Evaluation of chromosomal abnormalities by cIg-FISH and association with proliferative and apoptotic indexes in multiple myeloma

Eighty-six newly diagnosed multiple myeloma (MM) patients from a public hospital of São Paulo (Brazil) were evaluated by cIg-FISH for the presence of del(13)(q14), t(4;14)(p16.3;q32) and del(17)(p13). These abnormalities were observed in 46.5, 9.3, and 7.0% of the patients, respectively. In order to identify the possible role of del(13)(q14) in the physiopathology of MM, we investigated the association between this abnormality and the proliferative and apoptotic indexes of plasma cells. When cases demonstrating t(4;14)(p16.3;q32) and del(17)(p13) were excluded from the analysis, we observed a trend towards a positive correlation between the proportion of cells carrying del(13)(q14) and plasma cell proliferation, determined by Ki-67 expression (r = 0.23, P = 0.06). On the other hand, no correlation between the proportion of cells carrying del(13)(q14) and apoptosis, determined by annexin-V staining, was detected (r = 0.05, P = 0.69). In general, patients carrying del(13)(q14) did not have lower survival than patients without del(13)(q14) (P = 0.15), but patients with more than 80% of cells carrying del(13)(q14) showed a lower overall survival (P = 0.033). These results suggest that, when del(13)(q14) is observed in a high proportion of malignant cells, it may have a role in determining MM prognosis. Another finding was a statistically significant lower overall survival of patients with t(4;14)(p16.3;q32) (P = 0.026). In the present study, almost half the patients with t(4;14)(p16.3;q32) died just after diagnosis, before starting treatment. This fact suggests that, in São Paulo, there may be even more patients with this chromosomal abnormality, but they probably die before being diagnosed due to unfavorable socioeconomic conditions. This could explain the low prevalence of this chromosomal abnormality observed in the present study.

Periodontopathogens levels and clinical response to periodontal therapy in individuals with the interleukin-4 haplotype associated with susceptibility to chronic periodontitis

Periodontitis is an inflammatory disease that results from an interaction between dental biofilm agents and the host immune-inflammatory response. Periodontopathogenic organisms, such as Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola, as well as the host's susceptibility, represented by the host's genetic makeup, are the key factors that influence this complex disease. Recently, we identified haplotypes in the IL4 gene that were associated with chronic periodontitis (CP). This study aimed to evaluate whether subjects with different IL4 haplotypes (TCI/CCI and TTD/CTI) would be differentially colonized by periodontopathogens and whether they would respond differently to non-surgical periodontal therapy. Thirty-nine patients carrying the IL4 haplotype of genetic susceptibility to CP (IL4+) or protection against CP (IL4-) were evaluated. Those groups were further subdivided into individuals with CP (CP IL4+ or CP IL4-) and those that were periodontally healthy (H) (H IL4+ or H IL4-). CP patients were submitted to non-surgical periodontal therapy. Clinical and microbiological analyses were performed considering the data at baseline and 45 and 90 days after periodontal therapy. Periodontopathogens levels were evaluated by absolute quantitative polymerase chain reaction (qPCR). The baseline data revealed that the total levels of periodontopathogens were higher in the CP IL4+ than in the CP IL4- groups. Clinical analyses revealed that the periodontal therapy was equally effective, independent of the subject's IL4 genetic load. The TCI/CCI IL4 haplotype, previously associated with genetic susceptibility to CP, was also associated with increased levels of periodontopathogenic bacteria, but this genetic background did not influence the response to non-surgical periodontal treatment.

