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Physical and projected pairs of quasars.
No full textQuasars are the most luminous type of active galactic nuclei and their study is fundamental to shed light on the high redshift Universe. In this work we take advantage of the rare cases of close projected and physical quasar pairs to detect the presence of the cool enriched gas and of the dark mass that surround quasars.
This Thesis consists of two parts. In the first one we present optical spectroscopy of projected quasar pairs (i.e., two quasars that are close in the sky but have discordant redshifts) that represents a powerful tool to investigate the metal absorption lines imprinted on the spectrum of the background quasar by the gaseous halo surrounding the foreground one (Chapter 1). In the last decades, this technique has been extensively used to detect the haloes of non–active galaxies, but to date no systematic study has been performed in the case of quasars. We selected a sample of 46 projected pairs, with separations ranging from ∼ 50 kpc to ∼ 200 kpc, aimed to assess the presence of Mg II and CIV absorptions systems associated to foreground quasars. In Chapter 2 we show the first outcomes of our programme, based on the spectra of 13 pairs partially collected by me at the ESO Very Large Telescope (VLT). These data reveal a high covering fraction for the Mg II absorbers and a non isotropical distribution of the gas. Our results are compared to those of similar works performed on non–active galaxies available in the literature. We propose that the cool gaseous haloes of quasars and of normal galaxies are similar, once the stellar mass of the systems is taken into account. Observations of the remaining 33 pairs are ongoing at the Gran Telescopio Canarias (GTC). The preparatory analysis of the SDSS spectra of these systems show that a 10m class telescope is mandatory for this kind of studies (Chapter 3).
The second part of the Thesis addresses with physical systems of quasars (i.e., two or more quasars that have small projected and redshift separations) that are an expected outcome of the current hierarchical framework of galaxy formation and evolution (Chapter 4). In Chapter 5 we accurately study the dynamics of six low redshift quasar pairs and we found that the dynamical mass required for this systems to be gravitationally bound is larger of than the stellar masses of the host galaxies. This suggests that these pairs are hosted by galaxies with massive dark haloes or that they reside in a group/cluster of galaxies. In Chapter 6, we present our discovery of the second physical quasar triplet known to date. We estimate that these systems are extremely rare in terms of simple accidental superposition of the three quasars, suggesting a possible common origin for their ignition. Finally, conclusions and future perspectives of this study are presented in Chapter 7.
The present work principally deals with the analysis and studies presented in Farina et al. (2012b, on the gaseous haloes of quasars), in Farina et al. (2011, on the dynamical study of low redshift quasar pairs), and in Farina et al. (2012c, on the discovery of a new quasar triplet). A complete list of works published during my PhD is provided in Appendix C
Development and validation of an algorithm to predict the success of the ablation of macroreentrant atrial tachycardia.
No full textAblation of macroreentrant atrial tachycardia (MRAT) is challenging because of complex anatomy and multiple reentrant loops. In order to define an effective ablation strategy, 3-dimensional electroanatomic mapping proved very useful. To identify predictors of ablation procedure failure may be helpful for patients treatment. In the first part of our study we analyzed into details the electroanatomical features of the reentry circuit in MRAT and we compared the characteristics of successfully versus unsuccessfully consecutive treated patients undegoing electroanatomic mapping and ablation of MRAT in order to identify variables predicting the ablation outcome. Ablation was linearly placed at the mid-diastolic isthmus (MDI) to achieve arrhythmia interruption and conduction block. Variables were analyzed for predictors of both procedural failure and cumulative failure. We demonstrated a significant difference as to the electroanatomic mapping characteristics: successfully treated cases showed a narrower target isthmus with a slower conduction velocity across the isthmus itself. In the second part of our research, we analyzed the relation between the strongest predictors of procedure outcome identified in part I (MDI width and conduction velocity across the MDI) and the chance of success of the ablation procedure. In order to analyze this relation and to predict the difficulty of the ablation procedure, we developed an algorithm and we validated prospectively the accuracy of the developed model in a second patient series
Extensive characterization of EGFR and of its downstream pathways may help integrating the use of EGFR-targeted therapies in patients. WITH SQUAMOUS CELL ANAL CANCER
No full textSquamous cell anal cancer (SCAC) is a rare disease. Patients are managed with chemoradiation therapy. When non-response occur, abdominoperitoneal resection is recommended. New therapeutic options are wondered to overcome the severe effects of this procedure.
