Biological and Physical Factors Affecting the Natural History and Evolution of Encapsulated Development

The evolution of reproductive strategies involves a complex calculus of costs and benefits to both parents and offspring. Many marine animals produce embryos packaged in tough egg capsules or gelatinous egg masses attached to benthic surfaces. While these egg structures can protect against environmental stresses, the packaging is energetically costly for parents to produce. In this series of studies, I examined a variety of ecological factors affecting the evolution of benthic development as a life history strategy. I used marine gastropods as my model system because they are incredibly diverse and abundant worldwide, and they exhibit a variety of reproductive and developmental strategies.

The first study examines predation on benthic egg masses. I investigated: 1) behavioral mechanisms of predation when embryos are targeted (rather than the whole egg mass); 2) the specific role of gelatinous matrix in predation. I hypothesized that gelatinous matrix does not facilitate predation. One study system was the sea slug Olea hansineensis, an obligate egg mass predator, feeding on the sea slug Haminoea vesicula. Olea fed intensely and efficiently on individual Haminoea embryos inside egg masses but showed no response to live embryos removed from gel, suggesting that gelatinous matrix enables predation. This may be due to mechanical support of the feeding predator by the matrix. However, Haminoea egg masses outnumber Olea by two orders of magnitude in the field, and each egg mass can contain many tens of thousands of embryos, so predation pressure on individuals is likely not strong. The second system involved the snail Nassarius vibex, a non-obligate egg mass predator, feeding on the polychaete worm Clymenella mucosa. Gel neither inhibits nor promotes embryo predation for Nassarius, but because it cannot target individual embryos inside an egg mass, its feeding is slow and inefficient, and feeding rates in the field are quite low. However, snails that compete with Nassarius for scavenged food have not been seen to eat egg masses in the field, leaving Nassarius free to exploit the resource. Overall, egg mass predation in these two systems likely benefits the predators much more than it negatively affects the prey. Thus, selection for environmentally protective aspects of egg mass production may be much stronger than selection for defense against predation.

In the second study, I examined desiccation resistance in intertidal egg masses made by Haminoea vesicula, which preferentially attaches its flat, ribbon-shaped egg masses to submerged substrata. Egg masses occasionally detach and become stranded on exposed sand at low tide. Unlike adults, the encased embryos cannot avoid desiccation by selectively moving about the habitat, and the egg mass shape has high surface-area-to-volume ratio that should make it prone to drying out. Thus, I hypothesized that the embryos would not survive stranding. I tested this by deploying individual egg masses of two age classes on exposed sand bars for the duration of low tide. After rehydration, embryos midway through development showed higher rates of survival than newly-laid embryos, though for both stages survival rates over 25% were frequently observed. Laboratory desiccation trials showed that >75% survival is possible in an egg mass that has lost 65% of its water weight, and some survival (<25%) was observed even after 83% water weight lost. Although many surviving embryos in both experiments showed damage, these data demonstrate that egg mass stranding is not necessarily fatal to embryos. They may be able to survive a far greater range of conditions than they normally encounter, compensating for their lack of ability to move. Also, desiccation tolerance of embryos may reduce pressure on parents to find optimal laying substrata.

The third study takes a big-picture approach to investigating the evolution of different developmental strategies in cone snails, the largest genus of marine invertebrates. Cone snail species hatch out of their capsules as either swimming larvae or non-dispersing forms, and their developmental mode has direct consequences for biogeographic patterns. Variability in life history strategies among taxa may be influenced by biological, environmental, or phylogenetic factors, or a combination of these. While most prior research has examined these factors singularly, my aim was to investigate the effects of a host of intrinsic, extrinsic, and historical factors on two fundamental aspects of life history: egg size and egg number. I used phylogenetic generalized least-squares regression models to examine relationships between these two egg traits and a variety of hypothesized intrinsic and extrinsic variables. Adult shell morphology and spatial variability in productivity and salinity across a species geographic range had the strongest effects on egg diameter and number of eggs per capsule. Phylogeny had no significant influence. Developmental mode in Conus appears to be influenced mostly by species-level adaptations and niche specificity rather than phylogenetic conservatism. Patterns of egg size and egg number appear to reflect energetic tradeoffs with body size and specific morphologies as well as adaptations to variable environments. Overall, this series of studies highlights the importance of organism-scale biotic and abiotic interactions in evolutionary patterns.

