A new cell labelling strategy to study the biogenesis of exosomes and their role in human melanoma progression driven by microenvironmental acidic pH

Gli esosomi sono nanovescicole di origine endosomiale, che rappresentano un mezzo importante di comunicazione cellula-cellula. Essendo coinvolti in diversi tipi di processi, sia fisiologici che patologici, sono diventati oggetto di sperimentazione clinica, nonostante molti meccanismi alla base della loro biogenesi rimangono tuttora sconosciuti. Quindi nel nostro laboratorio è stata messa a punto una metodica atta a produrre esosomi fluorescenti, in modo da poterne seguire la biogenesi nei diversi compartimenti intracellulari, il rilascio nell’ambiente extracellulare e la fusione con la membrana delle cellule riceventi. È stato quindi studiato, in un modello cellulare di melanoma umano, come questo precursore lipidico fluorescente è assorbito dalla cellula, accumulato all’interno del reticolo endoplasmatico, per poi diventare parte integrante della membrana degli esosomi. Queste nanovescicole fluorescenti sono state caratterizzate per dimensione, densità e presenza di marcatori proteici e successivamente è stata studiata con esperimenti di cinetica (5min – 24h) la secrezione. In seguito tale metodica è stata applicata allo studio della progressione del melanoma in condizioni di acidità microambientale. Nel melanoma è già noto che il pH microambientale è in grado di promuovere invasione e migrazione cellulare, e in questa tesi abbiamo analizzato il ruolo degli esosomi in tali processi. Quindi abbiamo studiato come cellule di melanoma umano, corrispondente a uno stadio intermedio, coltivate in ambiente acido producano una maggior quantità di esosomi, con un’aumentata capacità di trasferimento in cellule bersaglio. Inoltre queste vescicole presentano un profilo proteico che permette alle cellule riceventi di acquisire capacità migratorie e invasive. Infine, attraverso studi di meta-analisi ed ex vivo su biopsie di pazienti, è stato confermato che le molecole arricchite negli esosomi secreti in condizioni di acidità possano rappresentare dei marcatori della progressione del melanoma, convalidando quindi il valore diagnostico e prognostico degli esosomi.Exosomes, nanosized vesicles of endosomal origin, are worldwide recognized for their ability to transfer biological molecules, from cell to cell, crucial for both physiological and pathological processes. Hundred studies have been focused on exosome application also to clinics although biogenesis modalities are under investigation. Therefore, we set up a new effective fluorescent labelling strategy to trace exosome biogenesis and release with the aim to seek in human melanoma cell lines the impact that tumor progression may exert on their secretion and composition. Cells exposed to a fluorescent analogue of palmitic acid (Bodipy FL C16) were able to promptly synthetize fluorescent phospholipids, constituents of exosome membrane bilayer. Afterwards, we were able to follow exosome biogenesis from the intracellular sites of origin to cell secretion, chasing over time by direct cytofluorimetric analysis. To get insight into their function, we focused our studies on exosomes derived from melanoma cells maintained at low pH, which is a microenvironmental leverage for primary tumor to be transformed into widespread metastasis. When melanoma cells at specific intermediate stage were subjected to an acidic microenvironment, showed an increase in exosome release and transfer capability. Most importantly, when control melanoma cells were incubated with exosomes secreted in acidic medium acquired migratory and invasive capacities, demonstrating that exosomes carrying molecular payload can modify recipient cell program. Finally, meta-analysis and ex vivo studies confirmed the importance of acidic exosomes molecule content as marker of melanoma progression and so exosomes prognostic and diagnostic value

Luteinizing Hormone Action in Human Oocyte Maturation and Quality: Signaling Pathways, Regulation, and Clinical Impact

The ovarian follicle luteinizing hormone (LH) signaling molecules that regulate oocyte meiotic maturation have recently been identified. The LH signal reduces preovulatory follicle cyclic nucleotide levels which releases oocytes from the first meiotic arrest. In the ovarian follicle, the LH signal reduces cyclic nucleotide levels via the CNP/NPR2 system, the EGF/EGF receptor network, and follicle/oocyte gap junctions. In the oocyte, reduced cyclic nucleotide levels activate the maturation promoting factor (MPF). The activated MPF induces chromosome segregation and completion of the first and second meiotic divisions. The purpose of this paper is to present an overview of the current understanding of human LH signaling regulation of oocyte meiotic maturation by identifying and integrating the human studies on this topic. We found 89 human studies in the literature that identified 24 LH follicle/oocyte signaling proteins. These studies show that human oocyte meiotic maturation is regulated by the same proteins that regulate animal oocyte meiotic maturation. We also found that these LH signaling pathway molecules regulate human oocyte quality and subsequent embryo quality. Remarkably, in vitro maturation (IVM) prematuration culture (PMC) protocols that manipulate the LH signaling pathway improve human oocyte quality of cultured human oocytes. This knowledge has improved clinical human IVM efficiency which may become a routine alternative ART for some infertile patients

