Effetti degli ormoni steroidei nell'omeostasi intracellulare del calcio e nella regolazione della crescita cellulare in cellule derivate da cervelletto di feto bovino

ABSTRACT Steroid hormones differently affect various physiological processes by genomic or non-genomic mechanisms, in both excitable and non-excitable cells. Neurosteroids play a key role in the regulation of many cellular processes, such as cell growth, cell differentiation and apoptosis, exerting their effect in different ways (i.e. promoting or inhibiting a particular event) depending on the brain region of action. In this work two different themes were investigated: 1) the slow genomic effects exerted by estradiol (E2) and testosterone (T) in the regulation of intracellular calcium (Ca2+) homeostasis in a non-excitable cellular model; 2) the trophic effects promoted by estrogens in neuronal cells and in their dendritic branches. Recent experimental evidences highlight the involvement of estrogens and androgens in the regulation of intracellular homeostasis of calcium ion (Ca2+), one of the most important second messengers mediating steroid hormones action. The effects of neurosteroids on excitable cells are well documented; however, these steroids also influence numerous physiological events in non-excitable cells, such as fibroblasts or vascular endothelial cells. In the first part of this work I have focused my attention on an immortalized endothelial-like cell line derived from fetal bovine cerebellum. E2 (10nM) and T (10nM) effects on intracellular Ca2+ homeostasis were tested by varying the exposure time to the hormones (8, 24, 48 h). Ca2+ measurements were performed with genetically encoded Ca2+ probes (Cameleons) targeted to the main subcellular compartments involved in intracellular Ca2+ homeostasis (cytosol, endoplasmic reticulum, mitochondria). While T treatment had not any effect on intracellular Ca2+ fluxes, mitochondrial Ca2+ uptake significantly decreased after 48-h exposure to E2, whereas cytosolic and endoplasmic reticulum responses were unaffected. The effect of E2 on mitochondrial Ca2+ handling was blocked by ICI 182,780 (10nM), a pure estrogen receptor antagonist, suggesting that the effect was estrogen-receptor–mediated. To investigate further the effect of E2 on mitochondria, we tested two other concentrations of E2 (1 nM and 100 nM) and we observed a similar effect on the Ca2+ peak decrease indicating that saturation was possibly reached at 1 nM. To evaluate whether the decrease of Ca2+ affected mitochondrial membrane potential (ΔΨm), cells were monitored in the presence of tetra-methyl-rhodamine-methylester (TMRM): no significant changes were seen between cells treated with E2 and controls. To investigate a mechanism of action, we assessed the possibile involvement of the permeability transition pore (PTP), an inner mitochondrial membrane channel influencing energy metabolism and cell viability. We treated cells with CyclosporinA (CsA) (0.8μM), which binds to the matrix chaperone cyclophilin-D and regulates PTP opening. CsA reversed the effects of a 48-h treatment with E2, suggesting a possible transcriptional modulation of proteins involved in the mitochondrial permeability transition process. Taken together, these data reveal that, in this immortalized endothelial-like cell line, the genomic effect exerted by E2 can affect PTP opening without causing a collapse in ΔΨm. In the second part of this study I examined the effects of estradiol in the regulation of the growth of neuronal cells and of their dendritic branches. Dendrites are the principal cellular sites where neurons receive, process, and integrate inputs from their multiple pre-synaptic partners. It is widely known that, in early developmental stages, estrogens modulate neuronal morphology through a regulation of dendritic development. Estrogens regulate neuronal plasticity, synaptogenesis and cellular growth in many brain regions, such as cortex and hypothalamus. Few papers focused on what estrogens cause in the morphology of cerebellum, a region of the brain with a crucial role in controlling balance, posture, motor coordination as well as many other cognitive processes. To characterize the trophic effects of E2, primary neuronal cells from bovine cerebellum of both male and female fetuses were analyzed. To highlight differences in the morphological organization of neuronal cells we performed fluorescent-immunocytochemical analysis using a specific marker of immature neurons (βIII-tubulin). The whole area and the whole perimeter of neuronal cells, the number, the length and the diameter of dendritic branches were estimated and comparisons between E100nM samples and control cells were performed; differences intra sex were also considered. Results indicate that E100nM increases all the parameters in cells from females, while in males the trophic effect is restricted only to some values. Moreover, females have greater values than males in the soma dimensions, in the total number of branches and in the length of dendritic branches. On the contrary, males show superior values in the diameter of dendrites. Taken together, these preliminary data suggest that E100nM produces a trophic effect in both male and female samples; since several differences between males and females emerged, it could be supposed that neurons of the cerebellum in this embryonic stage exhibit dimorphic properties

