Biochemistry in healthy and neoplastic human tissues: metabolic alteration revealed by HR-MAS nuclear magnetic resonance spectroscopy

This thesis is focused on the metabolomic study of human cancer tissues by ex vivo High Resolution-Magic Angle Spinning (HR-MAS) nuclear magnetic resonance (NMR) spectroscopy. This new technique allows for the acquisition of spectra directly on intact tissues (biopsy or surgery), and it has become very important for integrated metabonomics studies. The objective is to identify metabolites that can be used as markers for the discrimination of the different types of cancer, for the grading, and for the assessment of the evolution of the tumour. Furthermore, an attempt to recognize metabolites, that although involved in the metabolism of tumoral tissues in low concentration, can be important modulators of neoplastic proliferation, was performed. In addition, NMR data was integrated with statistical techniques in order to obtain semi-quantitative information about the metabolite markers. In the case of gliomas, the NMR study was correlated with gene expression of neoplastic tissues. Chapter 1 begins with a general description of a new “omics” study, the metabolomics. The study of metabolism can contribute significantly to biomedical research and, ultimately, to clinical medical practice. This rapidly developing discipline involves the study of the metabolome: the total repertoire of small molecules present in cells, tissues, organs, and biological fluids. Metabolomic approaches are becoming increasingly popular in disease diagnosis and will play an important role on improving our understanding of cancer mechanism. Chapter 2 addresses in more detail the basis of NMR Spectroscopy, presenting the new HR-MAS NMR tool, that is gaining importance in the examination of tumour tissues, and in the assessment of tumour grade. Some advanced chemometric methods were used in an attempt to enhance the interpretation and quantitative information of the HR-MAS NMR data are and presented in chapter 3. Chemometric methods seem to have a high potential in the study of human diseases, as it permits the extraction of new and relevant information from spectroscopic data, allowing a better interpretation of the results. Chapter 4 reports results obtained from HR-MAS NMR analyses performed on different brain tumours: medulloblastoma, meningioms and gliomas. The medulloblastoma study is a case report of primitive neuroectodermal tumor (PNET) localised in the cerebellar region by Magnetic Resonance Imaging (MRI) in a 3-year-old child. In vivo single voxel 1H MRS shows high specificity in detecting the main metabolic alterations in the primitive cerebellar lesion; which consist of very high amounts of the choline-containing compounds and of very low levels of creatine derivatives and N-acetylaspartate. Ex vivo HR-MAS NMR, performed at 9.4 Tesla on the neoplastic specimen collected during surgery, allows the unambiguous identification of several metabolites giving a more in-depth evaluation of the metabolic pattern of the lesion. The ex vivo HR-MAS NMR spectra show higher detail than that obtained in vivo. In addition, the spectroscopic data appear to correlate with some morphological features of the medulloblastoma. The present study shows that ex vivo HR-MAS 1H NMR is able to strongly improve the clinical possibility of in vivo MRS and can be used in conjunction with in vivo spectroscopy for clinical purposes. Three histological subtypes of meningiomas (meningothelial, fibrous and oncocytic) were analysed both by in vivo and ex vivo MRS experiments. The ex vivo HR-MAS investigations are very helpful for the assignment of the in vivo resonances of human meningiomas and for the validation of the quantification procedure of in vivo MR spectra. By using one- and two dimensional experiments, several metabolites in different histological subtypes of meningiomas, were identified. The spectroscopic data confirmed the presence of the typical metabolites of these benign neoplasms and, at the same time, that meningomas with different morphological characteristics have different metabolic profiles, particularly regarding macromolecules and lipids. The profile of total choline metabolites (tCho) and the expression of the Kennedy pathway genes in biopsies of human gliomas were also investigated using HR-MAS NMR, and microfluidic genomic cards. 