Exosomal and non-exosomal circulatory miRNAs in bovine follicular fluid : potential role of exosomal miRNAs in oocyte development

Growth and development of bovine follicle and oocyte is the result of series of complex and coordinated processes that involves extensive cell-to-cell communication in the follicle. This phenomenon involves enormous complex and heterogeneous biochemical substances existing in the oocytes and its surrounding cells and the follicular fluid. Cell-cell communication within the follicle involves many signaling molecules, and this process may be mediated by secretion and uptake of exosomes that contain several bioactive molecules including extra-cellular miRNAs. The molecular mechanism of oocytes development and interaction between oocyte and follicular cells in the follicular micro-environment remains vague. Follicular fluid and cells from individual follicles of cattle were grouped based on Brilliant Cresyl Blue Pro staining of the corresponding oocytes. Both ExoquickTM precipitation and differential ultracentrifugation were used to separate the exosome and non-exosomal portion of follicular fluid. Following miRNA isolation from both fractions, the human miRCURY LNATM Universal RT miRNA PCR array system was used to profile miRNA expression. Western blot analysis against specific protein and electron microscopy imaging confirms the efficient separation of exosomal and non-exosomal fraction of follicular fluid. The real time qPCR array analysis revealed that a handful number of miRNAs are present in both exosomal and non-exosomal portion of bovine follicular fluid. Results revealed 25 miRNAs differentially expressed (16 up and 9 down) in exosomes and 30 miRNAs differentially expressed (21 up and 9 down) in non-exosomal portion of follicular fluid in comparison of BCB- versus BCB+ oocyte groups. Expression of selected miRNAs was detected in theca, granulosa and cumulus cells that may indicate the origin of extra-cellular miRNAs in follicular fluid. To further explore the potential roles of these extra-cellular miRNAs in follicular fluid, the potential targets were predicted using in silico based analysis, and functional annotation and pathway analysis revealed most of these pathways are known regulators of follicular development and oocyte growth. In order to validate exosome mediated cell-cell communication within follicular microenvironment, we demonstrated uptake of exosomes and resulting increase of endogenous miRNA level and subsequent alteration of mRNA levels in follicular cells in vitro. The present study demonstrates for the first time, the presence of exosome or non-exosome mediated transfer of miRNA in the bovine follicular fluid, and oocyte growth dependent variation in extra-cellular miRNA signatures in the follicular environment.Exosomale und non-exosomale zirkulierende miRNAs in der Follikelflüssigkeit beim Rind: Die Rolle von exosomalen miRNAs in der Oozytenentwicklung Wachstum und Entwicklung der Rinder Follikel und Eizellen ist das Ergebnis einer Reihe von komplexen und koordinierten Prozessen, die umfangreiche Zell-Zell-Kommunikation innerhalb der Follikel beinhaltet. Diese Interaktion beruht auf komplexen und heterogenen biochemischen Stoffen sowohl in den Eizellen, den benachbarten Zellen als auch in der Follikelflüssigkeit. Die Zell-Zell-Kommunikation innerhalb der Follikel ist geprägt durch viele Signalmoleküle. Dieser Prozess kann durch Sekretion und Aufnahme von Exosomen die mehrere bioaktive Moleküle einschließlich extrazellulärer miRNAs enthalten, vermittelt werden. Bisher jedoch, sind die molekularen Mechanismen der Eizellenentwicklung und der Interaktion zwischen Eizellen und Follikelzellen in der follikulären Umgebung noch nicht voll aufgeklärt. Follikelflüssigkeit und Zellen aus einzelnen Rinder Follikeln wurden auf der Grundlage einer Vitalfärbung der entsprechenden Eizellen mittels Brillant Cresyl blau Pro gruppiert. Sowohl eine Exoquick-Präzipitation als auch differentielle Ultrazentrifugation wurde verwendet, um die exosomalen und non-exosomalen Bestandteile der Follikelflüssigkeit zu trennen. Nach miRNA Isolation von beiden Fraktionen wurde mittels des humanen miRCURY LNATMUniversal RT miRNA PCR-Array-System, ein miRNA Expressionsprofil erstellt. Durch Western-Blot-Analysen und Elektronen-Mikroskopie konnte die effiziente Trennung von exosomalen und non-exosomalen Bestandteilen in der Follikelflüssigkeit bestätigt werden. Mittels Echtzeit-qPCR-Array-Analysen konnte einige miRNAs im exosomalen und non-exosomalen Teil der Rinder Follikelflüssigkeit nachgewiesen werden. Von diesen waren 25 miRNAs im exosomalen Teil differentiell exprimiert (16 rauf- und 9 runter reguliert) und 30 miRNAs waren im non-exosomalen Teil der Follikelflüssigkeit differentiell exprimiert (21 rauf- und 9 runter reguliert) im Vergleich zu den BCB- versus BCB+ Oozyten Gruppen. Im Anschluss daran, wurde die Expression ausgewählter miRNAs in Thecazellen, Granulosa- und Kumuluszellen ermittelt. Das Ergebnis könnte ein Hinweis auf die Herkunft der extrazellulären miRNAs in der Follikelflüssigkeit sein. Für eine genauere Erklärung der möglichen Funktion dieser extrazellulären miRNAs in der Follikelflüssigkeit wurden potenziellen Zielgene mit in silico Analyse, funktioneller Annotation und Pathway-Analysen untersucht. Dies ergab, dass die miRNAs überwiegend an Signalwegen der Regulation der follikulären Entwicklung und des Eizellen Wachstums beteiligt sind. Um die Ergebnisse in Bezug auf die exosomal vermittelte Zell-Zell-Kommunikation in der follikulären Mikroumgebung zu überprüfen, konnten wir nachweisen, dass aus der Aufnahme des Exosoms in die Zelle eine Erhöhung des miRNA-Levels mit nachträglicher Veränderung des mRNA-Niveaus von Targetgenen in Follikel-Zellen in vitro resultiert. Die vorliegende Studie zeigt zum ersten Mal, den von exosomalen oder non-exosomalen Bestandteilen vermittelten Transfer von miRNA in die bovine Follikelflüssigkeit. Weiterhin ist das Wachstum der Eizellen von den Variationen der extrazellulären miRNA Signaturen in der follikulären Umgebung abhängig

