Asynchrone Replikation geprägter und nicht geprägter Chromosomenregionen und Expression relevanter Entwicklungsgene in Präimplantationsembryonen der Maus

Zum besseren Verständnis der epigenetischen Reprogrammierung nach der Befruchtung, wurde in der vorliegenden Studie unter Verwendung eines Interphase-FISH-Assays eine systematische Analyse des Replikationsverhaltens geprägter und nicht geprägter Chromosomenregionen in Präimplantationsembryonen der Maus durchgeführt. Dabei konnte erstmalig gezeigt werden, dass sowohl geprägte als auch nicht geprägte Chromosomen-regionen direkt nach der Befruchtung asynchron replizieren. Vier von fünf nicht geprägten Chromosomenregionen replizierten erst nach dem Zweizell-Embryostadium synchron. Eine asynchrone Replikation geprägter Regionen wurde während der gesamten Präimplantationsentwicklung und in differenzierten Zellen beobachtet. In Morula-Embryonen zeigten der in diesem Stadium nicht exprimierte Dlk1-Gtl2-Locus sowie der biallelisch exprimierte Igf2r-Locus jedoch eine Relaxation der asynchronen Replikation. In einem weiteren Projekt konnte mit Hilfe eines Multiplex-RT-PCR-Ansatzes die sensitive Detektion von multiplen Transkripten in einzelnen Zellen etabliert werden. Anschließend wurden Expressionsmuster von 17 für die epigenetische Reprogrammierung relevanten Entwicklungs-genen in Präimplantationsembryonen sowie in einzelnen Morula-Blastomeren analysiert. Der Transkriptionsfaktor Pou5f1 wurde in allen Präimplantationsembryonen und allen Morula-Blastomeren detektiert, was auf eine uniforme Reaktivierung der Pluripotenz hinweist. Dagegen variierte die mRNA-Expression verschiedener DNA-Cytosin-5-Methyltransferasen, 5-methyl-CpG-Bindeproteine sowie Enzyme der Basenexzisionsreparatur stark zwischen individuellen Zellen des gleichen Embryos und noch stärker zwischen Zellen verschiedener Embryonen. Diese Ergebnisse zeigen, dass sich das für die Reprogrammierungsmaschinerie kodierende Transkriptom zu bestimmten Entwicklungs-zeitpunkten zwischen einzelnen Blastomeren unterscheidet.

Valutazione del ruolo dell'espressione di IRS-1 nel differenziamento osteoblastico di cellule di osteosarcoma ed MSCs

L’osteosarcoma (OS) è il tumore primitivo dell’osso più comune in età pediatrica e adolescenziale. L’OS è stato recentemente riconsiderato come una patologia da de-differenziamento, legata all’interruzione del processo cui vanno incontro i precursori osteoblastici, quali le cellule staminali mesenchimali (MSCs), per trasformarsi in osteoblasti maturi. Il sistema IGF è coinvolto nella regolazione della proliferazione e del differenziamento di cellule di OS. IRS-1 è un mediatore critico di tale via di segnalazione e il suo livello di espressione modula il differenziamento di cellule ematopoietiche. Lo scopo di questa tesi è stato quello di definire il ruolo di IRS-1 nel differenziamento osteoblastico di MSCs e cellule di OS. Il potenziale differenziativo di cellule di OS umano e murino e di MSCs derivate da midollo osseo è stato valutato tramite Alizarin Red staining e Real Time-PCR. Dai dati ottenuti è emerso come i livelli di espressione di IRS-1 diminuiscano durante il differenziamento osteoblastico. Conseguentemente, i livelli di espressione di IRS-1 sono stati manipolati utilizzando shRNA per down-regolare l’espressione della proteina o un plasmide per sovra-esprimerla. Sia la down-regolazione sia la sovra-espressione di IRS-1 hanno inibito il differenziamento osteoblastico delle linee cellulari considerate. Allo scopo di valutare il contributo di IRS-1 nella via di segnalazione di IGF-1R è stato utilizzato l’inibitore di tale recettore, αIR-3. Anche in questo caso è stata osservata una riduzione della capacità differenziativa. L’inibitore del proteasoma MG-132 ha portato ad un aumento dei livelli di IRS-1, portando nuovamente all’inibizione del differenziamento osteoblastico e suggerendo che l’ubiquitinazione di questa proteina potrebbe avere un ruolo importante nel mantenimento di appropriati livelli di espressione di IRS-1. I risultati ottenuti indicano la criticità dei livelli di espressione di IRS-1 nella determinazione della capacità differenziativa sia di cellule di OS umano e murino, sia delle MSCs.

