Evaluation of fluid transport processes in dental enamel. Methods to assess the relevance of enamel permeability in caries prevention and etching treatments.

This thesis evaluated in vivo and in vitro enamel permeability in different physiological and clinical conditions by means of SEM inspection of replicas of enamel surface obtained from polyvinyl siloxane impressions subsequently later cast in polyether impression ma-terial. This technique, not invasive and risk-free, allows the evaluation of fluid outflow from enamel surface and is able to detect the presence of small quantities of fluid, visu-alized as droplets. Fluid outflow on enamel surface represents enamel permeability. This property has a paramount importance in enamel physiolgy and pathology although its ef-fective role in adhesion, caries pathogenesis and prevention today is still not fully under-stood. The aim of the studies proposed was to evaluate enamel permeability changes in differ-ent conditions and to correlate the findings with the actual knowledge about enamel physiology, caries pathogenesis, fluoride and etchinhg treatments. To obtain confirmed data the replica technique has been supported by others specific techniques such as Ra-man and IR spectroscopy and EDX analysis. The first study carried out visualized fluid movement through dental enamel in vivo con-firmed that enamel is a permeable substrate and demonstrated that age and enamel per-meability are closely related. Examined samples from subjects of different ages showed a decreasing number and size of droplets with increasing age: freshly erupted permanent teeth showed many droplets covering the entire enamel surface. Droplets in permanent teeth were prominent along enamel perikymata. These results obtained through SEM inspection of replicas allowed innovative remarks in enamel physiology. An analogous testing has been developed for evaluation of enamel permeability in primary enamel. The results of this second study showed that primary enamel revealed a substantive permeability with droplets covering the entire enamel sur-face without any specific localization accordingly with histological features, without changes during aging signs of post-eruptive maturation. These results confirmed clinical data that showed a higher caries susceptibility for primary enamel and suggested a strong relationship between this one and enamel permeability. Topical fluoride application represents the gold standard for caries prevention although the mechanism of cariostatic effect of fluoride still needs to be clarified. The effects of topical fluoride application on enamel permeability were evaluated. Particularly two dif-ferent treatments (NaF and APF), with different pH, were examined. The major product of topical fluoride application was the deposition of CaF2-like globules. Replicas inspec-tion before and after both treatments at different times intervals and after specific addi-tional clinical interventions showed that such globule formed in vivo could be removed by professional toothbrushing, sonically and chemically by KOH. The results obtained in relation to enamel permeability showed that fluoride treatments temporarily reduced enamel water permeability when CaF2-like globules were removed. The in vivo perma-nence of decreased enamel permeability after CaF2 globules removal has been demon-strated for 1 h for NaF treated teeth and for at least 7 days for APF treated teeth. Important clinical consideration moved from these results. In fact the caries-preventing action of fluoride application may be due, in part, to its ability to decrease enamel water permeability and CaF2 like-globules seem to be indirectly involved in enamel protection over time maintaining low permeability. Others results obtained by metallographic microscope and SEM/EDX analyses of or-thodontic resins fluoride releasing and not demonstrated the relevance of topical fluo-ride application in decreasing the demineralization marks and modifying the chemical composition of the enamel in the treated area. These data obtained in both the experiments confirmed the efficacy of fluoride in caries prevention and contribute to clarify its mechanism of action. Adhesive dentistry is the gold standard for caries treatment and tooth rehabilitation and is founded on important chemical and physical principles involving both enamel and dentine substrates. Particularly acid etching of dental enamel enamel has usually employed in bonding pro-cedures increasing microscopic roughness. Different acids have been tested in the litera-ture suggesting several etching procedures. The acid-induced structural transformations in enamel after different etching treatments by means of Raman and IR spectroscopy analysis were evaluated and these findings were correlated with enamel permeability. Conventional etching with 37% phosphoric acid gel (H3PO4) for 30 s and etching with 15 % HCl for 120 s were investigated. Raman and IR spectroscopy showed that the treatment with both hydrochloric and phosphoric acids induced a decrease in the carbonate content of the enamel apatite. At the same time, both acids induced the formation of HPO42- ions. After H3PO4 treatment the bands due to the organic component of enamel decreased in intensity, while in-creased after HCl treatment. Replicas of H3PO4 treated enamel showed a strongly reduced permeability while replicas of HCl 15% treated samples showed a maintained permeability. A decrease of the enamel organic component, as resulted after H3PO4 treatment, involves a decrease in enamel permeability, while the increase of the organic matter (achieved by HCl treat-ment) still maintains enamel permeability. These results suggested a correlation between the amount of the organic matter, enamel permeability and caries. The results of the different studies carried out in this thesis contributed to clarify and improve the knowledge about enamel properties with important rebounds in theoretical and clinical aspects of Dentistry.

