DEVELOPMENT OF A LARGE EDDY SIMULATION MODEL FOR THE STUDY OF POLLUTANT DISPERSION IN URBAN AREAS

2008/2009In this thesis, a new large-eddy simulation solver, LES-AIR, has been developed, tested and applied to a practical situation of flow and pollutant dispersion in urban environments. The novelty of the present research resides in the application of a high resolution, accurate, CFD technique to the simulation of real-life flows. The code uses a body fitted curvilinear grid to account for the macro geometry such as terrain slopes, and is thus able to reproduce in detail the complex conditions typical of urban areas; by utilizing the technique of immersed boundaries, the code is also able to mimic the presence the micro complexities such as anthropic structures (i.e. buildings). The first part of the thesis presents a detailed description of the mathematical and numerical model on which the code is based. An extensive set of validation tests was performed in flow configurations having an increasing degree of complexity in terms of forcing and geometry. The numerical model thus validated is applied for obtaining flow and pollutant dispersion in the Servola-Valmaura suburban area of the city of Trieste in Italy. The pollutant was introduced into the domain from a line source near the ground, mimicking the emission from vehicular traffic. In spite of the idealizations inherent to the model, LES-AIR is able to predict the flow and dispersion patterns well, and has proven to be a reliable tool for adaptation in urban pollution studies.Nella presente tesi è stato sviluppato, testato ed applicato ad un caso studio applicativo un nuovo solutore numerico, chiamato codice LES-AIR, capace di predire i campi di vento e la dispersione di nquinanti in ambienti urbani. La maggiore novità di questo lavoro risiede nell’utilizzo di una tecnica fluidodinamica molto accurata e ad alta risoluzione per la simulazione di flussi reali. Il codice LES-AIR è capace di riprodurre con grande dettaglio le geometrie complesse tipiche delle aree urbane tramite l’utilizzo congiunto di una griglia curvilinea, che si adatta all’ orografia del terreno, e della tecnica dei corpi immersi, con la quale vengono riprodotti gli ostacoli antropici, quali gli edifici. Nella prima parte della tesi viene fornita una descrizione dettagliata del modello matematico e numerico su cui si basa il codice. Il modello è stato validato per mezzo di un esteso set di casi test, aventi un grado crescente di complessit à in termini di forzanti e di configurazione geometriche. Il modello così validato è stato applicato alla riproduzione di un caso applicativo nel quale i campi di vento e la dispersione di un inquinante nella zona di Servola-Valmaura, situata nella periferia di Trieste, sono stati simulati. L’ inquinante è stato introdotto da una sorgente lineare posta in prosimità del terreno e rappresentante l’emissione derivante dal traffico cittadino. Nonostante le condizioni idealizzate di vento considerate, il codice LES-AIR si è dimostrato molto efficace nella predizione del flusso e della dispersione dell’inquinante e quindi si è attestato essere un valido strumento negli studi d’ inquinamento urbani.XXII Ciclo198

Impact of contrast-enhanced ultrasound in patients with renal function impairment

To investigate the role of contrast enhanced ultrasound (CEUS) in evaluating patients with renal function impairment (RFI) showing: (1) acute renal failure (ARF) of suspicious vascular origin; or (2) suspicious renal lesions

In developing hippocampal neurons, NR2B-containing N-methyl-d-aspartate receptors (NMDARs) can mediate signaling to neuronal survival and synaptic potentiation, as well as neuronal death

