Thyroid hormone and gland status of Greenland sledge dogs (Canis lupus familiaris) exposed to contaminated blubber

As a model of high trophic level carnivores, sledge dogs were fed from 2 to 18 months of age with minke whale blubber containing organohalogen compounds (OHC) corresponding to 128 µg PCB/day. Controls were fed uncontaminated porcine fat. Thyroid hormone levels were assessed in 7 exposed and 7 control sister bitches (sampled at age 6-18 months) and 4 exposed and 4 control pups, fed the same diet as their mothers (sampled age 3-12 months). Lower free and total T3 and T4 were seen in exposed vs. control bitches beyond 10 months of age, and total T3 was lower through 3-12 months of age in exposed pups. A negative correlation with thyroid gland weight was significant for SumDDT, as was a positive association with total T3 for dieldrin. This study therefore supports observational data that OHCs may adversely affect thyroid functions, and it suggests that OHC exposure duration of 10 months or more may be required for current OHC contamination levels to result in detectable adverse effects on thyroid hormone dynamics.

Dissolved Iron, Aluminium, and Dissolved Inorganic Phosphorus in the (Sub-)Tropical Atlantic Surface Ocean

Inorganic nitrogen depletion restricts productivity in much of the low-latitude oceans, generating a selective advantage for diazotrophic organisms capable of fixing atmospheric dinitrogen (N2). However, the abundance and activity of diazotrophs can in turn be controlled by the availability of other potentially limiting nutrients, including phosphorus (P) and iron (Fe). Here we present high-resolution data (~0.3°) for dissolved iron, aluminum, and inorganic phosphorus that confirm the existence of a sharp north-south biogeochemical boundary in the surface nutrient concentrations of the (sub)tropical Atlantic Ocean. Combining satellite-based precipitation data with results from a previous study, we here demonstrate that wet deposition in the region of the intertropical convergence zone acts as the major dissolved iron source to surface waters. Moreover, corresponding observations of N2 fixation and the distribution of diazotrophic Trichodesmium spp. indicate that movement in the region of elevated dissolved iron as a result of the seasonal migration of the intertropical convergence zone drives a shift in the latitudinal distribution of diazotrophy and corresponding dissolved inorganic phosphorus depletion. These conclusions are consistent with the results of an idealized numerical model of the system. The boundary between the distinct biogeochemical systems of the (sub)tropical Atlantic thus appears to be defined by the diazotrophic response to spatial-temporal variability in external Fe inputs. Consequently, in addition to demonstrating a unique seasonal cycle forced by atmospheric nutrient inputs, we suggest that the underlying biogeochemical mechanisms would likely characterize the response of oligotrophic systems to altered environmental forcing over longer timescales.

Effects of CO2 and iron availability on rbcL gene expression in Bering Sea diatoms

Iron (Fe) can limit phytoplankton productivity in approximately 40% of the global ocean, including in high-nutrient, low-chlorophyll (HNLC) waters. However, there is little information available on the impact of CO2-induced seawater acidification on natural phytoplankton assemblages in HNLC regions. We therefore conducted an on-deck experiment manipulating CO2 and Fe using Fe-deficient Bering Sea water during the summer of 2009. The concentrations of CO2 in the incubation bottles were set at 380 and 600 ppm in the non-Fe-added (control) bottles and 180, 380, 600, and 1000 ppm in the Fe-added bottles. The phytoplankton assemblages were primarily composed of diatoms followed by haptophytes in all incubation bottles as estimated by pigment signatures throughout the 5-day (control) or 6-day (Fe-added treatment) incubation period. At the end of incubation, the relative contribution of diatoms to chlorophyll a biomass was significantly higher in the 380 ppm CO2 treatment than in the 600 ppm treatment in the controls, whereas minimal changes were found in the Fe-added treatments. These results indicate that, under Fe-deficient conditions, the growth of diatoms could be negatively affected by the increase in CO2 availability. To further support this finding, we estimated the expression and phylogeny of rbcL (which encodes the large subunit of RuBisCO) mRNA in diatoms by quantitative reverse transcription polymerase chain reaction (PCR) and clone library techniques, respectively. Interestingly, regardless of Fe availability, the transcript abundance of rbcL decreased in the high CO2 treatments (600 and 1000 ppm). The present study suggests that the projected future increase in seawater pCO2 could reduce the RuBisCO transcription of diatoms, resulting in a decrease in primary productivity and a shift in the food web structure of the Bering Sea.