Meccanismi replicativi e di regolazione dell'espressione genica di Parvovirus B19

Il Parvovirus B19, virus patogeno umano della famiglia Parvoviridae, mostra uno specifico tropismo per i precursori eritroidi e una limitata replicazione in alcune linee cellulari megacarioblastoidi. Allo scopo di sviluppare sistemi utili allo studio delle caratteristiche biologiche del virus, diversi laboratori si sono occupati della costruzione di cloni genomici di B19 dotati di competenza funzionale e capaci di generare virus infettante. Parte del presente lavoro ha riguardato l’analisi funzionale di diversi cloni genomici di B19 e ha permesso di caratterizzare le regioni terminali del virus e di identificare requisiti essenziali per la loro funzionalità. Nel contesto intracellulare, esistono differenti livelli di restrizione in relazione alla capacità della cellula di supportare la replicazione virale, non ancora del tutto caratterizzati. Inoltre si sono accumulate evidenze circa la capacità del B19 di instaurare persistenza in numerosi tessuti. Non sono ancora note le caratteristiche funzionali del genoma virale in questo stato, è possibile che il virus persista in forma silente e meccanismi epigenetici possano regolare tale silenziamento. In questo studio è stato analizzato lo stato di metilazione del genoma di B19 e il suo possibile effetto sul ciclo replicativo virale ed è stata investigata la possibile associazione del DNA virale agli istoni cellulari nel corso di infezione in vitro. I risultati ottenuti confermano la presenza di questi meccanismi epigenetici, potendo ipotizzare che giochino un importante ruolo nella regolazione della funzionalità virale e nell’interazione B19-cellula e siano un elemento critico per l’adattamento del virus nell’ambiente in cui si trova. Inoltre l’ipotesi che anche i microRNA possano assumere un importante significato nell’interazione B19-cellula è stata proposta da diversi lavori e nel presente studio è stata valutata la produzione di queste piccole molecole durante l'infezione in vitro, ricercando microRNA (cellulari e/o virali) con omologia di sequenza per il genoma di B19 e quindi specifici per il virus.

The prehistoric inhabitants of the Wadi Howar : an anthropological study of human skeletal remains from the Sudanese part of the Eastern Sahara

All currently available human skeletal remains from the Wadi Howar (Eastern Sahara, Sudan) were employed in an anthropological study. The study’s first aim was to describe this unique 5th to 2nd millennium BCE material, which comprised representatives of all three prehistoric occupation phases of the region. Detecting diachronic differences in robusticity, occupational stress levels and health within the spatially, temporally and culturally heterogeneous sample was its second objective. The study’s third goal was to reveal metric and non-metric affinities between the different parts of the series and between the Wadi Howar material and other relevant prehistoric as well as modern African populations. rnThe reconstruction and comprehensive osteological analysis of 23 as yet unpublished individuals, the bulk of the Wadi Howar series, constituted the first stage of the study. The analyses focused on each individual’s in situ position, state of preservation, sex, age at death, living height, living weight, physique, biological ancestry, epigenetic traits, robusticity, occupational stress markers, health and metric as well as morphological characteristics. Building on the results of these efforts and the re-examination of the rest of the material, the Wadi Howar series as a whole, altogether 32 individuals, could be described. rnA wide variety of robusticity, occupational stress and health variables was evaluated. The pre-Leiterband (hunter-gatherer-fisher/hunter-gatherer-fisher-herder) and the Leiterband (herder-gatherer) data of over a third of these variables differed statistically significantly or in tendency from each other. The Leiterband sub-sample was characterised by higher enamel hypoplasia frequencies, lower mean ages at death and less pronounced expressions of occupational stress traits. This pattern was interpreted as evidence that the adoption and intensification of animal husbandry did probably not constitute reactions to worsening conditions. Apart from that, the relevant observations, noteworthy tendencies and significant differences were explained as results of a broader spectrum of pre-Leiterband subsistence activities and the negative side effects of the increasingly specialised herder-gatherer economy of the Leiterband phase. rnUsing only the data which could actually be collected from it, multiple, separate, individualised discriminant function analyses were carried out for each Wadi Howar skeleton to determine which prehistoric and which modern comparative sample it was most similar to. The results of all individual analyses were then summarised and examined as a whole. Thus it became possible to draw conclusions about the affinities the Wadi Howar material shared with prehistoric as well as modern populations and to answer questions concerning the diachronic links between the Wadi Howar’s prehistoric populations. When the Wadi Howar remains were positioned in the context of the selected prehistoric (Jebel Sahaba/Tushka, A-Group, Malian Sahara) and modern comparative samples (Southern Sudan, Chad, Mandinka, Somalis, Haya) in this fashion three main findings emerged. Firstly, the series as a whole displayed very strong affinities with the prehistoric sample from the Malian Sahara (Hassi el Abiod, Kobadi, Erg Ine Sakane, etc.) and the modern material from Southern Sudan and, to a lesser extent, Chad. Secondly, the pre-Leiterband and the Leiterband sub-sample were closer to the prehistoric Malian as well as the modern Southern Sudanese material than they were to each other. Thirdly, the group of pre-Leiterband individuals approached the Late Pleistocene sample from Jebel Sahaba/Tushka under certain circumstances. A theory offering explanations for these findings was developed. According to this theory, the entire prehistoric population of the Wadi Howar belonged to a Saharo-Nilotic population complex. The Jebel Sahaba/Tushka population constituted an old Nilotic and the early population of the Malian Sahara a younger Saharan part of this complex. The pre-Leiterband groups probably colonised the Wadi Howar from the east, either during or soon after the original Saharo-Nilotic expansion. Unlike the pre-Leiterband groups, the Leiterband people originated somewhere west of the Wadi Howar. They entered the region in the context of a later, secondary Saharo-Nilotic expansion. In the process, the incoming Leiterband groups absorbed many members of the Wadi Howar’s older pre-Leiterband population. The increasing aridification of the Wadi Howar region ultimately forced its prehistoric inhabitants to abandon the wadi. Most of them migrated south and west. They, or groups closely related to them, probably were the ancestors of the majority of the Nilo-Saharan-speaking pastoralists of modern-day Southern Sudan and Eastern Chad.

