Parallel Processing in the Leech Central Nervous System

The aim of this thesis is to shed light on the parallel processing in the leech nervous system. Two main problems have been addressed: 1) how a mechanical stimulus is coded by mechanosensory neurons at the first stage of the information processing in the central nervous system (CNS). 2) which statistical properties of the electrical activity of neurons in the segmental ganglion are relevant for infom1ation processing and in particular for motor reactions. A semi-intact preparation consisting of a single segment innervated by its own ganglion and the adjacent ganglia was used. Sensory stimulation was delivered by touching the skin with an appropriate device. Previous observations indicate that each point of the skin surface is innervated by several mechanosensory neurons (Nicholls and Baylor, 1968; Yau, 1976; Lewis and Kristan, 1998-c). In my thesis I analyzed how sensory information is coded in the electrical activity of a population of mechanosensory neurons. I developed and used an experimental set-up allowing the recording of the simultaneous activation of all mechanosensory neurons belonging to three consecutive segmental ganglia. The experiments were performed in high Mg2+ Ringer fluid in order to block all chemical synapses so as to eliminate synaptic responses and muscle contraction leading to a motion of the skin resulting in an additional sensory feedback. In these conditions it is possible isolate the responses of sensory neurons directly activated by the stimulus. The extent of the activation of mechanosensory neurons in three adjacent ganglia after a stimulation was analyzed. The results of this mapping show that all mechanosensory neurons are consistently activated also in their accessory receptive fields. The reproducibility of their response is the same as that observed in mechanosensory neurons responding in major receptive fields. T cells activation presents the same characteristics for stimulation \ub7of the major or minor receptive fields, indeed they respond to the transient phase of the stimulus. P cells show a different dynamics of activation if stimulated in their major or minor receptive field. In presence of a prolonged stimulation, P cells of the central ganglion fire action potentials continuously while P cells of accessory ganglia adapt. N cells are always activated in major and minor receptive fields showing approximately the same dynamics. When the skin is touched with a moderate tactile stimulus, i.e. exerting a force ofless than 20 mN, many different neurons (T and P cells) of the three ganglia fire action potentials. Thus sensory coding is initially redundant. If the stimulation is prolonged, many of these sensory neurons rapidly adapt and only P cells of the central ganglion respond. Thus sensory coding is dynamical and becomes very sharply tuned. In the second part of my PhD work, a different stage of the information flow has been investigated. The aim of the research was to study which statistical properties of the electrical activity of neurons in the leech ganglion are important for neural processing. In particular I analyzed the statistics of the evoked activity in response to a sensory input. The ganglion was considered as a black box with inputs and outputs; inputs are represented by sensory neurons and outputs by motor neurons or other neurons innervating roots and com1ectives. The responses of mechanosensory neurons to touch stimuli show trial-to-trial reproducibility, i.e. spikes in sensory neurons occur at very precise times. This does not happen for motor neurons. The experiment consisted in stimulating repeatedly a particular mechanosensory neuron by evoking action potentials with an intracellular electrode. The evoked activity in response to this input was recorded by suction electrodes. The extracellular recordings contain the superimposed activity of many neurons which can be separated by spike sorting procedure. The first and second order statistics were studied. Two main results were obtained by this analysis: 1) the response is distributed on many neurons; many neurons respond to the same stimulus and the same neurons are recruited for different stimuli. 2) the response is characterized by spatio-temporal variability, i.e. different neurons respond in different trials and neurons respond with jitter in time occurrences of spikes. The comparison between reproducibility of mechanosensory neurons evoked by touch stimulus and motor neurons evoked by mechanosensory neurons. activation shows that sensory cells are very reproducible, therefore the origin of motor neuron variability has to be found in synaptic transmission. The analysis of the second order statistics was used to check correlation among coactivated neurons. Joint entropies and joint probabilities for each pair of neurons were computed and compared with the sum of individual entropies and the product of individual probabilities, respectively. In all cases these quantities are consistently equal. This means that coactivated neurons are statistically independent. This property of the system can be functional for neural proeessmg. If the response of the network is distributed, then the pooling of the electrical activity of all individual responses is important and may be the key feature of the network. In this perspective, a distributed process consisting in a large number of statistically independent unreliable elements leads to a reproducible response, when the response is pooled over all elements. There is theoretical justification for this statement, indeed it is demonstrated that the pooling of a large number of statistical independent stochastic processes affected by high variability lead to a stochastic process with low variability. In conclusion, increasing the number of elements pooled, the response is more and more reliable

