"Ex vivo" and "in vivo" studies to assess chemical effects on steroidogenesis in fish: development and application of methods

The aquatic environment receives many chemical substances of natural or anthropogenic origin, which can influence the endocrine functions and health of wildlife. Various examples of endocrine disruption in wildlife were documented in aquatic organisms, for which associations between reproductive and developmental effects and exposure to endocrine-disrupting chemicals (EDCs) have been demonstrated. Since most of the endocrine-disrupting effects reported appear to be a consequence of feminization of males, most ecotoxicological research has been directed to identify estrogenic chemicals. However, the endocrine-disrupting effects exerted by EDCs can result from different mechanisms such as agonism or antagonism of endogenous steroid hormones via interaction with steroid hormone-receptors, or interference with the sex steroid hormone synthesis. Given the potential threat of these EDCs for wildlife, effective testing methods are required by regulatory agencies and industry to identify and assess the different mechanisms of action by which the EDCs exert their adverse effects. Testing strategies for endocrine disruption are being developed, in particular with fish test assays. These strategies are based on tiered approach, starting with fish in vitro and in vivo screening assays that identify and inform on potential endocrine mechanisms and effects. The results of the screening assays have then to be confirmed by higher tiered fish in vivo assays that characterize any apical adverse effect resulting from endocrine mode of action. Although the assays to screen for chemicals interacting with sex steroid receptor are widely available, tests to identify and inform on effects of chemicals that act via disrupting sex steroid biosynthesis still need to be developed. The aim of this thesis was therefore to develop and evaluate the potential of different fish test methods focused on chemicals that may interfere with the sex steroid biosynthesis. In a first step, an ex vivo gonad assay from juvenile brown trout (Salmo trutta fario) was developed to specifically identify substances that disrupt the activity of enzymes involved in the sex steroid biosynthesis. The ex vivo gonad assay was applied to test model chemicals, known or suspected to inhibit sex steroid biosynthesis: 1,4,6-androstatriene-3,17-dione (ATD), an aromatase inhibitor pharmaceutical; prochloraz, an imidazole fungicide; tributyltin (TBT), an organotin compound and persistent organic pollutant. Their effects in the ex vivo gonad assay were assessed by measuring 17ß-estradiol and testosterone concentrations from the culture medium. The different profile of sex steroid concentrations obtained for each chemical exposure showed that the ex vivo gonad assay cannot only identify the chemicals disrupting the steroidogenesis, but has also the potential to inform on their specific mechanism of action. To further evaluate the ex vivo gonad assay and its potential to inform on in vivo effects, the responses to prochloraz and TBT exposure were compared in the ex vivo and in vivo exposure assays of juvenile brown trout. The effects were again assessed by measuring 17ß-estradiol and testosterone concentrations, and also by analyzing somatic indices and histopathology of gonads from fish exposed in vivo to the test chemicals. The results of this study demonstrated that the ex vivo gonad assay has the potential to inform on in vivo effects of chemicals disrupting the steroidogenesis and accordingly on their potential to affect sexual development of fish. This study highlights the potential of the ex vivo gonad assay to be a sensitive and informative tool for such EDCs. The ex vivo gonad assay was then used to further analyze the potential of the steroidogenic inhibitors to impair the regulation of early sexual development of fish. This was investigated by comparing cellular and molecular effects of ex vivo and in vivo exposures to ATD, prochloraz and TBT. The ex vivo 17ß-estradiol and testosterone concentrations were measured and ex vivo/in vivo gene expression of the aromatase and insulin-like growth factors (IGFs), involved in the regulation of sexual development, were compared. It was shown that the test chemicals could interfere with both the sex steroid and IGF systems and potentially lead to altered sexual development. Finally, to confirm the potential of steroidogenic inhibitors to impair sex differentiation and development, a higher tier fish in vivo test, a Fish Sexual Development Test (FSDT), was applied. Two model fish species, zebrafish (Danio rerio) and fathead minnow (Pimephales promelas) were exposed, from embryo to sexual maturity, to prochloraz and the effects on their sexual differentiation were compared by assessing the sex ratios, the histology of gonads, and the vitellogenin concentration. The results of this last study demonstrated that, although the different strategies of sexual differentiation of zebrafish and fathead minnow influence the response of their gonad morphology and their sensitivity to prochloraz exposure, the exposure to steroidogenic inhibitors has the potential to alter their sexual development and subsequently the reproductive success and population structure of fish. To conclude, we suggest that the evaluation of the ex vivo and in vivo methods in our different studies are sensitive and valuable tools for application in environmental risk assessment of chemicals interfering with the sex steroid biosynthesis. Although further characterization and validation studies of the ex vivo gonad and FSDT assays are still required, the combination of both ex vivo and in vivo assays represents a good testing approach

