Computational methods for genome screening

Motivation An actual issue of great interest, both under a theoretical and an applicative perspective, is the analysis of biological sequences for disclosing the information that they encode. The development of new technologies for genome sequencing in the last years, opened new fundamental problems since huge amounts of biological data still deserve an interpretation. Indeed, the sequencing is only the first step of the genome annotation process that consists in the assignment of biological information to each sequence. Hence given the large amount of available data, in silico methods became useful and necessary in order to extract relevant information from sequences. The availability of data from Genome Projects gave rise to new strategies for tackling the basic problems of computational biology such as the determination of the tridimensional structures of proteins, their biological function and their reciprocal interactions. Results The aim of this work has been the implementation of predictive methods that allow the extraction of information on the properties of genomes and proteins starting from the nucleotide and aminoacidic sequences, by taking advantage of the information provided by the comparison of the genome sequences from different species. In the first part of the work a comprehensive large scale genome comparison of 599 organisms is described. 2,6 million of sequences coming from 551 prokaryotic and 48 eukaryotic genomes were aligned and clustered on the basis of their sequence identity. This procedure led to the identification of classes of proteins that are peculiar to the different groups of organisms. Moreover the adopted similarity threshold produced clusters that are homogeneous on the structural point of view and that can be used for structural annotation of uncharacterized sequences. The second part of the work focuses on the characterization of thermostable proteins and on the development of tools able to predict the thermostability of a protein starting from its sequence. By means of Principal Component Analysis the codon composition of a non redundant database comprising 116 prokaryotic genomes has been analyzed and it has been showed that a cross genomic approach can allow the extraction of common determinants of thermostability at the genome level, leading to an overall accuracy in discriminating thermophilic coding sequences equal to 95%. This result outperform those obtained in previous studies. Moreover, we investigated the effect of multiple mutations on protein thermostability. This issue is of great importance in the field of protein engineering, since thermostable proteins are generally more suitable than their mesostable counterparts in technological applications. A Support Vector Machine based method has been trained to predict if a set of mutations can enhance the thermostability of a given protein sequence. The developed predictor achieves 88% accuracy.

An experimental and theoretical approach to correct for the scattered radiation in an X-ray computer tomography system for industrial applications

The main problem connected to cone beam computed tomography (CT) systems for industrial applications employing 450 kV X-ray tubes is the high amount of scattered radiation which is added to the primary radiation (signal). This stray radiation leads to a significant degradation of the image quality. A better understanding of the scattering and methods to reduce its effects are therefore necessary to improve the image quality. Several studies have been carried out in the medical field at lower energies, whereas studies in industrial CT, especially for energies up to 450 kV, are lacking. Moreover, the studies reported in literature do not consider the scattered radiation generated by the CT system structure and the walls of the X-ray room (environmental scatter). In order to investigate the scattering on CT projections a GEANT4-based Monte Carlo (MC) model was developed. The model, which has been validated against experimental data, has enabled the calculation of the scattering including the environmental scatter, the optimization of an anti-scatter grid suitable for the CT system, and the optimization of the hardware components of the CT system. The investigation of multiple scattering in the CT projections showed that its contribution is 2.3 times the one of primary radiation for certain objects. The results of the environmental scatter showed that it is the major component of the scattering for aluminum box objects of front size 70 x 70 mm2 and that it strongly depends on the thickness of the object and therefore on the projection. For that reason, its correction is one of the key factors for achieving high quality images. The anti-scatter grid optimized by means of the developed MC model was found to reduce the scatter-toprimary ratio in the reconstructed images by 20 %. The object and environmental scatter calculated by means of the simulation were used to improve the scatter correction algorithm which could be patented by Empa. The results showed that the cupping effect in the corrected image is strongly reduced. The developed CT simulation is a powerful tool to optimize the design of the CT system and to evaluate the contribution of the scattered radiation to the image. Besides, it has offered a basis for a new scatter correction approach by which it has been possible to achieve images with the same spatial resolution as state-of-the-art well collimated fan-beam CT with a gain in the reconstruction time of a factor 10. This result has a high economic impact in non-destructive testing and evaluation, and reverse engineering.