Insights on surface analysis techniques to study glass primary packaging

During the forming process of a vial by tubing glass, temperatures of up to 1200◦C are applied to adjust the glass viscosity. This process causes the release of volatile components such as alkali borates. Consequently, the percentage of sodium and boron measured on the inner surface of the vial can be higher than that measured on the corresponding glass tube. This study aimed to characterize the inner surface of two different borosilicate glass tubes of type I before and after the vial forming process at the nanoscale level. Quantitative elemental analysis of the surface along the vertical axis of glass tubes and vials was performed by X-ray photoelectron spectroscopy, whereas the topographical investigation was carried out by scanning electron microscopy (SEM). In the near-bottom region of a vial, which is usually the area most prone to corrosion, the SEM micrographs showed the appearance of bulges on the surface. The latter were then analyzed by time-of-flight secondary ion mass spectrometry to characterize their molecular composition. The purpose of this work is to identify possible new strategies for faster identification of factors that eventually influence chemical resistance of pharmaceutical glasses and to provide useful information needed to improve industrial processes

Analisi delle problematiche connesse al raddoppio della linea adduttrice tra Quero e San Sebastiano

Analisi della problematiche relative al posizionamento di una condotta acquedottistica su un versante in forte pendenza. Progetto delle opere di mitigazione per il drenaggio del pendio e l'allontanamento delle acque meteoriche.openEmbargo per motivi di segretezza e/o di proprietà dei risultati e informazioni di enti esterni o aziende private che hanno partecipato alla realizzazione del lavoro di ricerca relativo alla tes

ARRAY-CGH COME ESAME DI PRIMO LIVELLO NELLA DIAGNOSI MOLECOLARE DI RITARDO MENTALE E ANOMALIE CONGENITE

The array-CGH technique has emerged in recent years as a powerful tool for the identification of molecular causes underlying complex phenotypes characterized by intellectual disability, autism, epilepsy, psychiatric disorders and multiple congenital anomalies. Over the past 10 years, it has become clear that the conventional cytogenetic analysis is unable to detect rearrangements less than 5-10Mb which can be responsible for these clinical phenotypes. This limit has been exceeded with array-CGH technique which has increased by 15-20% the detection rate of cryptic chromosomal imbalances (deletions or duplications). The possibility to have a genome wide technique with a high resolution led to the proposal, in 2010, by the International Standard Cytogenomic Array (ISCA) Consortium, to use this technique as the first-line test in individuals with intellectual disabilities and congenital anomalies. From studies with microarray technology it has become clear that there are chromosomal regions in which aberrant recombination are particularly frequent, due to the presence of segments with elevated sequence homology, that cause a high degree of genomic instability. Moreover the use of the array-CGH showed the presence in the genome of a large number of structural variations, larger than 1Kb, defined copy number variation or CNVs, that does not always represent a direct cause of disease because they have also been identified in healthy individuals. This complexity in the interpretation of pathogenic CNVs is even more relevant in prenatal diagnosis because it leads to uncertainty in terms of prognosis for the fetal health. For this reason, despite the advantages of the technique, the array-CGH analysis in prenatal diagnosis is considered as a second-line test to be used in association to conventional cytogenetics analysis. In this study were evaluated with array-CGH analysis, 1051 patients with mental and / or developmental disabilities, autism, multiple congenital anomalies and dimorphisms. The main purpose was to verify the presence of cryptic chromosomal rearrangements in order to demonstrate the utility of genomic microarray as first-line test for the characterization of the molecular causes underlying the phenotype of individuals. Then the mechanisms of formation of anomalies were hypothesized by the analysis of the breakpoints, to verify the presence of regions of homology that may have predisposed to the rearrangement. So it was examined whether the mechanism of formation and the clinical significance of the identified CNVs could be related to the pattern of inheritance, the type or the size of the imbalance. The results show that 15.8% of the patients has at least one pathological anomaly or VOUS (variant of uncertain significance) that is likely pathological, and that these are more frequently deletions and CNVs arisen de novo. It was also highlighted that both the clinical significance of CNVs and their mechanism of formation may be related to the size of the imbalance. It was later analyzed the distribution of CNVs in different chromosomes and it was found that in some of them the density of anomalies is greater than the others. The application of the array-CGH in a high number of patients has also allowed to estimate the sensitivity to detect mosaicism, although they have a frequency less than 1% in individuals with learning disabilities. It was observed that the technique is able to detect anomalies present in up to 10% of cells. Finally some fetal samples of chorionic villi and amniotic fluid were analyzed to evaluate the possible use of genomic microarray in prenatal diagnosis. The small number of analyzed samples did not allow us to draw conclusions, but the difficulties in the interpretation of the clinical significance of CNVs, make it a second-line test to be used in association with standard karyotype