Performance insights: Site-specific theatre and performance with special reference to Deborah Warner, Peter Brook and Ariane Mnouchkine

This thesis aims to develop a critical vocabulary for dealing with site-specific performances. It focuses on their association with dereliction and decay and assesses the implications of this. A central claim is that these performance modes are best understood in terms of their critical reception. I argue that site-specific performances redefine the language of criticism while profoundly questioning theatre's cultural location. Even in the cases of site-specific performances that flagrantly negate traditional theatre forms, the theatre text and critical frameworks, these return in said performances as fragmented, spectral or unconscious. The thesis divides into two parts. Part 1 deals with the emergence of site-specific performance at the intersection of trends in art and theatre in the 1960s. It outlines the role of decay and the 'found' object/space in creating a genealogy for site-specific performances, while showing how critical writing changed to map this new terrain (Chapter 1). Furthermore, it argues that site-specific performances are characterized by distinctive modes of critical writing, in which the critic is self-reflexive and creative (Chapter 2). Arguing that critics are deeply implicated in the production of site-specific performances, Part 1 ends with a critical and creative reconstruction of Deborah Warner's use of abandoned sites for performances in London in the 1990s (Chapter 3). Part 2 of the thesis re-reads the creation of Peter Brook's Bouffes du Nord and Ariane Mnouchkine's Cartoucherie de Vincennes as site-specific events which were subsequently reinscribed as the defining moment in each theatre's history. I show how site-specificity changed from being a counter-cultural gesture into a constantly redeployed marker of cultural identification. Chapter 4 examines narratives of the discovery of the two theatre venues by their directors and critics, showing how site-specificity is produced at the intersection of individual, cultural and aesthetic discourses. Analysis of the Bouffes du Nord in Chapter 5 charts the critical uses of the theatre's decay, while Chapter 6 views the Cartoucherie as the culmination of the Théâtre du Soleil's quest for group identity through identification with workers