Natural patterns and anthropogenic disturbance in north Adriatic marine benthic assemblages: descriptive and methodological studies

Marine soft bottom systems show a high variability across multiple spatial and temporal scales. Both natural and anthropogenic sources of disturbance act together in affecting benthic sedimentary characteristics and species distribution. The description of such spatial variability is required to understand the ecological processes behind them. However, in order to have a better estimate of spatial patterns, methods that take into account the complexity of the sedimentary system are required. This PhD thesis aims to give a significant contribution both in improving the methodological approaches to the study of biological variability in soft bottom habitats and in increasing the knowledge of the effect that different process (both natural and anthropogenic) could have on the benthic communities of a large area in the North Adriatic Sea. Beta diversity is a measure of the variability in species composition, and Whittaker’s index has become the most widely used measure of beta-diversity. However, application of the Whittaker index to soft bottom assemblages of the Adriatic Sea highlighted its sensitivity to rare species (species recorded in a single sample). This over-weighting of rare species induces biased estimates of the heterogeneity, thus it becomes difficult to compare assemblages containing a high proportion of rare species. In benthic communities, the unusual large number of rare species is frequently attributed to a combination of sampling errors and insufficient sampling effort. In order to reduce the influence of rare species on the measure of beta diversity, I have developed an alternative index based on simple probabilistic considerations. It turns out that this probability index is an ordinary Michaelis-Menten transformation of Whittaker's index but behaves more favourably when species heterogeneity increases. The suggested index therefore seems appropriate when comparing patterns of complexity in marine benthic assemblages. Although the new index makes an important contribution to the study of biodiversity in sedimentary environment, it remains to be seen which processes, and at what scales, influence benthic patterns. The ability to predict the effects of ecological phenomena on benthic fauna highly depends on both spatial and temporal scales of variation. Once defined, implicitly or explicitly, these scales influence the questions asked, the methodological approaches and the interpretation of results. Problem often arise when representative samples are not taken and results are over-generalized, as can happen when results from small-scale experiments are used for resource planning and management. Such issues, although globally recognized, are far from been resolved in the North Adriatic Sea. This area is potentially affected by both natural (e.g. river inflow, eutrophication) and anthropogenic (e.g. gas extraction, fish-trawling) sources of disturbance. Although few studies in this area aimed at understanding which of these processes mainly affect macrobenthos, these have been conducted at a small spatial scale, as they were designated to examine local changes in benthic communities or particular species. However, in order to better describe all the putative processes occurring in the entire area, a high sampling effort performed at a large spatial scale is required. The sedimentary environment of the western part of the Adriatic Sea was extensively studied in this thesis. I have described, in detail, spatial patterns both in terms of sedimentary characteristics and macrobenthic organisms and have suggested putative processes (natural or of human origin) that might affect the benthic environment of the entire area. In particular I have examined the effect of off shore gas platforms on benthic diversity and tested their effect over a background of natural spatial variability. The results obtained suggest that natural processes in the North Adriatic such as river outflow and euthrophication show an inter-annual variability that might have important consequences on benthic assemblages, affecting for example their spatial pattern moving away from the coast and along a North to South gradient. Depth-related factors, such as food supply, light, temperature and salinity play an important role in explaining large scale benthic spatial variability (i.e., affecting both the abundance patterns and beta diversity). Nonetheless, more locally, effects probably related to an organic enrichment or pollution from Po river input has been observed. All these processes, together with few human-induced sources of variability (e.g. fishing disturbance), have a higher effect on macrofauna distribution than any effect related to the presence of gas platforms. The main effect of gas platforms is restricted mainly to small spatial scales and related to a change in habitat complexity due to a natural dislodgement or structure cleaning of mussels that colonize their legs. The accumulation of mussels on the sediment reasonably affects benthic infauna composition. All the components of the study presented in this thesis highlight the need to carefully consider methodological aspects related to the study of sedimentary habitats. With particular regards to the North Adriatic Sea, a multi-scale analysis along natural and anthopogenic gradients was useful for detecting the influence of all the processes affecting the sedimentary environment. In the future, applying a similar approach may lead to an unambiguous assessment of the state of the benthic community in the North Adriatic Sea. Such assessment may be useful in understanding if any anthropogenic source of disturbance has a negative effect on the marine environment, and if so, planning sustainable strategies for a proper management of the affected area.