Path planning for image based visual servoing

This thesis deals with Visual Servoing and its strictly connected disciplines like projective geometry, image processing, robotics and non-linear control. More specifically the work addresses the problem to control a robotic manipulator through one of the largely used Visual Servoing techniques: the Image Based Visual Servoing (IBVS). In Image Based Visual Servoing the robot is driven by on-line performing a feedback control loop that is closed directly in the 2D space of the camera sensor. The work considers the case of a monocular system with the only camera mounted on the robot end effector (eye in hand configuration). Through IBVS the system can be positioned with respect to a 3D fixed target by minimizing the differences between its initial view and its goal view, corresponding respectively to the initial and the goal system configurations: the robot Cartesian Motion is thus generated only by means of visual informations. However, the execution of a positioning control task by IBVS is not straightforward because singularity problems may occur and local minima may be reached where the reached image is very close to the target one but the 3D positioning task is far from being fulfilled: this happens in particular for large camera displacements, when the the initial and the goal target views are noticeably different. To overcame singularity and local minima drawbacks, maintaining the good properties of IBVS robustness with respect to modeling and camera calibration errors, an opportune image path planning can be exploited. This work deals with the problem of generating opportune image plane trajectories for tracked points of the servoing control scheme (a trajectory is made of a path plus a time law). The generated image plane paths must be feasible i.e. they must be compliant with rigid body motion of the camera with respect to the object so as to avoid image jacobian singularities and local minima problems. In addition, the image planned trajectories must generate camera velocity screws which are smooth and within the allowed bounds of the robot. We will show that a scaled 3D motion planning algorithm can be devised in order to generate feasible image plane trajectories. Since the paths in the image are off-line generated it is also possible to tune the planning parameters so as to maintain the target inside the camera field of view even if, in some unfortunate cases, the feature target points would leave the camera images due to 3D robot motions. To test the validity of the proposed approach some both experiments and simulations results have been reported taking also into account the influence of noise in the path planning strategy. The experiments have been realized with a 6DOF anthropomorphic manipulator with a fire-wire camera installed on its end effector: the results demonstrate the good performances and the feasibility of the proposed approach.

Retrieval of trace gases vertical profile in the lower atmosphere combining. Differential Optical Absorption Spectroscopy with radiative transfer models

The motivation for the work presented in this thesis is to retrieve profile information for the atmospheric trace constituents nitrogen dioxide (NO2) and ozone (O3) in the lower troposphere from remote sensing measurements. The remote sensing technique used, referred to as Multiple AXis Differential Optical Absorption Spectroscopy (MAX-DOAS), is a recent technique that represents a significant advance on the well-established DOAS, especially for what it concerns the study of tropospheric trace consituents. NO2 is an important trace gas in the lower troposphere due to the fact that it is involved in the production of tropospheric ozone; ozone and nitrogen dioxide are key factors in determining the quality of air with consequences, for example, on human health and the growth of vegetation. To understand the NO2 and ozone chemistry in more detail not only the concentrations at ground but also the acquisition of the vertical distribution is necessary. In fact, the budget of nitrogen oxides and ozone in the atmosphere is determined both by local emissions and non-local chemical and dynamical processes (i.e. diffusion and transport at various scales) that greatly impact on their vertical and temporal distribution: thus a tool to resolve the vertical profile information is really important. Useful measurement techniques for atmospheric trace species should fulfill at least two main requirements. First, they must be sufficiently sensitive to detect the species under consideration at their ambient concentration levels. Second, they must be specific, which means that the results of the measurement of a particular species must be neither positively nor negatively influenced by any other trace species simultaneously present in the probed volume of air. Air monitoring by spectroscopic techniques has proven to be a very useful tool to fulfill these desirable requirements as well as a number of other important properties. During the last decades, many such instruments have been developed which are based on the absorption properties of the constituents in various regions of the electromagnetic spectrum, ranging from the far infrared to the ultraviolet. Among them, Differential Optical Absorption Spectroscopy (DOAS) has played an important role. DOAS is an established remote sensing technique for atmospheric trace gases probing, which identifies and quantifies the trace gases in the atmosphere taking advantage of their molecular absorption structures in the near UV and visible wavelengths of the electromagnetic spectrum (from 0.25 μm to 0.75 μm). Passive DOAS, in particular, can detect the presence of a trace gas in terms of its integrated concentration over the atmospheric path from the sun to the receiver (the so called slant column density). The receiver can be located at ground, as well as on board an aircraft or a satellite platform. Passive DOAS has, therefore, a flexible measurement configuration that allows multiple applications. The ability to properly interpret passive DOAS measurements of atmospheric constituents depends crucially on how well the optical path of light collected by the system is understood. This is because the final product of DOAS is the concentration of a particular species integrated along the path that radiation covers in the atmosphere. This path is not known a priori and can only be evaluated by Radiative Transfer Models (RTMs). These models are used to calculate the so called vertical column density of a given trace gas, which is obtained by dividing the measured slant column density to the so called air mass factor, which is used to quantify the enhancement of the light path length within the absorber layers. In the case of the standard DOAS set-up, in which radiation is collected along the vertical direction (zenith-sky DOAS), calculations of the air mass factor have been made using “simple” single scattering radiative transfer models. This configuration has its highest sensitivity in the stratosphere, in particular during twilight. This is the result of the large enhancement in stratospheric light path at dawn and dusk combined with a relatively short tropospheric path. In order to increase the sensitivity of the instrument towards tropospheric signals, measurements with the telescope pointing the horizon (offaxis DOAS) have to be performed. In this circumstances, the light path in the lower layers can become very long and necessitate the use of radiative transfer models including multiple scattering, the full treatment of atmospheric sphericity and refraction. In this thesis, a recent development in the well-established DOAS technique is described, referred to as Multiple AXis Differential Optical Absorption Spectroscopy (MAX-DOAS). The MAX-DOAS consists in the simultaneous use of several off-axis directions near the horizon: using this configuration, not only the sensitivity to tropospheric trace gases is greatly improved, but vertical profile information can also be retrieved by combining the simultaneous off-axis measurements with sophisticated RTM calculations and inversion techniques. In particular there is a need for a RTM which is capable of dealing with all the processes intervening along the light path, supporting all DOAS geometries used, and treating multiple scattering events with varying phase functions involved. To achieve these multiple goals a statistical approach based on the Monte Carlo technique should be used. A Monte Carlo RTM generates an ensemble of random photon paths between the light source and the detector, and uses these paths to reconstruct a remote sensing measurement. Within the present study, the Monte Carlo radiative transfer model PROMSAR (PROcessing of Multi-Scattered Atmospheric Radiation) has been developed and used to correctly interpret the slant column densities obtained from MAX-DOAS measurements. In order to derive the vertical concentration profile of a trace gas from its slant column measurement, the AMF is only one part in the quantitative retrieval process. One indispensable requirement is a robust approach to invert the measurements and obtain the unknown concentrations, the air mass factors being known. For this purpose, in the present thesis, we have used the Chahine relaxation method. Ground-based Multiple AXis DOAS, combined with appropriate radiative transfer models and inversion techniques, is a promising tool for atmospheric studies in the lower troposphere and boundary layer, including the retrieval of profile information with a good degree of vertical resolution. This thesis has presented an application of this powerful comprehensive tool for the study of a preserved natural Mediterranean area (the Castel Porziano Estate, located 20 km South-West of Rome) where pollution is transported from remote sources. Application of this tool in densely populated or industrial areas is beginning to look particularly fruitful and represents an important subject for future studies.