DNA-Reparatur-assoziierte genetische und epigenetische Faktoren für die Zweittumorentstehung nach Tumoren im Kindes- und Jugendalter

Die Ursachen der Zweittumorentwicklung bei Personen, die eine Krebserkrankung in der Kindheit überlebten, sind weitgehend unklar. Strahlenexposition oder Chemotherapie führen in normalen somatischen Zellen zu DNA-Schäden, welche bei fehlerhafter Reparatur eine Karzinogenese auslösen können. Es ist denkbar, dass genetische Unterschiede z. B. in den Signalwegen der Zellzykluskontrolle und der DNA-Reparatur nach therapieinduzierten DNA-Schäden eine entscheidende Rolle bei der Zweittumorentwicklung spielen. Im Rahmen dieser Arbeit wurden 20 Personen, die eine Krebserkrankung in der Kindheit überlebten und einen unabhängigen Zweittumor entwickelten, mit 20 gematchten Kontrollpersonen ohne Zweittumorentwicklung verglichen. Die primären Fibroblasten der Patienten wurden auf somatische, genetische und/oder epigenetische Unterschiede in DNA-Reparaturnetzwerken untersucht. Die biologisch relevantesten Ergebnisse lieferten Proteinuntersuchungen mittels Antikörper-Microarrays. Hierbei wurde eine konstitutiv erniedrigte Menge an RAD9A und einigen anderen DNA-Reparatur-Proteinen (BRCA1, DDIT3, MSH6, p53, RAD51) in den Zweittumorpatienten im Vergleich zu den Eintumorpatienten festgestellt. Nach einer DNA-Schädigung durch 1 Gray Bestrahlung erhöhte sich die RAD9A-Proteinmenge, wobei die Zweittumorpatienten eine geringere Induktion als die Eintumorpatienten zeigten. Bei der Quantifizierung der mRNA-Expression mittels RTq-PCR wurde ein niedrigerer RAD9A-mRNA-Level sowohl in den unbehandelten und als auch in den 1 Gray bestrahlten Zellen der Zweittumorpatienten festgestellt. SNP-Array und Methylierungsanalysen konnten keine Auffälligkeiten im RAD9A-Lokus nachweisen. Diese Ergebnisse unterstützen die Hypothese, dass Modulationen von RAD9A und anderen Zellzyklusarrest- und DNA-Reparaturproteinen zum Risiko einer Zweittumorentwicklung in Kinderkrebspatienten beitragen. Bei einem diskordanten monozygoten Zwillingspaar wurde in ca. 20% der Zellen des Zweittumorzwillings eine Hypermethylierung des Tumorsuppressorgens BRCA1 festgestellt, die mit einer konstitutiv erniedrigten BRCA1-Proteinexpression einhergeht und einen möglichen Krebsrisikofaktor darstellt. Die partielle Deletion des Gens RSPO3, die wahrscheinlich als somatisches Zellmosaik beim Zweittumorzwilling vorliegt, korreliert mit einer niedrigeren RSPO3-mRNA-Expression und ist vermutlich auch mit einer erhöhten Krebsprädisposition assoziiert.

