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Effetto della modulazione di HIF-1 sulla risposta di colture bi- e tri-dimensionali di cellule di adenocarcinoma colorettale a 5-fluorouracile, oxaliplatino e doxorubicina.
No full textI carcinomi colorettali (CRC) rappresentano la seconda causa di morte fra tutti i tipi di tumore. I fattori eziologici coinvolti nell’insorgenza della patologia hanno origini sia genetiche che di carattere ambientale e comportamentale. I tre approcci terapeutici tradizionalmente utilizzati per la cura dei carcinomi colorettali sono costituiti dall’intervento chirurgico, dalla radioterapia e dalla chemioterapia. La refrattarietà di questo tipo di tumore alla radioterapia ed alla chemioterapia è ascrivibile a diversi fattori, tra i quali sembra rivestire un ruolo non trascurabile la presenza di regioni ipossiche all’interno della massa tumorale: da qui nasce l’idea che colpire fattori coinvolti nella risposta del tumore all’ipossia possa produrre la chemosensibilizzazione, oltre che il trattamento, del tumore stesso.
Il principale mediatore delle risposte attuate a seguito dell’ipossia è il fattore trascrizionale HIF-1 (fattore inducibile dall’ipossia). Si tratta di un eterodimero costituito da una subunità , regolabile attraverso meccanismi ossigeno-dipendenti e/o indipendenti, e da una sub unità , espressa costitutivamente. La riduzione della pressione parziale di ossigeno nel microambiente cellulare determina una riduzione della degradazione proteica di HIF-1, che porta alla stabilizzazione dei suoi livelli intracellulari. L’aumento dei livelli intracellulari di HIF-1può essere inoltre determinato da un aumento della sua biosintesi, mediato da meccanismi ossigeno-indipendenti (p.e. attivazione di vie di trasduzione del segnale da parte di fattori di crescita e prodotti oncogenici, perdita di funzione di geni oncosoppressori). In ambedue i casi, all’aumento dei livelli di HIF-1 fa seguito la sua traslocazione nel nucleo, dove avviene la formazione del dimero HIF-1(), in grado di legare le sequenze HRE (Hypoxia Response Element) presenti nei promotori dei geni bersaglio, attivandone la trascrizione. Molti dei geni controllati da HIF-1 codificano per proteine coinvolte nell’angiogenesi, nel trasporto dell’ossigeno e del glucosio, nella glicolisi, nella proliferazione e nella sopravvivenza cellulare e possono di conseguenza mediare la transizione delle cellule neoplastiche verso un fenotipo altamente maligno e resistente alla chemioterapia.
Data l’importanza della proteina HIF-1 nell’indurre le risposte adattative cellulari alla mancanza di ossigeno, scopo del presente lavoro è stato valutare se questo fattore trascrizionale eterodimerico potesse rappresentare un valido bersaglio per interventi volti ad aumentare l’efficacia della chemioterapia nei confronti dei CRC.
Grazie ad esperimenti preliminari è stato confermato che l’induzione di una condizione di ipossia moderata (pO2 1 %) è in grado di determinare, in misura diversa in relazione alle diverse caratteristiche delle tre linee cellulari utilizzate (HT29, HCT16, H630) e al diverso meccanismo d’azione dei farmaci esaminati (5-FU, DOX, OxPt), a) un aumento dei livelli di HIF-1, nonché dell’attività trascrizionale di HIF- 1; e b) una ridotta risposta a tre farmaci citotossici, noti per agire attraverso meccanismi diversi a livello molecolare.
Nella seconda parte del lavoro è stato dimostrato, attraverso la manipolazione genetica della linea cellulare HCT116 utilizzando: a) un shRNA in grado di ridurre l’espressione di HIF-1 b) e una forma mutata di HIF-1(HIF-1MUT), resistente alla degradazione in normossia e quindi in grado di indurre l’attivazione costitutiva di HIF-1, che esiste un possibile ruolo causale di HIF-1 nella mancata risposta ai farmaci in ipossia.
