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Mechanical and functional characterization of the diaphragmatic lymphatic network.
No full textThe development of transplantation procedures and increased clinical problems involving edema and cancer have created a growing need for detailed studies of the lymphatic system due to its role as a component in the circulatory system and as a component in the immunological system.
In the initial phase of the present study, we measured the compliance of the lymphatic vessel wall in vivo and performed ex vivo mechanical tests on diaphragmatic tissue strips.
A finite element model (FEM) was thus developed, using the vessel’s actual dimensions, its compliance Measurement and the diaphragmatic tissue’s elastic module. Visualization of the model’s stress distribution then showed the functional differences between superficial, intermediate and deep lymphatic vessels.
The next step was to delve into the study of the lymphatic network with vertical connections draining from superficial capillaries to deeper collecting vessels though, given their anatomical position, these are rather challenging to assess experimentally.
The experimental data thus obtained was then processed in the lumped parameter model, clarifying significantly the way in which the lymph is drained by the diaphragmatic lymphatic network considering both superficial and deep lymphatic vessels.
In part III of the present thesis, we developed a mathematical model that include ribs diaphragm and lungs to quantify and visualize the stress distribution that are generated on the diaphragm, and therefore on the lymphatic vessels, during a whole breathing cycle.
The result is a simulation of a normal breathing
Malattia mentale e imputabilità penale nel dibattito giuridico e medico-scientifico tra Otto e Novecento.
No full textQuesta ricerca si propone di analizzare alcuni aspetti del problema dell'imputabilità penale, con particolare riguardo al vizio di mente giuridicamente rilevante come causa scriminante o scusante, in un periodo di grande fermento, quello dei lavori per il primo codice penale unitario del Regno d'Italia, durante il quale l'intero ambiente giuridico italiano è teso nel massimo sforzo di elaborazione di norme in grado di amalgamare le diverse tradizioni del Paese.
Dopo un breve excursus storico sulle disposizioni preunitarie in materia, si passa ad un esame approfondito della dottrina giuridica e del dibattito interno ad essa, che non solo si concentra sui problemi di formulazione del dettato codicistico, ma indaga sui fondamenti stessi dell'istituto dell'infermità mentale. Il dibattito è influenzato in modo significativo dai numerosi contributi provenienti dall'ambiente medico-scientifico e che per più di venti anni accompagnano il percorso legislativo per supportarlo o, più spesso, per criticarlo e influenzarlo: si è rivelato essenziale, quindi, riportare non solo gli interventi degli esperti del diritto, ma anche le opinioni dei maggiori alienisti italiani della seconda metà dell'Ottocento. Per offrire un'indagine complessiva sulla problematica e sulle principali difficoltà di recepimento della nuova normativa vengono poi illustrate le principali questioni pratiche emerse dopo l’entrata in vigore dell’articolo 46 del Codice Zanardelli, facendo riferimento al corposo materiale giurisprudenziale di fine secolo. Oltre a ciò, data la rilevanza delle problematiche poste al confine tra scienza e diritto, alcune pagine sono dedicate ad un approfondimento della controversa questione della follia morale ed all'influenza sulla responsabilità penale di patologie come la monomania e l'epilessia.
La seconda parte dell'elaborato si concentra, invece, sulla parziale responsabilità di mente, controverso punto di discussione tra Positivisti ed esponenti della Scuola classica, prestando una specifica attenzione alle questioni processuali di compatibilità con alcune cause aggravanti, in particolare la premeditazione.
Infine, il tema del terzo capitolo è il tentativo di Zanardelli, rivelatosi poi fallimentare, di introdurre il manicomio criminale (pensato come un istituto di reclusione per gli imputati assolti per vizio di mente) all'interno del codice in via di promulgazione, e sul dibattito suscitato da una simile proposta nell'ambiente medico e giuridico, con l'insanabile contrasto tra le due grandi Scuole del diritto penale italiano che emerge con prepotenza dalle pagine dei resoconti parlamentari e dai numerosi contributi dottrinali
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
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
Amino acid oxidases in red biotechnologies: a target and a tool.
