Different routes to pure food allergens: extraction, recombinant techniques and total chemical synthesis for obtaining the plant-food pan-allergen LTP

La richiesta di allergeni puri è in continuo aumento per scopi diagnostici, come standard per metodi di rilevamento e di quantificazione, per l'immunoterapia e per lo studio a livello molecolare dei meccanismi delle reazioni allergiche, al fine di facilitare lo sviluppo di possibili cure. In questa tesi di dottorato sono descritte diverse strategie per l’ottenimento di forme pure di non-specific Lipid Transfer Proteins (nsLTPs), le quali sono state riconosciute essere rilevanti allergeni alimentari in molti frutti e verdure comunemente consumati e sono state definite come modello di veri allergeni alimentari. Una LTP potenzialmente allergenica, non nota in precedenza, è stata isolata dalle mandorle, mentre una LTP dall’allergenicità nota contenuta nelle noci è stata prodotta mediante tecniche di DNA ricombinante. Oltre a questi approcci classici, metodi per la sintesi chimica totale di proteine sono stati applicati per la prima volta alla produzione di un allergene, utilizzando Pru p 3, la LTP prototipica e principale allergene della pesca nell'area mediterranea, come modello. La sintesi chimica totale di proteinepermette di controllarne completamente la sequenza e di studiare la loro funzione a livello atomico. La sua applicazione alla produzione di allergeni costituisce perciò un importante passo avanti nel campo della ricerca sulle allergie alimentari. La proteina Pru p 3 è stata prodotta nella sua intera lunghezza e sono necessari solo due passaggi finali di deprotezione per ottenere il target nella sua forma nativa. Le condizioni sperimentali per tali deprotezioni sono state messe a punto durante la produzione dei peptidi sPru p 3 (1-37) e sPru p 3 (38-91), componenti insieme l'intera proteina. Tecniche avanzate di spettrometria di massa sono state usate per caratterizzare tutti i composti ottenuti, mentre la loro allergenicità è stata studiata attraverso test immunologici o approcci in silico.

Altered blood vessel responses in the eye and finger in coronary artery disease

Cardiac function, such as heart rate variability, is abnormal in coronary artery disease, but its relation with the function of ocular and nail-fold blood vessels is unknown. The hypothesis was that there is abnormal retinal and peripheral microvascular endothelial function compared with large blood vessel and cardiac function. Twenty-four patients with coronary artery disease (CAD) and 30 healthy, age- and sex-matched control subjects were enrolled in the study.

Size frequency distributions of the florid prion protein aggregates in variant Creutzfeldt-Jakob disease follow a power-law function

The objective was to test the hypothesis that the size frequency distributions of the prion protein (PrP) plaques in cases of variant Creutzfeldt-Jakob disease (vCJD) follow a power-law function. The design was a retrospective neuropathological study. The patients were 11 cases of clinically and neuropathologically verified vCJD. Size distributions of the diffuse and florid-type plaques were measured in several areas of the cerebral cortex and hippocampus from each case and a power-law function fitted to each distribution. The size distributions of the florid and diffuse plaques were fitted successfully by a powerlaw function in 100% and 42% of brain areas investigated respectively. Processes of aggregation/disaggregation may be more important than surface diffusion in the pathogenesis of the florid plaques. By contrast, surface diffusion may be a more significant factor in the development of the diffuse plaques. © Springer-Verlag Italia 2006.