Mechanisms of cell senescence: p21 and DNA damage response in aging and longevity

Theory of aging postulates that aging is a remodeling process where the body of survivors progressively adapts to internal and external damaging agents they are exposed to during several decades. Thus , stress response and adaptation mechanisms play a fundamental role in the aging process where the capability of adaptating effects, certainly, also is related the lifespan of each individual. A key gene linking aging to stress response is indeed p21, an induction of cyclin-dependent kinase inhibitor which triggers cell growth arrest associated with senescence and damage response and notably is involved in the up-regulation of multiple genes that have been associated with senescence or implicated in age-related . This PhD thesis project that has been performed in collaboration with the Roninson Lab at Ordway Research Institute in Albany, NY had two main aims: -the testing the hypothesis that p21 polymorphisms are involved in longevity -Evaluating age-associated differences in gene expression and transcriptional response to p21 and DNA damage In the first project, trough PCR-sequencing and Sequenom strategies, we we found out that there are about 30 polymorphic variants in the p21 gene. In addition, we found an haplotpype located in -5kb region of the p21 promoter whose frequency is ~ 2 fold higher in centenarians than in the general population (Large-scale analysis of haplotype frequencies is currently in progress). Functional studies I carried out on the promoter highilighted that the ―centenarian‖ haplotype doesn’t affect the basal p21 promoter activity or its response to p53. However, there are many other possible physiological conditions in which the centenarian allele of the p21 promoter may potentially show a different response (IL6, IFN,progesterone, vitamin E, Vitamin D etc). In the second part, project #2, trough Microarrays we seeked to evaluate the differences in gene expression between centenarians, elderly, young in dermal fibroblast cultures and their response to p21 and DNA damage. Microarray analysis of gene expression in dermal fibroblast cultures of individuals of different ages yielded a tentative "centenarian signature". A subset of genes that were up- or downregulated in centenarians showed the same response to ectopic expression of p21, yielding a putative "p21-centenarian" signature. Trough RQ-PCR (as well Microarrays studies whose analysis is in progress) we tested the DNA damage response of the p21-centenarian signature genes showing a correlation stress/aging in additional sets of young and old samples treated with p21-inducing drug doxorubicin thus finding for a subset of of them , a response to stress age-related.