No full textD-Amino acid oxidase (DAAO; EC 1.4.3.3) has been proposed to play a main role in the degradation of D-serine, an allosteric activator of the N-methyl-D-aspartate-type glutamate receptor in the human brain, associated with the onset of schizophrenia. To prevent excessive D-serine degradation, novel drugs for schizophrenia treatment based on DAAO inhibition were designed and tested on rats. The properties of rat DAAO (rDAAO) are unknown and various in vivo trials reported on the effects of DAAO inhibitors on D-serine concentration in rats. rDAAO was efficiently expressed in Escherichia coli. The recombinant enzyme was purified as an active, 40 kDa monomeric flavoenzyme showing the basic properties of the dehydrogenase-oxidase class of flavoproteins. rDAAO differs significantly from the human enzyme because it: 1)) possesses a different substrate specificity; 2) shows a lower kinetic efficiency (because of a low substrate affinity); 3) differs in affinity for binding of classical inhibitors; 4) is a stable monomer; 5) interacts with the mammalian protein modulator pLG72 yielding a 100 kDa complex in addition to the 200 kDa, as formed by the human DAAO. Interestingly, the concentration of endogenous D-serine in U87 glioblastoma cells was not affected by transfection with rDAAO whereas it was significantly decreased when expressing the human homologue. These results raise doubt on the use of rat as model system for testing new drugs against schizophrenia and indicate a different physiological function of DAAO in rodents and humans.
During past years, a number of variants of D-amino acid oxidase from the yeast Rhodotorula gracilis (RgDAAO) with altered substrate specificity (e.g., active on acidic, or hydrophobic, or on all D-amino acids) both by rational design and directed evolution methods have been produced in our laboratory. RgDAAO is the most suitable biotechnological tool for the detection of D-amino acids and in this work we evaluated the capability of some mutant forms of this flavoenzyme previously produced in our laboratory in order to determine D-amino acid content in different biological samples. The kinetic constants for a number of natural and unnatural D-amino acids have been investigated. This information constitutes the basis for considering potential analytical applications of these variants of RgDAAO.
Glycine is implicated in several physiological functions, e.g. as a biosynthetic precursor or neurotransmitter in the central nervous system. Glycine is an important coagonist of NMDA receptor and it is putatively involved in schizophrenia susceptibility and other neurological diseases, such as congenital nonketotic hyperglycinemia. With the final aim to produce an optimized enzyme that can be employed in a specific biosensor for glycine detection, glycine oxidase from Bacillus subtilis (GO) was engineered to improve its kinetic efficiency on this small amino acid. Based on in silico analysis, site saturation mutagenesis was independently performed at positions Met49, Gly51, Ala54, Met95, Tyr241, His244, Tyr246, Met261, Arg302, Arg329 and Asn330. The GO variants were screened by employing a rapid colorimetric assay on 96 well-plates based on the determination of hydrogen peroxide produced on glycine as substrate: seven GO variants were selected. Significant alteration of kinetic parameters was observed for H244K and H244R GO variants: kcat increased about twice and Km decreased 3-5-fold, yielding a 7-12-fold higher kinetic efficiency on glycine, as compared to the wild-type GO. Screening of GO variants at position 49 also identified an improved enzyme (M49I) showing a 1.4-fold decreased Km. Combination of information gathered from the site saturation mutagenesis approach could be useful to obtain an evolved GO variant suitable for biotechnological applications
Sviluppo di materiali nanostrutturati e apparati per applicazioni in catalisi eterogenea.
No full textThe doctoral research period was devoted to the development of new ideas in the field of heterogeneous catalysis, involving both the realisation of novel apparatus for the study of catalysts and the synthesis of new catalytic materials.
A new apparatus for the study of selectivity in oxidative dehydrogenation reaction was initially setup aiming at studying the performances of new saponite catalysts. These materials have been shown to lead to coke productivity during ODH reaction.