Die Rolle von Ephrin-B2 in der Invasion maligner Gliome

Das Glioblastoma multiforme zählt zu den häufigsten glialen Neoplasien des Menschen und weist zudem unter den Gliomen die höchste Malignität auf. Glioblastompatienten haben trotz aggressiver therapeutischer Ansätze eine mittlere Überlebenszeit von weniger als einem Jahr. Die diffuse Invasion in das umliegende Hirngewebe ist einer der Hauptgründe für die Rezidivbildung und die infauste Prognose von Glioblastompatienten. Neuere Untersuchungen lassen vermuten, dass die starke Invasion auch einer der Gründe für die beobachtete anti-angiogene Resistenz bei der Behandlung von Glioblastomen ist. Das bidirektionale EphB/Ephrin-B-System wurde bei der axonalen Wegfindung als Vermittler repulsiver Signale identifiziert und auch im Zusammenhang der Migration und Invasion von Zellen überprüft. In der vorliegenden Arbeit sollte daher die Funktion der bidirektionalen Eph- und Ephrin-Signaltransduktion in Bezug auf die Glioblastominvasion und Progression untersucht werden. rn Genetische und epigenetische Untersuchungen der EphB/Ephrin-B-Familie in einer Kohorte von Gliompatienten unterschiedlicher Malignitätsgrade identifizierten Ephrin-B2 als mögliches Tumorsuppressorgen. In Übereinstimmung damit führte die Inaktivierung von Ephrin-B2 in einem murinen Gliommodell zu einer verstärkten Invasion und einem erhöhtem Tumorwachstum in vivo. Dies konnte in verschiedenen Invasion-Assays in vitro bestätigt werden. Weiterhin zeigten unsere Untersuchungen, dass Ephrin-B2 transkriptionell durch das hypoxische Mikromilieu HIF-1α-vermittelt reprimiert wird. Da HIF-1α als transkriptioneller Aktivator Ephrin-B2 nicht direkt reprimieren kann, wurden potentielle HIF-1α-regulierte Repressoren untersucht, die für die Ephrin-B2 Herunterregulation verantwortlich sein könnten. Dabei wurde anhand von Ephrin-B2-Promotoranalysen und ChIP-Assays ZEB2 als HIF-1α-induzierbarer Repressor von Ephrin-B2 identifiziert. Zur Bestätigung der Hypothese, dass ZEB2 ein wichtiger Regulator der Tumorinvasion ist, wurden humane ZEB2-Knockdown-Glioblastomzellen generiert und in vitro sowie in vivo untersucht. Im Hinblick auf mögliche therapeutische Anwendungen wurden die ZEB2-Knockdown-Glioblastomzellen zusätzlich im Zusammenhang anti-Angiogenese-induzierter Invasion analysiert. Der Verlust von ZEB2 führte dabei zu einer verringerten Glioblastominvasion und Progression in einem Maus-Xenograft Modell. Die Behandlung der Tumoren mit dem anti-VEGF-Antikörper Avastin resultierte in einer stark erhöhten Invasion, die durch die Inaktivierung von ZEB2 und der dadurch reaktivierten repulsiven Signale von Ephrin-B2 wieder aufgehoben werden konnte. Zusammenfassend konnte in der vorliegenden Arbeit erstmals gezeigt werden, dass Ephrin-B2 als Tumorsuppressor in Gliomen agiert und durch verschiedene Mechanismen wie der genetischen und epigenetischen Kontrolle, aber auch der HIF-1α-vermittelten, ZEB2-abhängigen Repression inaktiviert wird. Dies resultiert in einer Blockade repulsiver Signale, so dass Tumorzellen diffus in das Parenchym und zu den Blutgefäßen migrieren können. Der in dieser Arbeit neu identifizierte Signalweg stellt ein attraktives therapeutisches Ziel zur Inhibition der Tumorzellinvasion dar und ermöglicht darüber hinaus der Ausbildung von Resistenzen gegenüber anti-angiogener Behandlung entgegenzuwirken. rn