It has been suggested that NR2B-containing NMDA receptors have a selective tendency to promote pro-death signalling and synaptic depression, compared to the survival promoting, synapse potentiating properties of NR2A-containing NMDA receptors. A preferential localization of NR2A-containing NMDA receptors at the synapse in maturing neurons could thus explain differences in synaptic vs. extrasynaptic NMDA receptor signalling. We have investigated whether NMDA receptors can mediate signalling to survival, death, and synaptic potentiation, in neurons at a developmental stage prior to significant NR2A expression and subunit-specific differences between synaptic and extrasynaptic NMDA receptors. We show that in developing hippocampal neurons, the progressive reduction in sensitivity of NMDA receptor currents to the NR2B antagonist ifenprodil applies to both synaptic and extrasynaptic locations. However, the reduction is less acute in extrasynaptic currents, indicating that NR2A does partition preferentially, but not exclusively, into synaptic locations at DIV>12. We then studied NMDA receptor signalling at DIV10, when both synaptic and extrasynaptic NMDA receptors are both overwhelmingly and equally NR2B-dominated. To analyse pro-survival signalling we studied the influence of synaptic NMDA receptor activity on staurosporine-induced apoptosis. Blockade of spontaneous NMDAR activity with MK-801, or ifenprodil exacerbated the apoptotic insult. Furthermore, MK-801 and ifenprodil both antagonized neuroprotection promoted by enhancing synaptic activity. Pro-death signalling induced by a toxic dose of NMDA is also blocked by NR2B-specific antagonists. Using a cell culture model of synaptic NMDA receptor-dependent synaptic potentiation, we find that this is mediated exclusively by NR2B-containing NMDARs, as implicated by NR2B-specific antagonists and the use of selective vs. non-selective doses of the NR2A-preferring antagonist NVP-AAM077. Therefore, within a single neuron, NR2B-NMDA receptors are able to mediate both survival and death signalling, as well as model of NMDA receptor-dependent synaptic potentiation. In this instance, subunit differences cannot account for the dichotomous nature of NMDA receptor signalling

Induction and expression of GluA1 (GluR-A)-independent LTP in the hippocampus

Long-term potentiation (LTP) at hippocampal CA3–CA1 synapses is thought to be mediated, at least in part, by an increase in the postsynaptic surface expression of α-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA) receptors induced by N-methyl-d-aspartate (NMDA) receptor activation. While this process was originally attributed to the regulated synaptic insertion of GluA1 (GluR-A) subunit-containing AMPA receptors, recent evidence suggests that regulated synaptic trafficking of GluA2 subunits might also contribute to one or several phases of potentiation. However, it has so far been difficult to separate these two mechanisms experimentally. Here we used genetically modified mice lacking the GluA1 subunit (Gria1−/− mice) to investigate GluA1-independent mechanisms of LTP at CA3–CA1 synapses in transverse hippocampal slices. An extracellular, paired theta-burst stimulation paradigm induced a robust GluA1-independent form of LTP lacking the early, rapidly decaying component characteristic of LTP in wild-type mice. This GluA1-independent form of LTP was attenuated by inhibitors of neuronal nitric oxide synthase and protein kinase C (PKC), two enzymes known to regulate GluA2 surface expression. Furthermore, the induction of GluA1-independent potentiation required the activation of GluN2B (NR2B) subunit-containing NMDA receptors. Our findings support and extend the evidence that LTP at hippocampal CA3–CA1 synapses comprises a rapidly decaying, GluA1-dependent component and a more sustained, GluA1-independent component, induced and expressed via a separate mechanism involving GluN2B-containing NMDA receptors, neuronal nitric oxide synthase and PKC

CXCL12 inhibits expression of the NMDA receptor's NR2B subunit through a histone deacetylase-dependent pathway contributing to neuronal survival