The effects of formulation and processing on surface characteristics and functional properties of dairy powders

The objectives of this thesis were to (i) study the effect of increasing protein concentration in milk protein concentrate (MPC) powders on surface composition and sorption properties; (ii) examine the effect of increasing protein content on the rehydration properties of MPC; (iii) study the physicochemical properties of spraydried emulsion-containing powders having different water and oil contents; (iv) analyse the effect of protein type on water sorption and diffusivity properties in a protein/lactose dispersion, and; (v) characterise lactose crystallisation and emulsion stability of model infant formula containing intact or hydrolysed whey proteins. Surface composition of MPC powders (protein contents 35 - 86 g / 100 g) indicated that fat and protein were preferentially located on the surface of powders. Low protein powder (35 g / 100 g) exhibited lactose crystallisation, whereas powders with higher protein contents did not, due to their high protein: lactose ratio. Insolubility was evident in high protein MPCs and was primarily related to insolubility of the casein fraction. High temperature (50 °C) was required for dissolution of high protein MPCs (protein content > 60 g / 100 g). The effect of different oil types and spray-drying outlet temperature on the physicochemical properties of the resultant fat-filled powders was investigated and showed that increasing outlet temperature reduced water content, water activity and tapped bulk density, irrespective of oil type, and increased solvent-extractable free fat for all oil types and onset of glass transition (Tg) and crystallisation (Tcr) temperature. Powder dispersions of protein/lactose (0.21:1), containing either intact or hydrolysed whey protein (12 % degree of hydrolysis; DH), were spray-dried at pilot scale. Moisture sorption analysis at 25 °C showed that dispersions containing intact whey protein exhibited lactose crystallisation at a lower relative humidity (RH). Dispersions containing hydrolysed whey protein had significantly higher (P < 0.05) water diffusivity. Finally, a spray-dried model infant formula was produced containing hydrolysed or intact whey as the protein with sunflower oil as the fat source. Reconstituted, hydrolysed formula had a significantly (P < 0.05) higher fat globule size and lower emulsion stability than intact formula. Lactose crystallisation in powders occurred at higher RH for hydrolysed formula. In conclusion, this research has shown the effect of altering the protein type, protein composition, and oil type on the surface composition and physical properties of different dairy powders, and how these variations greatly affect their rehydration characteristics and storage stability.

The Effect of Water Consumption on Schoolchildren's Fine Motor Skills, Cognitive Function and Mood

Previous research has suggested that dehydration may have a negative effect on some aspects of mood, cognitive performance and motor skills (Benton, 2011). Furthermore, a large proportion of children arrive at school in a dehydrated state (Baron, Courbebaisse, Lepicard, & Friedlander, 2015). The present work investigated whether supplementing children with water may, as a consequence of reducing dehydration, improve their cognitive performance and motor skills. In studies 1, 2, 3 and 5, it was found that tasks that predominantly tested motor skills, were improved in children who had a drink, compared to those who did not. Furthermore, study 3 showed that this effect was moderated by hydration status. One theoretical explanation for the poorer performance of dehydrated children is that they may lack the neurological resources to sustain their effort and thus performance does not improve over time. In support of this, these studies showed that, when re-hydrated, performance on these tasks improves to the level of non-dehydrated children. Study 2 showed that the number of errors increased in a StopSignal task in children that had high self-rated levels of thirst, compared to low levels: and hydration status did not moderate this effect. A possible explanation for the increased number of errors in children with high self-rated thirst is that the thirst sensation diverts attention away from the task, causing task performance to deteriorate. In study 4, it was observed that there was a large variation in intra-individual and inter-individual hydration scores throughout the day, which was not related to volume drank or levels of thirst. Further studies should use imaging techniques to study brain activity during dehydration and rehydration, and during periods of high thirst, to help to further elucidate the mechanism underlying the negative effect of dehydration on motor performance, and the effect of self-rated thirst on attention.