RNA Interference and cyclooxygenase-2 (COX-2) regulation in colon cancer cells

Despite new methods and combined strategies, conventional cancer chemotherapy still lacks specificity and induces drug resistance. Gene therapy can offer the potential to obtain the success in the clinical treatment of cancer and this can be achieved by replacing mutated tumour suppressor genes, inhibiting gene transcription, introducing new genes encoding for therapeutic products, or specifically silencing any given target gene. Concerning gene silencing, attention has recently shifted onto the RNA interference (RNAi) phenomenon. Gene silencing mediated by RNAi machinery is based on short RNA molecules, small interfering RNAs (siRNAs) and microRNAs (miRNAs), that are fully o partially homologous to the mRNA of the genes being silenced, respectively. On one hand, synthetic siRNAs appear as an important research tool to understand the function of a gene and the prospect of using siRNAs as potent and specific inhibitors of any target gene provides a new therapeutical approach for many untreatable diseases, particularly cancer. On the other hand, the discovery of the gene regulatory pathways mediated by miRNAs, offered to the research community new important perspectives for the comprehension of the physiological and, above all, the pathological mechanisms underlying the gene regulation. Indeed, changes in miRNAs expression have been identified in several types of neoplasia and it has also been proposed that the overexpression of genes in cancer cells may be due to the disruption of a control network in which relevant miRNA are implicated. For these reasons, I focused my research on a possible link between RNAi and the enzyme cyclooxygenase-2 (COX-2) in the field of colorectal cancer (CRC), since it has been established that the transition adenoma-adenocarcinoma and the progression of CRC depend on aberrant constitutive expression of COX-2 gene. In fact, overexpressed COX-2 is involved in the block of apoptosis, the stimulation of tumor-angiogenesis and promotes cell invasion, tumour growth and metastatization. On the basis of data reported in the literature, the first aim of my research was to develop an innovative and effective tool, based on the RNAi mechanism, able to silence strongly and specifically COX-2 expression in human colorectal cancer cell lines. In this study, I firstly show that an siRNA sequence directed against COX-2 mRNA (siCOX-2), potently downregulated COX-2 gene expression in human umbilical vein endothelial cells (HUVEC) and inhibited PMA-induced angiogenesis in vitro in a specific, non-toxic manner. Moreover, I found that the insertion of a specific cassette carrying anti-COX-2 shRNA sequence (shCOX-2, the precursor of siCOX-2 previously tested) into a viral vector (pSUPER.retro) greatly increased silencing potency in a colon cancer cell line (HT-29) without activating any interferon response. Phenotypically, COX-2 deficient HT-29 cells showed a significant impairment of their in vitro malignant behaviour. Thus, results reported here indicate an easy-to-use, powerful and high selective virus-based method to knockdown COX-2 gene in a stable and long-lasting manner, in colon cancer cells. Furthermore, they open up the possibility of an in vivo application of this anti-COX-2 retroviral vector, as therapeutic agent for human cancers overexpressing COX-2. In order to improve the tumour selectivity, pSUPER.retro vector was modified for the shCOX-2 expression cassette. The aim was to obtain a strong, specific transcription of shCOX-2 followed by COX-2 silencing mediated by siCOX-2 only in cancer cells. For this reason, H1 promoter in basic pSUPER.retro vector [pS(H1)] was substituted with the human Cox-2 promoter [pS(COX2)] and with a promoter containing repeated copies of the TCF binding element (TBE) [pS(TBE)]. These promoters were choosen because they are partculary activated in colon cancer cells. COX-2 was effectively silenced in HT-29 and HCA-7 colon cancer cells by using enhanced pS(COX2) and pS(TBE) vectors. In particular, an higher siCOX-2 production followed by a stronger inhibition of Cox-2 gene were achieved by using pS(TBE) vector, that represents not only the most effective, but also the most specific system to downregulate COX-2 in colon cancer cells. Because of the many limits that a retroviral therapy could have in a possible in vivo treatment of CRC, the next goal was to render the enhanced RNAi-mediate COX-2 silencing more suitable for this kind of application. Xiang and et al. (2006) demonstrated that it is possible to induce RNAi in mammalian cells after infection with engineered E. Coli strains expressing Inv and HlyA genes, which encode for two bacterial factors needed for successful transfer of shRNA in mammalian cells. This system, called “trans-kingdom” RNAi (tkRNAi) could represent an optimal approach for the treatment of colorectal cancer, since E. Coli in normally resident in human intestinal flora and could easily vehicled to the tumor tissue. For this reason, I tested the improved COX-2 silencing mediated by pS(COX2) and pS(TBE) vectors by using tkRNAi system. Results obtained in HT-29 and HCA-7 cell lines were in high agreement with data previously collected after the transfection of pS(COX2) and pS(TBE) vectors in the same cell lines. These findings suggest that tkRNAi system for COX-2 silencing, in particular mediated by pS(TBE) vector, could represent a promising tool for the treatment of colorectal cancer. Flanking the studies addressed to the setting-up of a RNAi-mediated therapeutical strategy, I proposed to get ahead with the comprehension of new molecular basis of human colorectal cancer. In particular, it is known that components of the miRNA/RNAi pathway may be altered during the progressive development of colorectal cancer (CRC), and it has been already demonstrated that some miRNAs work as tumor suppressors or oncomiRs in colon cancer. Thus, my hypothesis was that overexpressed COX-2 protein in colon cancer could be the result of decreased levels of one or more tumor suppressor miRNAs. In this thesis, I clearly show an inverse correlation between COX-2 expression and the human miR- 101(1) levels in colon cancer cell lines, tissues and metastases. I also demonstrate that the in vitro modulating of miR-101(1) expression in colon cancer cell lines leads to significant variations in COX-2 expression, and this phenomenon is based on a direct interaction between miR-101(1) and COX-2 mRNA. Moreover, I started to investigate miR-101(1) regulation in the hypoxic environment since adaptation to hypoxia is critical for tumor cell growth and survival and it is known that COX-2 can be induced directly by hypoxia-inducible factor 1 (HIF-1). Surprisingly, I observed that COX-2 overexpression induced by hypoxia is always coupled to a significant decrease of miR-101(1) levels in colon cancer cell lines, suggesting that miR-101(1) regulation could be involved in the adaption of cancer cells to the hypoxic environment that strongly characterize CRC tissues.