Development of Clinical Decision Support Systems based on Mathematical Models of Physiological Systems

In the last years of research, I focused my studies on different physiological problems. Together with my supervisors, I developed/improved different mathematical models in order to create valid tools useful for a better understanding of important clinical issues. The aim of all this work is to develop tools for learning and understanding cardiac and cerebrovascular physiology as well as pathology, generating research questions and developing clinical decision support systems useful for intensive care unit patients. I. ICP-model Designed for Medical Education We developed a comprehensive cerebral blood flow and intracranial pressure model to simulate and study the complex interactions in cerebrovascular dynamics caused by multiple simultaneous alterations, including normal and abnormal functional states of auto-regulation of the brain. Individual published equations (derived from prior animal and human studies) were implemented into a comprehensive simulation program. Included in the normal physiological modelling was: intracranial pressure, cerebral blood flow, blood pressure, and carbon dioxide (CO2) partial pressure. We also added external and pathological perturbations, such as head up position and intracranial haemorrhage. The model performed clinically realistically given inputs of published traumatized patients, and cases encountered by clinicians. The pulsatile nature of the output graphics was easy for clinicians to interpret. The manoeuvres simulated include changes of basic physiological inputs (e.g. blood pressure, central venous pressure, CO2 tension, head up position, and respiratory effects on vascular pressures) as well as pathological inputs (e.g. acute intracranial bleeding, and obstruction of cerebrospinal outflow). Based on the results, we believe the model would be useful to teach complex relationships of brain haemodynamics and study clinical research questions such as the optimal head-up position, the effects of intracranial haemorrhage on cerebral haemodynamics, as well as the best CO2 concentration to reach the optimal compromise between intracranial pressure and perfusion. We believe this model would be useful for both beginners and advanced learners. It could be used by practicing clinicians to model individual patients (entering the effects of needed clinical manipulations, and then running the model to test for optimal combinations of therapeutic manoeuvres). II. A Heterogeneous Cerebrovascular Mathematical Model Cerebrovascular pathologies are extremely complex, due to the multitude of factors acting simultaneously on cerebral haemodynamics. In this work, the mathematical model of cerebral haemodynamics and intracranial pressure dynamics, described in the point I, is extended to account for heterogeneity in cerebral blood flow. The model includes the Circle of Willis, six regional districts independently regulated by autoregulation and CO2 reactivity, distal cortical anastomoses, venous circulation, the cerebrospinal fluid circulation, and the intracranial pressure-volume relationship. Results agree with data in the literature and highlight the existence of a monotonic relationship between transient hyperemic response and the autoregulation gain. During unilateral internal carotid artery stenosis, local blood flow regulation is progressively lost in the ipsilateral territory with the presence of a steal phenomenon, while the anterior communicating artery plays the major role to redistribute the available blood flow. Conversely, distal collateral circulation plays a major role during unilateral occlusion of the middle cerebral artery. In conclusion, the model is able to reproduce several different pathological conditions characterized by heterogeneity in cerebrovascular haemodynamics and can not only explain generalized results in terms of physiological mechanisms involved, but also, by individualizing parameters, may represent a valuable tool to help with difficult clinical decisions. III. Effect of Cushing Response on Systemic Arterial Pressure. During cerebral hypoxic conditions, the sympathetic system causes an increase in arterial pressure (Cushing response), creating a link between the cerebral and the systemic circulation. This work investigates the complex relationships among cerebrovascular dynamics, intracranial pressure, Cushing response, and short-term systemic regulation, during plateau waves, by means of an original mathematical model. The model incorporates the pulsating heart, the pulmonary circulation and the systemic circulation, with an accurate description of the cerebral circulation and the intracranial pressure dynamics (same model as in the first paragraph). Various regulatory mechanisms are included: cerebral autoregulation, local blood flow control by oxygen (O2) and/or CO2 changes, sympathetic and vagal regulation of cardiovascular parameters by several reflex mechanisms (chemoreceptors, lung-stretch receptors, baroreceptors). The Cushing response has been described assuming a dramatic increase in sympathetic activity to vessels during a fall in brain O2 delivery. With this assumption, the model is able to simulate the cardiovascular effects experimentally observed when intracranial pressure is artificially elevated and maintained at constant level (arterial pressure increase and bradicardia). According to the model, these effects arise from the interaction between the Cushing response and the baroreflex response (secondary to arterial pressure increase). Then, patients with severe head injury have been simulated by reducing intracranial compliance and cerebrospinal fluid reabsorption. With these changes, oscillations with plateau waves developed. In these conditions, model results indicate that the Cushing response may have both positive effects, reducing the duration of the plateau phase via an increase in cerebral perfusion pressure, and negative effects, increasing the intracranial pressure plateau level, with a risk of greater compression of the cerebral vessels. This model may be of value to assist clinicians in finding the balance between clinical benefits of the Cushing response and its shortcomings. IV. Comprehensive Cardiopulmonary Simulation Model for the Analysis of Hypercapnic Respiratory Failure We developed a new comprehensive cardiopulmonary model that takes into account the mutual interactions between the cardiovascular and the respiratory systems along with their short-term regulatory mechanisms. The model includes the heart, systemic and pulmonary circulations, lung mechanics, gas exchange and transport equations, and cardio-ventilatory control. Results show good agreement with published patient data in case of normoxic and hyperoxic hypercapnia simulations. In particular, simulations predict a moderate increase in mean systemic arterial pressure and heart rate, with almost no change in cardiac output, paralleled by a relevant increase in minute ventilation, tidal volume and respiratory rate. The model can represent a valid tool for clinical practice and medical research, providing an alternative way to experience-based clinical decisions. In conclusion, models are not only capable of summarizing current knowledge, but also identifying missing knowledge. In the former case they can serve as training aids for teaching the operation of complex systems, especially if the model can be used to demonstrate the outcome of experiments. In the latter case they generate experiments to be performed to gather the missing data.

