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Synthesis of novel nitrogen ligands and their application to transition metal catalyzed reactions.
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Mitochondrial network fragmentation initiates Purkinje cells degeneration via Ca2+ buffering impairment: dissecting the molecular events from AFG3L2 mutation to defective mitochondrial dynamics.
Get PDFSpinocerebellar ataxia type 28 (SCA28) is a neurodegenerative disorder characterized by unbalanced standing, gait incoordination, nystagmus, ophthalmoparesis and pyramidal signs. Several disease-causing mutations have been identified in the AFG3L2 gene. The encoded protein, AFG3L2, coassembles with paraplegin into multimeric complexes, called the m-AAA proteases, in the inner mitochondrial membrane. These complexes are crucial components of the mitochondrial protein quality control system and regulate mitochondrial morphology.
The haploinsufficient Afg312 mouse recapitulates the features of SCA28 patients, displaying motor incoordination due to dark degeneration of Purkinje cells (PC-DCD). This is a form of degeneration characterized by toxic levels of intracellular Ca2+and activation of calpains. Differently from other forms of SCA, where this phenomenon is associated to excitotoxicity, in the SCA28 mouse is unique since it originates from a mitochondrial dysfunction.
We established, that Afg312-depleted mitochondria ineffectively buffer the evoked peaks of Ca2+.This enhances cytoplasmic Ca2+ levels in PC, thus triggering PC-DCD. We demonstrated that this defect is caused by the negative synergism between the mitochondrial network fragmentation and the altered trafficking of the organelles to PC dendrites.
To determine the molecular mechanism that induces mitochondrial network fragmentation in the absence of AFG3L2, we studied the regulatory pathway involved in mitochondrial dynamics. Previous work demonstrated that mitochondrial morphology alteration, in the absence of AFG3L2, is caused by an increased OMA1-mediated OPA1 processing. Studying the regulatory pathway that drives OMA1 activity, we discovered that AFG3L2 is deeply involved in this pathway. Full length OMA1 protein is 60 kD, while the proposed active form is 40kD. We identified AFG3L2 as the most important protease involved in OMA1 60/40 kD processing although, additional unknown protease(s) contribute to alternative pathway during stress conditions.
We found that the absence of the m-AAA protease induces a striking accumulation of the 60 kD band and a reduction of the 40kD. To clarify the role of AFG3L2 in OMA1 processing we performed co-immunoprecipitation experiments, which disclosed a physical-interaction between the two proteins. Interestingly, we were able to rescue OMA1 processing in Afg3l2 ko murine embryonic fibroblasts by overexpression of wild type AFG3L2, but not with the proteolytic inactive mutant (AFG3L2-E575Q).
Moreover, we showed that OMA1 40kD form is a substrate of the the i-AAA protease demonstrating that the m-AAA proteases and the i-AAA proteases work coordinately in the regulation of OMA1.
These data, besides disclosing the pathogenetic mechanism of SCA28, demonstrate for the first time the impact of defective mitochondrial Ca2+uptake on local Ca2+ signaling in a physiopathological condition of the nervous system. We demonstrated that the altered Ca2+ homeostasis is caused by the mitochondrial network fragmentation, via OPA1 over processing OMA1-mediated consequent to the absence of AFG3L2.
Moreover, we demonstrated for the first time the fundamental role of the m-AAA and the i-AAA complexes in the regulation of OMA1 processing and confirming their fundamental role in mitochondrial dynamics
Estimating the galaxy power spectrum of the VIPERS galaxy distribution.
Get PDFThe power spectrum is a statistic that describes the clustering of matter in Fourier space. The shape and amplitude of the power are directly predicted by theory and direct measurements of the power can constrain cosmological parameters.
The large size of recent surveys has opened new windows in the observation of secondary features of the galaxy power spectrum such as the Baryonic Acoustic Oscillations (BAO), which used as standard ruler can infer dark energy properties, and Redshift-Space Distortions (RSD), which thanks to the anisotropic observed clustering in redshift space can discriminate between different theories of gravity.
In this thesis, we apply the power spectrum statistic to the VIMOS Public Extragalactic Redshift Survey (VIPERS) distribution of galaxies in order to study the underlying clustering of matter.