1H HR-MAS spectra, allowed the resolution and relative quantification by LCModel of the resonances from choline (Cho), phosphorylcholine (PC) and glycerolphorylcholine (GPC), the three main components of the combined tCho peak observed in gliomas by in vivo 1H MRS spectroscopy. All glioma biopsies depicted an increase in tCho as calculated from the addition of Cho, PC and GPC HR-MAS resonances. However, the increase was constantly derived from augmented GPC in low grade NMR gliomas or increased PC content in the high grade gliomas, respectively. This circumstance allowed the unambiguous discrimination of high and low grade gliomas by 1H HR-MAS, which could not be achieved by calculating the tCho/Cr ratio commonly used by in vivo 1H MR spectroscopy. The expression of the genes involved in choline metabolism was investigated in the same biopsies. The present findings offer a convenient procedure to classify accurately glioma grade using 1H HR-MAS, providing in addition the genetic background for the alterations of choline metabolism observed in high and low gliomas grade. Chapter 5 reports the study on human gastrointestinal tract (stomach and colon) neoplasms. The human healthy gastric mucosa, and the characteristics of the biochemical profile of human gastric adenocarcinoma in comparison with that of healthy gastric mucosa were analyzed using ex vivo HR-MAS NMR. Healthy human mucosa is mainly characterized by the presence of small metabolites (more than 50 identified) and macromolecules. The adenocarcinoma spectra were dominated by the presence of signals due to triglycerides, that are usually very low in healthy gastric mucosa. The use of spin-echo experiments enable us to detect some metabolites in the unhealthy tissues and to determine their variation with respect to the healthy ones. Then, the ex vivo HR-MAS NMR analysis was applied to human gastric tissue, to obtain information on the molecular steps involved in the gastric carcinogenesis. A microscopic investigation was also carried out in order to identify and locate the lipids in the cellular and extra-cellular environments. Correlation of the morphological changes detected by transmission (TEM) and scanning (SEM) electron microscopy, with the metabolic profile of gastric mucosa in healthy, gastric atrophy autoimmune diseases (AAG), Helicobacter pylori-related gastritis and adenocarcinoma subjects, were obtained. These ultrastructural studies of AAG and gastric adenocarcinoma revealed lipid intra- and extra-cellularly accumulation associated with a severe prenecrotic hypoxia and mitochondrial degeneration. A deep insight into the metabolic profile of human healthy and neoplastic colon tissues was gained using ex vivo HR-MAS NMR spectroscopy in combination with multivariate methods: Principal Component Analysis (PCA) and Partial Least Squares Discriminant Analysis (PLS-DA). The NMR spectra of healthy tissues highlight different metabolic profiles with respect to those of neoplastic and microscopically normal colon specimens (these last obtained at least 15 cm far from the adenocarcinoma). Furthermore, metabolic variations are detected not only for neoplastic tissues with different histological diagnosis, but also for those classified identical by histological analysis. These findings suggest that the same subclass of colon carcinoma is characterized, at a certain degree, by metabolic heterogeneity. The statistical multivariate approach applied to the NMR data is crucial in order to find metabolic markers of the neoplastic state of colon tissues, and to correctly classify the samples. Significant different levels of choline containing compounds, taurine and myoinositol, were observed. Chapter 6 deals with the metabolic profile of normal and tumoral renal human tissues obtained by ex vivo HR-MAS NMR. The spectra of human normal cortex and medulla show the presence of differently distributed osmolytes as markers of physiological renal condition. The marked decrease or disappearance of these metabolites and the high lipid content (triglycerides and cholesteryl esters) is typical of clear cell renal carcinoma (RCC), while papillary RCC is characterized by the absence of lipids and very high amounts of taurine. This research is a contribution to the biochemical classification of renal neoplastic pathologies, especially for RCCs, which can be evaluated by in vivo MRS for clinical purposes. Moreover, these data help to gain a better knowledge of the molecular processes envolved in the onset of renal carcinogenesis.