Physicochemical characterisation of protein ingredients prepared from milk by ultrafiltration or microfiltration for application in formulated nutritional products

Formulated food systems are becoming more sophisticated as demand grows for the design of structural and nutritional profiles targeted at increasingly specific demographics. Milk protein is an important bio- and techno-functional component of such formulations, which include infant formula, sports supplements, clinical beverages and elderly nutrition products. This thesis outlines research into ingredients that are key to the development of these products, namely milk protein concentrate (MPC), milk protein isolate (MPI), micellar casein concentrate (MCC), β-casein concentrate (BCC) and serum protein concentrate (SPC). MPC powders ranging from 37 to 90% protein (solids basis) were studied for properties of relevance to handling and storage of powders, powder solubilisation and thermal processing of reconstituted MPCs. MPC powders with ≥80% protein were found to have very poor flowability and high compressibility; in addition, these high-protein MPCs exhibited poor wetting and dispersion characteristics during rehydration in water. Heat stability studies on unconcentrated (3.5%, 140°C) and concentrated (8.5%, 120°C) MPC suspensions, showed that suspensions prepared from high-protein MPCs coagulated much more rapidly than lower protein MPCs. β-casein ingredients were developed using membrane processing. Enrichment of β-casein from skim milk was performed at laboratory-scale using ‘cold’ microfiltration (MF) at <4°C with either 1000 kDa molecular weight cut-off or 0.1 µm pore-size membranes. At pilot-scale, a second ‘warm’ MF step at 26°C was incorporated for selective purification of micellised β-casein from whey proteins; using this approach, BCCs with β-casein purity of up to 80% (protein basis) were prepared, with the whey protein purity of the SPC co-product reaching ~90%. The BCC ingredient could prevent supersaturated solutions of calcium phosphate (CaP) from precipitating, although the amorphous CaP formed created large micelles that were less thermo-reversible than those in CaP-free systems. Another co-product of BCC manufacture, MCC powder, was shown to have superior rehydration characteristics compared to traditional MCCs. The findings presented in this thesis constitute a significant advance in the research of milk protein ingredients, in terms of optimising their preparation by membrane filtration, preventing their destabilisation during processing and facilitating their effective incorporation into nutritional formulations designed for consumers of a specific age, lifestyle or health status

