. The differential expression of a subset of genes was confirmed in additional larynx carcinoma samples and in carcinomas from a distinct head and neck subsite. This result suggests the existence of potential common biomarkers for prognosis and targeted-therapy development in this heterogeneous type of tumor.

The Modified Card Sorting Test: Test-retest stability and relationships with demographic variables in a healthy older adult sample

Objectives. To investigate the test-retest stability of a standardized version of Nelson's (1976) Modified Card Sorting Test (MCST) and its relationships with demographic variables in a sample of healthy older adults. Design. A standard card order and administration were devised for the MCST and administered to participants at an initial assessment, and again at a second session conducted a minimum of six months later in order to examine its test-retest stability. Participants were also administered the WAIS-R at initial assessment in order to provide a measure of psychometric intelligence. Methods. Thirty-six (24 female, 12 male) healthy older adults aged 52 to 77 years with mean education 12.42 years (SD = 3.53) completed the MCST on two occasions approximately 7.5 months (SD = 1.61) apart. Stability coefficients and test-retest differences were calculated for the range of scores. The effect of gender on MCST performance was examined. Correlations between MCST scores and age, education and WAIS-R IQs were also determined. Results. Stability coefficients ranged from .26 for the percent perseverative errors measure to .49 for the failure to maintain set measure. Several measures were significantly correlated with age, education and WAIS-R IQs, although no effect of gender on MCST performance was found. Conclusions. None of the stability coefficients reached the level required for clinical decision making. The results indicate that participants' age, education, and intelligence need to be considered when interpreting MCST performance. Normative studies of MCST performance as well as further studies with patients with executive dysfunction are needed.

Searching behaviour and diet of Oystercatcher Haematopus ostralegus pairs feeding in their territories

Search path, searching behaviour and diet of pairs of Oystercatchers feeding in mudflat territories were studied during spring. females ate Nereis, Mya, small unidentified prey, probably Corophium, and a few Macoma, whereas males primarily ate Macoma. Even when female and male foraged in the same site, they often caught different prey. The combination of 'The Search-rate/Detection Model' (Gendron & Staddon 1983) and 'The Harvestable Prey Model' (Zwarts & Wanink 1993) provide the theoretical framework in which to explain these differences in diet. Macoma are thought to be more cryptic than Nereis, Mya and Corophium. Therefore females, while searching at a faster rate than their respective mates, caught far fewer cryptic prey, but a greater number of more conspicuous prey than their mates. On the basis of distances moved before and after capturing prey, males exhibited area-restricted searching for Macoma and Corophium. In contrast, females did not exhibit any area-restricted searching. it is suggested that the distribution of Macoma and Corophium available to males searching slowly was more clumped than that of these two prey species available to females searching more quickly.

Executive dysfunction in children and adolescents with temporal lobe epilepsy: Is the Wisconsin Card Sorting Test enough?

The Wisconsin Card Sorting Test (WCST) is the gold standard in the evaluation of executive dysfunction (ED) in patients with temporal lobe epilepsy (TLE). We evaluated 35 children with TLE and 25 healthy controls with the WCST and with a more comprehensive battery. Among the children with TLE, 77.14% showed impairment on the WCST. On other tests (Wechsler Intelligence Scale for Children-Digit Forward, Matching Familiar Figures Test, Trail Making Test, Word Fluency, Finger Windows, and Number-Letter Memory), impairment was demonstrated in 94.29%. The authors concluded that the WCST is a good paradigm to measure executive impairment in children with TLE: however, it may be not enough. Evaluation performed only with the WCST not only underestimated the number of patients with ED, but also missed relevant information regarding the type of ED. (C) 2009 Elsevier Inc. All rights reserved.

Differential sorting and fate of endocytosed GPI-anchored proteins

In this paper, we studied the fate of endocytosed glycosylphosphatidyl inositol anchored proteins (GPI-APs) in mammalian cells, using aerolysin, a bacterial toxin that binds to the GPI anchor, as a probe. We find that GPI-APs are transported down the endocytic pathway to reducing late endosomes in BHK cells, using biochemical, morphological and functional approaches. We also find that this transport correlates with the association to raft-like membranes and thus that lipid rafts are present in late endosomes (in addition to the Golgi and the plasma membrane). In marked contrast, endocytosed GPI-APs reach the recycling endosome in CHO cells and this transport correlates with a decreased raft association. GPI-APs are, however, diverted from the recycling endosome and routed to late endosomes in CHO cells, when their raft association is increased by clustering seven or less GPI-APs with an aerolysin mutant. We conclude that the different endocytic routes followed by GPI-APs in different cell types depend on the residence time of GPI-APs in lipid rafts, and hence that raft partitioning regulates GPI-APs sorting in the endocytic pathway.

