Effect-based proteomic detection of growth promoter abuse

Unregulated growth promoter use in food-producing animals is an issue of concern both from food safety and animal welfare perspectives. However, the monitoring of such practices is analytically challenging due to the concerted actions of users to evade detection. Techniques based on the monitoring of biological responses to exogenous administrations have been proposed as more sensitive methods to identify treated animals. This study has, for the first time, profiled plasma proteome responses in bovine animals to treatment with nortestosterone decanoate and 17 beta-oestradiol benzoate, followed by dexamethasone administration. Two-dimensional fluorescence differential in-gel electrophoresis analysis revealed a series of hepatic and acute-phase proteins within plasma whose levels were up- or down-regulated within phases of the treatment regime. Surface plasmon resonance (SPR) immuno-assays were developed to quantify responses of identified protein markers during the experimental treatment study with a view to developing methods which can be used as screening tools for growth promoter abuse detection. SPR analysis demonstrated the potential for plasma proteins to be used as indicative measures of growth promoter administrations and concludes that the sensitivity and robustness of any detection approach based on plasma proteome analysis would benefit from examination of a range of proteins representative of diverse biological processes rather being reliant on specific individual markers.

The Role of Growth Trajectories in Classifying Fetal Growth Restriction

OBJECTIVE: To examine the validity of a growth trajectory method to discriminate between pathologically and constitutionally undergrown fetuses using repeated measures of estimated fetal weight.

METHODS: In a prospective, observational, multicenter study in Ireland, 1,116 women with a growth-restricted fetus diagnosed participated with the objective of evaluating ultrasound findings as predictors of pediatric morbidity and mortality. Fetal growth trajectories were based on estimated fetal weight.

RESULTS: Between 22 weeks of gestation and term, two fetal growth trajectories were identified: normal (96.7%) and pathologic (3.3%). Compared with the normal trajectory, the pathologic trajectory was associated with an increased risk for preeclampsia (odds ratio [OR] 8.1, 95% confidence interval [CI] 2.6–23.4), increased umbilical artery resistance at 30 weeks of gestation (OR 12.6, 95% CI 4.6–34.1) or 34 weeks of gestation (OR 28.0, 95% CI 8.9–87.7), reduced middle cerebral artery resistance at 30 weeks of gestation (OR 0.33, 95% CI 0.12–0.96) or 34 weeks of gestation (OR 0.14, 95% CI 0.03–0.74), lower gestational age at delivery (mean 32.02 weeks of gestation compared with 38.02 weeks of gestation; P<.001), and higher perinatal complications (OR 21.5, 95% CI 10.5–44.2). In addition, 89.2% of newborns with pathologic fetal growth were admitted to neonatal intensive care units compared with 25.9% of those with normal growth.

CONCLUSIONS: Fetal growth trajectory analysis reliably differentiated fetuses with a pathologic growth pattern among a group of women with growth-restricted fetuses. With further development, this approach could provide clarity to how we define, identify, and ultimately manage pathologic fetal growth.

LEVEL OF EVIDENCE: II

Youth in Contexts of Political Violence: A Developmental Approach to the Study of Youth Identity and Emotional Security in Their Communities

Going beyond the association between youth exposure to political violence and psychopathology, the current article examines within-person change in youth strength of identity with their ethno-political group and youth reports of the insecurity in their communities. Conceptually related but growing out of different paradigms, both group identity and emotional insecurity have been examined as key variables impacting youth responses to threats from other group members. The goal of the current study is to review previous studies examining these two key variables and to contribute new analyses, modeling within-person change in both variables and examining covariation in their growth. The current article uses data from 823 Belfast adolescents over 4 years. The results suggest youth are changing linearly over age in both constructs and that there are ethno-political group differences in how youth are changing. The results also indicate that change in insecurity is related to strength of identity at age 18, and strength of identity and emotional insecurity are related at age 18. Implications and directions for future work in the area of youth and political violence are discussed. © 2014 American Psychological Association.

Fibroblast growth factor receptor 4 (FGFR4): a targetable regulator of drug resistance in colorectal cancer