Host cellular regulatory networks in dengue virus-human interactions

Dengue fever is one of the most important mosquito-borne diseases worldwide and is caused by infection with dengue virus (DENV). The disease is endemic in tropical and sub-tropical regions and has increased remarkably in the last few decades. At present, there is no antiviral or approved vaccine against the virus. Treatment of dengue patients is usually supportive, through oral or intravenous rehydration, or by blood transfusion for more severe dengue cases. Infection of DENV in humans and mosquitoes involves a complex interplay between the virus and host factors. This results in regulation of numerous intracellular processes, such as signal transduction and gene transcription which leads to progression of disease. To understand the mechanisms underlying the disease, the study of virus and host factors is therefore essential and could lead to the identification of human proteins modulating an essential step in the virus life cycle. Knowledge of these human proteins could lead to the discovery of potential new drug targets and disease control strategies in the future. Recent advances of high throughput screening technologies have provided researchers with molecular tools to carry out investigations on a large scale. Several studies have focused on determination of the host factors during DENV infection in human and mosquito cells. For instance, a genome-wide RNA interference (RNAi) screen has identified host factors that potentially play an important role in both DENV and West Nile virus replication (Krishnan et al. 2008). In the present study, a high-throughput yeast two-hybrid screen has been utilised in order to identify human factors interacting with DENV non-structural proteins. From the screen, 94 potential human interactors were identified. These include proteins involved in immune signalling regulation, potassium voltage-gated channels, transcriptional regulators, protein transporters and endoplasmic reticulum-associated proteins. Validation of fifteen of these human interactions revealed twelve of them strongly interacted with DENV proteins. Two proteins of particular interest were selected for further investigations of functional biological systems at the molecular level. These proteins, including a nuclear-associated protein BANP and a voltage-gated potassium channel Kv1.3, both have been identified through interaction with the DENV NS2A. BANP is known to be involved in NF-kB immune signalling pathway, whereas, Kv1.3 is known to play an important role in regulating passive flow of potassium ions upon changes in the cell transmembrane potential. This study also initiated a construction of an Aedes aegypti cDNA library for use with DENV proteins in Y2H screen. However, several issues were encountered during the study which made the library unsuitable for protein interaction analysis. In parallel, innate immune signalling was also optimised for downstream analysis. Overall, the work presented in this thesis, in particular the Y2H screen provides a number of human factors potentially targeted by DENV during infection. Nonetheless, more work is required to be done in order to validate these proteins and determine their functional properties, as well as testing them with infectious DENV to establish a biological significance. In the long term, data from this study will be useful for investigating potential human factors for development of antiviral strategies against dengue.

Secagem da microalga Spirulina em camada delgada utilizando secador com bomba de calor

A Spirulina apresenta propriedades antioxidantes o que favorece seu uso como alimento funcional, fato que tem motivado a sua comercialização para a formulação de alimentos diversos e com finalidades terapêuticas. A secagem ganha importância durante produção de Spirulina, uma vez que a umidade necessária, para garantir que não ocorra degradação da biomassa desidratada durante o armazenamento, é alcançada através do conhecimento dos parâmetros que caracterizam a operação. Neste estudo foi utilizada a secagem com bomba de calor, um método alternativo, pois viabiliza a operação com temperaturas inferiores as tradicionalmente utilizadas, além de seu funcionamento ser independente das condições meteorológicas do ambiente. O trabalho experimental da secagem de Spirulina sp. foi iniciado com um estudo comparativo entre a secagem com bomba de calor (SBC) e a secagem tradicional (ST). O efeito dos diferentes métodos utilizados sob a amostra foi comparado em relação à cinética da operação e as características da microalga desidratada (cor, ficocianina, compostos fenólicos totais e atividade antioxidante total). As temperaturas do ar foram de 50 e 60ºC e a umidade absoluta da SBC foi dez vezes inferior a utilizada durante a ST. Os parâmetros que caracterizam a secagem foram influenciados pela temperatura do ar, bem como, pela baixa umidade absoluta na SBC. Os valores do tempo total da SBC foram 40% inferiores aos encontrados para a secagem ST, em ambas as temperaturas do ar. A maior preservação das características da Spirulina foi obtida na SBC e temperatura do ar de 50°C, e nesta condição os valores foram 14% (ficocianina), 60% (compostos fenólicos) e 10% (atividade antioxidante) superiores aos encontrados na mesma condição para a ST. Isto evidencia que o método de secagem é determinante na qualidade do produto desidratado. Posteriormente, foi realizado o estudo da cinética da SBC, bem como a otimização da operação de secagem e a reidratação das amostras desidratadas nas diferentes condições de secagem. O estudo foi realizado através de um planejamento fatorial 32, tendo como fatores de estudo a temperatura do ar (30, 40 e 50ºC) e a espessura da bandeja (1, 3 e 5 mm). As respostas utilizadas foram ficocianina, compostos fenólicos, atividade antioxidante total e cor da microalga desidratadas. Também foram realizadas a microscopia eletrônica de varredura (MEV) e as curvas termogravimétricas (DSC) das amostras desidratadas. A secagem apresentou um curto período de taxa constante, delimitado pela umidade crítica, sendo que, seus valores foram influenciados apenas pela temperatura do ar de secagem. O modelo Logarítmico forneceu elevados valores de R2ajust e os menores valores de soma dos erros quadráticos (SSE) e de critério informativo de Akaike (AIC). Os valores das energias de ativação para as espessuras de 1, 3 e 5 mm, foram na faixa de 20-23 kJ mol-1. A condição de operação mais adequada, para a secagem de Spirulina sp. com bomba de calor, foi obtida na temperatura do ar de 50°C e espessura da bandeja de 5 mm, com valores de ficocianina, compostos fenólicos, atividade antioxidante total e diferença de cor de 19,60 mg g-1, 1508 µgEAG g-1, 52,6% e 5,71, respectivamente. Os termogramas (DSC) evidenciaram que em 50 ºC e espessura de 5 mm, o produto apresentou maior estabilidade térmica. As amostras de Spirulina sp. desidratadas apresentaram estrutura morfológica (MEV), aparentemente, rígida e heterogênea, e os seus percentuais de reidratação corresponderam a 85-91% da umidade da microalga in natura.