These data gave us the idea to use saponite materials as polymer flame retardant. The clay material flame retardant effect is due to two synergic effect: the creation of a protective inorganic layer on the surface of the polymer matrix and the formation of char laminar structures. Our purpose was to increase the catalytic conversion performances of propene to coke by exploiting both acid saponite sites and vanadium species. The catalytic performances of different saponite materials toward propene ODH reaction was studied. Different metal-free acidic saponites (containing different amount of Brønsted acid sites) and V-containing saponite both acidic and non-acidic were investigated.
We found that the synergic effect of acidic site and Vanadium presence allow to the system (HV-Sap) to reach the highest low temperature activities and it is the only system that still maintain a positive coke production at 773 K.
The second research field is the development of apparatus suitable for planar catalyst libraries tests. A reactor which is able to scan small active catalytic surfaces using very low gas flow was developed and successfully tested in our laboratories. The reactor is designed to allow a continuous scanning test on a reduced circular area without any sealing system. The system consists of two main parts: the sample holder and the reactor head. The sample holder is mounted on a movable XYZ stage to set the relative 3D position with respect the fixed reactor head position. The reactor head features a little open chamber at the bottom; the reactant gas mixture is fed by a hose in the middle of the chamber, whereas the reaction gases are extracted by a side hose.
The entire system (reactor head and sample holder) is located in a controlled argon atmosphere chamber. The product gas mixture is confined and diluted in-situ by a flow of inert leakage gas from the controlled external atmosphere. The product gas mixtures are analysed by a downstream quadrupole-based mass spectrometer. Two model reactions were used to develop the scanning reactor: the hydrogenation of 1-3 butadiene over Pd nano-structured and the CO oxidation reaction over gold supported on titania
Finally, the possibility to obtain hydrogen from the photoproduction of renewable chemicals is investigated. At present, hydrogen is mainly produced from fossil fuels; a very interesting hydrogen source is water-splitting but, in spite of the intense research activities registered in the last decades, pure water splitting is still far from any technological application due to the complexity of the process and its inherent drawbacks. In particular, the main issue is the fast recombination between H2 and O2 (thermodynamically favourable inverse reaction) or between photogenerated electrons and holes.
A different approach to photoproduction is the use of organic compound as sacrificial agent for the hole sequestration: this reaction is accordingly called photoreforming. In particular, the catalytic photoreforming of ethanol and glycerol is investigated in this study. The first is obtained from carbohydrates fermentation and glycerol is the main byproduct of trans-etherification reaction for biodiesel production, being both cheap and widely available chemicals.
We want to study bimetallic gold-platinum systems supported over titania; a number of application of monometallic system in photoreforming are known in the literature and bimetallic systems are known to show optimal performances as catalytic system for a large number of reactions but they have never been used as photocatalysts.
The bimetallic system showed the highest activity during the photoreforming reaction of ethanol but during the reactions with glycerol solution the presence of both metal didn’t improve the reactivity of the system. Also the activity of photodeposited system were investigated on glycerol photoreforming with UV-A light. The hydrogen production rate of photodeposited gold monometallic system is close to the impregnated bimetallic system; the photodeposited bimetallic system showed a hydrogen production rate of 3400 μmol g-1 h-1 after 6 reaction hours,1000 μmol g-1 h-1 higher than bimetallic impregnated system
X-ray telescope mirrors from surface profile to point spread function.
No full textX-ray astronomy was born on 1962 when Giacconi decided to put a Geiger counter onto a rocket, in hope of measuring the X-ray emission from the Sun. Even if the Sun emission was disappointing, he made the discovery that changed our view on the Universe. An unknown background of X-ray emission that later turned out to contain millions of X-ray sources, both galactic and extra-galactic. Owing to the development of increasingly sophisticated instruments, the sensitivity and the resolution to detect X-ray sources has improved significantly over the last 50 years.
One of the major technological improvement was the development of focusing telescope, which allowed to enhance the angular resolution and sensitivity of several orders of magnitude. The angular resolution of an X-ray imaging telescope is mainly determined by the quality of its focusing optics. These generally consist of a number of nested shells of grazing incidence mirrors. The typical configuration used, which minimizes the effect of coma aberration and reduces the focal length, is the so-called Wolter-I (paraboloid-hyperboloid mirror configuration). In order to keep the mass to levels comparable with the launcher (because X-ray absorption in the atmosphere prevents observation from ground) the optics have to be lightweight, hence the mirrors have to be thin.