Transkriptom des Meristemgewebes des Kolumnarapfels

Kolumnare Apfelbäume (Malus x domestica) stellen aufgrund ihres auffälligen Phänotyps eine ökonomisch interessante Wuchsform dar. Diese extreme Form des Kurztriebwuchses zeichnet sich durch einen insgesamt sehr schlanken, säulenförmigen Habitus aus, welcher eine dichte Pflanzung und damit einhergehend Ertragssteigerungen im Vergleich zu normalwüchsigen Bäumen ermöglicht. Verursacht wird der Phänotyp durch die Anwesenheit eines einzelnen, dominanten Allels des Columnar (Co)-Gens. Bis auf die approximative Lokalisation des Gens auf Chromosom 10 ist über mögliche Identität und Funktion bislang nichts bekannt.rnIn der vorliegenden Arbeit wurde ein erster Versuch unternommen, mit Hilfe von Next Generation Sequencing (NGS) Technologien und RNA-Seq Einblicke in das Transkriptom des Sprossapikalmeristems (SAM) kolumnarer Apfelbäume zu gewinnen. So konnte gezeigt werden, dass unabhängig vom Zeitpunkt der Entnahme des Materials mehrere hundert Gene differentiell reguliert werden. Diese lassen sich funktional in mehrere überrepräsentierte Kategorien gruppieren, von denen sich einige wiederum mit dem kolumnaren Phänotyp assoziieren lassen. Durch den Einsatz weiterer Expressionsstudien (Microarrays, qRT-PCR) konnten frühere Ergebnisse bezüglich des Hormonhaushalts auf Genebene bestätigt und neue Erkenntnisse gewonnen werden, die eine mögliche Erklärung für den Phänotyp darstellen. Weiterhin ergab der Vergleich aller durchgeführten Expressionsstudien eine Anreicherung signifikant differentiell regulierter Gene auf Chromosom 10, was auf einen „selective sweep“ hindeutet. Eine potentielle epigenetische Regulation dieser Gene durch das Genprodukt von Co könnte daher möglich sein. Mehr als die Hälfte dieser Gene lassen sich darüber hinaus aufgrund ihrer Funktion direkt mit dem kolumnaren Phänotyp assoziieren.rnDiese Ergebnisse zeigen, dass die Anwesenheit des Co-Allels massive Veränderungen in der Genregulation des SAMs mit sich bringt, wobei einige dieser differentiell regulierten Gene mit großer Wahrscheinlichkeit an der Etablierung des kolumnaren Phänotyps beteiligt sind. Auch wenn die Funktion des Co-Genproduktes nicht abschließend geklärt werden konnte, sind doch anhand der Resultate schlüssige Hypothesen diesbezüglich möglich.rn

Cancer and aging: a multidisciplinary medicinal chemistry approach on relevant biological targets such as proteasome, sirtuins and interleukin 6

It is well known that ageing and cancer have common origins due to internal and environmental stress and share some common hallmarks such as genomic instability, epigenetic alteration, aberrant telomeres, inflammation and immune injury. Moreover, ageing is involved in a number of events responsible for carcinogenesis and cancer development at the molecular, cellular, and tissue levels. Ageing could represent a “blockbuster” market because the target patient group includes potentially every person; at the same time, oncology has become the largest therapeutic area in the pharmaceutical industry in terms of the number of projects, clinical trials and research and development (R&D) spending, but cancer remains one of the leading causes of mortality worldwide. The overall aim of the work presented in this thesis was the rational design of new compounds able to modulate activity of relevant targets involved in cancer and aging-related pathologies, namely proteasome and immunoproteasome, sirtuins and interleukin 6. These three targets play different roles in human cells, but the modulation of its activity using small molecules could have beneficial effects on one or more aging-related diseases and cancer. We identified new moderately active and selective non-peptidic compounds able to inhibit the activity of both standard and immunoproteasome, as well as novel and selective scaffolds that would bind and inhibit SIRT6 selectively and can be used to sensitize tumor cells to commonly used anticancer agents such gemcitabine and olaparib. Moreover, our virtual screening approach led us also to the discovery of new putative modulators of SIRT3 with interesting in-vitro and cellular activity. Although the selectivity and potency of the identified chemical scaffolds are susceptible to be further improved, these compounds can be considered as highly promising leads for the development of future therapeutics.

Design and Synthesis of Naphthalene Diimides Based Small Molecules as Anticancer Agents: Targeting the Polyamine Transporter, G-quadruplex Structures and HDAC

Cancer is a multifactorial disease characterized by a very complex etiology. Basing on its complex nature, a promising therapeutic strategy could be based by the “Multi-Target-Directed Ligand” (MTDL) approach, based on the assumption that a single molecule could hit several targets responsible for the pathology. Several agents acting on DNA are clinically used, but the severe deriving side effects limit their therapeutic application. G-quadruplex structures are DNA secondary structures located in key zones of human genome; targeting quadruplex structures could allow obtaining an anticancer therapy more free from side effects. In the last years it has been proved that epigenetic modulation can control the expression of human genes, playing a crucial role in carcinogenesis and, in particular, an abnormal expression of histone deacetylase enzymes are related to tumor onset and progression. This thesis deals with the design and synthesis of new naphthalene diimide (NDI) derivatives endowed with anticancer activity, interacting with DNA together with other targets implicated in cancer development, such as HDACs. NDI-polyamine and NDI-polyamine-hydroxamic acid conjugates have been designed with the aim to provide potential MTDLs, in order to create molecules able simultaneously to interact with different targets involved in this pathology, specifically the G-quadruplex structures and HDAC, and to exploit the polyamine transport system to get selectively into cancer cells. Macrocyclic NDIs have been designed with the aim to improve the quadruplex targeting profile of the disubstituted NDIs. These compounds proved the ability to induce a high and selective stabilization of the quadruplex structures, together with cytotoxic activities in the micromolar range. Finally, trisubstituted NDIs have been developed as G-quadruplex-binders, potentially effective against pancreatic adenocarcinoma. In conclusion, all these studies may represent a promising starting point for the development of new interesting molecules useful for the treatment of cancer, underlining the versatility of the NDI scaffold.