The discovery of underlying mechanisms of drug resistance, and the development of novel agents to target these pathways, is a priority for patients with advanced colorectal cancer (CRC). We previously undertook a systems biology approach to design a functional genomic screen and identified fibroblast growth factor receptor 4 (FGFR4) as a potential mediator of drug resistance. The aim of this study was to examine the role of FGFR4 in drug resistance using RNAi and the small-molecule inhibitor BGJ398 (Novartis). We found that FGFR4 is highly expressed at the RNA and protein levels in colon cancer tumour tissue compared with normal colonic mucosa and other tumours. Silencing of FGFR4 reduced cell viability in a panel of colon cancer cell lines and increased caspase-dependent apoptosis. A synergistic interaction was also observed between FGFR4 silencing and 5-fluorouracil (5-FU) and oxaliplatin chemotherapy in colon cancer cell lines. Mechanistically, FGFR4 silencing decreased activity of the pro-survival STAT3 transcription factor and expression of the anti-apoptotic protein c-FLIP. Furthermore, silencing of STAT3 resulted in downregulation of c-FLIP protein expression, suggesting that FGFR4 may regulate c-FLIP expression via STAT3. A similar phenotype and downstream pathway changes were observed following FGFR4 silencing in cell lines resistant to 5-FU, oxaliplatin and SN38 and upon exposure of parental cells to the FGFR small-molecule inhibitor BGJ398. Our results indicate that FGFR4 is a targetable regulator of chemo-resistance in CRC, and hence inhibiting FGFR4 in combination with 5-FU and oxaliplatin is a potential therapeutic strategy for this disease.

Working with fathers to improve children's well-being: Results of a survey exploring service provision and intervention approach in the UK

Interventions for fathers are a recent growth area in family services. Although some specific approaches are beginning to be evaluated, there is little known about what kinds of interventions are more generally being used in practice. A web-based survey of practitioners was conducted in the UK, with contact being made via local authority service managers. Two hundred and twenty-one responses were received from 53% of local authorities. Both interventions specifically for fathers and services for both parents were targeted in the survey. Results are reported on organisational location; targeting of services; type of intervention; numbers and percentages of men attending services, recruitment of fathers; evaluation strategies; and ideological and theoretical approaches. Numbers of fathers engaged are relatively low - e.g. the median annual number of fathers attending structured parenting courses was eight and in courses for both parents, 21% of those attending were men. Responses on ideological and theoretical approaches suggest that overt gender politics play only a small part, but that the dominant views of practitioners are in line with mainstream approaches to parenting support. Cognitive and behavioral approaches were the most popular.

Sphingosine Kinase 1 Promotes Malignant Progression in Colon Cancer and Independently Predicts Survival of Patients With Colon Cancer by Competing Risk Approach in South Asian Population

OBJECTIVES: Sphingosine kinase 1 (SphK1) phosphorylates the membrane sphingolipid, sphingosine, to sphingosine-1-phosphate (S1P), an oncogenic mediator, which drives tumor cell growth and survival. Although SphK1 has gained increasing prominence as an oncogenic determinant in several cancers, its potential as a therapeutic target in colon cancer remains uncertain. We investigated the clinical relevance of SphK1 expression in colon cancer as well as its inhibitory effects in vitro.

METHODS: SphK1 expression in human colon tumor tissues was determined by immunohistochemistry and its clinicopathological significance was ascertained in 303 colon cancer cases. The effects of SphK1 inhibition on colon cancer cell viability and the phosphoinositide 3-kinase (PI3K)/Akt cell survival pathway were investigated using a SphK1-selective inhibitor-compound 5c (5c). The cytotoxicity of a novel combination using SphK1 inhibition with the chemotherapeutic drug, 5-fluorouracil (5-FU), was also determined.

RESULTS: High SphK1 expression correlated with advanced tumor stages (AJCC classification). Using a competing risk analysis model to take into account disease recurrence, we found that SphK1 is a significant independent predictor for mortality in colon cancer patients. In vitro, the inhibition of SphK1 induced cell death in colon cancer cell lines and attenuated the serum-dependent PI3K/Akt signaling. Inhibition of SphK1 also enhanced the sensitivity of colon cancer cells to 5-FU.

CONCLUSION: Our findings highlight the impact of SphK1 in colon cancer progression and patient survival, and provide evidence supportive of further development in combination strategies that incorporate SphK1 inhibition with current chemotherapeutic agents to improve colon cancer outcomes.

Control of biofilm growth through photodynamic treatments combined with chemical inhibitors: in vitro evaluation methods

The rock/atmosphere interface is inhabited by a complex microbial community including bacteria, algae and fungi. These communities are prominent biodeterioration agents and remarkably influence the status of stone monuments and buildings. Deeper comprehension of natural biodeterioration processes on stone surfaces has brought about a concept of complex microbial communities referred to as "subaerial biofilms". The practical implications of biofilm formation are that control strategies must be devised both for testing the susceptibility of the organisms within the biofilm and treating the established biofilm. Model multi-species biofilms associated with mineral surfaces that are frequently refractory to conventional treatment have been used as test targets. A combination of scanning microscopy with image analysis was applied along with traditional cultivation methods and fluorescent activity stains. Such a polyphasic approach allowed a comprehensive quantitative evaluation of the biofilm status and development. Effective treatment strategies incorporating chemical and physical agents have been demonstrated to prevent biofilm growth in vitro. Model biofilm growth on inorganic support was significantly reduced by a combination of PDT and biocides