Design and Computation of Warped Time-Frequency Transforms

In this work we introduce an analytical approach for the frequency warping transform. Criteria for the design of operators based on arbitrary warping maps are provided and an algorithm carrying out a fast computation is defined. Such operators can be used to shape the tiling of time-frequency plane in a flexible way. Moreover, they are designed to be inverted by the application of their adjoint operator. According to the proposed mathematical model, the frequency warping transform is computed by considering two additive operators: the first one represents its nonuniform Fourier transform approximation and the second one suppresses aliasing. The first operator is known to be analytically characterized and fast computable by various interpolation approaches. A factorization of the second operator is found for arbitrary shaped non-smooth warping maps. By properly truncating the operators involved in the factorization, the computation turns out to be fast without compromising accuracy.

Conformation and Stereodynamic of Hindered Aromatic System

The preparation of conformationally hindered molecules and their study by DNMR and computational methods are my thesis’s core. In the first chapter, the conformations and the stereodynamics of symmetrically ortho-disubstituted aryl carbinols and aryl ethers are described. In the second chapter, the structures of axially chiral atropisomers of hindered biphenyl carbinols are studied. In the third chapter, the steric barriers and the -barrier of 1,8-di-aylbiphenylenes are determined. Interesting atropisomers are found in the cases of arylanthrones, arylanthraquinones and arylanthracenes and are reported in the fourth chapter. By the combined use of dynamic NMR, ECD spectroscopy and DFT computations, the conformations and the absolute configurations of 2-Naphthylalkylsulfoxides are studied in the fifth chapter. In the last chapter, a new synthetic route to ,’-arylated secondary or tertiary alcohols by lithiated O-benzyl-carbamates carrying an N-aryl substituent and DFT calculations to determinate the cyclic intermediate are reported. This work was done in the research group of Prof. Jonathan Clayden, at the University of Manchester.

Language and Embodiment: sensory-motor and linguistic-social experience. Evidence on sentence comprehension

In this work I address the study of language comprehension in an “embodied” framework. Firstly I show behavioral evidence supporting the idea that language modulates the motor system in a specific way, both at a proximal level (sensibility to the effectors) and at the distal level (sensibility to the goal of the action in which the single motor acts are inserted). I will present two studies in which the method is basically the same: we manipulated the linguistic stimuli (the kind of sentence: hand action vs. foot action vs. mouth action) and the effector by which participants had to respond (hand vs. foot vs. mouth; dominant hand vs. non-dominant hand). Response times analyses showed a specific modulation depending on the kind of sentence: participants were facilitated in the task execution (sentence sensibility judgment) when the effector they had to use to respond was the same to which the sentences referred. Namely, during language comprehension a pre-activation of the motor system seems to take place. This activation is analogous (even if less intense) to the one detectable when we practically execute the action described by the sentence. Beyond this effector specific modulation, we also found an effect of the goal suggested by the sentence. That is, the hand effector was pre-activated not only by hand-action-related sentences, but also by sentences describing mouth actions, consistently with the fact that to execute an action on an object with the mouth we firstly have to bring it to the mouth with the hand. After reviewing the evidence on simulation specificity directly referring to the body (for instance, the kind of the effector activated by the language), I focus on the specific properties of the object to which the words refer, particularly on the weight. In this case the hypothesis to test was if both lifting movement perception and lifting movement execution are modulated by language comprehension. We used behavioral and kinematics methods, and we manipulated the linguistic stimuli (the kind of sentence: the lifting of heavy objects vs. the lifting of light objects). To study the movement perception we measured the correlations between the weight of the objects lifted by an actor (heavy objects vs. light objects) and the esteems provided by the participants. To study the movement execution we measured kinematics parameters variance (velocity, acceleration, time to the first peak of velocity) during the actual lifting of objects (heavy objects vs. light objects). Both kinds of measures revealed that language had a specific effect on the motor system, both at a perceptive and at a motoric level. Finally, I address the issue of the abstract words. Different studies in the “embodied” framework tried to explain the meaning of abstract words The limit of these works is that they account only for subsets of phenomena, so results are difficult to generalize. We tried to circumvent this problem by contrasting transitive verbs (abstract and concrete) and nouns (abstract and concrete) in different combinations. The behavioral study was conducted both with German and Italian participants, as the two languages are syntactically different. We found that response times were faster for both the compatible pairs (concrete verb + concrete noun; abstract verb + abstract noun) than for the mixed ones. Interestingly, for the mixed combinations analyses showed a modulation due to the specific language (German vs. Italian): when the concrete word precedes the abstract one responses were faster, regardless of the word grammatical class. Results are discussed in the framework of current views on abstract words. They highlight the important role of developmental and social aspects of language use, and confirm theories assigning a crucial role to both sensorimotor and linguistic experience for abstract words.