The gaseous environment of radio galaxies: a new perspective from high-resolution x-ray spectroscopy

It is known that massive black holes have a profound effect on the evolution of galaxies, and possibly on their formation by regulating the amount of gas available for the star formation. However, how black hole and galaxies communicate is still an open problem, depending on how much of the energy released interacts with the circumnuclear matter. In the last years, most studies of feedback have primarily focused on AGN jet/cavity systems in the most massive galaxy clusters. This thesis intends to investigate the feedback phenomena in radio--loud AGNs from a different perspective studying isolated radio galaxies, through high-resolution spectroscopy. In particular one NLRG and three BLRG are studied, searching for warm gas, both in emission and absorption, in the soft X-ray band. I show that the soft spectrum of 3C33 originates from gas photoionized by the central engine. I found for the first time WA in 3C382 and 3C390.3. I show that the observed warm emitter/absorbers is not uniform and probably located in the NLR. The detected WA is slow implying a mass outflow rate and kinetic luminosity always well below 1% the L(acc) as well as the P(jet). Finally the radio--loud properties are compared with those of type 1 RQ AGNs. A positive correlation is found between the mass outflow rate/kinetic luminosity, and the radio loudness. This seems to suggest that the presence of a radio source (the jet?) affects the distribution of the absorbing gas. Alternatively, if the gas distribution is similar in Seyferts and radio galaxies, the M(out) vs rl relation could simply indicate a major ejection of matter in the form of wind in powerful radio AGNs.

Ruolo del test da sforzo cardiopolmonare nei pazienti affetti da cardiomiopatia ipertrofica in età pediatrica

Background Decreased exercise capacity, and reduction in peak oxygen uptake are present in most patients affected by hypertrophic cardiomyopathy (HCM) . In addition an abnormal blood pressure response during a maximal exercise test was seen to be associated with high risk for sudden cardiac death in adult patients affected by HCM. Therefore exercise test (CPET) has become an important part of the evaluation of the HCM patients, but data on its role in patients with HCM in the pediatric age are quite limited. Methods and results Between 2004 and 2010, using CPET and echocardiography, we studied 68 children (mean age 13.9 ± 2 years) with HCM. The exercise test was completed by all the patients without adverse complications. The mean value of achieved VO2 max was 31.4 ± 8.3 mL/Kg/min which corresponded to 77.5 ± 16.9 % of predicted range. 51 patients (75%) reached a subnormal value of VO2max. On univariate analysis the achieved VO2 as percentage of predicted and the peak exercise systolic blood pressure (BP) Z score were inversely associated with max left ventricle (LV) wall thickness, with E/Ea ratio, and directly related with Ea and Sa wave velocities No association was found with the LV outflow tract gradient. During a mean follow up of 2.16 ± 1.7 years 9 patients reached the defined clinical end point of death, transplantation, implanted cardioverter defibrillator (ICD) shock, ICD implantation for secondary prevention or myectomy. Patients with peak VO2 < 52% or with peak systolic BP Z score < -5.8 had lower event free survival at follow up. Conclusions Exercise capacity is decreased in patients with HCM in pediatric age and global ventricular function seems being the most important determinant of exercise capacity in these patients. CPET seems to play an important role in prognostic stratification of children affected by HCM.