Impact of dark matter models on the EoR 21-cm signal bispectrum

The nature of dark matter sets the timeline for the formation of first collapsed haloes and thus affects the sources of reionization. Here, we consider two different models of dark matter: cold dark matter (CDM) and thermal warm dark matter (WDM), and study how they impact the epoch of reionization (EoR) and its 21-cm observables. Using a suite of simulations, we find that in WDM scenarios, the structure formation on small scales gets suppressed, resulting in a smaller number of low-mass dark matter haloes compared to the CDM scenario. Assuming that the efficiency of sources in producing ionizing photons remains the same, this leads to a lower number of total ionizing photons produced at any given cosmic time, thus causing a delay in the reionization process. We also find visual differences in the neutral hydrogen (H I) topology and in 21-cm maps in case of the WDM compared to the CDM. However, differences in the 21-cm power spectra, at the same neutral fraction, are found to be small. Thus, we focus on the non-Gaussianity in the EoR 21-cm signal, quantified through its bispectrum. We find that the 21-cm bispectra (driven by the HI topology) are significantly different in WDM models compared to the CDM, even for the same mass-averaged neutral fractions. This establishes that the 21-cm bispectrum is a unique and promising way to differentiate between dark matter models, and can be used to constrain the nature of the dark matter in the future EoR observations

Effects of Quasar Feedback in Galaxy Groups

We study the effect of quasar feedback on distributions of baryons and properties of intracluster medium in galaxy groups using high-resolution numerical simulations. We use the entropy-conserving Gadget code that includes gas cooling and star formation, modified to include a physically-based model of quasar feedback. For a sample of ten galaxy group-sized dark matter halos with masses in the range of 1 to $5\times 10^{13} M_{\odot}/h$, star formation is suppressed by more than 50% in the inner regions due to the additional pressure support by quasar feedback, while gas is driven from the inner region towards the outer region of the halos. As a result, the average gas density is 50% lower in the inner region and 10% higher in the outer region in the simulation, compared to a similar simulation with no quasar feedback. Gas pressure is lowered by about 40% in the inner region and higher in the outer region, while temperature and entropy are enhanced in the inner region by about 20-40%. The total group gas fraction in the two simulations generally differs by less than 10%. We also find a small change of the total thermal Sunyaev-Zeldovich distortion, leading to 10% changes in the microwave angular power spectrum at angular scales below two arcminutes.Comment: 12 pages, 9 figures, 8 tables. MNRAS accepted June 10 2008. Matches the accepted version. Error in the implementation has been corrected leading to few minor changes in conclusion

Experimental and Numerical Analysis of a Non-Newtonian Fluids Processing Pump

Abstract Centrifugal pumps are used in many applications in which non-Newtonian fluids are involved: food processing industry, pharmaceutical and oil/gas applications. In addition to pressure and temperature, the viscosity of a non-Newtonian fluid depends on the shear rate and usually is several orders of magnitude higher than water. High values of viscosity cause a derating of pump performance with respect to water. Nowadays, pumping and mixing non-Newtonian fluids is a matter of increasing interest, but there is still lack of a detailed analysis of the fluid-dynamic phenomena occurring within these machines. A specific design process should take into account these effects in order to define the proper pump geometry, able to operate with non-Newtonian fluids with specific characteristics. Only few approaches are available for correcting the pump performance based on the Hydraulic Institute method. In this work, an experimental and numerical campaign is presented for a semi–open impeller centrifugal pump elaborating non-Newtonian fluids. An on-purpose test bench was built and used to investigate the influence on pump performance of three different non-Newtonian fluids. Each pump performance test was accompanied by the rheological characterization of the fluid, in order to detect modifications of the rheological phenomena and allow a proper Computation Fluid Dynamics (CFD) modeling. The performance of the machine handling both Newtonian and non-Newtonian fluids are highlighted in relation with the internal flow field