Infine, l’oligonucleotide antisenso EZN2968, diretto contro il mRNA di HIF-1, e l’inibitore della tioredossina-1 PMX290, noto per inibire l’attività trascrizionale di HIF-1, si sono dimostrati in grado di potenziare l’azione citotossica dei tre chemioterapici in esame in una o più delle linee cellulari testate.
È stato inoltre messo a punto un modello di coltura tridimensionale, facendo crescere le cellule HCT116 sotto forma di sferoidi, in modo da riprodurre le condizioni di ossigenazione cellulare che si creano all’interno di una massa tumorale in modo più fedele rispetto a quanto si osservi nelle colture bidimensionali. Utilizzando sferoidi ottenuti da cellule HCT116/HRP-EGFP, in cui l’espressione della EGFP è regolata da HIF-1, abbiamo visualizzato in microscopia a fluorescenza la presenza di aree ipossiche all’interno degli sferoidi e quantificato l’attività trascrizionale di HIF-1 mediante citofluorimetria a flusso. I dati ottenuti hanno mostrato che il 5-FU è significativamente meno potente nell’inibire la capacità di formare cloni delle cellule derivanti da sferoidi rispetto al monostrato e che, anche in questo modello, esiste un effetto chemiosensibilizzante del composto PMX290.
Il fatto che l’inibizione di HIF-1 induca sensibilizzazione all’effetto dei farmaci in cellule ipossiche suggerisce che strategie polichemioterapiche che includano composti in grado di inibire l’espressione e/o l’attività di HIF-1 possano rappresentare un valido approccio al trattamento di tumori solidi refrattari alla chemioterapia e in cui si riscontri frequentemente la presenza di aree ipossiche
Chemometrics for complex data handling and process optimization in environmental sciences.
No full textScientists involved in environmental studies are called to investigate the effects of pollutants in environment compartments and on human beings. On one hand they are required to develop adequate environmental analytical methods, on the other hand they are charged with collecting and evaluating complex and often inexplicit data characterized by an extremely varying character. In this thesis successful applications of chemometric techniques in these field of environmental analysis are described.
Chapter one focuses on the study of a complex groundwater system situated on the Lake Como area. Geochemical methods and exploratory Principal Component Analysis (PCA) were applied on the chemical data and their results were finally compared. In the second chapter the study concerning some of the main tropospheric pollutants in the Lake Como area from 1992 to 2007 is presented. The pros and cons of the use of unfolding PCA and multiway methods (Tucker3) were discussed. Chapter three and four show how PCA and PLS (Partial Least Square Discriminans Analysis) could be used in classification matters characterized by a limited samples number.
Chapter three shows a new strategy for the unambiguous establishment of the link between the chemical and isotope fingerprint of historical copper mines and ancient copper artifacts. In chapter four multivariate analysis analysis was employed for the volatile profile discrimination of different Italian Pecorino cheese samples. Chapter five and chapter six show how multivariate regression could be employed in the process optimization using the “Experimental Design" strategy. The chapter five with the study of a new low cost analytical method for the platinum determination by Anodic Stripping Voltammetry (ASV) while the chapter six illustrates the optimization of a prevention strategy against Candida Albicans biofilm based on a molecule with antifouling effect
Interaction between human oncogenic retrovirus and cellular factors of infected cells.
No full textThe purpose of this PhD thesis was to investigate the cellular and molecular interactions between oncogenic retroviruses, particularly HTLV-2 (Human T cell ymphotropic Virus 2), and host factors with potential inhibitory action on retroviral replication. Special attention was concentrated n the host factor CIITA, the HLA class II transactivator.