Intrinsic uncoupling in the ATP synthase of Escherichia coli. Studies on WT and ε-truncated mutants

The H+/ATP ratio in the catalysis of ATP synthase has generally been considered a fixed parameter. However, Melandri and coworkers have recently shown that, in the ATP synthase of the photosynthetic bacterium Rb.capsulatus, this ratio can significantly decrease during ATP hydrolysis when the concentration of either ADP or Pi is maintained at a low level (Turina et al., 2004). The present work has dealt with the ATP synthase of E.coli, looking for evidence of this phenomenon of intrinsic uncoupling in this organism as well. First of all, we have shown that the DCCD-sensitive ATP hydrolysis activity of E.coli internal membranes was strongly inhibited by ADP and Pi, with a half-maximal effect in the submicromolar range for ADP and at 140 µM for Pi. In contrast to this monotonic inhibition, however, the proton pumping activity of the enzyme, as estimated under the same conditions by the fluorescence quenching of the ΔpH-sensitive probe ACMA, showed a clearly biphasic progression, both for Pi, increasing from 0 up to approximately 200 µM, and for ADP, increasing from 0 up to a few µM. We have interpreted these results as indicating that the occupancy of ADP and Pi binding sites shifts the enzyme from a partially uncoupled state to a fully coupled state, and we expect that the ADP- and Pi-modulated intrinsic uncoupling is likely to be a general feature of prokaryotic ATP synthases. Moreover, the biphasicity of the proton pumping data suggested that two Pi binding sites are involved. In order to verify whether the same behaviour could be observed in the isolated enzyme, we have purified the ATP synthase of E.coli and reconstituted it into liposomes. Similarly as observed in the internal membrane preparation, in the isolated and reconstituted enzyme it was possible to observe inhibition of the hydrolytic activity by ADP and Pi (with half-maximal effects at few µM for ADP and at 400 µM for Pi) with a concomitant stimulation of proton pumping. Both the inhibition of ATP hydrolysis and the stimulation of proton pumping as a function of Pi were lost upon ADP removal by an ADP trap. These data have made it possible to conclude that the results obtained in E.coli internal membranes are not due to the artefactual interference of enzymatic activities other than the ones of the ATP synthase. In addition, data obtained with liposomes have allowed a calibration of the ACMA signal by ΔpH transitions of known extent, leading to a quantitative evaluation of the proton pumping data. Finally, we have focused our efforts on searching for a possible structural candidate involved in the phenomenon of intrinsic uncoupling. The ε-subunit of the ATP-synthase is known as an endogenous inhibitor of the hydrolysis activity of the complex and appears to undergo drastic conformational changes between a non-inhibitory form (down-state) and an inhibitory form (up-state)(Rodgers & Wilce, 2000; Gibbons et al., 2000). In addition, the results of Cipriano & Dunn (2006) indicated that the C-terminal domain of this subunit played an important role in the coupling mechanism of the pump, and those of Capaldi et al. (2001), Suzuki et al. (2003) were consistent with the down-state showing a higher hydrolysis-to-synthesis ratio than the up-state. Therefore, we decided to search for modulation of pumping efficiency in a C-terminally truncated ε mutant. A low copy number expression vector has been built, carrying an extra copy of uncC, with the aim of generating an ε-overexpressing E.coli strain in which normal levels of assembly of the mutated ATP-synthase complex would be promoted. We have then compared the ATP hydrolysis and the proton pumping activity in membranes prepared from these ε-overexpressing E.coli strains, which carried either the WT ε subunit or the ε88-stop truncated form. Both strains yielded well energized membranes. Noticeably, they showed a marked difference in the inhibition of hydrolysis by Pi, this effect being largely lost in the truncated mutant. However, pre-incubation of the mutated enzyme with ADP at low nanomolar concentrations (apparent Kd = 0.7nM) restored the hydrolysis inhibition, together with the modulation of intrinsic uncoupling by Pi, indicating that, contrary to wild-type, during membrane preparation the truncated mutant had lost the ADP bound at this high-affinity site, evidently due to a lower affinity (and/or higher release) for ADP of the mutant relative to wild type. Therefore, one of the effects of the C-terminal domain of ε appears to be to modulate the affinity of at least one of the binding sites for ADP. The lack of this domain does not appear so much to influence the modulability of coupling efficiency, but instead the extent of this modulation. At higher preincubated ADP concentrations (apparent Kd = 117nM), the only observed effects were inhibition of both hydrolysis and synthesis, providing a direct proof that two ADP-binding sites on the enzyme are involved in the inhibition of hydrolysis, of which only the one at higher affinity also modulates the coupling efficiency.