The final performance of a mirror module is always subject to degradation, provided in the realization phase. During the different stages of production (under construction and integration) there may be distortions. In addition, the mirror surface is not ideally smooth but is characterized by a certain roughness topography. Both these types of imperfections combine to determine the degradation of the Point Spread Function (PSF), i.e. the annular integral of focused intensity around the focal spot, which generally characterizes the quality of the optics.
Regarding the characterization of an X-ray mirror, one of the basic objectives is to establish the relationship between the imperfections of the mirrors and their PSFas a function of the incident wave energy. The aimis to predict the angular resolution of a mirror, given measurements of profiles and microroughness, or to establish the level of tolerable imperfections of a mirror given a certain angular resolution required by the project specifications.
The study of the topography of the mirror surface is done through several methods.
It is generally divided into two different kinds of analysis: the study of the profile, i.e. large spatial wavelengths (comparable with the mirror length) and the study of microroughness, i.e. short spatial wavelengths.
The first ones, also called the figure errors, are often due to deformation of the mirror that occurs during construction and integration and are responsible for the degradation of the PSF and can be treated by geometrical optics. The second ones are due, for example, to the limits of the polishing mandrel methods, from which the shells are replicated, and the deposition technology of the reflective coating. These imperfections are responsible for a diffusion (called scattering), which degrades the PSF at increasing energy and can not be treated by geometrical optics, but using physical optics under some assumptions. This problem is much more important in Xray than in optical astronomy, because X-ray have a 1000 times smaller wavelengths and are sensitive to surface defects 1000 times smaller. The surface polishing is thereby a fundamental point in X-ray mirrors.
The characterization of the microroughness is made in terms of power spectrum as a function of the spatial frequency on the surface (PSD - Power Spectral Density).
The PSD is a fundamental quantity in the characterization of X-ray telescope optics because is proportional to the scattering. The measure of roughness is done with different instruments in order to have a range of spatial frequencies as more wide as possible, from a few millimetres to a few tens of nanometres.
There is also a range of intermediate frequencies, at the limit of microroughness, which generates a degradation of the PSF that can neither be predicted by geometrical optics and nor by the scattering theory. For this reason, it is difficult in general to predict accurately its effect on the PSF.
My PhD activity is included in the mission project NHXM financed by ASI and in the development of X-ray mirrors for ATHENA mission project financed by ESA.
The first part of my PhD project has been therefore aimed at the characterization of microroughness and reflectivity of the mirrors, at INAF/OAB, in order to determine the topography of the surface and to support the industry (Media Lario Technology, leader company of manufacturing optical components) in setting the process.
The second part of the project was instead dedicated to the development of a selfconsistent general method, based on physical optics, to compute the PSF of X-ray 2 mirrors from their profile metrology. The third part, which is the merging of these two parts of the project, consist in the applications to real cases through verifications with calibration tests.
My research work can be divided into three phases:
• First phase - I performed measurements of mirror profiles and roughness of several samples of mirrors for the missions NHXMand ATHENA, using different instruments available at INAF/OAB. The roughness measurements, at spatial scales smaller than 1 mm, can be achieved with different instruments that have different spatial wavelength ranges (i.e. optical interferometer WYKO and Atomic Force Microscope). However, the effects of roughness can also be directly observed by performing X-ray reflectivity measurements using an X-ray diffractometer. I used the X-ray diffractometer available at INAF/OAB for scattering measurements, with particular attention to the effects in large angle scattering and modulation interference introduced by the multilayer. By merging these different data I derived the complete roughness surface PSD.
Bymeans of the X-ray diffractometer Imade reflectivity measurements of samples of mirrors with multilayer coating, obtaining the reflectivity curve as a function of the angle of incidence and as a function of the energy. Using a program to fit the reflectivity curves (PPM), I estimated the thickness of the layers and their uniformity, then assessing the compliance with design specifications.