Sorting and convergence of primary olfactory axons are independent of the olfactory bulb

Primary olfactory axons expressing the same odorant receptor gene sort out and converge to fixed sites in the olfactory bulb. We examined the guidance of axons expressing the P2 odorant receptor when they were challenged with different cellular environments in vivo. In the mutant extratoes mouse, the olfactory bulb is lacking and is replaced by a fibrocellular mass. In these animals, primary olfactory axons form glomerular-like loci despite the absence of normal postsynaptic targets. P2 axons are able to sort out from other axons in this fibrocellular mass and converge to form loci of like axons. The sites of these loci along mediolateral and ventrodorsal axes were highly variable. Similar convergence was observed for larger subpopulations of axons expressing the same cell surface carbohydrates. The sorting out and convergence of like axons also occurred during regeneration following bulbectomy. Olfactory axon behaviour in these models demonstrates that sorting and convergence of axons are independent of the target, which instead provides distinct topographic cues for guidance. (C) 2003 Wiley-Liss, Inc.

Intracellular sorting and transport of proteins

The secretory and endocytic pathways of eukaryotic organelles consist of multiple compartments, each with a unique set of proteins and lipids. Specific transport mechanisms are required to direct molecules to defined locations and to ensure that the identity, and hence function, of individual compartments are maintained. The localisation of proteins to specific membranes is complex and involves multiple interactions. The recent dramatic advances in understanding the molecular mechanisms of membrane transport has been due to the application of a multi-disciplinary approach, intergrating membrane biology, genetics, imaging, protein and lipid biochemistry and structural biology. The aim of this review is to summarise the general principles of protein sorting in the secretory and endocytic pathways and to highlight the dynamic nature of these processes. The molecular mechanisms involved in this transport along the secretory and endocytic pathways are discussed along with the signals responsible for targeting proteins to different intracellular locations. (C) 2003 Elsevier Science Ltd. All rights reserved.

Vps20p and Vta1p interact with Vps4p and function in multivesicular body sorting and endosomal transport in Saccharomyces cerevisiae

Vps4p (End13p) is an AAA-family ATPase that functions in membrane transport through endosomes, sorting of soluble vacuolar proteins to the vacuole, and multivesicular body (MVB) sorting of membrane proteins to the vacuole lumen. In a yeast two-hybrid screen with Vps4p as bait we isolated VPS20 (YMR077c) and the novel open reading frame YLA181c, for which the name VTA1 has recently been assigned (Saccharomyces Genome Database). Vps4p directly binds Vps20p and Vta1p in vitro and binding is not dependent on ATP-conversely, Vps4p binding to Vps20p is partially sensitive to ATP hydrolysis. Both ATP binding [Vps4p-(K179A)] and ATP hydrolysis [Vps4p-(E233Q)] mutant proteins exhibit enhanced binding to Vps20p and Vta1p in vitro. The Vps4p-Vps20p interaction involves the coiled-coil domain of each protein, whereas the Vps4p-Vta1p interaction involves the (non-coiled-coil) C-terminus of each protein. Deletion of either VPS20 (vps20Delta) or VTA1 (vta1Delta) leads to similar class E Vps(-) phenotypes resembling those of vps4Delta, including carboxypeptidase Y (CPY) secretion, a block in ubiquitin-dependent MVB sorting, and a delay in both post-internalisation endocytic transport and biosynthetic transport to the vacuole. The vacuole resident membrane protein Sna3p (whose MVB sorting is ubiquitin-independent) does not appear to exit the class E compartment or reach the vacuole in cells lacking Vps20p, Vta1p or Vps4p, in contrast to other proteins whose delivery to the vacuole is only delayed. We propose that Vps20p and Vta1p regulate Vps4p function in vivo.