Interplay between the charge transport phenomena and the charge-transfer phase transition in RbxMn[Fe(CN)6]y.zH2O

Charge transport and dielectric measurements were carried out on compacted powder and single-crystal samples of bistable RbxMn[Fe(CN)6]y·zH2O in the two valence-tautomeric forms (MnIIFeIII and MnIIIFeII) as a function of temperature (120-350 K) and frequency (10-2-106 Hz). The complex conductivity data reveal universal conductivity behavior and obey the Barton-Nakajima-Namikawa relationship. The charge transport is accompanied by dielectric relaxation that displays the same thermal activation energy as the conductivity. Surprisingly, the activation energy of the conductivity was found very similar in the two valence-tautomeric forms (0.55 eV), and the conductivity change between the two phases is governed mainly by the variation of the preexponential factor in each sample. The phase transition is accompanied by a large thermal hysteresis of the conductivity and the dielectric constant. In the hysteresis region, however, a crossover occurs in the charge transport mechanism at T < 220 K from an Arrhenius-type to a varying activation energy behavior, conferring an unusual “double-loop” shape to the hysteresis.

Modelling trade offs between public and private conservation policies

To reduce global biodiversity loss, there is an urgent need to determine the most efficient allocation of conservation resources. Recently, there has been a growing trend for many governments to supplement public ownership and management of reserves with incentive programs for conservation on private land. This raises important questions, such as the extent to which private land conservation can improve conservation outcomes, and how it should be mixed with more traditional public land conservation. We address these questions, using a general framework for modelling environmental policies and a case study examining the conservation of endangered native grasslands to the west of Melbourne, Australia. Specifically, we examine three policies that involve i) spending all resources on creating public conservation areas; ii) spending all resources on an ongoing incentive program where private landholders are paid to manage vegetation on their property with 5-year contracts; and iii) splitting resources between these two approaches. The performance of each strategy is quantified with a vegetation condition change model that predicts future changes in grassland quality. Of the policies tested, no one policy was always best and policy performance depended on the objectives of those enacting the policy. Although policies to promote conservation on private land are proposed and implemented in many areas, they are rarely evaluated in terms of their ecological consequences. This work demonstrates a general method for evaluating environmental policies and highlights the utility of a model which combines ecological and socioeconomic processes.

Frontoparietal control of spatial attention and motor intention in human EEG

Relations between spatial attention and motor intention were investigated by means of an EEG potential elicited by shifting attention to a location in space as well as by the selection of a hand for responding. High-density recordings traced this potential to a common frontoparietal network activated by attentional orienting and by response selection. Within this network, parietal and frontal cortex were activated sequentially, followed by an anterior-to-posterior migration of activity culminating in the lateral occipital cortex. Based on temporal and polarity information provided by EEG, we hypothesize that the frontoparietal activation, evoked by directional information, updates a task-defined preparatory state by deselecting or inhibiting the behavioral option competing with the cued response side or the cued direction of attention. These results from human EEG demonstrate a direct EEG manifestation of the frontoparietal attention network previously identified in functional imaging. EEG reveals the time course of activation within this network and elucidates the generation and function of associated directing-attention EEG potentials. The results emphasize transient activation and a decision-related function of the frontoparietal attention network, contrasting with the sustained preparatory activation that is commonly inferred from neuroimaging.

Vergence analysis reveals the influence of axial distances on accommodation with age and axial ametropia

Despite numerous investigations, the aetiology and mechanism of accommodation and presbyopia remains equivocal. Using Gaussian first-order ray tracing calculations, we examine the contribution that ocular axial distances make to the accommodation response. Further, the influence of age and ametropia are also considered. The data show that all changes in axial distances during accommodation reduce the accommodation response, with the reduction in anterior chamber depth contributing most to this overall attenuation. Although the total power loss due to the changes in axial distances remained constant with increasing age, hyperopes exhibited less accommodation than myopes. The study, therefore, enhances our understanding of biometric accommodative changes and demonstrates the utility of vergence analysis in the assessment of accommodation.

Organizational and textual structuring of radio news discourse in English and Arabic.