It has been demonstrated that the majority of SCAC are characterized by EGFR deregulation. Recently, the anti-EGFR monoclonal antibody cetuximab has been FDA and EMA approved for the treatment of colorectal cancer (CRC). Only sporadic studies investigated its use in SCAC.
In SCAC little is known about EGFR and EGFR-downstream members alterations which are predictive markers of anti-EGFR therapies efficacy or impairment in patients affected by CRC.
The aim of this study was to characterize EGFR, KRAS, BRAF and PIK3CA alterations in a series of patients with SCAC.
We investigated 93 patients. EGFR gene copy number was evaluated by FISH. Mutations in KRAS, BRAF and PIK3CA genes were investigated by sequencing. EGFR gene copy number gain (FISH+) was found in 33/90 (37%) patients. KRAS mutations were found in 4/91 (4%) patients. BRAF was always wild-type (wt). PIK3CA mutations were found in 13/89 (15%) cases (10 in ex.9 and 3 in ex.20). Among 33 FISH+ cases, 3 (10%) patients showed a mutation in KRAS and 3 (10%) in PIK3CA ex.9.
Considering studies on CRC demonstrating that a EGFR FISH+/ KRAS wt/PIK3CA wt or ex.9 mutated status is associated with clinical benefit from cetuximab, it can be hypothesized that a subgroup of SCAC (approximately 33%) might have a proficient molecular profile with respect to anti-EGFR treatments. Our results, therefore, suggest a possible integration of EGFRtargeted therapies in SCAC and emphasize the need of molecular analyses for a better patients’ selection
Self-adjointe extensions for symmetric Laplacians on polygons.
No full textBy the results contained in the paper by Birman and Skvortsov “On the square summability of the highest derivatives of the solution to the Dirichlet problem in a region with piecewise smooth boundary" the Laplace operator o on a plane curvilinear polygon with domain the Sobolev space H2() and homogeneous Dirichlet boundary conditions is a closed symmetric operator with deficiency indices (n, n), where n is the number of non-convex corners. Therefore on a non-convex polygon, o has infinite self-adjoint extensions. Such extensions have been recently determined by means of Kreǐn's resolvent formula. The purpose of this thesis is to extend such results to the case of different, more general, boundary conditions. In the first part of the thesis we consider the case of mixed Dirichlet-Neumann conditions, thus allowing each side j of the polygon boundary to support either a Dirichlet or a Neumann homogeneous boundary condition. In this case, building on results by Grisvard, while in the pure Neumann case the dimensions of the defect spaces is the same as in the case of the pure Dirichlet case already studied by Birman and Skvortsov, the mixed case has a different behavior, allowing both convex cases and non-convex cases with double vertex contribution. After explicitly characterizing the defect sub- space we determined the self-adjoint extensions by a Kreǐn's resolvent formula proceeding analogously to the pure Dirichlet case, however taking into account the double contribution due to the vertices with mixed boundary conditions.
In the second part of the thesis we further extend our analysis by allowing some sides j to support Robin boundary conditions. While this is a deformation of the case considered in the first part, some not completely trivial calculations are necessary in order to get results similar to the ones concerning the mixed Dirichlet-Neumann case. By such calculations, one can recover results anologous to the ones in the first part. However also different behaviors are possible:
1. for any > 0, for any 0 < j < , there are parameter values which give dj = 1;
2. for any x < j (3/2) , x 1.43, there are parameter values which give dj = 2.
Moreover, as expected, the dj 's converge to the ones corresponding to the mixed Dirichlet-Neumann case as the j 's converge to either 0 or 1 accordingly to the different possible cases and as in the mixed Dirichlet-Neumann a Kreǐn's formula giving the classification of all the self-adjoint extension is provided in Chapter 4
Silkworm larval midgut: a striking example of tissue remodeling.
No full textBombyx mori is a pivotal model organism among Lepidoptera, a group of insects that is important for both commercial and agronomic purposes. The study of midgut development during larval period and metamorphosis is helpful to obtain a background useful for many applications concerning these contexts.
In these insects, metamorphosis involves a series of highly ordered mechanisms and passes through a well-defined sequence of events to eliminate tissues and organs that are functional only in larval stages (Meléndez and Neufeld, 2008). In Lepidoptera, midgut tissue undergoes extensive remodelling (Vilaplana et al., 2007) during the development and these modifications lead to cell death of the larval epithelium and to its replacement with a new pupal epithelium, which becomes the adult midgut epithelium. For this reason midgut remodelling has been chosen as preferential model to study cell death mechanisms and regeneration processes, and their regulation. In particular, although features of apoptosis and autophagy have been reported in the larval organs of Lepidoptera during metamorphosis, solid experimental evidence for autophagy is still lacking. Moreover, the role of the two cell death processes and the nature of their relationship are still cryptic.