Homeostatic chemokines, such as CXCL12, can affect neuronal activity by the regulation of inhibitory and excitatory neurotransmission, but the mechanisms involved are still undefined. Our previous studies have shown that CXCL12 protects cortical neurons from excitotoxicity by promoting the function of the gene-repressor protein Rb, which is involved in the recruitment of chromatin modifiers (such as histone deacetylases (HDACs)) to gene promoters. In neurons, Rb controls activity-dependent genes essential to neuronal plasticity and survival, such as the N-methyl--aspartic acid (NMDA) receptor's subunit NR2B, the expression of which in the tetrameric ion channel largely affects calcium signaling by glutamate. In this study, we report that CXCL12 differentially modulates intracellular responses after stimulation of synaptic and extrasynaptic NMDA receptors, by a specific regulation of the NR2B gene that involves HDACs. Our results show that CXCL12 selectively inhibits NR2B expression in vitro and in vivo altering NMDA-induced calcium responses associated with neuronal death, while promoting prosurvival pathways that depend on stimulation of synaptic receptors. Along with previous studies, these findings underline the role of CXCL12/CXCR4 in the regulation of crucial components of glutamatergic transmission. These novel effects of CXCL12 may be involved in the physiological function of the chemokine in both developing and mature brains

Reelin Secreted by GABAergic Neurons Regulates Glutamate Receptor Homeostasis

BACKGROUND: Reelin is a large secreted protein of the extracellular matrix that has been proposed to participate to the etiology of schizophrenia. During development, reelin is crucial for the correct cytoarchitecture of laminated brain structures and is produced by a subset of neurons named Cajal-Retzius. After birth, most of these cells degenerate and reelin expression persists in postnatal and adult brain. The phenotype of neurons that bind secreted reelin and whether the continuous secretion of reelin is required for physiological functions at postnatal stages remain unknown. METHODOLOGY/PRINCIPAL FINDINGS: Combining immunocytochemical and pharmacological approaches, we first report that two distinct patterns of reelin expression are present in cultured hippocampal neurons. We show that in hippocampal cultures, reelin is secreted by GABAergic neurons displaying an intense reelin immunoreactivity (IR). We demonstrate that secreted reelin binds to receptors of the lipoprotein family on neurons with a punctate reelin IR. Secondly, using calcium imaging techniques, we examined the physiological consequences of reelin secretion blockade. Blocking protein secretion rapidly and reversibly changes the subunit composition of N-methyl-D-aspartate glutamate receptors (NMDARs) to a predominance of NR2B-containing NMDARs. Addition of recombinant or endogenously secreted reelin rescues the effects of protein secretion blockade and reverts the fraction of NR2B-containing NMDARs to control levels. Therefore, the continuous secretion of reelin is necessary to control the subunit composition of NMDARs in hippocampal neurons. CONCLUSIONS/SIGNIFICANCE: Our data show that the heterogeneity of reelin immunoreactivity correlates with distinct functional populations: neurons synthesizing and secreting reelin and/or neurons binding reelin. Furthermore, we show that continuous reelin secretion is a strict requirement to maintain the composition of NMDARs. We propose that reelin is a trans-neuronal messenger secreted by GABAergic neurons that regulates NMDARs homeostasis in postnatal hippocampus. Defects in reelin secretion could play a major role in the development of neuropsychiatric disorders, particularly those associated with deregulation of NMDARs such as schizophrenia

Disease-associated missense mutations in GluN2B subunit alter NMDA receptor ligand binding and ion channel properties.

Genetic and bioinformatic analyses have identified missense mutations in GRIN2B encoding the NMDA receptor GluN2B subunit in autism, intellectual disability, Lennox Gastaut and West Syndromes. Here, we investigated several such mutations using a near-complete, hybrid 3D model of the human NMDAR and studied their consequences with kinetic modelling and electrophysiology. The mutants revealed reductions in glutamate potency; increased receptor desensitisation; and ablation of voltage-dependent Mg block. In addition, we provide new views on Mg and NMDA channel blocker binding sites. We demonstrate that these mutants have significant impact on excitatory transmission in developing neurons, revealing profound changes that could underlie their associated neurological disorders. Of note, the NMDAR channel mutant GluN2B unusually allowed Mg permeation, whereas nearby N615I reduced Ca permeability. By identifying the binding site for an NMDAR antagonist that is used in the clinic to rescue gain-of-function phenotypes, we show that drug binding may be modified by some GluN2B disease-causing mutations