VIPERS is an ongoing spectroscopic survey, composed of two fields, W1 and W4, with the aim of mapping the spatial distribution of galaxies within a large volume of the z ∼ 1 Universe. The main properties of VIPERS are the high redshift range surveyed 0.4 < z < 1.2 and the high sampling rate at those redshifts.
Its main goal is the measurement of the anisotropy of the galaxy clustering in redshift space in order to constrain the growth rate of structure at a mean redshift hzi ∼ 0.8, never reached by other surveys.
Even the measure of the galaxy power spectrum at these redshifts is something new. Our goal is to estimate the clustering of matter in Fourier space constraining the cosmological quantities that mainly affect the power spectrum shape such as the matter density and the baryonic fraction. The approach used to estimate the galaxy power spectrum is quite classical and has been used in past surveys such as the Two Degree Field Galaxy Redshift Survey (2dFGRS).
We describe the power spectrum estimator using a novel simulation approach called “Γ”-simulation, including the main properties of a real survey such as the cone-like geometry and the density gradient function of the redshift.
The main factor that alters our power spectrum measurement is the window function, due to the limited size of the sample, which is not negligible even for a box geometry.
We show a method to correctly take into account the window function effects including it with a three dimensional convolution with the input model of the simulation.
To analyse the potential of the VIPERS sample for extracting cosmological information, we use a set of VIPERS mock catalogues drawn from the MultiDark simulation. We analyse all the selection function effects of the survey, such as the sampling strategy and the decreasing mean density, in our estimation and study their effect on the recovered power spectrum.
The VIPERS window function is problematic due to the small angular coverage, strongly suppressing the clustering in Fourier space at large scales and completely damping the BAO signal. We measure the window function of VIPERS which will be used to perform a three dimensional convolution with the theoretical model generated from the tested cosmology.
RSD also determine the observed clustering and, in the simplest case, their impact could be divided in two regimes. Small scale effects due to the high non-linear velocity of galaxies inside virialised structures are corrected with a simple velocity dispersion model, an empirical prediction of the behaviour of the clustering in redshift space. Large scale effects due to the linear in-fall of galaxies in high density peaks, which increase the observed clustering of matter, are included with the Kaiser term dependent on the linear growth rate of matter. We include these two terms before applying the 3D convolution of the model with the window function.
Some tests are performed on VIPERS mock catalogues to recover the known cosmological information of the simulation with a chi-square technique and, in all cases, we were able to extract the input cosmology with lower systematics errors than statistical ones.
Finally we apply this approach to the data dividing the full survey into four subsamples, two redshift bins, 0.6 < z1 < 0.9 and 0.9 < z2 < 1.1, for each field. Despite the different window functions in each subsample due to small differences in volume and in geometry, the four measurements are consistent with each other. This is due to the fact that, at large scales, statistical fluctuations related to cosmic variance are much bigger with respect to small differences in the window. Even the overall amplitude is similar between the two redshift bins because the lower amplitude expected for the higher redshift power spectrum (because structures were less compact in the past) is compensated by the higher bias factor.
We compare the measured power spectrum from data with some theoretical models in order to extract the baryonic fraction and the amount of matter density in the Universe. The probability contours display evidence of a well-known degeneracy previously observed in other surveys (such as 2dFGRS) between the two recovered parameters in the determination of the overall shape of the matter power spectrum. Increasing the matter density of the Universe in fact shifts the power spectrum toward higher modes enhancing the clustering at the scales sampled by VIPERS. In this case to compensate the increase of power, the good fit is given by models with high baryonic fraction necessary to suppress the power at those scales. Vice-versa a low-M Universe does not need a high baryonic fraction. This degeneracy is broken combining the two redshift bins with a joint likelihood.
Finally, we provide an estimation of the matter density fixing all the other cosmological parameters to the best known values. We evaluate the matter density to be M = 0.272+0.027 −0.031 in perfect agreement with Planck (once we rescale the Hubble factor from the Hubble Space Telescope used for the estimation of M to be consistent with the Planck one) and with the matter density estimated from the VIPERS Public Data Release-1 measurements of the clustering ratio
Adrenergetic modulation of the intrinsic myogenic activity of peripheral diaphragmatic lymphatics.