Classificazione di brani musicali mediante reti neuronali artificiali

L'informatica musicale è una disciplina in continua crescita che sta ottenendo risultati davvero interessanti con l'impiego di sistemi artificiali intelligenti, come le reti neuronali, che permettono di emulare capacità umane di ascolto e di esecuzione musicale. Di particolare interesse è l'ambito della codifica di informazioni musicali tramite formati simbolici, come il MIDI, che permette un'analisi di alto livello dei brani musicali e consente la realizzazione di applicazioni sorprendentemente innovative. Una delle più fruttifere applicazioni di questi nuovi strumenti di codifica riguarda la classificazione di file audio musicali. Questo elaborato si propone di esporre i fondamentali aspetti teorici che concernono la classificazione di brani musicali tramite reti neuronali artificiali e descrivere alcuni esperimenti di classificazione di file MIDI. La prima parte fornisce alcune conoscenze di base che permettono di leggere gli esperimenti presenti nella seconda sezione con una consapevolezza teorica più profonda. Il fine principale della prima parte è quello di sviluppare una comparazione da diversi punti di vista disciplinari tra le capacità di classificazione musicale umane e quelle artificiali. Si descrivono le reti neuronali artificiali come sistemi intelligenti ispirati alla struttura delle reti neurali biologiche, soffermandosi in particolare sulla rete Feedforward e sull'algoritmo di Backpropagation. Si esplora il concetto di percezione nell'ambito della psicologia cognitiva con maggiore attenzione alla percezione uditiva. Accennate le basi della psicoacustica, si passa ad una descrizione delle componenti strutturali prima del suono e poi della musica: la frequenza e l'ampiezza delle onde, le note e il timbro, l'armonia, la melodia ed il ritmo. Si parla anche delle illusioni sonore e della rielaborazione delle informazioni audio da parte del cervello umano. Si descrive poi l'ambito che interessa questa tesi da vicino: il MIR (Music Information Retrieval). Si analizzano i campi disciplinari a cui questa ricerca può portare vantaggi, ossia quelli commerciali, in cui i database musicali svolgono ruoli importanti, e quelli più speculativi ed accademici che studiano i comportamenti di sistemi intelligenti artificiali e biologici. Si descrivono i diversi metodi di classificazione musicale catalogabili in base al tipo di formato dei file audio in questione e al tipo di feature che si vogliono estrarre dai file stessi. Conclude la prima sezione di stampo teorico un capitolo dedicato al MIDI che racconta la storia del protocollo e ne descrive le istruzioni fondamentali nonchè la struttura dei midifile. La seconda parte ha come obbiettivo quello di descrivere gli esperimenti svolti che classificano file MIDI tramite reti neuronali mostrando nel dettaglio i risultati ottenuti e le difficoltà incontrate. Si coniuga una presentazione dei programmi utilizzati e degli eseguibili di interfaccia implementati con una descrizione generale della procedura degli esperimenti. L'obbiettivo comune di tutte le prove è l'addestramento di una rete neurale in modo che raggiunga il più alto livello possibile di apprendimento circa il riconoscimento di uno dei due compositori dei brani che le sono stati forniti come esempi.

L'eccezione è la regola: ricerca espolorativa sul fenomeno del commissariamento nel settore culturale italiano

Di fronte al moltiplicarsi di episodi di commissariamento nella pubblica amministrazione italiana e in particolare nel settore culturale, la presente ricerca mira a comprendere gli obiettivi, le modalità di intervento ed i risultati raggiunti mediante la nomina di commissari straordinari. Si tratta di un intervento anomalo ed inusuale a livello internazionale (la stessa voce commissariamento risulta intraducibile in inglese), dove la ricerca di possibili termini di paragone, effettuata passando in rassegna la letteratura di disaster management, quella sul riaccentramento amministrativo e quella sugli interventi di turnaround nel settore pubblico, restituisce un quadro estremamente specifico del fenomeno, per lo più interno ai confini nazionali. Nello studio, caratterizzato da un forte approccio esplorativo e phenomenon driven, vengono analizzati quattro casi di commissariamento nel settore culturale italiano: due aree archeologiche (quella di Napoli e Pompei e quella di Roma e Ostia) e due Fondazioni Liriche (Teatro Carlo Felice di Genova e Arena di Verona). Dalla ricerca emerge un quadro profondamente critico delle gestioni commissariali. Gli obiettivi ambigui e l’elevata discrezionalità concessa al commissario non sono accompagnati da un’adeguata trasparenza nei processi di nomina, proroga e sostituzione dei commissari, con la prevalenza di rapporti di tipo personale o ‘feudale’. Dal punto di vista dei risultati il commissariamento non incide sulla routine dell’amministrazione ordinaria, rappresentando nel migliore dei casi una parentesi temporanea di buona gestione, o, nel peggiore, perseguendo interventi non legittimi rispetto ai valori professionali che regolano le organizzazioni analizzate. Se considerato alla luce del più generale processo di riforma in senso manageriale che ha coinvolto le istituzioni analizzate dalla fine degli anni ’90, il commissariamento esalta ed intensifica gli aspetti maggiormente critici dell’approccio italiano al New Public Management, in termini di scarsa trasparenza e accountability, elevata influenza della politica nei processi decisionali e generale incoerenza dei disegni di riforma.