Synthesis, Modification, and Application of Siloxane Materials for Environmental Sensing

As human populations and resource consumption increase, it is increasingly important to monitor the quality of our environment. While laboratory instruments offer useful information, portable, easy to use sensors would allow environmental analysis to occur on-site, at lower cost, and with minimal operator training. We explore the synthesis, modification, and applications of modified polysiloxane in environmental sensing. Multiple methods of producing modified siloxanes were investigated. Oligomers were formed by using functionalized monomers, producing siloxane materials containing silicon hydride, methyl, and phenyl side chains. Silicon hydride-functionalized oligomers were further modified by hydrosilylation to incorporate methyl ester and naphthyl side chains. Modifications to the siloxane materials were also carried out using post-curing treatments. Methyl ester-functionalized siloxane was incorporated into the surface of a cured poly(dimethylsiloxane) film by siloxane equilibration. The materials containing methyl esters were hydrolyzed to reveal carboxylic acids, which could later be used for covalent protein immobilization. Finally, the siloxane surfaces were modified to incorporate antibodies by covalent, affinity, and adsorption-based attachment. These modifications were characterized by a variety of methods, including contact angle, attenuated total reflectance Fourier transform infrared spectroscopy, dye labels, and 1H nuclear magnetic resonance spectroscopy. The modified siloxane materials were employed in a variety of sensing schemes. Volatile organic compounds were detected using methyl, phenyl, and naphthyl-functionalized materials on a Fabry-Perot interferometer and a refractometer. The Fabry-Perot interferometer was found to detect the analytes upon siloxane extraction by deformation of the Bragg reflectors. The refractometer was used to determine that naphthyl-functionalized siloxanes had elevated refractive indices, rendering these materials more sensitive to some analytes. Antibody-modified siloxanes were used to detect biological analytes through a solid phase microextraction-mediated enzyme linked immunosorbent assay (SPME ELISA). The SPME ELISA was found to have higher analyte sensitivity compared to a conventional ELISA system. The detection scheme was used to detect Escherichia coli at 8500 CFU/mL. These results demonstrate the variety of methods that can be used to modify siloxanes and the wide range of applications of modified siloxanes has been demonstrated through chemical and biological sensing schemes.

Patterns of Behaviour, Group Structure and Reproductive Status Predict Levels of Glucocorticoid Metabolites in Zoo-Housed Ring-Tailed Lemurs, Lemur catta

In ring-tailed lemurs, Lemur catta, the factors modulating hypothalamic-pituitaryadrenal (HPA) activity differ between wild and semi-free-ranging populations. Here we assess factors modulating HPA activity in ring-tailed lemurs housed in a third environment: the zoo. First we validate an enzyme immunoassay to quantify levels of glucocorticoid (GC) metabolites in the faeces of L. catta . We determine the nature of the femalefemale dominance hierarchies within each group by computing David's scores and examining these in relation to faecal GC (fGC). Relationships between female age and fGC are assessed to evaluate potential age-related confounds. The associations between fGC, numbers of males in a group and reproductive status are explored. Finally, we investigate the value of 7 behaviours in predicting levels of fGC. The study revealed stable linear dominance hierarchies in females within each group. The number of males in a social group together with reproductive status, but not age, influenced fGC. The 7 behavioural variables accounted for 68% of the variance in fGC. The amounts of time an animal spent locomoting and in the inside enclosure were both negative predictors of fGC. The study highlights the flexibility and adaptability of the HPA system in ring-tailed lemurs.

Surface plasmon resonance biosensing: Approaches for screening and characterising antibodies for food diagnostics