In order to analyze the remodelling processes in B. mori midgut tissues during larvaladult transformation, we performed a morpho-functional analysis at different developmental stages. In addition, we accomplished a cellular, biochemical and molecular analysis of the degeneration process that occurs in the larval midgut, with the aim to analyze autophagy and apoptosis in cells that dye under physiological conditions.
We found that stem cells proliferate actively since the wandering stage, leading to the formation of a new pupal midgut which is progressively remodelled until adult ecdysis. Larval midgut cells undergo progressive degradation, forming a compact mass called yellow body, that progressively degenerates inside the lumen of the new pupal midgut and finally disappears.
Through histochemical analysis we showed marked changes in metabolic activity in both larval and pupal epithelium at different stages of the midgut renewal process.
We demonstrated that larval midgut degradation is a gradual process due to the concerted action of autophagic and apoptotic mechanisms, which occur at different times and have different functions. In particular, autophagy is activated from the wandering stage and reaches a high level of activity during the spinning and prepupal stages. Our data showed also that the process of autophagy can recycle molecules from the degenerating cells and supply nutrients to the animal during the non-feeding period. Apoptosis intervenes later. In fact, although genes encoding caspases are transcribed at the end of the larval period, the activity of these proteases is not appreciable until the second day of spinning and apoptotic features are observable from prepupal phase. The abundance of apoptotic features during the pupal phase, when the majority of the cells die, indicates that apoptosis is actually responsible for cell death and for the disappearance of larval midgut cells
Spectroscopic signatures of sub-parsec massive black hole.
No full textThe focus of this thesis is on the electromagnetic signatures associated with massive black hole (MBH) binaries (MBHBs). In current models of structure formation, galaxies assemble in a bottom-up hierarchical fashion. Observations show that MBHs are hosted in the nuclei of almost all nearby massive galaxies. The existence of close MBHBs is therefore expected as the result of galaxy mergers, although clear observational evidences of MBHBs remain elusive. Most of the current sub-pc MBHB candidates have been spectroscopically selected through the Doppler-shift technique. This method consists in the identification of broad emission lines (BELs) shifted with respect to the galaxy reference frame, as traced by the set of narrow lines (NELs). This shift is due to the hypothesized motion of the active MBH orbiting in a close binary. However velocity off-sets between BELs and NELs are not a unique feature of close MBHBs, and cannot be searched for beyond z ∼ 0.8 since most NELs fall out of the wavelength range covered by current optical surveys.
This work presents our contribution to (i) the prediction of new spectroscopic features associated with MBHBs at different evolutionary stages in the aftermath of a gas-rich merger, and to (ii) the search for new secure candidates at different redshifts, ruling out other possible interpretations of their physical nature.
In the first part of this thesis we discuss a MBHB model envisaging a set of narrow lines characterized by the same Doppler-shift displayed by the BELs.
This additional NEL system originates from the lower density gas orbiting in the so called gap region excavated by the gravitational interaction between the sub-pc MBHB and a surrounding thin circum-binary disk. We propose this model to interpret the features observed in the spectrum of the quasar SDSSJ092712.65+294344.0 (SDSSJ0927 hereafter). For the case of this peculiar source we show that the binary hypothesis is favored against the other possible physical scenarios of a recoiling AGN or of a chance superposition of two distinct objects in a galaxy cluster. However further follow up observations are mandatory to monitor on longer timescales (∼ > 10 yr) the periodic drift of the BELs expected over the binary orbital period, and to gather additional information on the quasar host galaxy and its close environment. Our study on SDSSJ0927 contributed to the realization of a systematic search for MBHB candidates at z ∼ < 0.8 through the development of automatic procedures by different groups including ours. The new algorithm implemented by our group will be also adapted to look for new candidates at higher redshifts based on the presence of two displaced peaks in the profile of the broad CIV line interpreted in terms of two active binary MBHs.