In summary, from one hand surface roughness (from direct topography measurements and scattering measurements) to obtain the PSD, on the other hand, measurements of reflectivity (as a function of both the incidence angle and the energy) for the characterization of the structure of the multilayer. The feedback provided to the industry in a commons way and the isolation of the critical points has lead to the deposition of coatings with excellent reflectivity.
I performed reflectivity measurements also within the study of the crystallization of gold during the evaporation process, which contributes to worsen the surface roughness. The gold layer is deposited on the mandrel, which is then electroformed a Nickel-Cobalt shell (the mirror). The gold layer serves to detach the shell from mandrel and it should minimize the microroughness increasing.
In this regard, I performed diffraction measurements of different gold deposits with different thickness. Studying the Bragg peaks I obtained an estimation of the size of the gold crystallites as a function of the thickness.
Larger are the crystallites, higher is the value of the microroughness. The conclusion is that more the gold layer is thick, larger are the crystallites and larger crystallites means microroughness increasing.
• Second phase - I developed a new method to calculate the PSF of an X-ray mirror (e.g. Wolter-I configuration, in double reflection) at any energy by applying the principle of Huygens-Fresnel from real profile and roughness data.
In other words, the X-ray reflection is treated by the undulatory theory, building the wavefront deformed by the mirror imperfections. In this interpretation, even the deformed geometry are treated by the physical optics. This allows to obtain the PSF determined from both contributions (figure error and scattering) at any energy in a self-consistent way, without considering different separated energy regimes treated with different methods. This method, never used before, ultimately solves the problem of PSF computation, starting from the complete surface topography of an X-ray mirror.
• Third phase - I performed several calibrations over mirror shells in different configurations as demonstrator for the NHXM hard X-ray imaging telescope (0.3 - 80 keV). Prototypes of NHXM mirror modules with a few mirror shells were manufactured, aiming at demonstrating the feasibility of mirrors. Imade the direct performance verification by measuring the X-ray PSF (Point Spread Function) up to 50 keV in full-illumination setup at PANTER (MPE, Germany) and in pencil-beam set up at monochromatic X-ray energies from 15 to 63 keV at the BL20B2 beamline of the SPring-8 synchrotron radiation facility. Moreover, I simulated PSF from the metrology profile of mirror shell using Fresnel diffraction method. The calibration measured data and the simulated data (obtained with my Fresnel method) match perfectly. This provide the experimental proof of the correctness of the method, that therefore will represent, from now on, a powerful prediction tool in X-ray optics.
The Fresnel diffraction method is easily extendible to other optical systems, also out of astrophysical applications, even with a number of more than two reflections, e.g Syncrotron and FEL facilities.
For the future, I plan to implement the Fresnel diffraction method improving the simulations of mirrors coated with multilayer. In this case in order to increase the prediction accuracy, we have to taking into account the scattering from multilayer interfaces
Molecular physiological studies on the peptide transporter-1, PepT1.
No full textThe peptide transporter-1, PepT1, is responsible for the uptake of peptides in the mammalian intestine after protein digestion. It has also been shown to transport a wide range of pharmacological agents; thus making it important in drug design and delivery. Many studies have addressed structure-function relationships in the transporter, using biochemical and uptake assays; however PepT1 is an electrogenic transporter whose activities could be more clearly studied using electrophysiological methods.
In this work therefore, the transporter has been studied using the two electrode voltage clamp technique on the transporter expressed in Xenopus laevis oocytes.
Temperature effects on the kinetics of the three isoforms studied (rabbit, zebrafish, seabass) is reported here with a suggestion of a possible structural adaptation in the transporter. Since the transporter is also of interest in animal feed formulation, transport characteristics of a range of dipeptide combinations are also presented; with a recommendation on the possible optimal lysine supplementation in feeds that require essential amino acids supplementation.
Reverse operation in transporters may be the result of or lead to pathophysiological states. Experimental data here show that mutants in Arginine 282 and aspartate 341 exhibit properties that make them suitable as models to study possible reverse operation in PepT1. To understand the physiological significance of reverse operation, the structural and functional basis of this phenomenon was also explored