Synthesis and characterization of polymers containing oligothiophenes

In the present work a series of thiophene oligomers of three and six thiophene units were synthesized, starting from thiophene, and characterized. Polymers containing these electroative side groups were then prepared by two strategies. The oligomers were attached to existing polymer systems and were connected to a polymerizable unit leading to monomer containing the oligothiophenes as side groups. Subsequently the properties of the monomers and the polymers were investigated. A butylcellulose derivative carrying terthienyl side chains (BCTTE, 26) was synthesized starting from cellulose acetate and 5-(2-chloroethyl)-2,2':5',2'-terthiophene (4). The polymer had a degree of substitution (DS) of the butyl and terthienyl side chains of DSbutyl = 1.9 and DSterth = 0.35, respectively. It was successfully spread on a Langmuir-Blodgett (LB) trough and then transferred to several solid substrates. X-rays reflectometry showed an ordered architecture of the cellulose backbones. However, the terthiophene side groups were found as isotropically aligned by polarized UV-Vis spectroscopy. When used as anode material in the electropolymerization of 3-pentylthiophene (28), polythiophene was grafted onto the cellulose backbone through the terthienyl side groups. The polythiophene chains showed an average anisotropic alignment of 20 % along the LB dipping direction, calculated by means of polarized UV-Vis spectroscopy. A second butylcellulose derivative carrying sexithienyl side chains (BCST) was synthesized and investigated, starting from butylcellulose and 2-[b ', b ''-dipentyl-5'''-(2-hydroxyethyl)-2,2': 5',2':5',2'':5'',2'':5'',2'''-sexithiophen-5-yl]-ethyl p-toluensulfonate (7). The polymer showed formation of stable LB monolayers at the air-water interface, but its transfer onto solid substrates was not successful. A poly(p-phenylene-ethynylene) bearing sexithienyl side chains (BzAcST, 31) was prepared by reaction of the two monomers 2-[b ', b ''-dipentyl-5'''-(2-hydroxyethyl)-2,2': 5',2':5',2'':5'',2'':5'',2'''-sexithiophen-5-yl]-ethyl 2,5-diiodobenzoate (15) and 2-[b', b ''-dipentyl-5'''-(2-hydroxyethyl)-2,2':5',2':5',2'':5'',2'':5'',2'''-sexithiophen-5-yl]-ethyl 2,5-diethynylbenzoate (18). The polymer was obtained as insoluble product. Upon oxidation with FeCl3 (doping) of the polymer suspension, BzAcST showed an electrical conductivity of ó = 2.5 . 10 -6 S/cm, a typical value for semiconductors. The IR spectrum of the doped polymer presented the diagnostic bands of oxidized sexithiophene in good agreement with literature results. Along with the monomer and polymer synthesis, an a,a '-disubstituted sexithiophene, b ', b ''-dipentyl-5,5'''-bis-(2-hydroxyethyl)-2,2':5',2':5',2'':5'',2'':5'',2'''-sexithiophene (6a),was synthesized and characterized. The UV-Vis absorption of the chromophore wasinvestigated as a function of temperature and different solvents, showing a blue-shift of the absorption maximum with increasing temperature and a red-shift changing the solvent from hexane to ethanol to toluene. Monitoring the change of the UV-Vis spectrum upon electrochemical oxidation, the oxidized chromophore showed a new broad absorption band, red shifted with respect to the p -p* transition of the neutral state. Upon reduction, the new band disappeared and the UV-Vis spectrum of the chromophore was restored. Such oxidation-reduction cycles were totally reversible. This feature, together with the absorption maximum falling in the visible region, makes this chromophore a suitable compound for the development of an electrochemical sensor.Attempts to polymerize acrylic monomers carrying sexythienyl side chains both via radical polymerization, as in the case of 2-[b ', b ''-dipentyl-5'''-(2-hydroxyethyl)-2,2': 5',2':5',2'':5'',2'':5'',2'''-sexithiophen-5-yl]-ethyl acrylate (8), and anionic polymerization, as in the case of 2-{b ', b ''-dipentyl-5'''-[2-(tertbutyldimethylsiloxy)ethyl]-2,2':5',2':5',2'':5'',2'': 5'',2''' -sexithiophen-5-yl}-ethylacrylate (29), were not successful, probably due to the steric hindrance of the oligothiophene side group. However, due to the time consuming and therefore restricted availability of the monomers, a screening of the polymerization conditions towards the formation of polymeric material was not possible.

Molecular and biological analysis on the horizontal transfer of insect transposons in Baculoviruses

Summary During the infection of Lepidoptera larvae with baculoviruses the horizontal escape of Tc1-like transposons, termed TCl4.7 and TCp3.2, from the genome of the host Cryptophlebia leucotreta and Cydia pomonella into the genome of Cydia pomonella granulovirus was observed. In this study we addressed the question whether the transposon harboring viruses had a replication advantage over the wild-type and became dominant in the virus population or whether the activity of the host transposable elements is stimulated by virus infection. Biological characterization studies demonstrated that the transposon containing viruses killed C. pomonella larvae slower than CpGV-M. In co-infection experiments of C. pomonella larvae using a mixture of CpGV-M and mutant viruses as inoculum, it was shown that the transposon carrying mutants had a significant selection disadvantage compared to CpGV-M. Transcription levels of the transposase gene of TCp3.2 were investigated in virus infected and uninfected larvae. These experiments demonstrated that a higher level of transposase transcription was detectable in CpGV-M infected than in mock infected control larvae. This observation gave strong evidence that CpGV-M infection might trigger the activity of transposon TCp3.2 within the genome of Cydia pomonella. Our results suggest that the horizontal transfer of insect host transposons into baculovirus genomes might be induced by virus infection.