Effect of pre-treatments and post-treatments on drying products

Freeze drying technology can give good quality attributes of vegetables and fruits in terms of color, nutrition, volume, rehydration kinetics, stability during storage, among others, when compared with solely air dried ones. However, published scientific works showed that treatments applied before and after air dehydration are effective in food attributes, improving its quality. Therefore, the hypothesis of the present thesis was focus in a vast research of scientific work that showed the possibility to apply a pre-treatment and a post-treatment to food products combined with conventional air drying aiming being close, or even better, to the quality that a freeze dried product can give. Such attributes are the enzymatic inactivation, stability during storage, drying and rehydration kinetics, color, nutrition, volume and texture/structure. With regard to pre-treatments, the ones studied along the present work were: water blanching, steam blanching, ultrasound, freezing, high pressure and osmotic dehydration. High electric pulsed field was also studied but the food attributes were not explained on detailed. Basically, water and steam blanching showed to be adequate to inactivate enzymes in order to prevent enzymatic browning and preserve the product quality during long storage periods. With regard to ultrasound pre-treatment the published results pointed that ultrasound is an effective pre-treatment to reduce further drying times, improve rehydration kinetics and color retention. On the other hand, studies showed that ultrasound allow sugars losses and, in some cases, can lead to cell disruption. For freezing pre-treatment an overall conclusion was difficult to draw for some food attributes, since, each fruit or vegetable is unique and freezing comprises a lot of variables. However, for the studied cases, freezing showed to be a pre-treatment able to enhance rehydration kinetics and color attributes. High pressure pre-treatment showed to inactivate enzymes improving storage stability of food and showed to have a positive performance in terms of rehydration. For other attributes, when high pressure technology was applied, the literature showed divergent results according with the crops used. Finally, osmotic dehydration has been widely used in food processing to incorporate a desired salt or sugar present in aqueous solution into the cellular structure of food matrix (improvement of nutrition attribute). Moreover, osmotic dehydration lead to shorter drying times and the impregnation of solutes during osmose allow cellular strengthens of food. In case of post-treatments, puffing and a new technology denominated as instant controlled pressure drop (DIC) were reported in the literature as treatments able to improve diverse Abstract Effect of Pre-treatments and Post-treatments on Drying Products x food attributes. Basically, both technologies are similar where the product is submitted to a high pressure step and the process can make use of different heating mediums such as CO2, steam, air and N2. However, there exist a significant difference related with the final stage of both which can comprise the quality of the final product. On the other hand, puffing and DIC are used to expand cellular tissues improving the volume of food samples, helping in rehydration kinetics as posterior procedure, among others. The effectiveness of such pre and/or post-treatments is dependent on the state of the vegetables and fruits used which are also dependent of its cellular structure, variety, origin, state (fresh, ripe, raw), harvesting conditions, etc. In conclusion, as it was seen in the open literature, the application of pre-treatments and post-treatments coupled with a conventional air dehydration aim to give dehydrated food products with similar quality of freeze dried ones. Along the present Master thesis the experimental data was removed due to confidential reasons of the company Unilever R&D Vlaardingen