Conoscenza e consapevolezza sugli effetti biologici degli ultrasuoni nella pratica clinica in Italia: risultati di una inchiesta con questionario tra ecografisti della socetà italiana di ultrasonologia in medicina e biologia (S.I.U.M.B.)

Background New potential hazards in the use of ultrasound (US) are implied in new diagnostic applications of US, such as contrast enhanced US. Aim To assess the level of awareness and knowledge on safety issues of clinical use of US among physicians who are members of the Italian National Society for Ultrasound (SIUMB) Materials and methods A questionnaire including 11 multiple choice quiz was sent by e-mail to members of SIUMB, who preliminarly agreed to participate in this initiative. The answers were received anonimously and statistically analyzed. Results The number of returned valid questionnaires was 97 (8 were considered not valid for less than 10 answers filled). Mean age of the responders was 44 years old, and the average time the physician has been performing ultrasound examinations was 13 years. The principal workplace (70%) was a public Hospital. Physicians seemed to know the general definitions of principal safety-parameters, but few of them knew the definition of specific indexes. There was a general knowledge about the safe use of ultrasound in obstetrics, but there was a poor knowledge of biological effects of US: only about 37% answered correctly to questions about damage of vasculature of lung by high Mechanical Index US investigation and about the increase of temperature under the probe, according to the thermal indexes. Conclusion In conclusion the present findings indicate that greater efforts of National Ultrasound Societies are warranted in disseminating knowledge about the bio-effects of diagnostic ultrasound modalities among their members to prevent possible hazards.

A skin surface characterization system based on capacitive image analysis

During the last few years, several methods have been proposed in order to study and to evaluate characteristic properties of the human skin by using non-invasive approaches. Mostly, these methods cover aspects related to either dermatology, to analyze skin physiology and to evaluate the effectiveness of medical treatments in skin diseases, or dermocosmetics and cosmetic science to evaluate, for example, the effectiveness of anti-aging treatments. To these purposes a routine approach must be followed. Although very accurate and high resolution measurements can be achieved by using conventional methods, such as optical or mechanical profilometry for example, their use is quite limited primarily to the high cost of the instrumentation required, which in turn is usually cumbersome, highlighting some of the limitations for a routine based analysis. This thesis aims to investigate the feasibility of a noninvasive skin characterization system based on the analysis of capacitive images of the skin surface. The system relies on a CMOS portable capacitive device which gives 50 micron/pixel resolution capacitance map of the skin micro-relief. In order to extract characteristic features of the skin topography, image analysis techniques, such as watershed segmentation and wavelet analysis, have been used to detect the main structures of interest: wrinkles and plateau of the typical micro-relief pattern. In order to validate the method, the features extracted from a dataset of skin capacitive images acquired during dermatological examinations of a healthy group of volunteers have been compared with the age of the subjects involved, showing good correlation with the skin ageing effect. Detailed analysis of the output of the capacitive sensor compared with optical profilometry of silicone replica of the same skin area has revealed potentiality and some limitations of this technology. Also, applications to follow-up studies, as needed to objectively evaluate the effectiveness of treatments in a routine manner, are discussed.

Effetto di erbicidi su crescita ed efficienza fotosintetica della diatomea Skeletonema marinoi