Polyelectrolytes and their counterions studied by EPR spectroscopy

In this thesis methods of EPR spectroscopy were used to investigate polyion-counterion interactions in polyelectrolyte solutions. The fact that EPR techniques are local methods is exploited and by employing spin-carrying (i.e., EPR-active) probe ions it is possible to examine polyelectrolytes from the counterions’ point of view. It was possible to gain insight into i) the dynamics and local geometry of counterion attachment, ii) conformations and dynamics of local segments of the polyion in an indirect manner, and iii) the spatial distribution of spin probe ions that surround polyions in solution. Analysis of CW EPR spectra of dianion nitroxide spin probe Fremy’s salt (FS, potassium nitrosodisulfonate) in solutions of cationic PDADMAC polyelectrolyte revealed that FS ions and PDADMAC form transient ion pairs with a lifetime of less than 1 ns. This effect was termed as dynamic electrostatic attachment (DEA). By spectral simulation taking into account the rotational dynamics as a uniaxial Brownian reorientation, also the geometry of the attached state could be characterized. By variation of solvent, the effect of solvent viscosity and permittivity were investigated and indirect information of the polyelectrolyte chain motion was obtained. Furthermore, analysis of CW EPR data also indicates that in mixtures of organic solvent/water PDADMAC chains are preferentially solvated by the organic solvent molecules, while in purely aqueous mixtures the PDADMAC chain segments were found in different conformations depending on the concentration ratio R of FS counterions to PDADMAC repeat units.Broadenings in CW EPR spectra of FS ions were assigned to spin-exchange interaction and hence contain information on the local concentrations and distributions of the counterions. From analysis of these broadenings in terms of a modified cylindrical cell approach of polyelectrolyte theory, radial distribution functions for the FS ions in the different solvents were obtained. This approach breaks down in water above a threshold value of R, which again indicates that PDADMAC chain conformations are altered as a function of R. Double electron-electron resonance (DEER) measurements of FS ions were carried out to probe the distribution of attached counterions along polyelectrolyte chains. For a significant fraction of FS spin probes in solution with a rigid-rod model polyelectrolyte containing charged Ru2+-centers, a bimodal distance distribution was found that nicely reproduced the spacings of direct and next-neighbor Ru2+-centers along the polyelectrolyte: 2.35 and 4.7 nm. For the system of FS/PDADMAC, DEER data could be simulated by assuming a two-state distribution of spin probes, one state corresponding to a homogeneous (3-dimensional) distribution of spin probes in the polyelectrolyte bulk and the other to a linear (1-dimensional) distribution of spin probes that are electrostatically condensed along locally extended PDADMAC chain segments. From this analysis it is suggested that the PDADMAC chains form locally elongated structures of a size of at least ~5 nm.

Structural and functional analysis of centromeric chromatin

Animal neocentromeres are defined as ectopic centromeres that have formed in non-centromeric locations and avoid some of the features, like the DNA satellite sequence, that normally characterize canonical centromeres. Despite this, they are stable functional centromeres inherited through generations. The only existence of neocentromeres provide convincing evidence that centromere specification is determined by epigenetic rather than sequence-specific mechanisms. For all this reasons, we used them as simplified models to investigate the molecular mechanisms that underlay the formation and the maintenance of functional centromeres. We collected human cell lines carrying neocentromeres in different positions. To investigate the region involved in the process at the DNA sequence level we applied a recent technology that integrates Chromatin Immuno-Precipitation and DNA microarrays (ChIP-on-chip) using rabbit polyclonal antibodies directed against CENP-A or CENP-C human centromeric proteins. These DNA binding-proteins are required for kinetochore function and are exclusively targeted to functional centromeres. Thus, the immunoprecipitation of DNA bound by these proteins allows the isolation of centromeric sequences, including those of the neocentromeres. Neocentromeres arise even in protein-coding genes region. We further analyzed if the increased scaffold attachment sites and the corresponding tighter chromatin of the region involved in the neocentromerization process still were permissive or not to transcription of within encoded genes. Centromere repositioning is a phenomenon in which a neocentromere arisen without altering the gene order, followed by the inactivation of the canonical centromere, becomes fixed in population. It is a process of chromosome rearrangement fundamental in evolution, at the bases of speciation. The repeat-free region where the neocentromere initially forms, progressively acquires extended arrays of satellite tandem repeats that may contribute to its functional stability. In this view our attention focalized to the repositioned horse ECA11 centromere. ChIP-on-chip analysis was used to define the region involved and SNPs studies, mapping within the region involved into neocentromerization, were carried on. We have been able to describe the structural polymorphism of the chromosome 11 centromeric domain of Caballus population. That polymorphism was seen even between homologues chromosome of the same cells. That discovery was the first described ever. Genomic plasticity had a fundamental role in evolution. Centromeres are not static packaged region of genomes. The key question that fascinates biologists is to understand how that centromere plasticity could be combined to the stability and maintenance of centromeric function. Starting from the epigenetic point of view that underlies centromere formation, we decided to analyze the RNA content of centromeric chromatin. RNA, as well as secondary chemically modifications that involve both histones and DNA, represents a good candidate to guide somehow the centromere formation and maintenance. Many observations suggest that transcription of centromeric DNA or of other non-coding RNAs could affect centromere formation. To date has been no thorough investigation addressing the identity of the chromatin-associated RNAs (CARs) on a global scale. This prompted us to develop techniques to identify CARs in a genome-wide approach using high-throughput genomic platforms. The future goal of this study will be to focalize the attention on what strictly happens specifically inside centromere chromatin.