Profili di espressione genetica ed epigenetica ad impatto prognostico e predittivo nel carcinoma squamoso e nelle lesioni potenzialmente maligne del cavo orale

Il carcinoma squamoso orale (CSO) è spesso preceduto da lesioni definite potenzialmente maligne tra cui la leucoplachia e il lichen ma una diagnosi precoce avviene ancora oggi in meno della metà dei casi. Inoltre spesso un paziente trattato per CSO svilupperà secondi tumori. Scopo del lavoro di ricerca è stato: 1) Studiare, mediante metodica di next generation sequencing, lo stato di metilazione di un gruppo di geni a partire da prelievi brushing del cavo orale al fine di identificare CSO o lesioni ad alto rischio di trasformazione maligna. 2) Valurare la relazione esistente tra sovraespressione di p16INK4A e presenza di HPV in 35 pazienti affetti da lichen 3) Valutare la presenza di marker istopatologici predittivi di comparsa di seconde manifestazioni tumorali 4) valutare la relazione clonale tra tumore primitivo e metastasi linfonodale in 8 pazienti mediante 2 metodiche di clonalità differenti: l’analisi di mtDNA e delle mutazioni del gene TP53. I risultati hanno mostrato: 1) i geni ZAP70 e GP1BB hanno presentato un alterato stato di metilazione rispettivamente nel 100% e nel 90,9% di CSO e lesioni ad alto rischio, mentre non sono risultati metilati nei controlli sani; ipotizzando un ruolo come potenziali marcatori per la diagnosi precoce nel CSO. 2)Una sovraespressione di p16INK4A è risultata in 26/35 pazienti affetti da lichen ma HPV-DNA è stato identificato in soli 4 campioni. Nessuna relazione sembra essere tra sovraespressione di p16INK4A e virus HPV. 3)L’invasione perineurale è risultato un marker predittivo della comparsa di recidiva locale e metastasi linfonodale, mentre lo stato dei margini chirurgici si è rilevato un fattore predittivo per la comparsa di secondi tumori primitivi 4) Un totale accordo nei risultati c’è stato tra analisi di mtDNA e analisi di TP53 e le due metodiche hanno identificato la presenza di 4 metastasi linfonodali non clonalmente correlate al tumore primitivo.

Evolutionary epigenetics of modern human populations

Epigenetic variability is a new mechanism for the study of human microevolution, because it creates both phenotypic diversity within an individual and within population. This mechanism constitutes an important reservoir for adaptation in response to new stimuli and recent studies have demonstrated that selective pressures shape not only the genetic code but also DNA methylation profiles. The aim of this thesis is the study of the role of DNA methylation changes in human adaptive processes, considering the Italian peninsula and macro-geographical areas. A whole-genome analysis of DNA methylation profile across the Italian penisula identified some genes whose methylation levels differ between individuals of different Italian districts (South, Centre and North of Italy). These genes are involved in nitrogen compound metabolism and genes involved in pathogens response. Considering individuals with different macro-geographical origins (individuals of Asians, European and African ancestry) more significant DMRs (differentially methylated regions) were identified and are located in genes involved in glucoronidation, in immune response as well as in cell comunication processes. A "profile" of each ancestry (African, Asian and European) was described. Moreover a deepen analysis of three candidate genes (KRTCAP3, MAD1L and BRSK2) in a cohort of individuals of different countries (Morocco, Nigeria, China and Philippines) living in Bologna, was performed in order to explore genetic and epigenetic diversity. Moreover this thesis have paved the way for the application of DNA methylation for the study of hystorical remains and in particular for the age-estimation of individuals starting from biological samples (such as teeth or blood). Noteworthy, a mathematical model that considered methylation values of DNA extracted from cementum and pulp of living individuals can estimate chronological age with high accuracy (median absolute difference between age estimated from DNA methylation and chronological age was 1.2 years).