Research in biosensing approaches as alternative techniques for food diagnostics for the detection of chemical contaminants and foodborne pathogens has increased over the last twenty years. The key component of such tests is the biorecognition element whereby polyclonal or monoclonal antibodies still dominate the market. Traditionally the screening of sera or cell culture media for the selection of polyclonal or monoclonal candidate antibodies respectively has been performed by enzyme immunoassays. For niche toxin compounds, enzyme immunoassays can be expensive and/or prohibitive methodologies for antibody production due to limitations in toxin supply for conjugate production. Automated, self-regenerating, chip-based biosensors proven in food diagnostics may be utilised as rapid screening tools for antibody candidate selection. This work describes the use of both single channel and multi-channel surface plasmon resonance (SPR) biosensors for the selection and characterisation of antibodies, and their evaluation in shellfish tissue as standard techniques for the detection of domoic acid, as a model toxin compound. The key advantages in the use of these biosensor techniques for screening hybridomas in monoclonal antibody production were the real time observation of molecular interaction and rapid turnaround time in analysis compared to enzyme immunoassays. The multichannel prototype instrument was superior with 96 analyses completed in 2h compared to 12h for the single channel and over 24h for the ELISA immunoassay. Antibodies of high sensitivity, IC50's ranging from 4.8 to 6.9ng/mL for monoclonal and 2.3-6.0ng/mL for polyclonal, for the detection of domoic acid in a 1min analysis time were selected. Although there is a progression for biosensor technology towards low cost, multiplexed portable diagnostics for the food industry, there remains a place for laboratory-based SPR instrumentation for antibody development for food diagnostics as shown herein.

Immunoassays, optical and electrochemical immunosensorsfor tetrodotoxin determination in puffer fish samples

Tetrodotoxin (TTX) is a low molecular weight and potent marine neurotoxin which is usually present in some species of puffer fish. TTX selectively binds to voltage-sensitive sodium channels (VSGCs), blocking the influx of sodium into the cell and affecting neural transmission. The bioaccumulation of this toxin in seafood can poses a risk to human safety. With the purpose of achieving cheap, specific and reliable tools to determine TTX in puffer fish samples, a self-assembled dithiol-based immunoassay, an electrochemical immunosensor and an optical Surface Plasmon Resonance (SPR) immunosensor are proposed. The immunoassay for TTX based on the use of dithiols self-assembled on maleimide-plates (mELISA) has been able to detect as low as 2.28 μg/L of TTX. The effect of different puffer fish matrixes on this mELISA has been quantified and the corresponding correction factors have been established. This
mELISA has enabled to establish the cross-reactivity factors for four TTX analogues: 5,6,11-trideoxy-TTX, 5,6,11-trideoxy-4-anhydro-TTX, 11-nor-TTX-6-ol and 5,11-deoxy-TTX. The crossreactivity factors have also been established by the optical SPR immunosensor previously reported, which had a limit of detection (LOD) of 4.27 μg/L. The mELISA and the SPR immunosensor have then been tested with spiked-puffer fish matrixes, providing an effective
LOD of 0.23 and 0.43 mg/kg respectively, well below the limit set in Japan (2 mg/kg). The mELISA and the SPR immunosensor have also been applied to the analysis of naturally contaminated puffer fish samples, providing similar TTXs contents between techniques and also compared to LC-MS/MS. The suitability of these immunochemical techniques has been demonstrated not only for screening purposes, but also for research activities. Currently, given that dithiols could improve the electron transfer and the sensitivity of an electrochemical assay, the mELISA strategy is being transferred to gold electrodes for the electrochemical detection of TTX and the subsequent development of the multiplexed electrochemical immunosensor.

Fast and sensitive aflatoxin B1 and total aflatoxins ELISAs for analysis of peanuts, maize and feed ingredients

Aflatoxins are a group of carcinogenic compounds produced by Aspergillus fungi that can grow on different agricultural crops. Both acute and chronic exposure to these mycotoxins can cause serious illness. Due to the high occurrence of aflatoxins in crops worldwide fast and cost-effective analytical methods are required for the identification of contaminated agricultural commodities before they are processed into final products and placed on the market. In order to provide new tools for aflatoxin screening two prototype fast ELISA methods: one for the detection of aflatoxin B₁ and the other for total aflatoxins were developed. Seven monoclonal antibodies with unique high sensitivity and at the same time good cross-reactivity profiles were produced. The monoclonal antibodies were characterized and two antibodies showing IC₅₀ of 0.037 ng/mL and 0.031 ng/mL for aflatoxin B₁ were applied in simple and fast direct competitive ELISA tests. The methods were validated for peanut matrix as this crop is one of the most affected by aflatoxin contamination. The detection capabilities of aflatoxin B₁ and total aflatoxins ELISAs were 0.4 μg/kg and 0.3 μg/kg for aflatoxin B₁, respectively, which are one of the lowest reported values. Total aflatoxins ELISA was also validated for the detection of aflatoxins B₂, G₁ and G₂. The application of the developed tests was demonstrated by screening 32 peanut samples collected from the UK retailers. Total aflatoxins ELISA was further applied to analyse naturally contaminated maize porridge and distiller's dried grain with solubles samples and the results were correlated with these obtained by UHPLC-MS/MS method.