Influence of GluN2 subunit identity on NMDA receptor function

AbstractN-methyl-d-aspartate receptors (NMDARs) are ligand-gated ion channels (‘ionotropic’ receptors) activated by the major excitatory neurotransmitter, l-glutamate. While the term ‘the NMDAR’ is often used it obscures the fact that this class of receptor contains within it members whose properties are as different as they are similar. This heterogeneity was evident from early electrophysiological, pharmacological and biochemical assessments of the functional properties of NMDARs and while the molecular basis of this heterogeneity has taken many years to elucidate, it indicated from the outset that the diversity of NMDAR phenotypes could allow this receptor family to subserve a variety of functions in the mammalian central nervous system. In this review we highlight some recent studies that have identified structural elements within GluN2 subunits that contribute to the heterogeneous biophysical properties of NMDARs, consider why some recently described novel pharmacological tools may permit better identification of native NMDAR subtypes, examine the evidence that NMDAR subtypes differentially contribute to the induction of long-term potentiation and long-term depression and discuss how through the use of chimeric proteins additional insights have been obtained that account for NMDAR subtype-dependency of physiological and pathophysiological signalling.This article is part of the Special Issue entitled ‘Glutamate Receptor-Dependent Synaptic Plasticity’

Probing spatial and subunit-dependent signalling by the NMDA receptor

NMDARs are ligand-gated cation channels which are activated by the neurotransmitter glutamate. NMDARs are essential in coupling electrical activity to biochemical signalling as a consequence of their high Ca2+ permeability. This Ca2+ influx acts as a secondary messenger to mediate neurodevelopment, synaptic plasticity, neuroprotection and neurodegeneration. The biological outcome of NMDAR activation is determined by a complicated interrelationship between the concentration of Ca2+ influx, NMDAR location (synaptic vs. extrasynaptic) as well as the subtype of the GluN2 subunit. Despite the recognition that NMDAR mediated physiology is multifaceted, tools used to study subunit and location dependent signalling are poorly characterized and in other cases, non-existent. Therefore, the aim of this thesis is to address this issue. Firstly, I assessed the current pharmacological approach used to selectively activate extrasynaptic NMDARs. Here, synaptic NMDARs are first blocked with MK-801 during phasic activation and then extrasynaptic NMDARs are tonically activated. This approach relies on the continual irreversible blockade of synaptic NMDARs by MK-801 yet contrary to the current dogma, I demonstrate this blockade is unstable during tonic agonist exposure and even more so when physiologically relevant concentrations of Mg2+ are present. This confines a temporal limit in which selective activation of extrasynaptic NMDARs can occur with significant consequences for studying synaptic vs. extrasynaptic NMDAR signalling. Dissecting subunit-dependent signalling mediated by the two major GluN2 subunits in the forebrain, GluN2A and GluN2B, has been advanced significantly by selective GluN2B antagonism yet a reciprocal GluN2A selective antagonist has been lacking. Utilizing novel GluN2A-specific antagonists, I demonstrate a developmental upregulation of GluN2A-mediated NMDA currents which concurrently dilutes the contribution of GluN2B-mediated currents. Moreover, I tested the hypothesis that the Cterminus of GluN2A and GluN2B are essential in controlling the developmental switch of GluN2 subunits utilizing knock-in mice whereby the C-terminus of GluN2A is replaced with that of GluN2B. Surprisingly, the exchange of the C-terminus does not impede the developmental switch in subunits nor the proportion of NMDARs at synaptic vs extrasynaptic sites. However, replacing the C-terminus of GluN2A with that of GluN2B induces a greater neuronal vulnerability to NMDA-dependent excitotoxicity. Collectively, this work enhances our understanding of the complex physiology mediated by the NMDAR by determining how pharmacological tools are best utilized to study the roles of NMDAR location and subunit composition in addition to revealing the importance of the GluN2 C-terminus in development and excitotoxicity