Synthesis of new bioactive β-lactam compounds

New biologically active β-lactams were designed and synthesized, developing novel antibiotics and enzymatic inhibitors directed toward specific targets. Within a work directed to the synthesis of mimetics for RGD (Arg-Gly-Asp) sequence able to interact with αvβ3 and α5β1-type integrins, new activators were developed and their Structure-Activity Relationships (SAR) analysis deepened, enhancing their activity range towards the α4β1 isoform. Moreover, to synthesize novel compounds active both against bacterial infections and pulmonary conditions of cystic fibrosis patients, new β-lactam candidates were studied. Among the abundant library of β-lactams prepared, mainly with antioxidant and antibacterial double activities, it was identified a single lead to be pharmacologically tested in vivo. Its synthesis was optimized up to the gram-scale, and pretreatment method and HPLC-MS/MS analytical protocol for sub-nanomolar quantifications were developed. Furthermore, replacement of acetoxy group in 4-acetoxy-azetidinone derivatives was studied with different nucleophiles and in aqueous media. A phosphate group was introduced and the reactivity exploited using different hydroxyapatites, obtaining biomaterials with multiple biological activities. Following the same kind of reactivity, a small series of molecules with a β-lactam and retinoic hybrid structure was synthesized as epigenetic regulators. Interacting with HDACs, two compounds were respectively identified as an inhibitor of cell proliferation and a differentiating agent on steam cells. Additionally, in collaboration with Professor L. De Cola at ISIS, University of Strasbourg, some new photochemically active β-lactam Pt (II) complexes were designed and synthesized to be used as bioprobes or theranostics. Finally, it was set up and optimized the preparation of new chiral proline-derived α-aminonitriles through an enantioselective Strecker reaction, and it was developed a chemo-enzymatic oxidative method for converting alcohols to aldehydes or acid in a selective manner, and amines to relative aldehydes, amides or imines. Moreover, enzymes and other green chemistry methodologies were used to prepare Active Pharmaceutical Ingredients (APIs).

Analysis of Copy Number Variants identifies new candidate genes for Autism Spectrum Disorder and Intellectual Disability

Autism spectrum disorder (ASD) and Intellectual Disability (ID) are complex neuropsychiatric disorders characterized by extensive clinical and genetic heterogeneity and with overlapping risk factors. The aim of my project was to further investigate the role of Copy Numbers Variants (CNVs), identified through genome-wide studies performed by the Autism Geome Project (AGP) and the CHERISH consortium in large cohorts of ASD and ID cases, respectively. Specifically, I focused on four rare genic CNVs, selected on the basis of their impact on interesting ASD/ID candidate genes: a) a compound heterozygous deletion involving CTNNA3, predicted to cause the lack of functional protein; b) a 15q13.3 duplication containing CHRNA7; c) a 2q31.1 microdeletion encompassing KLHL23, SSB and METTL5; d) Lastly, I investigated the putative imprinting regulation of the CADPS2 gene, disrupted by a maternal deletion in two siblings with ASD and ID. This study provides further evidence for the role of CTNNA3, CHRNA7, KLHL23 and CADPS2 as ASD and/or ID susceptibility genes, and highlights that rare genetic variation contributes to disease risk in different ways: some rare mutations, such as those impacting CTNNA3, act in a recessive mode of inheritance, while other CNVs, such as those occurring in the 15q13.3 region, are implicated in multiple developmental and/or neurological disorders possibly interacting with other susceptibility variants elsewhere in the genome. On the other hand, the discovery of a tissue-specific monoallelic expression for the CADPS2 gene, implicates the involvement of epigenetic regulatory mechanisms as risk factors conferring susceptibility to ASD/ID.

Der Einfluss assistierter Reproduktionstechniken auf die epigenetische Reprogrammierung in der fruehen Embryogenese und Gametogenese im Mausmodell

Seit der Geburt von Louise J. Brown (1978) als erstem künstlich erzeugtem Kind hat sich die Nachfrage nach assistierten Reproduktionstechniken (ART) stark erhöht. Der Anteil der nach In-vitro-Fertilisation (IVF) oder Intrazytoplasmatischer Spermieninjektion (ICSI) geborenen Kinder macht mittlerweile abhängig vom betrachteten Industrieland zwischen 1-4% an der Gesamtgeburtenzahl aus. In zahlreichen Studien korreliert eine erhöhte Prävalenz für seltene Imprinting-Erkrankungen, wie z.B. Beckwith-Wiedemann oder Angelman-Syndrom, mit der Geburt nach assistierten Reproduktionstechniken. Es ist bekannt, dass die medizinischen Interventionen zur Behandlung von Sub- und Infertilität in sehr sensitive Phasen der epigenetischen Reprogrammierung des Embryos und der Keimzellen eingreifen. In der vorliegenden Arbeit wurde untersucht, ob die ovarielle Stimulation einen Einfluss auf die epigenetische Integrität von geprägten Genen in murinen Präimplantationsembryonen hat. Die in diesem Zusammenhang entwickelte digitale Bisulfitpyrosequenzierung gewährleistet die Analyse der DNA-Methylierung auf Einzelallelebene durch eine adäquate Verdünnung der Probe im Vorfeld der PCR. Die ovarielle Induktion führte zu einem erhöhten Rate an Epimutationen des paternalen H19-Allels, sowie des maternalen Snrpn-Allels. Zudem konnte festgestellt werden, dass die Expression von drei potentiellen Reprogrammierungsgenen (Apex1, Polb, Mbd3) in Embryonen aus hormonell stimulierten Muttertieren dereguliert ist. Whole-Mount Immunfluoreszenzfärbungen für APEX1 korrelierten dessen differentielle Genexpression mit dem Proteinlevel. Anzeichen früher apoptotischer Vorgänge äußerten sich in Embryonen aus hormonell induzierten Muttertieren in der hohen Rate an Embryonen, die keines der drei Transkripte exprimierten oder weniger APEX1-positive Blastomeren aufwiesen.In einer weiteren Fragestellung wurde untersucht, ob die Kryokonservierung muriner Spermatozoen den epigenetischen Status geprägter Gene in den Keimzellen beeinflusst. Die Analyse von F1-Zweizellembryonen, die durch IVF mit den jeweiligen Spermatozoen eines Männchens generiert wurden, diente der Aufklärung möglicher paternaler Transmissionen. Insgesamt konnten keine signifikanten Auswirkungen der Kryokonservierung auf den epigenetischen Status in Spermatozoen und F1-Embryonen ermittelt werden.