Fungal and microbial volatile organic compounds exposure assessment in a waste sorting plant

In the management of solid waste, pollutants over a wide range are released with different routes of exposure for workers. The potential for synergism among the pollutants raises concerns about potential adverse health effects, and there are still many uncertainties involved in exposure assessment. In this study, conventional (culture-based) and molecular real-time polymerase chain reaction (RTPCR) methodologies were used to assess fungal air contamination in a waste-sorting plant which focused on the presence of three potential pathogenic/toxigenic fungal species: Aspergillus flavus, A. fumigatus, and Stachybotrys chartarum. In addition, microbial volatile organic compounds (MVOC) were measured by photoionization detection. For all analysis, samplings were performed at five different workstations inside the facilities and also outdoors as a reference. Penicillium sp. were the most common species found at all plant locations. Pathogenic/toxigenic species (A. fumigatus and S. chartarum) were detected at two different workstations by RTPCR but not by culture-based techniques. MVOC concentration indoors ranged between 0 and 8.9 ppm (average 5.3 ± 3.16 ppm). Our results illustrated the advantage of combining both conventional and molecular methodologies in fungal exposure assessment. Together with MVOC analyses in indoor air, data obtained allow for a more precise evaluation of potential health risks associated with bioaerosol exposure. Consequently, with this knowledge, strategies may be developed for effective protection of the workers.

Assessment of fungal contamination in waste sorting and incineration: case study in Portugal

Organic waste is a rich substrate for microbial growth, and because of that, workers from waste industry are at higher risk of exposure to bioaerosols. This study aimed to assess fungal contamination in two plants handling solid waste management. Air samples from the two plants were collected through an impaction method. Surface samples were also collected by swabbing surfaces of the same indoor sites. All collected samples were incubated at 27◦C for 5 to 7 d. After lab processing and incubation of collected samples, quantitative and qualitative results were obtained with identification of the isolated fungal species. Air samples were also subjected to molecular methods by real-time polymerase chain reaction (RT PCR) using an impinger method to measure DNA of Aspergillus flavus complex and Stachybotrys chartarum. Assessment of particulate matter (PM) was also conducted with portable direct-reading equipment. Particles concentration measurement was performed at five different sizes (PM0.5; PM1; PM2.5; PM5; PM10). With respect to the waste sorting plant, three species more frequently isolated in air and surfaces were A. niger (73.9%; 66.1%), A. fumigatus (16%; 13.8%), and A. flavus (8.7%; 14.2%). In the incineration plant, the most prevalent species detected in air samples were Penicillium sp. (62.9%), A. fumigatus (18%), and A. flavus (6%), while the most frequently isolated in surface samples were Penicillium sp. (57.5%), A. fumigatus (22.3%) and A. niger (12.8%). Stachybotrys chartarum and other toxinogenic strains from A. flavus complex were not detected. The most common PM sizes obtained were the PM10 and PM5 (inhalable fraction). Since waste is the main internal fungal source in the analyzed settings, preventive and protective measures need to be maintained to avoid worker exposure to fungi and their metabolites.

Measuring and evaluating the impacts of TOD measures: searching for evidence of TOD characteristics in Azambuja train line

TOD (Transit Oriented Development) is typically defined as a high density mixed area (residential and commercial) within easy walking distance of a high capacity public transport station (typically within an 800m buffer area). TOO is viewed as a set of strategies to increase the use of public transport, increasing walking activity, containing urban sprawl, and creating more liveable places. It is believed that this type of combined strategies will improve sustainable growth. This work is an exploratory work for evidence of TOD characteristics in train station areas in Azambuja train line, setting further methodologies to evaluate the success of TOD areas.

Aging and Alzheimer's disease: Searching for novel molecular cues

Tese de Doutoramento em Ciências da Saúde

Prevalence of thyroiditis and immunohistochemistry study searching for a morphologic consensus in morphology of autoimmune thyroiditis in a 4613 autopsies series

We sought to verify the prevalence of lymphocytic thyroiditis (LT) and Hashimoto's thyroiditis (HT) in autopsy materials. Cases examined between 2003 and 2007 at the Department of Pathology of Faculty of Medicine of São Paulo University were studied. Immunohistochemical analyses were conducted in selected cases to characterize the type of infiltrating mononuclear cells; in addition, we evaluated the frequency of apoptosis by TUNEL assay technique and caspase-3 immunostaining. Significant increase in overall thyroiditis frequency was observed in the present series when compared with the previous report (2.2978% vs. 0.0392%). Thyroiditis was more prevalent among older people. Selected cases of LT and HT (5 cases each) had their infiltrating lymphocytes characterized by immunohistochemical analyses. Both LT and HT showed similar immunostaining patterns for CD4, CD8, CD68, thus supporting a common pathophysiology mechanism and indicating that LT and HT should be considered different presentations of a same condition, that is, autoimmune thyroiditis. Moreover, apoptosis markers strongly evidenced that apoptosis was present in all studied cases. Our results demonstrated an impressive increase in the prevalence of thyroiditis during recent years and our data support that the terminology of autoimmune thyroiditis should be used to designate both LT and HT. This classification would facilitate comparison of prevalence data from different series and studies.