Get PDFThe diaphragmatic lymphatic system drains fluids and solutes from diaphragmatic interstitium and from the pleural and peritoneal cavities. Initial lymphatics, devoid of smooth muscle cells (SMCs) in their walls, join into long linear vessels or complex loops, formed at the confluence of linear vessels. Both linear vessels and loops, visible below the pleural and peritoneal mesothelia over the diaphragmatic dome, may be segmented in functional units, called lymphangions, separated by intraluminal valves, which ensure unidirectional lymph flow. Lymph progression within the diaphragmatic lymphatic vessels is due to an hydraulic pressure gradient of between adjacent lymphatic segments whose generation and maintenance depends upon a system of extrinsic and intrinsic pumps. Extrinsic pumping is mainly due to the movement of the surrounding tissues which causes the contraction/expansion of the vessel, while the intrinsic pumping mechanism is due to the rhythmic contraction of the smooth muscle cells surrounding the wall of the lymphangions. Extrinsic pumping prevails in lymphatics of the medial diaphragm, while lymphatic loops located at the extreme diaphragmatic periphery do require an intrinsic pumping mechanism to propel lymph centripetally. Lymph propulsion within the most peripheral diaphragmatic lymphatics depends upon tissue displacements and contraction of smooth muscle cells that surround the collecting lymphatics. The aim of the present work was to investigate, in actively contracting sites of peripheral diaphragmatic lymphatic vessels, the contribution of single strokes and valves opening/closing dynamics to lymph propulsion, and to analyze how this phenomenon is modulated by epinephrine.
Anaesthetized rats received an intraperitoneal injection of a mixture of FITC-conjugated dextrans and TRITC-labeled microspheres (0.1-1 m diameter). After passive lymphatic vessels loading, microspheres movement were video recorded ex-vivo in excised pieces of diaphragm, kept superfused with warmed oxygenated Tyrode’s solution in a flow chamber on the stage of an upright microscope. Instantaneous and mean microsphere velocities were derived from microsphere trajectories along with vessel diameter changes due to spontaneous active strokes.
Data obtained show that active strokes exert a distance-dependent effect on microspheres progression from the contracting site. Their velocity profile results parabolic with a peak velocity of about 96 μm/sec. In the presence of intraluminal valves, microspheres show an oscillatory trajectory on the proximal side and monotonic outward directed flow on the distal side of the valve. Epinephrine administration has opposite effects in linear vessels and lymphatic loops: in particular, epinephrine determines an increase in contraction frequency of about 3 bpm and a greater distance traveled by microspheres in loops and an impairment of spontaneous activity in linear vessels
Correlative analysis of putative molecular predictive factors in patients with curatively resected stage III colon cancer, treated with adjuvant oxaliplatin-based chemotherapy.
Get PDFColon cancer is the second cause of death for neoplasm worldwide. In most cases it is diagnosed when still localized to the intestinal wall or in regional lymph nodes. Adjuvant therapy with 5-Fluorouracil (5FU) and folinic acid (FA), in combination with oxaliplatin (FOLFOX) are the standard options for patients with radically resected stage III disease. However, a proportion of patients will develop recurrence due to drug resistance and oxaliplatin-based chemotherapy is a regimen that may cause potentially disabling sensory neuropathy. Therefore there is an increase needing for a better selection of patients to be addressed to the most appropriate chemotherapeutic treatment, also in the adjuvant setting.
Several proteins and genetic markers have been described in an attempt to refine prognostic information and predict the benefit derived from systemic treatment. In particular TS protein expression, MSI, p53 expression, BRAF and TP53 mutations, have been described in several reports in relation to 5FU treatment, whereas ERCC1 polymorphism, ERCC1 expression and KRAS mutations, seem to be related to oxaliplatin efficacy in advanced colon cancer patients.