Co-branding: il caso Supreme x Louis Vuitton

ope

Optical delivery of liposome encapsulated chemical stimuli to neuronal cells

Spatially confined and precise time delivery of neuroactive molecules is an important issue in neurophysiology. In this work we developed a technique for delivering chemical stimuli to cultured neurons consisting in encapsulating the molecules of interest in liposomes. These vectors were then loaded in reservoirs consisting of glass capillaries. The reservoirs were placed in the recording chamber and single liposomes were trapped and transported out by optical tweezers to the site of stimulation on cultured neurons. Finally, the release of liposome content was induced by application of UV-pulses, breaking the liposome membrane. The efficiency of encapsulation and release were first evaluated by loading the liposomes with fluorescein. In order to test the effect of the UV-induced release, liposomes with diameter ranging from 1 to 10 μm (fL to pL volumes), were filled with KCl and tested on neuronal cells. Neuronal cultures, loaded with Ca(2+) dye, were monitored by imaging intracellular Ca(2+). An efficient release from the liposomes was demonstrated by detectable calcium signals, indicating stimulated depolarization of the neuronal cells by KCl. The present technique represents an alternative method for focal chemical stimulation of cultured cells that circumvents some of the limitations of microejection and photorelease of caged compounds

An expression signature of the angiogenic response in gastrointestinal neuroendocrine tumours: correlation with tumour phenotype and survival outcomes.

BACKGROUND: Gastroenteropancreatic neuroendocrine tumours (GEP-NETs) are heterogeneous with respect to biological behaviour and prognosis. As angiogenesis is a renowned pathogenic hallmark as well as a therapeutic target, we aimed to investigate the prognostic and clinico-pathological role of tissue markers of hypoxia and angiogenesis in GEP-NETs. METHODS: Tissue microarray (TMA) blocks were constructed with 86 tumours diagnosed from 1988 to 2010. Tissue microarray sections were immunostained for hypoxia inducible factor 1α (Hif-1α), vascular endothelial growth factor-A (VEGF-A), carbonic anhydrase IX (Ca-IX) and somatostatin receptors (SSTR) 1–5, Ki-67 and CD31. Biomarker expression was correlated with clinico-pathological variables and tested for survival prediction using Kaplan–Meier and Cox regression methods. RESULTS: Eighty-six consecutive cases were included: 51% male, median age 51 (range 16–82), 68% presenting with a pancreatic primary, 95% well differentiated, 51% metastatic. Higher grading (P=0.03), advanced stage (P<0.001), high Hif-1α and low SSTR-2 expression (P=0.03) predicted for shorter overall survival (OS) on univariate analyses. Stage, SSTR-2 and Hif-1α expression were confirmed as multivariate predictors of OS. Median OS for patients with SSTR-2+/Hif-1α-tumours was not reached after median follow up of 8.8 years, whereas SSTR-2-/Hif-1α+ GEP-NETs had a median survival of only 4.2 years (P=0.006). CONCLUSION: We have identified a coherent expression signature by immunohistochemistry that can be used for patient stratification and to optimise treatment decisions in GEP-NETs independently from stage and grading. Tumours with preserved SSTR-2 and low Hif-1α expression have an indolent phenotype and may be offered less aggressive management and less stringent follow up