Búsqueda de marcadores de evolución y tratamiento en la miocardiopatía por Enfermedad de Chagas. Searching for indicators of evolution and treatment of Chagas disease miocardiopathy.

El 30 por ciento de personas infectadas con T. cruzi desarrollará una cardiopatía chagásica de expresión clínica variada. Por esta razón es relevante identificar marcadores genéticos y de evolución de la miocardiopatía a fin de clasificar a los pacientes, acorde al grado de riesgo de desarrollar la enfermedad, así como es necesario investigar sobre mejores tratamientos.Los marcadores genéticos de riesgo (polimorfismos relacionados con enfermedades) colaboran identificando genes involucrados en enfermedades poligénicas. Analizaremos SNPs (single nucleotide polymorphism) localizados en zonas potencialmente funcionales de los genes de endotelina-1, su receptor A, de SOD-Mn, y de TNF alfa, factores que intervendrían en la expresión de severidad de la cardiopatía.El corazón es un órgano altamente dependiente de la energía provista por las mitocondrias y éstas son blanco de mediadores inflamatorios que se producen con el ingreso del parásito; por eso estudiaremos en corazones de ratones y de pacientes chagásicos las alteraciones genéticas, morfológicas y funcionales mitocondriales con el fin de determinar lesiones y evolución de las mismas.Existen controversias en tratar la Enfermedad de Chagas fuera de la etapa aguda por la toxicidad de las drogas. La clomipramina antidepresivo usado en siquiatría, demostró impedir la evolución de la infección aguda en modelos experimentales; proponemos el tratamiento con benznidazol a la mitad de la dosis habitual asociada a clomipramina en bajas concentraciones en modelos experimentales en el estadío crónico. Estos resultados aportarán a la fisiopatogenia de la miocardiopatía chagásica, al contar con marcadores de evolución, severidad y de probable riesgo de desarrollar la cardiopatía y serán un aporte a la prevención y nuevos tratamientos.

An analysis of the use of photogrammetric sorting to audit construction and demolition waste production on site

This study analyses the area of construction and demolition waste (C & D W) auditing. The production of C&DW has grown year after year since the Environmental Protection Agency (EPA) first published a report in 1996 which provided data for C&D W quantities for 1995 (EPA, 1996a). The most recent report produced by the EPA is based on data for 2005 (EPA, 2006). This report estimated that the quantity of C&DW produced for that period to be 14 931 486 tonnes. However, this is a ‘data update’ report containing an update on certain waste statistics so any total provided would not be a true reflection of the waste produced for that period. This illustrates that a more construction site-specific form of data is required. The Department of Building and Civil Engineering in the Galway-Mayo Institute of Technology have carried out two recent research projects (Grimes, 2005; Kelly, 2006) in this area, which have produced waste production indicators based on site-specific data. This involved the design and testing of an original auditing tool based on visual characterisation and the application of conversion factors. One of the main recommendations of these studies was to compare this visual characterisation approach with a photogrammetric sorting methodology. This study investigates the application of photogrammetric sorting on a residential construction site in the Galway region. A visual characterisation study is also carried out on the same project to compare the two methodologies and assess the practical application in a construction site environment. Data collected from the waste management contractor on site was also used to provide further evaluation. From this, a set of waste production indicators for new residential construction was produced: □ 50.8 kg/m2 for new residential construction using data provided by the visual characterisation method and the Landfill Levy conversion factors. □ 43 kg/m2 for new residential construction using data provided by the photogrammetric sorting method and the Landfill Levy conversion factors. □ 23.8 kg/m2 for new residential construction using data provided by Waste Management Contractor (WMC). The acquisition of the data from the waste management contractor was a key element for testing of the information produced by the visual characterisation and photogrammetric sorting methods. The actual weight provided by the waste management contractor shows a significant difference between the quantities provided.