It was previously shown in this laboratory that CIITA inhibits the transcriptional function of Tax-2 and, consequently, the replication of HTLV-2 virus in human target cells. We tested different fragments of CIITA in 293T cell line, used as a human physiologic system for functional assays with HTLV-2, for the inhibition of Tax-2. We confirm that the Nterminus of CIITA, encompassing the region 1-252, is involved in the block of the Tax-2-mediated transactivation of viral LTR promoter. We also investigated the molecular mechanism at the basis of this effect, and we an unprecedented interaction between CIITA
and Tax-2, that involves the N-term part of CIITA molecule. We identified two
independent regions necessary for the interaction with Tax-2, at the N-terminus and
another one at the C-terminus of CIITA, that are differentially available in the wild type
molecule. We conclude that CIITA could inhibit Tax-2 by directly binding the viral
transactivator, and that this effect may be enhanced by the presence of different factors in common between the host cell and the virus like p300, CBP or PCAF that, together with NF-YB, could help the formation of the inhibiting complex.
We repeatedly observed that when Tax-2 is co-transfected with CIITA, induces a
significant increase of the expression of CIITA, and this effect is evident also with the Nterm deletion mutants and fragments that belong to the central region of CIITA, but not with C-term fragments. We also demonstrate that the rate of CIITA protein increase is dependent on the amount of Tax-2. We identified a region of CIITA that is the target of Tax-2 –mediated increase, including the aminoacids from position 1 to 252.
We asked whether the interaction between the host factor CIITA and the viral transactivator and the increase of CIITA protein could affect also the activity of CIITA on HLA-II promoters. With flow cytometry analysis, we observed that Tax-2 alone does not affect HLA expression, whereas it increases CIITA-mediated expression of HLA-DR on the cell surface. Interestingly, we found also that in the presence of Tax-2 the interaction
between CIITA and NF-YB is significantly more efficient, indicating that CIITA when coexpressed with Tax-2 presents a better functionality on class II genes promoters. We ruled out a possible transcriptional effect of Tax-2 on CMV promoter driving the expression of CIITA, because Tax-2 does not increase the expression of other proteins transcribed from the same CMV promoter, such as GFP and NF-YB, but enhances a CIITA molecule whose expression is under the control of a RSV promoter.
Further studies allowed us to determine that Tax-2 effect is the result of two contributions: Tax-2 stabilizes CIITA mRNA and induces a modification of the kinetic of degradation of CIITA, extending its half-life from 2 to 3 hours.
Together with our previous observations that CIITA inhibits Tax-2 function, these findings
might indicate that HLTV-2 may enhance CIITA protein expression and functionality in
order to exploit its ability to inhibit Tax-2 LTR promoter transactivation and virus
replication. We hypothesize that this “feed back” inhibitory mechanism may control from
one side HTLV virus replication and spreading, but on the other side may also help the virus to maintain a latent state in the infected cells
Exploring accretion theory with a new subclass of high mass x-ray binaries: interpretation of integral observations of the supergiant fast x-ray transients.
No full textIn the last eight years the INTEGRAL satellite has allowed to discover a new class of transient X-ray binaries associated with OB supergiants called Supergiant Fast X-ray Transients (SFXTs). They display a high dynamic range, spanning 3-5 orders of magnitude from a quiescent luminosity of 1032 erg s-1 up to a peak luminosity of 1036 - 1037 erg s-1, and they spend most of the time in an intermediate aring level of emission at around 1033 - 1034 erg s-1. The enigmatic properties emerging from this class of sources raise interesting problems to the standard theories for the accretion of matter onto compact objects, and to the properties of OB supergiants in SFXTs. The aim of this thesis is to gain more information about the peculiar transient behaviour of SFXTs and to explore the accretion mechanisms involved in these enigmatic X-ray sources. I present a new clumpy wind model for OB supergiants, with both spherical and non-spherical geometry, that I have developed to investigate the e_ects of accretion from a clumpy wind on the X-ray variability of SFXTs. I assume that the clumps are con_ned by ram pressure of the ambient gas, and I assume that a fraction of the stellar wind is in form of clumps with power law mass and radius distributions. Then, I compute the expected X-ray lightcurves in the framework of the Bondi-Hoyle accretion theory, modi_ed to take into account the presence of clumps, and I apply this model to reproduce the X-ray lightcurves of _ve representative HMXBs: two persistent supergiant systems (Vela X-1 and 4U 1700-377), and the SFXTs IGR J16479-4514, IGR J11215-5952 and IGR J18483-0311. The model can reproduce the observed lightcurves well, but requiring in all cases an overall mass loss from the supergiants about a factor 3-10 smaller than the values inferred from UV lines studies that assume homogeneous wind, but in agreement with recent studies that assume clumpy winds.