Parasitic Infection and Immunity—A Special Biomedicines Issue

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PLGA Based Drug Carrier and Pharmaceutical Applications: The Most Recent Advances

Poly(lactic-co-glycolic acid) (PLGA) is one of the most successful polymers that has been used to produce medicines, such as drug carriers (DC) [...]</jats:p

Mining clinically relevant signatures from colony morphology images

Human pathogenic bacteria are a common cause of infection and a recurrent disease complication, causing millions of deaths and bringing considerable costs to clinical facilities. Timely identification of the bacteria involved in infection is essential to clinical diagnosis and thus, disease management. Often, colony morphology observation complements conventional microbial diagnosis as means to detect phenotypic heterogeneity. Colony morphology variants, including rough, small and mucoid phenotypes, have been isolated from several human infections. Notably, P. aeruginosa mucoid phenotype and S. aureus small colony variants (SCV) are of relevance in the study of cystic fibrosis, showing a markedly more resistant phenotype to antibiotics. Several other antibiotic resistant morphotypes have been identified in bacteria related to chronic and acute infections and even to device-associated infections. Clinical decision making relies on the ability to profile pathogenic behaviours, to interpret such behaviours adequately, and to monitor and control new occurrences. Therefore, colony morphology profiles could be useful to anticipate the response (at least, to a certain extent) to be expected of a bacterial community to antimicrobial treatment. Colony morphology features can be viewed as part of a pathogenic signature. By collecting signatures across diseases and species, predictive and associative data mining can be used to debrief these signatures regarding occurrence-consequence relationships. Moreover, the correlation of morphological manifestations with particular regulatory responses (and pathways) can be explored by further detailing the signatures in terms of genome and proteome. Overall, these efforts originate a rich and varied set of features for decision making. The aim of this work was to evaluate the morphotyping ability of image processing tools, both and to maximise characterisation abilities. The case study relates to previously described pathogenic morphotypes, publicly available at the colony morphology database MorphoCol (http://morphocol.org/). Evaluation addressed the inter-annotation agreement between expert visual inspection and automatic image processing using the publicly available ImageJ tool. The level of agreement obtained was sufficient to consider the creation of a semi-automatic image annotation pipeline. Since automatic processing is focused on technical aspects of the images (e.g. pixel intensity), it is unbiased towards the characterisation of multiple species or diseases and does not implicate expert knowledge. Then, MALDI-TOF MS analyses could be used to obtain molecular signatures and support the analysis of meaningful pathogenic regulatory cascades. Preliminary results on this side of the pipeline showed promising insights on the biology of pathogenic morphotypes. Financial support from IBB-CEB, FCT and FEDER (PTDC/SAU-ESA/646091/2006/FCOMP-01-0124-FEDER-007480) and Ana Sousa PhD Grant (SFRH/BD/72551/2010) are acknowledged

Impact of nutritional conditions on colony morphology variants isolated from P. aeruginosa and S. aureus biofilms