In questa tesi è stato studiato l’effetto dell’esposizione della diatomea Skeletonema marinoi, una specie molto comune nel Nord Adriatico e importante per il suo annuale contributo alla produzione primaria, agli erbicidi maggiormente utilizzati nella pianura Padana e riscontrati in acque dolci e salmastre di zone limitrofe al mare Adriatico. Gli erbicidi scelti consistono in terbutilazina e metolachlor, i più frequentemente riscontrati sia nelle acque superficiali che in quelle sotterranee dell’area Padana, noti per avere un effetto di inibizione su vie metaboliche dei vegetali; inoltre è stato valutato anche l’effetto di un prodotto di degradazione della terbutilazina, la desetilterbutilazina, presente anch’esso in concentrazioni pari al prodotto di origine e su cui non si avevano informazioni circa la tossicità sul fitoplancton. L’esposizione delle microalghe a questi erbicidi può avere effetti che si ripercuotono su tutto l’ecosistema: le specie fitoplanctoniche, in particolare le diatomee, sono i produttori primari più importanti dell’ecosistema: questi organismi rivestono un ruolo fondamentale nella fissazione del carbonio, rappresentando il primo anello della catena alimentari degli ambienti acquatici e contribuendo al rifornimento di ossigeno nell’atmosfera. L’effetto di diverse concentrazioni di ciascun composto è stato valutato seguendo l’andamento della crescita e dell’efficienza fotosintetica di S. marinoi. Per meglio determinare la sensibilità di questa specie agli erbicidi, l’effetto della terbutilazina è stato valutato anche al variare della temperatura (15, 20 e 25°C). Infine, dal momento che gli organismi acquatici sono solitamente esposti a una miscela di composti, è stato valutato l’effetto sinergico di due erbicidi, entrambi somministrati a bassa concentrazione. Le colture di laboratorio esposte a concentrazioni crescenti di diversi erbicidi e, in un caso, anche a diverse temperature, indicano che l’erbicida al quale la microalga mostra maggiore sensibilità è la Terbutilazina. Infatti a parità di concentrazioni, la sensibilità della microalga alla Terbutilazina è risultata molto più alta rispetto al suo prodotto di degradazione, la Desetilterbutilazina e all’erbicida Metolachlor. Attraverso l’analisi di densità algale, di efficienza fotosintetica, di biovolume e di contenuto intracellulare di Carbonio e Clorofilla, è stato dimostrato l’effetto tossico dell’erbicida Terbutilazina che, agendo come inibitore del trasporto degli elettroni a livello del PS-II, manifesta la sua tossicità nell’inibizione della fotosintesi e di conseguenza sulla crescita e sulle proprietà biometriche delle microalghe. E’ stato visto come la temperatura sia un parametro ambientale fondamentale sulla crescita algale e anche sugli effetti tossici di Terbutilazina; la temperatura ideale per la crescita di S. marinoi è risultata essere 20°C. Crescendo a 15°C la microalga presenta un rallentamento nella crescita, una minore efficienza fotosintetica, variazione nei valori biometrici, mostrando al microscopio forme irregolari e di dimensioni inferiori rispetto alle microalghe cresciute alle temperature maggiori, ed infine incapacità di formare le tipiche congregazioni a catena. A 25° invece si sono rivelate difficoltà nell’acclimatazione: sembra che la microalga si debba abituare a questa alta temperatura ritardando così la divisione cellulare di qualche giorno rispetto agli esperimenti condotti a 15° e a 20°C. Gli effetti della terbutilazina sono stati maggiori per le alghe cresciute a 25°C che hanno mostrato un calo più evidente di efficienza fotosintetica effettiva e una diminuzione di carbonio e clorofilla all’aumentare delle concentrazioni di erbicida. Sono presenti in letteratura studi che attestano gli effetti tossici paragonabili dell’atrazina e del suo principale prodotto di degradazione, la deetilatrazina; nei nostri studi invece non sono stati evidenziati effetti tossici significativi del principale prodotto di degradazione della terbutilazina, la desetilterbutilazina. Si può ipotizzare quindi che la desetilterbutilazina perda la propria capacità di legarsi al sito di legame per il pastochinone (PQ) sulla proteina D1 all’interno del complesso del PSII, permettendo quindi il normale trasporto degli elettroni del PSII e la conseguente sintesi di NADPH e ATP e il ciclo di riduzione del carbonio. Il Metolachlor non evidenzia una tossicità severa come Terbutilazina nei confronti di S. marinoi, probabilmente a causa del suo diverso meccanismo d’azione. Infatti, a differenza degli enzimi triazinici, metolachlor agisce attraverso l’inibizione delle elongasi e del geranilgeranil pirofosfato ciclasi (GGPP). In letteratura sono riportati casi studio degli effetti inibitori di Metolachlor sulla sintesi degli acidi grassi e di conseguenza della divisione cellulare su specie fitoplanctoniche d’acqua dolce. Negli esperimenti da noi condotti sono stati evidenziati lievi effetti inibitori su S. marinoi che non sembrano aumentare all’aumentare della concentrazione dell’erbicida. E’ interessante notare come attraverso la valutazione della sola crescita non sia stato messo in evidenza alcun effetto mentre, tramite l’analisi dell’efficienza fotosintetica, si possa osservare che il metolachlor determina una inibizione della fotosintesi.