Identification of target genes of the T-box proteins in Drosophila melanogaster

In a prior bioinformatic analysis by Hüyseyin Binbas, potential Tbx targets sequences in wing-related genes have been identified. Guided by this information, enhancer trap/reporter lacZ insertions were characterized by X-gal staining first in wildtype and then in l(1)omb imaginal discs.rnIn several lines I observed an increase in reporter expression in a l(1)omb mutant background. Since Omb is assumed to function predominantly as a transcriptional repressor, this may indicate direct regulation. Repression by Omb was observed e.g. for brk and tkv. These genes are negatively regulated by Dpp, while omb is induced by Dpp. Omb which mediates the effects of Dpp on proliferation could, thus, also mediate the Dpp effect on patterning of the wing disc. However, brk and tkv were not completely derepressed in l(1)omb indicating that Dpp represses these genes also by an Omb-independent mechanism.rnMore frequently I observed loss of reporter expression in an l(1)omb mutant background. In these cases, regulation by Omb presumably is indirect. For example, STAT92E-lacZ expression in the wildtype eye was symmetrically expressed at the dorsal and ventral margins. In l(1)omb, ventral expression was selectively lost. Loss of omb is known to cause ventral overproliferation of the eye by activation of the Jak/STAT pathway. STAT92E expression is negatively regulated by Jak/STAT signaling suggesting that loss of omb activates Jak/STAT further upstream in the pathway.rnRegional overproliferation of eye and wing in the l(1)omb mutant background proved a complicating issue in the search for Omb targets. This effect made it difficult to decide whether an expanded reporter expression pattern was due to tissue expansion or reporter gene derepression. For instance hth-lacZ appeared to expand along the ventral eye disc margin in l(1)omb. Without addtional experiments it cannot be concluded whether this is due to de-repression or to activation in association with the proliferative state. Parallel to my experiments, evidence accumulated in our laboratory that loss of omb may attenuate Wg and Hegehog signaling. Since these diffusible proteins are the main patterning molecules in the wing imaginal disc, with dpp being downstream of Hh, many of the observed effects could be secondary to reduced Wg and Hh activity. Examples are ab-lacZ, Dll-lacZ and vgBE-lacZ (reduced expression on the dorso-ventral boundary) and inv-lacZ (late larval expression in the anterior wing disc compartment is lost) or sal-lacZ. Epistasis experiment will be required to clarifiy these issues.rnFurthermore, loss of omb appeared to induce cell fate changes. It was reported previously that in an omb null mutant, the dorsal determinant apterous (ap) is ectopically expressed in the ventral compartment (an effect I did not observe with the strongly hypomorphic l(1)omb15, indicating strong dose dependence). Ventral repression of ap is maintained by epigenetic mechanisms. The patchy and variable nature of ectopic expression of ap or grn-1.1-lacZ points to an effect of omb on epigenetic stability.rnIn the second part of my thesis, an analysis of Omb expression in the Drosophila embryonic ventral nervous system was performed. Omb was found co-expressed with Eve in the medial aCC and RP2 motorneurons as well as the fpCC interneuron and the mediolateral CQ neurons. Additionally, Omb was detected in the Eg positive NB7-3 GW serotonergic motoneuron and the N2-4 neurons. Omb was not found in Repo positive glial cells. During embryonic stage 14, Omb showed some coepression with Dpn or Pros. At the embryonic stage 16, Omb was expressed in minor subset of Mid and Wg positive cells.