Elimination of urine in response to water intake is consistent in well-hydrated individuals

A simple method has been recently proposed to assess acute hydration status in humans; however, several questions remain regarding its reliability, validity, and practicality. Objective: Establish reliability of a simple method to assess euhydration, that is, to analyze whether this method can be used as a consistent indicator of a person´s hydration status. In addition, the study sought to assess the effect exercise has on urine volume when euhydration is maintained and a standardized volume of water is ingested. Methods: Five healthy physically active men and five healthy physically active women, 22.5 ± 2.3 years of age (mean ± standard deviation) reported to the laboratory after fasting for 10 hours or more on three occasions, each one week apart. During the two identical resting euhydration conditions (EuA and EuB), participants remained seated for 45 minutes. During the exercise condition (EuExer), participants exercised intermittently in an environmental chamber (average temperature and relative humidity = 32 ± 3°C and 65 ± 7%, respectively) for a period of 45 minutes and drank water to offset loss due to sweating. The order of treatments was randomized. Upon finishing the treatment period, they ingested a volume of water equivalent to 1.43% body mass (BM) within 30 minutes. Urine was collected and measured henceforth every 30 minutes for 3 hours. Results: Urine volume eliminated during EuExer (1205 ± 399.5 ml) was not different from EuB (1072.2±413.1 ml) or EuA (1068 ± 382.87 ml) (p-value = 0.44). Both resting conditions were practically identical (p-value = 0.98) and presented a strong intraclass correlation (r = 0.849, p-value = 0.001). Conclusions: This method, besides simple, proved to be consistent in all conditions; therefore, it can be used with the certainty that measurements are valid and reliable.

Rehidratación post-ejercicio con agua de coco: ¿igual o más efectiva que una bebida deportiva?

Objetivo: comparar la efectividad rehidratante del agua de coco fresca, con el agua embotellada y una bebida deportiva.Metodología: once participantes de 22,0 ± 1,9 años y 65,6 ± 13,0 kg de masa corporal (promedio ± D.E), asistieron al laboratorio en tres ocasiones, separadas entre sí por una semana. En cada sesión se deshidrataron por ejercicio en el calor hasta perder 1,84 ± 0,2% de la masa corporal. Luego fueron rehidratados en una hora, con agua comercial embotellada (AE), una bebida deportiva (BD) o agua de coco (AC), utilizando un volumen equivalente al 120% del peso perdido. Se evaluaron las sensaciones percibidas relacionadas con la tolerancia y la aceptación de las bebidas. Se recolectó la orina eliminada durante tres horas post-rehidratación.Resultados: Hubo interacción entre las bebidas y el tiempo sobre la orina excretada (p=0,003), siendo el AE estadísticamente diferente al AC y la BD a los 30 y 60 min post-rehidratación (p<0,05). El volumen total de orina fue mayor con agua embotellada (625 ± 183 mL), en comparación con AC y BD (390 ± 73 mL y 416 ± 200 mL, respectivamente, p<0,002); AC y BD no fueron distintas entre sí (p>0,05). Esto produjo porcentajes de conservación de líquido diferentes para AE (56%), con respecto a BD (71%) y AC (71%) (p<0,001). No hubo interacción entre las bebidas y el tiempo sobre las sensaciones percibidas de tolerancia (p>0,05).La percepción de la dulzura fue menor para AE (p= 0,024). BD obtuvo mejores puntajes de sabor y aceptación general (p<0,05).Conclusiones: el agua de coco fresca resulta tan efectiva como una bebida deportiva para conservar el líquido consumido, teniendo una buena tolerancia y aceptación. Podría ser usada como una bebida rehidratante, cuando la persona la tenga disponible y le agrade su sabor.