The present work studies the overall structuring of radio news discourse via investigating three metatextual/interactive functions: (1) Discourse Organizing Elements (DOEs), (2) Attribution and (3) Sentential and Nominal Background Information (SBI & NBI). An extended corpus of about 73,000 words from BBC and Radio Damascus news is used to study DOEs and a restricted corpus of 38,000 words for Attribution and S & NBI. A situational approach is adopted to assess the influence of factors such as medium and audience on these functions and their frequence. It is found that: (1) DOEs are organizational and their frequency is determined by length of text; (2) Attribution Function in accordance with the editor's strategy and its frequency is audience sensitive; and (3) BI provides background information and is determined by audience and news topics. Secondly, the salient grammatical elements in DOEs are discourse deictic demonstratives, address pronouns and nouns referring to `the news'. Attribution is realized in reporting/reported clauses, and BI in a sentence, a clause or a nominal group. Thirdly, DOEs establish a hierarchy of (1) news, (2) summary/expansion and (3) item: including topic introduction and details. While Attribution is generally, and SBI solely, a function of detailing, NBI and proper names are generally a function of summary and topic introduction. Being primarily addressed to audience and referring metatextually, the functions investigated support Sinclair's interactive and autonomous planes of discourse. They also shed light on the part(s) of the linguistic system which realize the metatextual/interactive function. Strictly, `discourse structure' inevitably involves a rank-scale; but news discourse also shows a convention of item `listing'. Hence only within the boundary of variety (ultimately interpreted across language and in its situation) can textual functions and discourse structure be studied. Finally, interlingual variety study provides invaluable insights into a level of translation that goes beyond matching grammatical systems or situational factors, an interpretive level which has to be described in linguistic analysis of translation data.

A study of cluster analysis techniques and their applications

This thesis seeks to describe the development of an inexpensive and efficient clustering technique for multivariate data analysis. The technique starts from a multivariate data matrix and ends with graphical representation of the data and pattern recognition discriminant function. The technique also results in distances frequency distribution that might be useful in detecting clustering in the data or for the estimation of parameters useful in the discrimination between the different populations in the data. The technique can also be used in feature selection. The technique is essentially for the discovery of data structure by revealing the component parts of the data. lhe thesis offers three distinct contributions for cluster analysis and pattern recognition techniques. The first contribution is the introduction of transformation function in the technique of nonlinear mapping. The second contribution is the us~ of distances frequency distribution instead of distances time-sequence in nonlinear mapping, The third contribution is the formulation of a new generalised and normalised error function together with its optimal step size formula for gradient method minimisation. The thesis consists of five chapters. The first chapter is the introduction. The second chapter describes multidimensional scaling as an origin of nonlinear mapping technique. The third chapter describes the first developing step in the technique of nonlinear mapping that is the introduction of "transformation function". The fourth chapter describes the second developing step of the nonlinear mapping technique. This is the use of distances frequency distribution instead of distances time-sequence. The chapter also includes the new generalised and normalised error function formulation. Finally, the fifth chapter, the conclusion, evaluates all developments and proposes a new program. for cluster analysis and pattern recognition by integrating all the new features.

The DHR1 domain of DOCK180 binds to SNX5 and regulates cation-independent mannose 6-phosphate receptor transport

DOCK180 is the archetype of the DOCK180-family guanine nucleotide exchange factor for small GTPases Rac1 and Cdc42. DOCK180-family proteins share two conserved domains, called DOCK homology region (DHR)-1 and -2. Although the function of DHR2 is to activate Rac1, DHR1 is required for binding to phosphoinositides. To better understand the function of DHR1, we searched for its binding partners by direct nanoflow liquid chromatography/tandem mass spectrometry, and we identified sorting nexins (SNX) 1, 2, 5, and 6, which make up a multimeric protein complex mediating endosome-to-trans-Golgi-network (TGN) retrograde transport of the cation-independent mannose 6-phosphate receptor (CI-MPR). Among these SNX proteins, SNX5 was coimmunoprecipitated with DOCK180 most efficiently. In agreement with this observation, DOCK180 colocalized with SNX5 at endosomes. The RNA interference-mediated knockdowns of SNX5 and DOCK180, but not Rac1, resulted in the redistribution of CI-MPR from TGN to endosomes. Furthermore, expression of the DOCK180 DHR1 domain was sufficient to restore the perturbed CI-MPR distribution in DOCK180 knockdown cells. These data suggest that DOCK180 regulates CI-MPR trafficking via SNX5 and that this function is independent of its guanine nucleotide exchange factor activity toward Rac1.