I motori di ricerca: algoritmi a confronto e sperimentazione in una classe di scuola superiore.

Tesi interdisciplinare che coniuga due importanti ambiti della Matematica: il Calcolo Numerico e la Didattica della Matematica. Alcuni algoritmi utilizzati per il web information retrieval sono stati introdotti all'interno di due classi di scuola superiore avvalendosi del programma di calcolo Matlab.

Un approccio per la concettualizzazione di insiemi di documenti

Introduzione a tecniche di web semantico e realizzazione di un approccio in grado di ricreare un ambiente familiare di un qualsiasi motore di ricerca con funzionalità semantico-lessicali e possibilità di estrazione, in base ai risultati di ricerca, dei concetti e termini chiave che costituiranno i relativi gruppi di raccolta per i vari documenti con argomenti in comune.

Content extraction: Identifying the main content in HTML documents

Except the article forming the main content most HTML documents on the WWW contain additional contents such as navigation menus, design elements or commercial banners. In the context of several applications it is necessary to draw the distinction between main and additional content automatically. Content extraction and template detection are the two approaches to solve this task. This thesis gives an extensive overview of existing algorithms from both areas. It contributes an objective way to measure and evaluate the performance of content extraction algorithms under different aspects. These evaluation measures allow to draw the first objective comparison of existing extraction solutions. The newly introduced content code blurring algorithm overcomes several drawbacks of previous approaches and proves to be the best content extraction algorithm at the moment. An analysis of methods to cluster web documents according to their underlying templates is the third major contribution of this thesis. In combination with a localised crawling process this clustering analysis can be used to automatically create sets of training documents for template detection algorithms. As the whole process can be automated it allows to perform template detection on a single document, thereby combining the advantages of single and multi document algorithms.

Named Entity Extraction: la piattaforma Gate/Annie

In questo lavoro si introducono i concetti di base di Natural Language Processing, soffermandosi su Information Extraction e analizzandone gli ambiti applicativi, le attività principali e la differenza rispetto a Information Retrieval. Successivamente si analizza il processo di Named Entity Recognition, focalizzando l’attenzione sulle principali problematiche di annotazione di testi e sui metodi per la valutazione della qualità dell’estrazione di entità. Infine si fornisce una panoramica della piattaforma software open-source di language processing GATE/ANNIE, descrivendone l’architettura e i suoi componenti principali, con approfondimenti sugli strumenti che GATE offre per l'approccio rule-based a Named Entity Recognition.

Temporal PageRank

The our reality is characterized by a constant progress and, to follow that, people need to stay up to date on the events. In a world with a lot of existing news, search for the ideal ones may be difficult, because the obstacles that make it arduous will be expanded more and more over time, due to the enrichment of data. In response, a great help is given by Information Retrieval, an interdisciplinary branch of computer science that deals with the management and the retrieval of the information. An IR system is developed to search for contents, contained in a reference dataset, considered relevant with respect to the need expressed by an interrogative query. To satisfy these ambitions, we must consider that most of the developed IR systems rely solely on textual similarity to identify relevant information, defining them as such when they include one or more keywords expressed by the query. The idea studied here is that this is not always sufficient, especially when it's necessary to manage large databases, as is the web. The existing solutions may generate low quality responses not allowing, to the users, a valid navigation through them. The intuition, to overcome these limitations, has been to define a new concept of relevance, to differently rank the results. So, the light was given to Temporal PageRank, a new proposal for the Web Information Retrieval that relies on a combination of several factors to increase the quality of research on the web. Temporal PageRank incorporates the advantages of a ranking algorithm, to prefer the information reported by web pages considered important by the context itself in which they reside, and the potential of techniques belonging to the world of the Temporal Information Retrieval, exploiting the temporal aspects of data, describing their chronological contexts. In this thesis, the new proposal is discussed, comparing its results with those achieved by the best known solutions, analyzing its strengths and its weaknesses.