Size-selective investigations of spectral and emission properties of small particles and nanotubes

In dieser Arbeit wurde die Elektronenemission von Nanopartikeln auf Oberflächen mittels spektroskopischen Photoelektronenmikroskopie untersucht. Speziell wurden metallische Nanocluster untersucht, als selbstorganisierte Ensembles auf Silizium oder Glassubstraten, sowie ferner ein Metall-Chalcogenid (MoS2) Nanoröhren-Prototyp auf Silizium. Der Hauptteil der Untersuchungen war auf die Wechselwirkung von fs-Laserstrahlung mit den Nanopartikeln konzentriert. Die Energie der Lichtquanten war kleiner als die Austrittsarbeit der untersuchten Proben, so dass Ein-Photonen-Photoemission ausgeschlossen werden konnte. Unsere Untersuchungen zeigten, dass ausgehend von einem kontinuierlichen Metallfilm bis hin zu Clusterfilmen ein anderer Emissionsmechanismus konkurrierend zur Multiphotonen-Photoemission auftritt und für kleine Cluster zu dominieren beginnt. Die Natur dieses neuen Mechanismus` wurde durch verschiedenartige Experimente untersucht. Der Übergang von einem kontinuierlichen zu einem Nanopartikelfilm ist begleitet von einer Zunahme des Emissionsstroms von mehr als eine Größenordnung. Die Photoemissions-Intensität wächst mit abnehmender zeitlicher Breite des Laserpulses, aber diese Abhängigkeit wird weniger steil mit sinkender Partikelgröße. Die experimentellen Resultate wurden durch verschiedene Elektronenemissions-Mechanismen erklärt, z.B. Multiphotonen-Photoemission (nPPE), thermionische Emission und thermisch unterstützte nPPE sowie optische Feldemission. Der erste Mechanismus überwiegt für kontinuierliche Filme und Partikel mit Größen oberhalb von mehreren zehn Nanometern, der zweite und dritte für Filme von Nanopartikeln von einer Größe von wenigen Nanometern. Die mikrospektroskopischen Messungen bestätigten den 2PPE-Emissionsmechanismus von dünnen Silberfilmen bei „blauer“ Laseranregung (hν=375-425nm). Das Einsetzen des Ferminiveaus ist relativ scharf und verschiebt sich um 2hν, wenn die Quantenenergie erhöht wird, wogegen es bei „roter“ Laseranregung (hν=750-850nm) deutlich verbreitert ist. Es zeigte sich, dass mit zunehmender Laserleistung die Ausbeute von niederenergetischen Elektronen schwächer zunimmt als die Ausbeute von höherenergetischen Elektronen nahe der Fermikante in einem Spektrum. Das ist ein klarer Hinweis auf eine Koexistenz verschiedener Emissionsmechanismen in einem Spektrum. Um die Größenabhängigkeit des Emissionsverhaltens theoretisch zu verstehen, wurde ein statistischer Zugang zur Lichtabsorption kleiner Metallpartikel abgeleitet und diskutiert. Die Elektronenemissionseigenschaften bei Laseranregung wurden in zusätzlichen Untersuchungen mit einer anderen Anregungsart verglichen, der Passage eines Tunnelstroms durch einen Metall-Clusterfilm nahe der Perkolationsschwelle. Die elektrischen und Emissionseigenschaften von stromtragenden Silberclusterfilmen, welche in einer schmalen Lücke (5-25 µm Breite) zwischen Silberkontakten auf einem Isolator hergestellt wurden, wurden zum ersten Mal mit einem Emissions-Elektronenmikroskop (EEM) untersucht. Die Elektronenemission beginnt im nicht-Ohmschen Bereich der Leitungsstrom-Spannungskurve des Clusterfilms. Wir untersuchten das Verhalten eines einzigen Emissionszentrums im EEM. Es zeigte sich, dass die Emissionszentren in einem stromleitenden Silberclusterfilm Punktquellen für Elektronen sind, welche hohe Emissions-Stromdichten (mehr als 100 A/cm2) tragen können. Die Breite der Energieverteilung der Elektronen von einem einzelnen Emissionszentrum wurde auf etwa 0.5-0.6 eV abgeschätzt. Als Emissionsmechanismus wird die thermionische Emission von dem „steady-state“ heißen Elektronengas in stromdurchflossenen metallischen Partikeln vorgeschlagen. Größenselektierte, einzelne auf Si-Substraten deponierte MoS2-Nanoröhren wurden mit einer Flugzeit-basierten Zweiphotonen-Photoemissions-Spektromikroskopie untersucht. Die Nanoröhren-Spektren wiesen bei fs-Laser Anregung eine erstaunlich hohe Emissionsintensität auf, deutlich höher als die SiOx Substratoberfläche. Dagegen waren die Röhren unsichtbar bei VUV-Anregung bei hν=21.2 eV. Eine ab-initio-Rechnung für einen MoS2-Slab erklärt die hohe Intensität durch eine hohe Dichte freier intermediärer Zustände beim Zweiphotonen-Übergang bei hν=3.1 eV.