At this purpose we enrolled 230 patients from Argentina and Switzerland who underwent surgical resection, followed by 6-months adjuvant treatment: 106 were treated with 5FU alone and 124 with FOLFOX. In all the cases we investigated the MSI status by fragment analysis, we analyzed BRAF, KRAS, TP53 mutations and ERCC1 codon 118 polymorphism by direct sequencing and we performed ERCC1 expression analysis at protein and mRNA levels by immunohistochemistry and real-time PCR, respectively. Finally, we correlated the molecular and immunohistochemical results with the clinical data.
Above all, a little advantage in survival was observed for patients treated with FOLFOX regimen if compared to those treated with 5FU (51.3 and 41.6 months, respectively, for DFS; 55.4 and 49.3 months, respectively, for OS), although the difference was not statistically significant, probably due to the low number of analyzed cases.
We found MSI in 12% of cases, BRAF mutations in 9% of cases, KRAS mutations in 28% of cases and ERCC1 resulted over-expressed in 40% of cases detected by IHC and in 49% of cases detected by real-time PCR. These percentages, as well as the types of alterations, are in line with those published in the literature.
Concerning the correlations among markers, we observed a significant association between MSI and BRAF mutations (in agreement with the literature) and absence of association between KRAS mutations and ERCC1 expression (at odds with the hypothesis proposed in a recent preclinical study).
When we matched the clinical data of the whole patients cohort with molecular alterations, we found a trend towards a better prognosis for patients with MSI than for those with a MSS status (p=0.17); we observed that KRAS mutations confer a worse prognosis to advanced colon cancer patients, borderline for the DFS (p=0.07) and statistically significant for the OS (p=0.004); finally we found a trend towards a better DFS (p=0.11) for patients showing low levels of ERCC1 mRNA expression.
When we subdivided the patients on the basis of the received treatment (5FU versus oxaliplatin-based chemotherapy), we observed similar percentages of alterations of all the markers between the two groups. By correlating the molecular alterations with clinical data, we found a trend towards a better survival for MSI patients treated 5FU (p=0.16 and p=0.37 for DFS and OS, respectively), while for FOLFOX patients no clinical differences were found between MSI and MSS cases.
As for KRAS mutations, in 5FU group we observed a statistical significant worse DFS (p=0.04) and a trend towards a worse OS (p=0.07) in KRAS mutated patients if compared to wild-type patients. In FOLFOX group, no statistical differences were identified between KRAS mutated and wild-type cases. Stratifying the population on the basis of KRAS mutational status, we noticed that in wild-type patients there was no difference in the clinical outcome in the two treatment modalities. On the contrary, in mutated cases a trend towards a better DFS (p=0.28) and OS (p=0.20) was observed in FOLFOX treated patients if compared to 5FU group.
As regards ERCC1 expression, we found only a trend toward a better DFS (p=0.17) in patients characterized by low ERCC1 mRNA levels when treated with FOLFOX.
As for the last markers, ERCC1 codon 118 polymorphism (AAT/AAC) and TP53 mutations, we found percentages of alterations in line with the literature (for ERCC1 polymorphism: TT genotype in 31% of cases, CC genotype in 21% of patients; for TP53: 44% of cases showed at least one mutation). The correlations between these two markers and the clinical outcome are now under evaluation.
In conclusion, looking at the whole cohort, we can confirm a better clinical outcome for adjuvant colon cancer patients treated with FOLFOX regimen with respect to 5FU treatment. MSI could be a useful tool indicating a better prognosis also for advanced colon cancer but its role in predicting 5FU or FOLFOX efficacy remains controversial. In addition, we propose to assess ERCC1 mRNA expression analysis before the administration of oxaliplatin-based chemotherapy, in order to early identify the patients who may benefit the most from this treatment.
Finally, we suggest that KRAS mutational status could help clinicians in selecting the best chemotherapeutic treatment in the adjuvant setting: only KRAS mutant patients should be treated with a platinum-based chemotherapy, while patients whose tumour is KRAS wild-type can be treated with 5FU alone, thus preventing adverse side effect in a consistent number of cases. Our results, of course, deserve confirmations
Array-CGH in the investigation of karyotype changes of CD34+ haematopoietic stem cells in lymphoma and multiple myeloma patients who underwent to autologous transplantation.