Proteomic identification of plasma proteins as markers of growth promoter abuse in cattle

Growth-promoting agents are continually misused for increasing animal growth and fraudulent gain in the meat industry, yet detection rates from conventional targeted testing for drug residues do not reflect this. This is because testing currently relies on direct detection of drugs or related metabolites and administrators of such compounds can take adaptive measures to avoid detection through the use of endogenous or unknown drugs, and low dose or combined mixtures. New detection methods are needed which focus on the screening of biological responses of an animal to such growth-promoting agents as it has been demonstrated that genomic, proteomic and metabolomics profiles are altered by xenobiotic intake. Therefore, an untargeted proteomics approach using comparative two-dimensional gel electrophoresis (2DE) was carried out to identify putative proteins altered in plasma after treatment with oestradiol, dexamethasone or prednisolone. Twenty-four male cattle were randomly assigned to four groups (n = 6) for experimental treatment over 40 days, namely a control group of non-treated cattle, and three groups administered 17β-oestradiol-3-benzoate (0.01 mg/kg, intramuscular), dexamethasone sodium phosphate (0.7 mg/day, per os) or prednisolone acetate (15 mg/day, per os), respectively. Plasma collected from each animal at day 25 post study initiation was subjected to proteomic analysis by 2DE for comparison of protein expression between treated and untreated animals. Analysis of acquired gel images revealed 22 plasma proteins which differed in expression by more than 50 % (p < 0.05) in treated animals compared to untreated animals. Proteins of interest underwent identification by LC–MS/MS analysis and were found to have associated roles in transport, blood coagulation, immune response and metabolism pathways. In this way, seven proteins are highlighted as novel biomarker candidates including transthyretin which is shown to be significantly increased in all treatment groups compared to control animals and potentially may find use as global markers of suspect anabolic practice.

Hypoxia response element-driven cytosine deaminase/5-fluorocytosine gene therapy system:a highly effective approach to overcome the dynamics of tumour hypoxia and enhance the radiosensitivity of prostate cancer cells in vitro

BACKGROUND: We proposed to exploit hypoxia-inducible factor (HIF)-1alpha overexpression in prostate tumours and use this transcriptional machinery to control the expression of the suicide gene cytosine deaminase (CD) through binding of HIF-1alpha to arrangements of hypoxia response elements. CD is a prodrug activation enzyme, which converts inactive 5-fluorocytosine to active 5-fluorouracil (5-FU), allowing selective killing of vector containing cells.

METHODS: We developed a pair of vectors, containing either five or eight copies of the hypoxia response element (HRE) isolated from the vascular endothelial growth factor (pH5VCD) or glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (pH8GCD) gene, respectively. The kinetics of the hypoxic induction of the vectors and sensitization effects were evaluated in 22Rv1 and DU145 cells in vitro.

RESULTS: The CD protein as selectively detected in lysates of transiently transfected 22Rv1 and DU145 cells following hypoxic exposure. This is the first evidence of GAPDH HREs being used to control a suicide gene therapy strategy. Detectable CD levels were sustained upon reoxygenation and prolonged hypoxic exposures. Hypoxia-induced chemoresistance to 5-FU was overcome in both cell lines treated with this suicide gene therapy approach. Hypoxic transfectants were sensitized to prodrug concentrations that were ten-fold lower than those that are clinically relevant. Moreover, the surviving fraction of reoxygenated transfectants could be further reduced with the concomitant delivery of clinically relevant single radiation doses.

CONCLUSIONS: This strategy thus has the potential to sensitize the hypoxic compartment of prostate tumours and improve the outcome of current therapies.

Punishment, youth justice and cultural contingency: Towards a balanced approach

Reflecting developments in the broader penological realm, accounts have been advanced over the last number of decades about a ‘punitive turn’ in the youth justice systems of Western democracies. Against the background of this work, this project seeks to identify convergent and divergent trends in the youth justice systems of England, the Republic of Ireland and Northern Ireland as well as the rationalities and discourses animating these. The results lend support to research emphasising the continued salience of national, regional and local factors on penal outcomes but also suggest the need to steer an analytical path somewhere between nomothetic (convergent) and idiographic (divergent) accounts.