The Universe 10 Billion years ago: Morphologies, Star-formation rates and Galactic-scale winds in z ∼ 2 Galaxies

This thesis is devoted to the study of the properties of high-redsfhit galaxies in the epoch 1 < z < 3, when a substantial fraction of galaxy mass was assembled, and when the evolution of the star-formation rate density peaked. Following a multi-perspective approach and using the most recent and high-quality data available (spectra, photometry and imaging), the morphologies and the star-formation properties of high-redsfhit galaxies were investigated. Through an accurate morphological analyses, the built up of the Hubble sequence was placed around z ~ 2.5. High-redshift galaxies appear, in general, much more irregular and asymmetric than local ones. Moreover, the occurrence of morphological k-­correction is less pronounced than in the local Universe. Different star-formation rate indicators were also studied. The comparison of ultra-violet and optical based estimates, with the values derived from infra-red luminosity showed that the traditional way of addressing the dust obscuration is problematic, at high-redshifts, and new models of dust geometry and composition are required. Finally, by means of stacking techniques applied to rest-frame ultra-violet spectra of star-forming galaxies at z~2, the warm phase of galactic-scale outflows was studied. Evidence was found of escaping gas at velocities of ~ 100 km/s. Studying the correlation of inter-­stellar absorption lines equivalent widths with galaxy physical properties, the intensity of the outflow-related spectral features was proven to depend strongly on a combination of the velocity dispersion of the gas and its geometry.

What are the components that contribute to variability in echocardiographic measurements in aortic stenosis?

INTRODUCTION Echocardiography is the standard clinical approach for quantification of the severity of aortic stenosis (AS). A comprehensive examination of its overall reproducibility and the simultaneous estimation of its variance components by multiple operators, readers, probe applications, and beats have not been undertaken. METHOD AND RESULTS Twenty-seven subjects with AS were scanned over 7 months in the echo-department by a median of 3 different operators. From each patient and each operator multiple runs of beats from multiple probe positions were stored for later analysis by multiple readers. The coefficient of variation was 13.3%, 15.9%, 17.6%, and 20.2% for the aortic peak velocity (Vmax), and velocity time integral (VTI), and left ventricular outflow tract (LVOT) Vmax and VTI respectively. The largest individual contributors to the overall variability were the beat-to-beat variability (9.0%, 9.3%, 9.5%, 9.4% respectively) and that of inability of an individual operator to precisely apply the probe to the same position twice (8.3%, 9.4%, 12.9%, 10.7% respectively). The tracing (inter-reader) and reader (inter-reader), and operator (inter-operator) contribution were less important. CONCLUSIONS Reproducibility of measurements in AS is poorer than often reported in the literature. The source of this variability does not appear, as traditionally believed, to result from a lack of training or operator and reader specific factors. Rather the unavoidable beat-to-beat biological variability, and the inherent impossibility of applying the ultrasound probe in exactly the same position each time are the largest contributors. Consequently, guidelines suggesting greater standardisation of procedures and further training for sonographers are unlikely to result in an improvement in precision. Clinicians themselves should be wary of relying on even three-beat averages as their expected coefficient of variance is 10.3% for the peak velocity at the aortic valve.

Multimodal (EEG-fMRI) functional connectivity study of levodopa effect in Parkinson’s disease

Aim: To assess if the intake of levodopa in patients with Parkinson’s Disease (PD) changes cerebral connectivity, as revealed by simultaneous recording of hemodynamic (functional MRI, or fMRI) and electric (electroencephalogram, EEG) signals. Particularly, we hypothesize that the strongest changes in FC will involve the motor network, which is the most impaired in PD. Methods: Eight patients with diagnosis of PD “probable”, therapy with levodopa exclusively, normal cognitive and affective status, were included. Exclusion criteria were: moderate-severe rest tremor, levodopa induced dyskinesia, evidence of gray or white matter abnormalities on structural MRI. Scalp EEG (64 channels) were acquired inside the scanner (1.5 Tesla) before and after the intake of levodopa. fMRI functional connectivity was computed from four regions of interest: right and left supplementary motor area (SMA) and right and left precentral gyrus (primary motor cortex). Weighted partial directed coherence (w-PDC) was computed in the inverse space after the removal of EEG gradient and cardioballistic artifacts. Results and discussion: fMRI group analysis shows that the intake of levodopa increases hemodynamic functional connectivity among the SMAs / primary motor cortex and: sensory-motor network itself, attention network and default mode network. w-PDC analysis shows that EEG connectivity among regions of the motor network has the tendency to decrease after the intake the levodopa; furthermore, regions belonging to the DMN have the tendency to increase their outflow toward the rest of the brain. These findings, even if in a small sample of patients, suggest that other resting state physiological functional networks, beyond the motor one, are affected in patients with PD. The behavioral and cognitive tasks corresponding to the affected networks could benefit from the intake of levodopa.