Aspectos da biologia reprodutiva de Eretmochelys imbricata (testudines, cheloniidae) no litoral sul do Rio Grande do Norte, Brasil

Female hawksbill turtles (Eretmochelys imbricata) nesting along the southeastcoastline of Rio Grande do Norte State, Brazil (6º13'40"S, 35º03'05"W) were captured and weighed during the four months from January to April 2007, in the course of the annual egglaying season, which extended from 06 rd November 2006 to 30 rd May 2007. In all, 99 weight measurements were performed. On first contact the females exhibited an average post-oviposition weight of 79.1 kg (range 56.2-98.9 kg, SD = 10.9 kg, n = 44 females). Those individuals which were subsequently recaptured showed a mean weight loss of 1.7 kg (range 0.7-4.5 kg, SD = 1.0 kg, n = 39 sets of measurements on 20 females) in the interval between two consecutive post-ovipositions, separated by a maximum time interval of 17 days. In the cases where the female aborted the nesting process, the pre-oviposition weight was measured. The clutch weight, that is to say, the weight loss between consecutive pre-oviposition and post-oviposition measurements (separated by a maximum time interval of 3 days), was found to be 5.2 kg (range 4.3-6.0 kg, SD = 0.9 kg, n = 6 sets of measurements). This value is significantly higher (t-test, p<0.001) than the loss between two consecutive post-oviposition measurements with the same female. The mean recovery in body weight, that is to say, the average gain in weight between successive post-oviposition and pre-oviposition captures of the same individual (separated by a time interval of 12 to 17 days), was found to be 3.0 kg (range 1.9-4.3 kg, SD = 1.0 kg, n = 4 sets of measurements) Although the small sample size makes it unwise to generalise, the recovery in body weight was found to be always significantly lower (t-test, p<0.005) than the clutch weight. This fact is in agreement with the observed weight loss tendency throughout the breeding season for this species. Considering the clutch weight and the internidal recovery in body weight we found that the total weight loss of the adult hawksbill females after three to five nesting events varied from 10.4% (range 8.7-11.9%, SD = 1.6%, n = 3) to 14.1% (range 11.8-15.4%, SD = 1.3%, n = 6) in relation to their initial pre-oviposition weight. If there were no body weight recovery during the internesting interval we estimate that a female that nests three to five times in the course of the season would lose from 19% to 31% of its initial weight. We emphasise that our clutch weight estimate was performed by weighing the females and not by multiplying the number of eggs in the nest by their average unit weight. In this way, our measurements take into account the loss of liquid during the oviposition. Despite the unequivocal evidence of body weight recovery during the internidal interval, it is not clear if the cause of this process is rehydration or feeding

Bimodal magneto-luminescent dysprosium (DyIII)-potassium (KI)-oxalate framework: magnetic switchability with high anisotropic barrier and solvent sensing

We report synthesis, characterization, and properties of a multifunctional oxalate framework, {KDy(C₂O₄)₂(H₂O)₄}_n (1) (C₂O₄^2- = oxalate dianion) composed of two absolutely different metal ions in terms of their size, charge, and electronic configuration. Dehydrated framework (1′) exhibits permanent porosity and interesting solvent (H₂O, MeOH, CH₃CN, and EtOH) vapor sorption characteristics based on specific interactions with unsaturated alkali metal sites on the pore surface. Compound 1 shows solvent responsive bimodal magnetic and luminescence properties related to the DyIII center. Compound 1 exhibits reversible ferromagnetic to antiferromagnetric phase transition upon dehydration and rehydration, hitherto unknown for any lanthanide based coordination polymer or metal-organic frameworks. Both the compounds 1 and 1′ exhibit slow magnetic relaxation with very high anisotropic barrier (417 ± 9 K for 1 and 418 ± 7 K for 1′) which has been ascribed to the single ion magnetic anisotropy of the Dy^III centers. Nevertheless, compound 1 shows a metal based luminescence property in the visible region and H₂O molecules exhibit the strongest quenching effect compared to other solvents MeOH, MeCN, and EtOH, evoking 1′ as a potential H₂O sensor.