Mitochondrial dysfunction in a cell model of thyroid oncocytoma

The role of mitochondrial dysfunction in cancer has long been a subject of great interest. In this study, such dysfunction has been examined with regards to thyroid oncocytoma, a rare form of cancer, accounting for less than 5% of all thyroid cancers. A peculiar characteristic of thyroid oncocytic cells is the presence of an abnormally large number of mitochondria in the cytoplasm. Such mitochondrial hyperplasia has also been observed in cells derived from patients suffering from mitochondrial encephalomyopathies, where mutations in the mitochondrial DNA(mtDNA) encoding the respiratory complexes result in oxidative phosphorylation dysfunction. An increase in the number of mitochondria occurs in the latter in order to compensate for the respiratory deficiency. This fact spurred the investigation into the presence of analogous mutations in thyroid oncocytic cells. In this study, the only available cell model of thyroid oncocytoma was utilised, the XTC-1 cell line, established from an oncocytic thyroid metastasis to the breast. In order to assess the energetic efficiency of these cells, they were incubated in a medium lacking glucose and supplemented instead with galactose. When subjected to such conditions, glycolysis is effectively inhibited and the cells are forced to use the mitochondria for energy production. Cell viability experiments revealed that XTC-1 cells were unable to survive in galactose medium. This was in marked contrast to the TPC-1 control cell line, a thyroid tumour cell line which does not display the oncocytic phenotype. In agreement with these findings, subsequent experiments assessing the levels of cellular ATP over incubation time in galactose medium, showed a drastic and continual decrease in ATP levels only in the XTC-1 cell line. Furthermore, experiments on digitonin-permeabilised cells revealed that the respiratory dysfunction in the latter was due to a defect in complex I of the respiratory chain. Subsequent experiments using cybrids demonstrated that this defect could be attributed to the mitochondrially-encoded subunits of complex I as opposed to the nuclearencoded subunits. Confirmation came with mtDNA sequencing, which detected the presence of a novel mutation in the ND1 subunit of complex I. In addition, a mutation in the cytochrome b subunit of complex III of the respiratory chain was detected. The fact that XTC-1 cells are unable to survive when incubated in galactose medium is consistent with the fact that many cancers are largely dependent on glycolysis for energy production. Indeed, numerous studies have shown that glycolytic inhibitors are able to induce apoptosis in various cancer cell lines. Subsequent experiments were therefore performed in order to identify the mode of XTC-1 cell death when subjected to the metabolic stress imposed by the forced use of the mitochondria for energy production. Cell shrinkage and mitochondrial fragmentation were observed in the dying cells, which would indicate an apoptotic type of cell death. Analysis of additional parameters however revealed a lack of both DNA fragmentation and caspase activation, thus excluding a classical apoptotic type of cell death. Interestingly, cleavage of the actin component of the cytoskeleton was observed, implicating the action of proteases in this mode of cell demise. However, experiments employing protease inhibitors failed to identify the specific protease involved. It has been reported in the literature that overexpression of Bcl-2 is able to rescue cells presenting a respiratory deficiency. As the XTC-1 cell line is not only respiration-deficient but also exhibits a marked decrease in Bcl-2 expression, it is a perfect model with which to study the relationship between Bcl-2 and oxidative phosphorylation in respiratory-deficient cells. Contrary to the reported literature studies on various cell lines harbouring defects in the respiratory chain, Bcl-2 overexpression was not shown to increase cell survival or rescue the energetic dysfunction in XTC-1 cells. Interestingly however, it had a noticeable impact on cell adhesion and morphology. Whereas XTC-1 cells shrank and detached from the growth surface under conditions of metabolic stress, Bcl-2-overexpressing XTC-1 cells appeared much healthier and were up to 45% more adherent. The target of Bcl-2 in this setting appeared to be the actin cytoskeleton, as the cleavage observed in XTC-1 cells expressing only endogenous levels of Bcl-2, was inhibited in Bcl-2-overexpressing cells. Thus, although unable to rescue XTC-1 cells in terms of cell viability, Bcl-2 is somehow able to stabilise the cytoskeleton, resulting in modifications in cell morphology and adhesion. The mitochondrial respiratory deficiency observed in cancer cells is thought not only to cause an increased dependency on glycolysis but it is also thought to blunt cellular responses to anticancer agents. The effects of several therapeutic agents were thus assessed for their death-inducing ability in XTC-1 cells. Cell viability experiments clearly showed that the cells were more resistant to stimuli which generate reactive oxygen species (tert-butylhydroperoxide) and to mitochondrial calcium-mediated apoptotic stimuli (C6-ceramide), as opposed to stimuli inflicting DNA damage (cisplatin) and damage to protein kinases(staurosporine). Various studies in the literature have reported that the peroxisome proliferator-activated receptor-coactivator 1(PGC-1α), which plays a fundamental role in mitochondrial biogenesis, is also involved in protecting cells against apoptosis caused by the former two types of stimuli. In accordance with these observations, real-time PCR experiments showed that XTC-1 cells express higher mRNA levels of this coactivator than do the control cells, implicating its importance in drug resistance. In conclusion, this study has revealed that XTC-1 cells, like many cancer cell lines, are characterised by a reduced energetic efficiency due to mitochondrial dysfunction. Said dysfunction has been attributed to mutations in respiratory genes encoded by the mitochondrial genome. Although the mechanism of cell demise in conditions of metabolic stress is unclear, the potential of targeting thyroid oncocytic cancers using glycolytic inhibitors has been illustrated. In addition, the discovery of mtDNA mutations in XTC-1 cells has enabled the use of this cell line as a model with which to study the relationship between Bcl-2 overexpression and oxidative phosphorylation in cells harbouring mtDNA mutations and also to investigate the significance of such mutations in establishing resistance to apoptotic stimuli.