In natural habitats, microorganisms are challenged all the time due to stress conditions imposed by the surrounding environment. To adapt to these environmental changes, bacteria alter their physiological and genetic traits. This adaptive behavior may be achieved by phenotype switching. This process consists in a reversible switch of phenotypes, as a mechanism ON/OFF, which occurs at high frequencies than spontaneous mutations. Colony morphology variation is the macroscopic feature of the phenotypic switching. Colony variation may have serious impact on bacterial virulence and antimicrobial resistance potentiating its ability to cause disease. Some colony variants are strongly associated to antibiotic resistance due to their presence in chronic infections despite antibiotic therapy. In cystic fibrosis, the switch of P. aeruginosa from non-mucoid to mucoid morphotype, which overproduce alginate, is a crucial stage to the establishment of this recalcitrant disease. Small colony variants (SCV) are other well-known resistant morphotype. These variants exhibited small size because its slow growth rate, pigmentation, haemolysis, reduced range of carbohydrate utilization and higher resistance to aminoglycosides antibiotics and cell-wall inhibitors. It has been growing the number of studies related with phenotypic switching and colony morphology characterization. However, normally each study reports the use of different solid growth media which makes the comparison between studies inaccurate. In order to clarify the role of nutritional conditions on bacterial colony morphologies and on its populational diversity, P. aeruginosa and S. aureus planktonic and biofilm-growing cells were spread onto the most common solid laboratory media (TSA, MHA, LB agar, MacConkey agar and Columbia horse blood agar). Additionally, the reproducibility of each medium was also inspected. Data showed that P. aeruginosa and S. aureus colony morphotypes are strongly influenced by the plating medium used. The main differences observed were the size, texture and form of colonies. The largest colonies were detected in TSA, MHA and LB agar. Colonies grown on MHA and LB agar were very similar possibly due to their identical nutritional composition. All the solid media tested showed reproducibility between assays except the Columbia horse blood agar which exhibited some inconsistency probably due to the presence of blood in its composition. Amongst the solid media tested, for planktonic and biofilm cultures, TSA gave rise to higher number of colony variants. Phenotype diversity seems to be more influenced by nutritional factors when bacteria derived from biofilms. This study allows concluding that, in contrast to fungi, bacterial colony appearance is influenced by the nutritional conditions of the solid media used to spread the cells. This evidence should be taking into account when important phenomena as phenotypic switching are going to be studied. The data obtained with this preliminary work may question the classification of colony morphotypes used until now.Fundação para a Ciência e Tecnologia (FCT) - Projecto PTDC/SAUESA/6460912006/FCOMP-01-0124-FEDER-007480, bolsa de Doutoramento SFRH/BD/31065/2006European CommunityFEDER - Program COMPET

Cancro do colo do útero: que rastreio?

O cancro do colo do útero é um dos cancros evitáveis mais frequente nas mulheres, Ferlay (2004) e Parkin(2005). O padrão epidemiológico da doença, caracterizado por um período longo entre a detecção das primeiras lesões e a instalação da doença, favorece a sua inclusão em programas de rastreio organizado, por serem custo-eficientes e terem ganhos de saúde associados, ver Anttila (2004), Castellsague (2006) e Koutsky (1998). O presente estudo teve como objectivo auxiliar os elementos da Comissão de Rastreio do Cancro do Colo do Útero na Região Norte na tomada de decisões relativas aos aspectos mais controversos dos rastreios ao cancro do colo do útero: limite de idade das mulheres rastreadas, intervalo entre exames e método utilizado. Assim, construiu-se um modelo matemático, com o intuito de simular os impactos económicos e de saúde, das combinações das várias alternativas de rastreio. Por fim, foi feita uma análise custo-benefício das soluções encontradas e determinou-se a solução, i.e. a opção de rastreio, mais vantajosa.Cervical cancer is one of the most frequent avoidable cancers in women, Ferlay (2004) and Parkin(2005). The natural history of the disease, characterized for a long period between the detection of the rst injuries and invasive cancer, favours its inclusion in organized screening programs, since they are cost-e ective and have clear advantages for the health of the population, see Anttila (2004), Castellsague (2006) and Koutsky (1998). The aim of this study is to construct a mathematical model that can help the elements of the Commission delegated for planning the screening program, to decide about the most controversial aspects: target population, interval between examinations and method to be used. The mathematical model allows to simulate the economical and health impact of all the possible solutions. Finally, a cost-bene t analysis of the combined screening alternatives was done to determine the solution, i.e, the more advantageous screening program

Warehouse design and product assignment and allocation: A mathematical programming model

Warehouses can be considered one of the most important nodes in supply chains. The dynamic nature of today's markets compels organizations to an incessant reassessment in an effort to respond to continuous challenges. Therefore warehouses must be continually re-evaluated to ensure that they are consistent with both market's demands and management's strategies. In this paper we discuss a mathematical programming model aiming to support product assignment and allocation to the functional areas as well as the size of each area. In particular a large mixed-integer programming model (MILP) is presented to capture the tradeoffs among the different warehouse costs in order to achieve global optimal design satisfying throughput requirements