In the second part of this work, we devise a new spectroscopic approach to select MBHBs based on peculiarly reduced flux ratios between broad lines of lower and higher ionization potentials. In particular, we show that MBHBs can display anomalously small flux ratios between the MgII and CIV BELs, i.e. FMgII/FCIV ∼ < 0.15, in a limited range of sub-pc separations due to the erosion of the BEL emitting material bound to the secondary active MBH. This erosion is due to the tidal field of the more massive binary companion.
The advantage of our newly proposed technique is twofold: (i) it can select new candidates at higher redshifts, z ∼ 2, where both the considered BELs are visible in the optical spectrum, and (ii) the detection of a reduced FMgII/FCIV would be a strong indication of the presence of a sub-pc MBHB since it is not expected in the other physical scenarios alternative to the binary hypothesis.
We show that it is possible to use our flux ratio criterion to select peculiar AGN spectra at z ∼ 2 from the database of the Sloan Sky Digital Survey.
These peculiar sources can be observed again in the IR band to look for a shift between the broad lines of Hβ, MgII and CIV and the narrow [OIII]λ5007.
We start this new MBHB search with a small sample of 9 targets observed in the NIR-H band with the Very Large Telescope. Two sources are selected as binary candidates because the lower ionization lines of the MgII and of the Hβ have reduced fluxes relative respectively to the higher ionization line of the CIV and of the [OIII], and the three observed BELs show moderate shifts up to 1000 kms−1 relative to the narrow [OIII]. Hence we plan to perform new optical observations of these two sources to confirm and monitor the measured shifts, and to complete the search for MBHBs at z ∼ 2 to check the reliability of our newly proposed spectroscopic approach
Proline dehydrogenase regulation regulation by the P53 family and the regulatiory circuit with HIF-1.
No full textProline differs from the other amino acids because its á-nitrogen is contained within a pyrrolidine ring. Therefore, it cannot be metabolized by the general transaminases and decarboxylases acting on other amino acids. Proline dehydrogenase (PRODH) is a stress-inducible, key enzyme in proline metabolism, catalyzing its conversion into Ä1-pyrroline-5-carboxylate, a crucial compound interconnecting proline metabolism with glutamate and á- ketoglutarate (á-KG) synthesis and with the Tricarboxylic Acids (TCA) and Urea cycles. Consequently, PRODH can influence various cellular pathways, including glutamatergic transmission, glutathione levels as well as the activity of a number of enzymes using á-KG as a substrate.
Proline can also be regarded as an emergency substrate, as abundant stores are released during degradation of intracellular or extracellular matrix proteins (especially collagens). PRODH is localized in the inner membrane of mitochondria and after reduction of the FAD cofactor bound to form the holoenzyme, it can directly transfer electrons to cytochrome C to generate ATP or it can oxidize O2 to generate reactive oxygen species (ROS). Thus when cells are under stress, PRODH has been proposed to act either as a survival factor, favouring maintenance of “survival energy levels”, or as a cell death effector, inducing ROS-dependent apoptosis.
Alterations in PRODH protein levels and catalytic activity have been implicated in diseases such as hyperprolinemia, DiGeorge syndrome, schizophrenia and cancer. For cancer in particular, several lines of evidence suggest a central role of PRODH as a mitochondrial tumor suppressor: 1) expression of PRODH is reduced in diverse colorectal and renal cancer cells as compared to normal counterparts; 2) restoration of PRODH expression in human hypo-expressing colon cancer cell lines suppresses their ability to form tumours when injected into SCID mice; 3) PRODH expression is regulated transcriptionally and posttranscriptionally by several cellular sensors of cell health and homeostasis, whose functions are deregulated during carcinogenesis, including p53, PPAR and mTOR (mammalian target of rapamycin). However, the exact mechanisms by which these proteins control PRODH function have been only partially elucidated.
Understanding transcriptional and post-transcriptional regulation of a gene and its product is clue to understanding its function. In the first two years of my PhD work we identified and characterized the p53 Response Elements (REs) in the PRODH gene, responsible for p53 binding and transactivation of this target. We confirmed p53-dependent induction of endogenous PRODH in response to genotoxic damage in cell lines of different histological origin and we established that overexpression of p73 or p63 is sufficient to induce PRODH expression in p53-null cells. The p53 family-dependent transcriptional activation of PRODH was linked to specific intronic response elements (REs), among those predicted by bioinformatics tools and experimentally validated by a yeast-based transactivation assay upon modulated expression of p53, p63 and p73 and by p53 occupancy measurements in HCT116 human cells by ChIP.