Inositol trisphosphate receptor-mediated Ca 2+ signalling stimulates mitochondrial function and gene expression in core myopathy patients

Core myopathies are a group of childhood muscle disorders caused by mutations of the ryanodine receptor (RyR1), the Ca2+ release channel of the sarcoplasmic reticulum. These mutations have previously been associated with elevated inositol trisphosphate receptor (IP3R) levels in skeletal muscle myotubes derived from patients. However, the functional relevance and the relationship of IP3R mediated Ca2+ signalling with the pathophysiology of the disease is unclear. It has also been suggested that mitochondrial dysfunction underlies the development of central and diffuse multi-mini-cores, devoid of mitochondrial activity, which is a key pathological consequence of RyR1 mutations. Here we used muscle biopsies of central core and multi-minicore disease patients with RyR1 mutations, as well as cellular and in vivo mouse models of the disease to characterize global cellular and mitochondrial Ca2+ signalling, mitochondrial function and gene expression associated with the disease. We show that RyR1 mutations that lead to the depletion of the channel are associated with increased IP3-mediated nuclear and mitochondrial Ca2+ signals and increased mitochondrial activity. Moreover, western blot and microarray analysis indicated enhanced mitochondrial biogenesis at the transcriptional and protein levels and was reflected in increased mitochondrial DNA content. The phenotype was recapitulated by RYR1 silencing in mouse cellular myotube models. Altogether, these data indicate that remodelling of skeletal muscle Ca2+ signalling following loss of functional RyR1 mediates bioenergetic adaptation

Impact of Cluster Physics on the Sunyaev-Zel'dovich Power Spectrum

We use an analytic model to investigate the theoretical uncertainty on the thermal Sunyaev-Zel'dovich (SZ) power spectrum due to astrophysical uncertainties in the thermal structure of the intracluster medium. Our model accounts for star formation and energy feedback (from supernovae and active galactic nuclei) as well as radially dependent non-thermal pressure support due to random gas motions, the latter calibrated by recent hydrodynamical simulations. We compare the model against X-ray observations of low redshift clusters, finding excellent agreement with observed pressure profiles. Varying the levels of feedback and non-thermal pressure support can significantly change both the amplitude and shape of the thermal SZ power spectrum. Increasing the feedback suppresses power at small angular scales, shifting the peak of the power spectrum to lower ell. On the other hand, increasing the non-thermal pressure support has the opposite effect, significantly reducing power at large angular scales. In general, including non-thermal pressure at the level measured in simulations has a large effect on the power spectrum, reducing the amplitude by 50% at angular scales of a few arcminutes compared to a model without a non-thermal component. Our results demonstrate that measurements of the shape of the power spectrum can reveal useful information on important physical processes in groups and clusters, especially at high-redshift where there exists little observational data. Comparing with the recent South Pole Telescope measurements of the small-scale cosmic microwave background power spectrum, we find our model reduces the tension between the values of sigma_8 measured from the SZ power spectrum and from cluster abundances.Comment: 15 Pages, 9 Figures, updated to match version accepted by Ap

Analysis of α-synuclein species enriched from cerebral cortex of humans with sporadic dementia with Lewy bodies.