Fecal androgen levels in common marmoset (Callithrix jacchus) males living in captive family groups

In captive common marmoset groups, the reproductive inhibition observed in subordinate female seems to be a result of olfactory, visual and behavioral cues from the dominant female. However, few studies have examined the relationship among adult males living in the same social group. These studies have shown that reproductive failure among peer males seems to be based on hormonal and behavioral mechanisms. New insights on sexual strategies in primates have been shown using fecal steroids, but so far no information is available for common marmoset males. In the present study, we evaluated the influence of light-dark cycle, age and reproductive condition on the profile of fecal androgens in males living in the same family group. Feces were collected from six fathers and six sons for androgen determination during the light phase of the 24-h cycle for eight days randomly distributed over a 4-week period. Androgen levels were determined by enzyme immunoassay technique. Adult sons showed higher androgen levels (166.97 ± 22.95 ng/g) than fathers (80.69 ± 44.38 ng/g) and juveniles (49.06 ± 23.15 ng/g; P < 0.05). No diurnal variation (P > 0.05) in fecal androgen profile was observed in adults or juveniles. No indication of androgen-mediated social competition between fathers and adult sons was demonstrable. These results provide basic information on fecal androgen profile useful to investigate the socioendocrinology of free-ranging common marmoset males and verify that, in contrast to daughters, the reproductive suppression of sons is not based on physiological inhibition of their gonads

Avaliação do padrão de resposta de imunoglobulinas em diferentes linhagens de camundongos frente à infecção por T.cruzi

Dissertação (Mestrado)

Fecal androgen levels in common marmoset (Callithrix jacchus) males living in captive family groups

In captive common marmoset groups, the reproductive inhibition observed in subordinate female seems to be a result of olfactory, visual and behavioral cues from the dominant female. However, few studies have examined the relationship among adult males living in the same social group. These studies have shown that reproductive failure among peer males seems to be based on hormonal and behavioral mechanisms. New insights on sexual strategies in primates have been shown using fecal steroids, but so far no information is available for common marmoset males. In the present study, we evaluated the influence of light-dark cycle, age and reproductive condition on the profile of fecal androgens in males living in the same family group. Feces were collected from six fathers and six sons for androgen determination during the light phase of the 24-h cycle for eight days randomly distributed over a 4-week period. Androgen levels were determined by enzyme immunoassay technique. Adult sons showed higher androgen levels (166.97 ± 22.95 ng/g) than fathers (80.69 ± 44.38 ng/g) and juveniles (49.06 ± 23.15 ng/g; P < 0.05). No diurnal variation (P > 0.05) in fecal androgen profile was observed in adults or juveniles. No indication of androgen-mediated social competition between fathers and adult sons was demonstrable. These results provide basic information on fecal androgen profile useful to investigate the socioendocrinology of free-ranging common marmoset males and verify that, in contrast to daughters, the reproductive suppression of sons is not based on physiological inhibition of their gonads

Development of a whole cell biochip for toxicant detection.

In the development of biosensors for ecotoxicity testing it is desirable to produce a small, portable system that can be used in the field. Toxicity testing using bioluminescence is widely used in the laboratory utilising natural and genetically modified (lux/ luc-marked) bacteria and other microorganisms. It is currently not possible to use genetically manipulated microorganisms in field testing and a biosensor, therefore, that incorporates naturally luminescent organisms may be preferred. In the development of a biosensor it is aimed to use the naturally luminescent bacterium Vibrio fischeri as a toxicity detection system on a chip. The bacterium will be immobilised in a polymeric matrix. Current work deals with the optimisation of light output and light preservation within the bacterium prior to immobilisation in polyvinyl alcohol. An examination of a range of physicochemical conditions within the polymer will be made, including cell density, thickness of polymer film, growth and light induction environment, and, preservation conditions, in order to develop a testing system giving consistent results over the lifetime of the biosensor. Data will be presented on light production using different culture media for the growth of V. fischeri and retention of light under immobilised conditions. .