Atomic structure of the two intermediate phase glasses SiSe4 and GeSe4

The microscopic origin of the intermediate phase in two prototypical covalently bonded AxB1-x network glass forming systems, where A=Ge or Si, B=Se, and 0=x=1, was investigated by combining neutron diffraction with first-principles molecular-dynamics methods. Specifically, the structure of glassy GeSe4 and SiSe4 was examined, and the calculated total structure factor and total pair-correlation function for both materials are in good agreement with experiment. The structure of both glasses differs markedly from a simple model comprising undefective AB4 corner-sharing tetrahedra in which all A atoms are linked by B2 dimers. Instead, edge-sharing tetrahedra occur and the twofold coordinated Se atoms form three distinct structural motifs, namely, Se-Se2, Se-SeGe (or Se-SeSi), and Se-Ge2 (or Se-Si2). This identifies several of the conformations that are responsible for the structural variability in GexSe1-x and SixSe1-x glasses, a quantity that is linked to the finite width of the intermediate phase window.

Adhesives and interfacial phenomena in wound healing

This chapter deals initially with the underlying principles of adhesion and adhesives and the understanding of interfacial behaviour. This provides a basis upon which to understand biological interactions (. Chapter 12). The two broad types of adhesive materials encountered in wound healing are pressure-sensitive adhesives (PSA) and tissue sealants. The function of pressure-sensitive adhesives is to form an adhesive bond between tissue and biomaterial under the influence of pressure. Tissue sealants are liquids that convert to solid form at the tissue surface and in so doing form either an effective seal against fluid leakage or a bond between adjacent tissue surfaces. The different requirements and characteristics of these systems are discussed. © 2011 Woodhead Publishing Limited All rights reserved.

Oxidative and inflammatory status in Type 2 diabetes patients with periodontitis

Aim: To determine the impact of periodontitis on oxidative/inflammatory status and diabetes control in Type 2 diabetes. Materials and Methods: A comparative study of 20 Type 2 diabetes patients with periodontitis [body mass index (BMI) 31+5], 20-age/gender-matched, non-periodontitis Type 2 diabetes controls (BMI 29+6) and 20 non-diabetes periodontitis controls (BMI 25+4) had periodontal examinations and fasting blood samples collected. Oxidative stress was determined by plasma small molecule antioxidant capacity (pSMAC) and protein carbonyl levels; inflammatory status by total/differential leucocytes, fibrinogen and high sensitivity C-reactive protein (hsCRP); diabetes status by fasting glucose, HbA1c, lipid profile, insulin resistance and secretion. Statistical analysis was performed using SPSS. Results: pSMAC was lower (p=0.03) and protein carbonyls higher (p=0.007) in Type 2 diabetes patients with periodontitis compared with those without periodontitis. Periodontitis was associated with significantly higher HbA1c (p=0.002) and fasting glucose levels (p=0.04) and with lower ß-cell function (HOMA-ß; p=0.01) in diabetes patients. Periodontitis had little effect on inflammatory markers or lipid profiles, but Type 2 diabetes patients with periodontitis had higher levels of hsCRP than those without diabetes (p=0.004) and the lowest levels of HDL-cholesterol of all groups. Conclusion: Periodontitis is associated with increased oxidative stress and compromised glycaemic control in Type 2 diabetes patients.