Then, I report the systematic analysis of all INTEGRAL observations from 2003 to 2009 of 14 SFXTs (con_rmed and candidates), implying a net exposure time of about 30 Ms. This analysis led to discover several new outbursts from SFXTs. I discuss the e_ects of X-ray photoionization on the accretion in close binary systems, and I show that, because of X-ray photoionization, there is a high probability of formation of transient accretion discs from the capture of angular momentum in IGR J16479-4514. This result suggests that the formation of transient accretion discs could be partly responsible for the aring activity in SFXTs with narrow orbits or in SFXTs with higher orbital periods and high eccentricities, when the neutron star is close to periastron. I also propose an alternative way to explain the origin of ares with peculiar shapes observed with INTEGRAL applying the intermittent accretion model of Lamb et al. (1977). Then, I report the study of the candidate SFXT IGR J16418-4532, for which I obtain a re_ned estimate of the orbital period from Swift/BAT data, and a re_ned estimate of the spin period of the neutron star from INTEGRAL data. I con_rm the presence of a region of the orbital lightcurve with a low ux, probably due to an eclipse, or due to the onset of the centrifugal inhibition of accretion. The uncertainties of the results from infrared observations do not allow an assessment of the spectral class of the counterpart of IGR J16418-4532, which could be a main sequence, giant, or O8.5 supergiant. From considerations involving the expected X-ray luminosities, the duration of the likely eclipse, and the onset of the centrifugal inhibition of accretion, I _nd that in all these cases it is possible to exclude the presence of a O8.5 V star, and the presence of a supergiant is favoured
Transition metal catalyzed reactions on allenic substrates for heterocyclic synthesis.
No full textThe main subject of this thesis are transition metal catalyzed reactions onto allenic substrates. The reactivity of allenic double bonds in presence or absence of transition metal was investigated and studied in order to favor intramolecular processes able to yield nitrogen-containing heterocyclic structures. We investigated the feasibility of cyclization processes onto allenamides that could have been built starting from α-aminoacids, that is, with a pre-introduced chiral information in the starting materials able to be kept till the obtainment of the final enantiopure products. At first we developed a procedure for building monocyclic five- and six-membered nitrogen containing heterocycles from under basic conditions. Next, we studied the Pd-catalyzed nitrogen addition onto the -carbon of the aforementioned allenes in order to obtain imidazolidinones structures, that are of great interest for organic chemists. Although the transition metal-catalyzed generation of heterocyclic compounds via reactions between allene bearing a nucleophilic functionality and organic halides is well established, such a strategy has never been applied to allenamides. Thus, we set up conditions for a synthesis of 2-styryl-imidazolidinone structures through a pure domino carbopalladation-amination sequence. The same protocol, under CO atmosphere, was able to lead to interesting enone structures, while the incorporation of an iodine atom in an appropriate position into the starting allenamides permitted to access imidazoisoquinolinones.
The second part of this thesis was spent in the laboratories of Prof. Giovanni Poli (Univ. Pierre et Marie Curie, Paris), where a new approach to 1,4-benzodiazepin-5-ones via a pure domino carbopalladation /allylic amination process starting from allenylamides of anthranilic acid was developed. The cyclization step is quite different from the previously described one, since the originality of this protocols lies in the fact that no phosphines as ligands are needed, as the Pd- species is stabilized by the conjoint action of BuLi and DMSO.
The shorter homologues of these allenylamides were the next substrate which we focused our attention. Submitted to the same protocol, they indeed gave rise to quinazolinone structures. However, a gold-catalyzed cyclization set up onto the same substrates was much more interesting, due to its ability to lead to vinyl-substituted products. Finally, the same products can be obtained through the first example of ruthenium catalyzed intramolecular hydroamination of allenes, though this reactions still needs to be investigated for its mechanism and scope