New prophylactic and therapeutic treatments to combat pathogenic Enterohaemorrhagic Escherichia coli

Bacterial diarrhoeal diseases have significant influence on global human health, and are a leading cause of preventable death in the developing world. Enterohaemorrhagic Escherichia coli (EHEC), pathogenic strains of E. coli that carry potent toxins, have been associated with a high number of large-scale outbreaks caused by contaminated food and water sources. This pathotype produces diarrhoea and haemorrhagic colitis in infected humans, and in some patients leads to the development of haemolytic uremic syndrome (HUS), which can result in mortality and chronic kidney disease. A major obstacle to the treatment of EHEC infections is the increased risk of HUS development that is associated with antibiotic treatment, and rehydration and renal support are often the only options available. New treatments designed to prevent or clear E. coli infections and reduce symptoms of illness would therefore have large public health and economic impacts. The three main aims of this thesis were: to explore mouse models for pre-clinical evaluation in vivo of small compounds that inhibit a major EHEC colonisation factor, to assess the production and role of two proteins considered promising candidates for a broad-spectrum vaccine against pathogenic E. coli, and to investigate a novel compound that has recently been identified as a potential inhibitor of EHEC toxin production. As EHEC cannot be safely tested in humans due to the risk of HUS development, appropriate small animal models are required for in vivo testing of new drugs. A number of different mouse models have been developed to replicate different features of EHEC pathogenesis, several of which we investigated with a focus on colonisation mediated by the Type III Secretion System (T3SS), a needle-like structure that translocates bacterial proteins into host cells, resulting in a tight, intimate attachment between pathogen and host, aiding colonisation of the gastrointestinal tract. As E. coli models were found not to depend significantly on the T3SS for colonisation, the Citrobacter rodentium model, a natural mouse pathogen closely related to E. coli, was deemed the most suitable mouse model currently available for in vivo testing of T3SS-targeting compounds. Two bacterial proteins, EaeH (an outer membrane adhesin) and YghJ (a putative secreted lipoprotein), highly conserved surface-associated proteins recently identified as III protective antigens against E. coli infection of mice, were explored in order to determine their suitability as candidates for a human vaccine against pathogenic E. coli. We focused on the expression and function of these proteins in the EHEC O157:H7 EDL933 strain and the adherent-invasive E. coli (AIEC) LF82 strain. Although expression of EaeH by other E. coli pathotypes has recently been shown to be upregulated upon contact with host intestinal cells, no evidence of this upregulation could be demonstrated in our strains. Additionally, while YghJ was produced by the AIEC strain, it was not secreted by bacteria under conditions that other YghJ-expressing E. coli pathotypes do, despite the AIEC strain carrying all the genes required to encode the secretion system it is associated with. While our findings indicate that a vaccine that raises antibodies against EaeH and YghJ may have limited effect on the EHEC and AIEC strains we used, recent studies into these proteins in different E. coli pathogens have suggested they are still excellent candidates for a broadly effective vaccine against E. coli. Finally, we characterised a small lead compound, identified by high-throughput screening as a possible inhibitor of Shiga toxin expression. Shiga toxin production causes both the symptoms of illness and development of HUS, and thus reduction of toxin production, release, or binding to host receptors could therefore be an effective way to treat infections and decrease the risk of HUS. Inhibition of Shiga toxin production by this compound was confirmed, and was shown to be caused by an inhibitory effect on activation of the bacterial SOS response rather than on the Shiga toxin genes themselves. The bacterial target of this compound was identified as RecA, a major regulator of the SOS response, and we hypothesise that the compound binds covalently to its target, preventing oligomerisation of RecA into an activated filament. Altogether, the results presented here provide an improved understanding of these different approaches to combating EHEC infection, which will aid the development of safe and effective vaccines and anti-virulence treatments against EHEC.