Based on the following pieces of evidence i) it has been proposed that during nutrient stress extracellular matrix (ECM) proteins may be degraded to provide substrates for energy production (ecophagy), ii) an abundant protein in ECM is collagen, that is very rich in proline and hydroxyproline, iii) the key enzyme in hydroxyproline metabolism is hydroxyproline dehydrogenase, homologous to PRODH, whose gene (PRODH2) was also shown, although less convincingly, to be a p53 target, we decided to characterize the p53 REs present in this gene as well. We demonstrated that the PRODH2 gene was not responsive to p63 nor p73 and was at best a weak p53 target, based on minimal levels of PRODH2 transcript induction by genotoxic stress observed only in one of four p53 wildtype cell lines tested. Consistently, all predicted p53 REs in PRODH2 were poor matches to the p53 RE consensus and showed limited responsiveness, only to p53, in the functional assay. Taken together, our results highlight that PRODH but not PRODH2 expression is likely under control of the entire p53 family members, supporting a deeper link between p53 proteins and metabolic pathways, as PRODH functions in modulating the balance of proline and glutamate levels and of their derivative alpha-keto-glutarate in the metabolism under normal and pathological (tumor) conditions.
Another important transcription factor that we considered for a possible role in regulation of PRODH, is the Hypoxia Inducible Factor 1 (HIF-1), whose function influences cellular metabolism and is altered during the tumourigenic process. HIF proteins are composed of two subunits, and , both constitutively expressed in cells. However, the á subunits are rapidly degraded by the proteasome at normal oxygen concentrations found in tissues. Key to HIF- degradation is its oxygen-dependent hydroxylation at specific residues (prolines 402 and 564) by Prolyl Hydroxylases (PHD), that target the protein for ubiquitylation and proteasomal degradation in presence of molecular oxygen, -KG and vitamin C. During hypoxia, HIF- subunits become stabilized, which enables them to form heterodimers with HIF-, that activate numerous cell survival pathways.
HIF-1 has been shown to control the expression of more than a hundred genes, either by direct transcriptional activation of protein coding genes and microRNAs (miRs), or by interacting or interfering with other transcription factors. HIF-1 activation results in profound alterations in tumour cell behaviour, which include triggering the angiogenic switch, shifting glucose metabolism towards glycolysis, promoting epithelial-to-mesenchymal transition and acquisition of an invasive phenotype, as well as increasing chemo- and radio-resistance. For this reason, tumour cells often maintain HIF-1 overexpression after they return to a normoxic environment.
We tested the hypothesis that an increase in PRODH activity, by increasing -KG, would provide substrate for the hydroxylation reaction catalyzed by PHDs, thus leading to a decrease in HIF-1 levels. indeed, ectopic expression of PRODH led to down-regulation of HIF- and VEGF protein levels in the U87glioblastoma cell line. This finding confirmed what was already reported to occur for colon cancer cell lines.
In addition to a role of PRODH in regulating HIF-1 stability in normoxia, we hypothesized that a regulatory circuit between PRODH and HIF-1 could exist. PRODH was found to be downregulated 2-fold in a transcriptomics analysis of genes regulated following induction of focal brain ischemia in rat. On the other hand, however, very recently PRODH was shown to be induced by hypoxia and this induction was AMPK-dependent and HIF-independent. Therefore, the question was still open for investigation. Our expectations are unbiased, because PRODH possesses the ability to promote either cell survival, in conditions in which energy levels are low, by producing ATP or inducing ROS dependent autophagy, or ROS induced apoptotic cell death. Of course a different outcome depending on the cell lines tested as well as on other types of stress acting on the cells concomitantly with the hypoxic stress may be expected.
In a first attempt to verify if PRODH transcript levels were modified by hypoxia, we exposed cancer cell lines of different histological origin (HCT116, colon; MCF7, breast; U87MG, glia; SHSY-5Y, neural crest) to 1% hypoxia, anoxia or to treatment with CoCl2, a PHDs inhibitor, and compared the levels of expression with those obtained in the same untreated cell lines, by using real time RTqPCR.
All cell lines showed a marked decrease in PRODH transcript, in particular after treatment with CoCl2, and a reduction also in protein levels, although of minor entity compared to transcript decrease. Preliminary results obtained during my PhD work confirm that PRODH-HIF-1 regulatory circuit does indeed exist, and lays the foundations for further investigations, to clarify the relationship between these two proteins to increase knowledge about PRODH regulation and its possible downregulation during the tumourigenic process