Reconstruction from image correspondences

A single picture provides a largely incomplete representation of the scene one is looking at. Usually it reproduces only a limited spatial portion of the scene according to the standpoint and the viewing angle, besides it contains only instantaneous information. Thus very little can be understood on the geometrical structure of the scene, the position and orientation of the observer with respect to it remaining also hard to guess. When multiple views, taken from different positions in space and time, observe the same scene, then a much deeper knowledge is potentially achievable. Understanding inter-views relations enables construction of a collective representation by fusing the information contained in every single image. Visual reconstruction methods confront with the formidable, and still unanswered, challenge of delivering a comprehensive representation of structure, motion and appearance of a scene from visual information. Multi-view visual reconstruction deals with the inference of relations among multiple views and the exploitation of revealed connections to attain the best possible representation. This thesis investigates novel methods and applications in the field of visual reconstruction from multiple views. Three main threads of research have been pursued: dense geometric reconstruction, camera pose reconstruction, sparse geometric reconstruction of deformable surfaces. Dense geometric reconstruction aims at delivering the appearance of a scene at every single point. The construction of a large panoramic image from a set of traditional pictures has been extensively studied in the context of image mosaicing techniques. An original algorithm for sequential registration suitable for real-time applications has been conceived. The integration of the algorithm into a visual surveillance system has lead to robust and efficient motion detection with Pan-Tilt-Zoom cameras. Moreover, an evaluation methodology for quantitatively assessing and comparing image mosaicing algorithms has been devised and made available to the community. Camera pose reconstruction deals with the recovery of the camera trajectory across an image sequence. A novel mosaic-based pose reconstruction algorithm has been conceived that exploit image-mosaics and traditional pose estimation algorithms to deliver more accurate estimates. An innovative markerless vision-based human-machine interface has also been proposed, so as to allow a user to interact with a gaming applications by moving a hand held consumer grade camera in unstructured environments. Finally, sparse geometric reconstruction refers to the computation of the coarse geometry of an object at few preset points. In this thesis, an innovative shape reconstruction algorithm for deformable objects has been designed. A cooperation with the Solar Impulse project allowed to deploy the algorithm in a very challenging real-world scenario, i.e. the accurate measurements of airplane wings deformations.

Insulin and TOR pathways regulate cellular and organismal growth through the myc oncogene in Drosophila