Since researchers identified α-synuclein as the principal component of Lewy bodies and Lewy neurites, studies have suggested that it plays a causative role in the pathogenesis of dementia with Lewy bodies and other 'synucleinopathies'. While α-synuclein dyshomeostasis likely contributes to the neurodegeneration associated with the synucleinopathies, few direct biochemical analyses of α-synuclein from diseased human brain tissue currently exist. In this study, we analysed sequential protein extracts from a substantial number of patients with neuropathological diagnoses of dementia with Lewy bodies and corresponding controls, detecting a shift of cytosolic and membrane-bound physiological α-synuclein to highly aggregated forms. We then fractionated aqueous extracts (cytosol) from cerebral cortex using non-denaturing methods to search for soluble, disease-associated high molecular weight species potentially associated with toxicity. We applied these fractions and corresponding insoluble fractions containing Lewy-type aggregates to several reporter assays to determine their bioactivity and cytotoxicity. Ultimately, high molecular weight cytosolic fractions enhances phospholipid membrane permeability, while insoluble, Lewy-associated fractions induced morphological changes in the neurites of human stem cell-derived neurons. While the concentrations of soluble, high molecular weight α-synuclein were only slightly elevated in brains of dementia with Lewy bodies patients compared to healthy, age-matched controls, these observations suggest that a small subset of soluble α-synuclein aggregates in the brain may drive early pathogenic effects, while Lewy body-associated α-synuclein can drive neurotoxicity

Simulating the impact of HI fluctuations on matched filter search for ionized bubbles in redshifted 21 cm maps

Extending the formalism of Datta, Bharadwaj & Choudhury (2007) for detecting ionized bubbles in redshifted 21 cm maps using a matched-filtering technique, we use different simulations to analyze the impact of HI fluctuations outside the bubble on the detectability of the bubble. In the first three kinds of simulations there is a spherical bubble of comoving radius R_b, the one that we are trying to detect, located at the center, and the neutral hydrogen (HI) outside the bubble traces the underlying dark matter distribution. We consider three different possible scenarios of reionization, i.e., (i) there is a single bubble (SB) in the field of view (FoV) and the hydrogen neutral fraction is constant outside this bubble (ii) patchy reionization with many small ionized bubbles in the FoV (PR1) and (iii) many spherical ionized bubbles of the same radius $R_b$ (PR2). The fourth kind of simulation uses more realistic maps based on semi-numeric modelling (SM) of ionized regions. We find that for both the SB and PR1 scenarios the fluctuating IGM restricts bubble detection to size R_b<= 6 Mpc and R_b<= 12 Mpc for the GMRT and the MWA respectively, however large be the integration time. These results are well explained by analytical predictions. Large uncertainty due to the HI fluctuations restricts bubble detection in the PR2 scenario for neutral fraction x_HI<0.6. The matched-filter technique works well even when the targeted ionized bubble is non-spherical due to surrounding bubbles and inhomogeneous recombination (SM). We find that determining the size and positions of the bubbles is not limited by the HI fluctuations in the SB and PR1 scenario but limited by the instrument's angular resolution instead, and this can be done more precisely for larger bubble (abridged).Comment: 14 pages, 15 Postscript figures, Revised to incorporate ionization maps produced by the semi-numeric approach. Accepted for publication in MNRA

Multi-state gene cluster switches determine the adaptive mitochondrial and metabolic landscape of breast cancer

Adaptive metabolic switches are proposed to underlie conversions between cellular states during normal development as well as in cancer evolution. Metabolic adaptations represent important therapeutic targets in tumors, highlighting the need to characterize the full spectrum, characteristics, and regulation of the metabolic switches. To investigate the hypothesis that metabolic switches associated with specific metabolic states can be recognized by locating large alternating gene expression patterns, we developed a method to identify interspersed gene sets by massive correlated biclustering (MCbiclust) and to predict their metabolic wiring. Testing the method on breast cancer transcriptome datasets revealed a series of gene sets with switch-like behavior that could be used to predict mitochondrial content, metabolic activity, and central carbon flux in tumors. The predictions were experimentally validated by bioenergetic profiling and metabolic flux analysis of 13C-labelled substrates. The metabolic switch positions also distinguished between cellular states, correlating with tumor pathology, prognosis, and chemosensitivity. The method is applicable to any large and heterogeneous transcriptome dataset to discover metabolic and associated pathophysiological states