The influence of water motion on the growth rate of the kelp Laminaria digitata

The shallow water kelp Laminaria digitata, abundant in coastal zones of the North Atlantic, is exposed to a range of hydrodynamic environments that makes it ideal for assessing the role of water motion on their growth rate. Here we quantify the growth of L. digitata, as a factor of blade and stipe elongation, at sites adjacent to Strangford Lough, Northern Ireland under different hydrodynamic conditions over a one year period. A modelling approach was used to numerically determine both the temporal and spatial variability of the hydrodynamic environment. Ambient seawater nutrient concentrations, temperature and irradiance were measured as well as the internal nutrient status of the L. digitata populations. Kelp populations growing in the greatest and lowest water motion showed the lowest growth rates. Differences observed in growth rate could not be attributed to seawater nutrient availability, temperature or light. The internal nutrient status also suggested no influence on the observed differences in growth rate. Therefore if there are minimal differences in light, temperature and nutrients between sites, then populations of L. digitata exposed to different water motions are likely to exhibit different growth rates. It is suggested that the growth rate differences observed were a function of water motion with the possibility that, in response to the hydrodynamic forces experienced by the algal cells, L. digitata kelps in the high energy environments were putting more energy into strengthening cell walls rather than blade elongation

Determinants of online travel purchase intentions: a holistic approach

Online travel shopping has attracted researchers due to its significant growth and there is a growing body of literature in this field. However, research on what drives consumers to purchase travel online has typically been fragmented. In fact, existing studies have largely concentrated on examining consumers’ online travel purchases either grounded on Davis’s Technology Acceptance Model, on the Theory of Reasoned Action and its extension, the Theory of Planned Behaviour or on Roger’s model of perceived innovation attributes, the Innovation Diffusion Theory. A thorough literature review has revealed that there is a lack of studies that integrate all theories to better understand online travel shopping. Therefore, based on relevant literature in tourism and consumer behaviour, this study proposes and tests an integrated model to explore which factors affect intentions to purchase travel online. Furthermore, it proposes a new construct, termed social media involvement, defined as a person’s level of interest or emotional attachment with social media, and examines its relationship with intentions to purchase travel online. To test the 18 hypotheses, a quantitative approach was followed by first collecting data through an online survey. With a sample of 1,532 Worldwide Internet users, Partial Least Squares analysis was than conducted to assess the validity and reliability of the data and empirically test the hypothesized relationships between the constructs. The results indicate that intentions to purchase travel online is mostly determined by attitude towards online shopping, which is influenced by perceived relative advantages of online travel shopping and trust in online travel shopping. In addition, the findings indicate that the second most important predictor of intentions to purchase travel online is compatibility, an attribute from the Innovation Diffusion Theory. Furthermore, even though online shopping is nowadays a common practice, perceived risk continues to negatively affect intentions to purchase travel online. The most surprising finding of this study was that Internet users more involved with social media for travel purposes did not have higher intentions to purchase travel online. The theoretical contributions of this study and the practical implications are discussed and future research directions are detailed.

An integrated and comparative approach to genetic selection of the aquaculture species Dicentrarchus labrax

The European sea bass, Dicentrarchus labrax, is one of the most important marine species cultivated in Southern Europe and has not benefited from selective breeding. One of the major goals in the sea bass (D. labrax) aquaculture industry is to understand and control the complexity of growth associated traits. The aim of the methodology developed for the studies reported in the thesis was not only to establish genetic and genomic resources for sea bass, but to also develop a conceptual strategy to efficiently create knowledge in a research environment that can easily be transferred to the aquaculture industry. The strategy involved; i) establishing an annotated sea bass transcriptome and then using it to, ii) identify new genetic markers for target QTL regions so that, iii) new QTL analysis could be performed and marker based resolution of the DNA regions of interest increased, and then iv) to merge the linkage map and the physical map in order to map the QTL confidence intervals to the sea bass genome and identify genes underlying the targeted traits. Finally to test if genes in the QTL regions that are candidates for divergent growth phenotypes have modified patterns of transcription that reflects the modified whole organism physiology SuperSAGE-SOLiD4 gene expression was used with sea bass with high growth heterogeneity. The SuperSAGE contributed to significantly increase the transcriptome information for sea bass muscle, brain and liver and also led to the identification of putative candidate genes lying in the genomic region of growth related QTL. Lastly all differentially expressed transcripts in brain, liver and muscle of the European sea bass with divergent specific growth rates were mapped to gene pathways and networks and the regulatory pathways most affected identified and established the tissue specific changes underlying the divergent SGR. Owing to the importance of European sea bass to Mediterranean aquaculture and the developed genomics resources from the present thesis and from other studies it should be possible to implement genetic selection programs using marker assisted selection.

From embryonic stem cells to sensory hair cells : a programming approach

Tese de doutoramento, Ciências Biomédicas (Biologia do Desenvolvimento), Universidade de Lisboa, Faculdade de Medicina, 2014