Get PDFHematopoietic stem cell transplantation (HSCT) represents an effective treatment strategy for a variety of hematologic and not hematologic malignancies.
In particular, autologous transplantation of haematopoietic stem cells (ASCT) from bone marrow of patients with hematologic malignancies is feasible and has low treatment-related mortality (Gribben JG, 2009). However, literature assessed late mortality in 29.4% of individuals who had survived 2 or more years after autologous haematopoietic cell transplantation (Burns, L.J., 2009; Bhatia S. and al, 2005). This could be due to the purified stem cells which might carry a mutation on a chromosome predisposing to the disease and lead to the risk of pathology recurrence.
Lymphoma and myeloma are perfect candidates for autologous transplantation after G-CSF stimulation and bone marrow ablation trough chemotherapy.
However, in this case too, pathology relapse or secondary malignancies are found in a high percentage of patients.
The aim of this project is to verify the existence of detectable imbalanced chromosome anomalies in stem cells before any ablative treatment for HSCT or developed after G-CSF stimulation or chemotherapy.
A cohort of 24 lymphoma and myeloma patients have been analyzed trough array-CGH to identify significant imbalanced chromosome anomalies also present in low percentage of mosaicism. The result showed anomalies in 8/24 patients: one patient affected by Hodgkin Lymphoma (HL) revealed a deletion of chromosome 2 in p16.1, where the REL gene is located and in part deleted; the amplification of chromosome 11 in q12.2q13.4 containing CCND1 gene (this patient was investigated both before and after transplantation) was found in one patient with multiple myeloma (MM); alterations of chromosome 14 in q32.31-33, where genes for variable chain of immune globulin are located, were found in five patients with Hodgkin and non Hodgkin lymphomas (HL/NHL). FISH on interphase nuclei has been used to confirm a-CGH data.
A short-time (36 months) clinical and haematological follow-up examination did not show a different trend between patients with chromosome imbalances and without but a long-term follow-up is needed to definitely correlate the imbalances with the clinical evolution and to have the indications of global survival of the considered population.
Work in progress is the extension of clinical and haematological observation to obtain evidence of a difference statistically significant and to reach the final goal of suggesting a possible protocol to candidate patients to purging treatments before the CD34+ cells re-infusion
A framework in support of emergency management for specified and unspecified emergencies.
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Synthesis and funcionalization of indole skeleton compunds via nitrosoarene-alkyne cycloadditions.
Get PDFIndoles are one of the most important and abundant classes of N-heterocycles, being present in the form of innumerable natural products and bioactive compounds. A class of indole derivatives which have received considerable attention in recent years are N-hydroxyindoles. The biological role of N-hydroxyindoles is still an area of active investigation since they have been shown to possess biological activity in certain cases. 3-Aroylindoles are another class of indole derivatives, known as bioactive compounds with interesting properties. Not many indolization procedures are known to afford directly 3-acylindoles starting from commercially available reactants.
Our general interest in this topic lead us to disclose a novel and regioselective indole synthesis by annulation reaction between nitro- and nitrosoarenes with alkynes. Indoles, N-hydroxy- and N-alkoxyindoles were afforded in moderate to excellent yields and good regioselectivity. Using conjugated alkynones as starting materials the reaction proceeded with the regioselective formation of 3-acylindoles and/or N-hydroxy-3-acylindoles. Highly functionalizable indole derivatives were synthesized and different derivatization reactions were developed
Profiling user interactions on online social networks.
Get PDFOver the last couple of years, there has been signi_cant research e_ort in mining user behavior on online social networks for applications ranging from sentiment analysis to marketing. In most of those applications, usually a snapshot of user attributes or user relationships are analyzed to build the data mining models, without considering how user attributes and user relationships can be utilized together.
In this thesis, we will describe how user relationships within a social network can be further augmented by information gathered from user generated texts to analyze large scale dynamics of social networks. Speci_cally, we aim at explaining social network interactions by using information gleaned from friendships, pro_les, and status posts of users. Our approach pro_les user interactions in terms of shared similarities among users, and applies the gained knowledge to help users in understanding the inherent reasons, consequences and bene_ts of interacting with other social network users