The Trypanosoma brucei MitoCarta and its regulation and splicing pattern during development

It has long been known that trypanosomes regulate mitochondrial biogenesis during the life cycle of the parasite; however, the mitochondrial protein inventory (MitoCarta) and its regulation remain unknown. We present a novel computational method for genome-wide prediction of mitochondrial proteins using a support vector machine-based classifier with approximately 90% prediction accuracy. Using this method, we predicted the mitochondrial localization of 468 proteins with high confidence and have experimentally verified the localization of a subset of these proteins. We then applied a recently developed parallel sequencing technology to determine the expression profiles and the splicing patterns of a total of 1065 predicted MitoCarta transcripts during the development of the parasite, and showed that 435 of the transcripts significantly changed their expressions while 630 remain unchanged in any of the three life stages analyzed. Furthermore, we identified 298 alternatively splicing events, a small subset of which could lead to dual localization of the corresponding proteins.

Position-dependent effects on stability in tricyclo-DNA modified oligonucleotide duplexes

A series of oligodeoxyribonucleotides and oligoribonucleotides containing single and multiple tricyclo(tc)-nucleosides in various arrangements were prepared and the thermal and thermodynamic transition profiles of duplexes with complementary DNA and RNA evaluated. Tc-residues aligned in a non-continuous fashion in an RNA strand significantly decrease affinity to complementary RNA and DNA, mostly as a consequence of a loss of pairing enthalpy DeltaH. Arranging the tc-residues in a continuous fashion rescues T(m) and leads to higher DNA and RNA affinity. Substitution of oligodeoxyribonucleotides in the same way causes much less differences in T(m) when paired to complementary DNA and leads to substantial increases in T(m) when paired to complementary RNA. CD-spectroscopic investigations in combination with molecular dynamics simulations of duplexes with single modifications show that tc-residues in the RNA backbone distinctly influence the conformation of the neighboring nucleotides forcing them into higher energy conformations, while tc-residues in the DNA backbone seem to have negligible influence on the nearest neighbor conformations. These results rationalize the observed affinity differences and are of relevance for the design of tc-DNA containing oligonucleotides for applications in antisense or RNAi therapy.

Synthesis, base pairing properties and trans-lesion synthesis by reverse transcriptases of oligoribonucleotides containing the oxidatively damaged base 5-hydroxycytidine

The synthesis of a caged RNA phosphoramidite building block containing the oxidatively damaged base 5-hydroxycytidine (5-HOrC) has been accomplished. To determine the effect of this highly mutagenic lesion on complementary base recognition and coding properties, this building block was incorporated into a 12-mer oligoribonucleotide for Tm and CD measurements and a 31-mer template strand for primer extension experiments with HIV-, AMV- and MMLV-reverse transcriptase (RT). In UV-melting experiments, we find an unusual biphasic transition with two distinct Tm's when 5-HOrC is paired against a DNA or RNA complement with the base guanine in opposing position. The higher Tm closely matches that of a C-G base pair while the lower is close to that of a C-A mismatch. In single nucleotide extension reactions, we find substantial misincorporation of dAMP and to a lesser extent dTMP, with dAMP almost equaling that of the parent dGMP in the case of HIV-RT. A working hypothesis for the biphasic melting transition does not invoke tautomeric variability of 5-HOrC but rather local structural perturbations of the base pair at low temperature induced by interactions of the 5-HO group with the phosphate backbone. The properties of this RNA damage is discussed in the context of its putative biological function.