Investigation of EDFA power transients in circuit-switched and packet-switched optical networks

Erbium-doped fibre amplifiers (EDFA’s) are a key technology for the design of all optical communication systems and networks. The superiority of EDFAs lies in their negligible intermodulation distortion across high speed multichannel signals, low intrinsic losses, slow gain dynamics, and gain in a wide range of optical wavelengths. Due to long lifetime in excited states, EDFAs do not oppose the effect of cross-gain saturation. The time characteristics of the gain saturation and recovery effects are between a few hundred microseconds and 10 milliseconds. However, in wavelength division multiplexed (WDM) optical networks with EDFAs, the number of channels traversing an EDFA can change due to the faulty link of the network or the system reconfiguration. It has been found that, due to the variation in channel number in the EDFAs chain, the output system powers of surviving channels can change in a very short time. Thus, the power transient is one of the problems deteriorating system performance. In this thesis, the transient phenomenon in wavelength routed WDM optical networks with EDFA chains was investigated. The task was performed using different input signal powers for circuit switched networks. A simulator for the EDFA gain dynamicmodel was developed to compute the magnitude and speed of the power transients in the non-self-saturated EDFA both single and chained. The dynamic model of the self-saturated EDFAs chain and its simulator were also developed to compute the magnitude and speed of the power transients and the Optical signal-to-noise ratio (OSNR). We found that the OSNR transient magnitude and speed are a function of both the output power transient and the number of EDFAs in the chain. The OSNR value predicts the level of the quality of service in the related network. It was found that the power transients for both self-saturated and non-self-saturated EDFAs are close in magnitude in the case of gain saturated EDFAs networks. Moreover, the cross-gain saturation also degrades the performance of the packet switching networks due to varying traffic characteristics. The magnitude and the speed of output power transients increase along the EDFAs chain. An investigation was done on the asynchronous transfer mode (ATM) or the WDM Internet protocol (WDM-IP) traffic networks using different traffic patterns based on the Pareto and Poisson distribution. The simulator is used to examine the amount and speed of the power transients in Pareto and Poisson distributed traffic at different bit rates, with specific focus on 2.5 Gb/s. It was found from numerical and statistical analysis that the power swing increases if the time interval of theburst-ON/burst-OFF is long in the packet bursts. This is because the gain dynamics is fast during strong signal pulse or with long duration pulses, which is due to the stimulatedemission avalanche depletion of the excited ions. Thus, an increase in output power levelcould lead to error burst which affects the system performance.

Hypoxia induces dilated cardiomyopathy in the chick embryo:mechanism, intervention, and long-term consequences

Background - Intrauterine growth restriction is associated with an increased future risk for developing cardiovascular diseases. Hypoxia in utero is a common clinical cause of fetal growth restriction. We have previously shown that chronic hypoxia alters cardiovascular development in chick embryos. The aim of this study was to further characterize cardiac disease in hypoxic chick embryos. Methods - Chick embryos were exposed to hypoxia and cardiac structure was examined by histological methods one day prior to hatching (E20) and at adulthood. Cardiac function was assessed in vivo by echocardiography and ex vivo by contractility measurements in isolated heart muscle bundles and isolated cardiomyocytes. Chick embryos were exposed to vascular endothelial growth factor (VEGF) and its scavenger soluble VEGF receptor-1 (sFlt-1) to investigate the potential role of this hypoxia-regulated cytokine. Principal Findings - Growth restricted hypoxic chick embryos showed cardiomyopathy as evidenced by left ventricular (LV) dilatation, reduced ventricular wall mass and increased apoptosis. Hypoxic hearts displayed pump dysfunction with decreased LV ejection fractions, accompanied by signs of diastolic dysfunction. Cardiomyopathy caused by hypoxia persisted into adulthood. Hypoxic embryonic hearts showed increases in VEGF expression. Systemic administration of rhVEGF165 to normoxic chick embryos resulted in LV dilatation and a dose-dependent loss of LV wall mass. Lowering VEGF levels in hypoxic embryonic chick hearts by systemic administration of sFlt-1 yielded an almost complete normalization of the phenotype. Conclusions/Significance - Our data show that hypoxia causes a decreased cardiac performance and cardiomyopathy in chick embryos, involving a significant VEGF-mediated component. This cardiomyopathy persists into adulthood.