A large body of literature documents in both mice and Drosophila the involvement of Insulin pathway in growth regulation, probably due to its role in glucose and lipid import, nutrient storage, and translation of RNAs implicated in ribosome biogenesis (Vanhaesebroeck et al. 2001). Moreover several lines of evidence implicate this pathway as a causal factor in cancer (Sale, 2008; Zeng and Yee 2007; Hursting et al., 2007; Chan et al., 2008). With regards to Myc, studies in cell culture have implied this family of transcription factors as regulators of the cell cycle that are rapidly induced in response to growth factors. Myc is a potent oncogene, rearranged and overexpressed in a wide range of human tumors and necessary during development. Its conditional knock-out in mice results in reduction of body weight due to defect in cell proliferation (Trumpp et al. 2001). Evidence from in vivo studies in Drosophila and mammals suggests a critical function for myc in cell growth regulation (Iritani and Eisenman 1999; Johnston et al. 1999; Kim et al. 2000; de Alboran et al. 2001; Douglas et al. 2001). This role is supported by our analysis of Myc target genes in Drosophila, which include genes involved in RNA binding, processing, ribosome biogenesis and nucleolar function (Orain et al 2003, Bellosta et al., 2005, Hulf et al, 2005). The fact that Insulin signaling and Myc have both been associated with growth control suggests that they may interact with each other. However, genetic evidence suggesting that Insulin signaling regulates Myc in vivo is lacking. In this work we were able to show, for the first time, a direct modulation of dMyc in response to Insulin stimulation/silencing both in vitro and in vivo. Our results suggest that dMyc up-regulation in response to DILPs signaling occurs both at the mRNA and potein level. We believe dMyc protein accumulation after Insulin signaling activation is conditioned to AKT-dependent GSK3β/sgg inactivation. In fact, we were able to demonstate that dMyc protein stabilization through phosphorylation is a conserved feature between Drosophila and vertebrates and requires multiple events. The final phosphorylation step, that results in a non-stable form of dMyc protein, ready to be degraded by the proteasome, is performed by GSK3β/sgg kinase (Sears, 2004). At the same time we demonstrated that CKI family of protein kinase are required to prime dMyc phosphorylation. DILPs and TOR/Nutrient signalings are known to communicate at several levels (Neufeld, 2003). For this reason we further investigated TOR contribution to dMyc-dependent growth regulation. dMyc protein accumulates in S2 cells after aminoacid stimulation, while its mRNA does not seem to be affected upon TORC1 inhibition, suggesting that the Nutrient pathway regulates dMyc mostly post-transcriptionally. In support to this hypothesis, we observed a TORC1-dependent GSK3β/sgg inactivation, further confirming a synergic effect of DILPs and Nutrients on dMyc protein stability. On the other hand, our data show that Rheb but not S6K, both downstream of the TOR kinase, contributes to the dMyc-induced growth of the eye tissue, suggesting that Rheb controls growth independently of S6K.. Moreover, Rheb seems to be able to regulate organ size during development inducing cell death, a mechanism no longer occurring in absence of dmyc. These observations suggest that Rheb might control growth through a new pathway independent of TOR/S6K but still dependent on dMyc. In order to dissect the mechanism of dMyc regulation in response to these events, we analyzed the relative contribution of Rheb, TOR and S6K to dMyc expression, biochemically in S2 cells and in vivo in morphogenetic clones and we further confirmed an interplay between Rheb and Myc that seems to be indipendent from TOR. In this work we clarified the mechanisms that stabilize dMyc protein in vitro and in vivo and we observed for the first time dMyc responsiveness to DILPs and TOR. At the same time, we discovered a new branch of the Nutrient pathway that appears to drive growth through dMyc but indipendently from TOR. We believe our work shed light on the mechanisms cells use to grow or restrain growth in presence/absence of growth promoting cues and for this reason it contributes to understand the physiology of growth control.

Morphogenesis of follicular epithelium in Drosophila melanogaster: function of von Hippel-Lindau tumour suppressor gene

During my PhD I have been involved in several projects regarding the morphogenesis of the follicular epithelium, such as the analysis of the pathways that correlate follicular epithelium patterning and eggshell genes expression. Moreover, I used the follicular epithelium as a model system to analyze the function of the Drosophila homolog of the human von Hippel-Lindau (d-VHL) during oogenesis, in order to gain insight into the role of h-VHL for the pathogenesis of VHL disease. h-VHL is implicated in a variety of processes and there is now a greater appreciation of HIF-independent h-VHL functions that are relevant to tumour development, including maintenance and organization of the primary cilium, maintenance of the differentiated phenotype in renal cells and regulation of epithelial-mesenchymal transition. However, the function of h-VHL gene during development has not been fully understood. It was previously shown that d-VHL down-regulates the motility of tubular epithelial cells (tracheal cells) during embryogenesis. Epithelial morphogenesis is important for organogenesis and pivotal for carcinogenesis, but mechanisms that control it are poorly understood. The Drosophila follicular epithelium is a genetically tractable model to understand these mechanisms in vivo. Therefore, to examine whether d-VHL has a role in epithelial morphogenesis and maintenance, I performed genetic and molecular analyses by using in vivo and in vitro approaches. From my analysis, I determined that d-VHL binds to and stabilizes microtubules. Loss of d-VHL depolymerizes the microtubule network during oogenesis, leading to a possible deregulation in the subcellular trafficking transport of polarity markers from Golgi apparatus to the different domains in which follicle cells are divided. The analysis carried out has allowed to establish a significant role of d-VHL in the maintenance of the follicular epithelium integrity.