Design And Implementation Of An End-To-End Simulator For The BepiColombo Rotation Experiment

Among the scientific objectives addressed by the Radio Science Experiment hosted on board the ESA mission BepiColombo is the retrieval of the rotational state of planet Mercury. In fact, the estimation of the obliquity and the librations amplitude were proven to be fundamental for constraining the interior composition of Mercury. This is accomplished by the Mercury Orbiter Radio science Experiment (MORE) via a strict interaction among different payloads thus making the experiment particularly challenging. The underlying idea consists in capturing images of the same landmark on the surface of the planet in different epochs in order to observe a displacement of the identified features with respect to a nominal rotation which allows to estimate the rotational parameters. Observations must be planned accurately in order to obtain image pairs carrying the highest information content for the following estimation process. This is not a trivial task especially in light of the several dynamical constraints involved. Another delicate issue is represented by the pattern matching process between image pairs for which the lowest correlation errors are desired. The research activity was conducted in the frame of the MORE rotation experiment and addressed the design and implementation of an end-to-end simulator of the experiment with the final objective of establishing an optimal science planning of the observations. In the thesis, the implementation of the singular modules forming the simulator is illustrated along with the simulations performed. The results obtained from the preliminary release of the optimization algorithm are finally presented although the software implemented is only at a preliminary release and will be improved and refined in the future also taking into account the developments of the mission.

Analysis of optimal control problems for the incompressible MHD equations and implementation in a finite element multiphysics code

This thesis deals with the study of optimal control problems for the incompressible Magnetohydrodynamics (MHD) equations. Particular attention to these problems arises from several applications in science and engineering, such as fission nuclear reactors with liquid metal coolant and aluminum casting in metallurgy. In such applications it is of great interest to achieve the control on the fluid state variables through the action of the magnetic Lorentz force. In this thesis we investigate a class of boundary optimal control problems, in which the flow is controlled through the boundary conditions of the magnetic field. Due to their complexity, these problems present various challenges in the definition of an adequate solution approach, both from a theoretical and from a computational point of view. In this thesis we propose a new boundary control approach, based on lifting functions of the boundary conditions, which yields both theoretical and numerical advantages. With the introduction of lifting functions, boundary control problems can be formulated as extended distributed problems. We consider a systematic mathematical formulation of these problems in terms of the minimization of a cost functional constrained by the MHD equations. The existence of a solution to the flow equations and to the optimal control problem are shown. The Lagrange multiplier technique is used to derive an optimality system from which candidate solutions for the control problem can be obtained. In order to achieve the numerical solution of this system, a finite element approximation is considered for the discretization together with an appropriate gradient-type algorithm. A finite element object-oriented library has been developed to obtain a parallel and multigrid computational implementation of the optimality system based on a multiphysics approach. Numerical results of two- and three-dimensional computations show that a possible minimum for the control problem can be computed in a robust and accurate manner.

Numerical Modelling of Graphene Nanoribbon-fets for Analog and Digital Applications

Graphene, that is a monolayer of carbon atoms arranged in a honeycomb lattice, has been isolated only recently from graphite. This material shows very attractive physical properties, like superior carrier mobility, current carrying capability and thermal conductivity. In consideration of that, graphene has been the object of large investigation as a promising candidate to be used in nanometer-scale devices for electronic applications. In this work, graphene nanoribbons (GNRs), that are narrow strips of graphene, for which a band-gap is induced by the quantum confinement of carriers in the transverse direction, have been studied. As experimental GNR-FETs are still far from being ideal, mainly due to the large width and edge roughness, an accurate description of the physical phenomena occurring in these devices is required to have valuable predictions about the performance of these novel structures. A code has been developed to this purpose and used to investigate the performance of 1 to 15-nm wide GNR-FETs. Due to the importance of an accurate description of the quantum effects in the operation of graphene devices, a full-quantum transport model has been adopted: the electron dynamics has been described by a tight-binding (TB) Hamiltonian model and transport has been solved within the formalism of the non-equilibrium Green's functions (NEGF). Both ballistic and dissipative transport are considered. The inclusion of the electron-phonon interaction has been taken into account in the self-consistent Born approximation. In consideration of their different energy band-gap, narrow GNRs are expected to be suitable for logic applications, while wider ones could be promising candidates as channel material for radio-frequency applications.