Structural and functional advances in the evolutionary studies of cells and viruses

Phylogenomics aims to describe evolutionary relatedness between organisms by analyzing genomic data. The common practice is to produce phylogenomic trees from molecular information in the sequence, order and content of genes in genomes. These phylogenies describe the evolution of life and have become valuable tools for taxonomy. The recent availability of structural and functional data for hundreds of genomes now offer the opportunity to study evolution using more conserved sets of molecular features. Here we report a phylogenomic (i.e. historical) and comparative (ahistorical) analysis that yields novel insights into the origin of cells (Chapters 1-3) and viruses (Chapters 4-6). We utilized conserved protein domain structure information (fold families [FFs] and fold superfamilies [FSFs]) and ontological definitions of gene products (Gene Ontology [GO]) to reconstruct rooted trees of life (ToL), taking advantage of a genomic census of molecular structure and function in the genomes of sampled organisms and viruses. The analysis revealed a global tendency in the proteomic repertories of cellular organisms to increase domain abundance. ToLs built directly from the census of molecular functions confirmed an early origin of Archaea relative to Bacteria and Eukarya, a conclusion further supported by comparative analysis. The analysis further revealed an ancient history of viruses and their evolution by gene loss. Despite the very high levels of variability seen in the replication strategies, morphologies, and host preferences of extant viruses, we recovered a conserved and ancient structural core of protein domains that was shared between cellular organisms and distantly related viruses. This core together with an analysis of the evolution of virion morphotypes strongly suggests an ancient origin for the viral supergroup. Moreover, a large number of viral proteins lacked cellular homologs and strongly negated the idea that viruses merely evolve by acquiring cellular genes. These virus-specific proteins confer pathogenic abilities to viruses and appeared late in evolution suggesting that the shift to parasitic mode of life happened later in viral evolution. The strong evolutionary association between viruses and cells is likely reminiscent of their ancient co-existence inside primordial cells. Moreover, the crucial dependency of viruses to replicate in an intracellular environment creates fertile grounds for genetic innovation. Interestingly, protein domains shared with viruses were widespread in the proteomes of all three cellular superkingdoms suggesting that viruses mediate gene transfer and crucially enhance biodiversity. The phylogenomic trees identify viruses as a ‘fourth supergroup’ along with cellular superkingdoms, Archaea, Bacteria, and Eukarya. The new model for the origin and evolution of viruses and cells is backed by strong molecular data and is compatible with the existing models of viral evolution. Our experiments indicate that structure and functionomic data represent a useful addition to the set of molecular characters used for tree reconstruction and that ToLs carry in deep branches considerable predictive power to explain the evolution of living organisms and viruses

SemNet: the knowledge representation of lolita

Many systems of Knowledge Representation exist, but none were designed specifically for general purpose large scale natural language processing. This thesis introduces a set of metrics to evaluate the suitability of representations for this purpose, derived from an analysis of the problems such processing introduces. These metrics address three broad categories of question: Is the representation sufficiently expressive to perform its task? What implications has its design on the architecture of the system using it? What inefficiencies are intrinsic to its design? An evaluation of existing Knowledge Representation systems reveals that none of them satisfies the needs of general purpose large scale natural language processing. To remedy this lack, this thesis develops a new representation: SemNet. SemNet benefits not only from the detailed requirements analysis but also from insights gained from its use as the core representation of the large scale general purpose system LOLITA (Large-scale Object-based Linguistic Interactor, Translator, and Analyser). The mapping process between Natural language and representation is presented in detail, showing that the representation achieves its goals in practice

Goal Reasoning: Papers from the ACS workshop

This technical report contains the 11 accepted papers presented at the Workshop on Goal Reasoning, which was held as part of the 2013 Conference on Advances in Cognitive Systems (ACS-13) in Baltimore, Maryland on 14 December 2013. This is the third in a series of workshops related to this topic, the first of which was the AAAI-10 Workshop on Goal-Directed Autonomy while the second was the Self-Motivated Agents (SeMoA) Workshop, held at Lehigh University in November 2012. Our objective for holding this meeting was to encourage researchers to share information on the study, development, integration, evaluation, and application of techniques related to goal reasoning, which concerns the ability of an intelligent agent to reason about, formulate, select, and manage its goals/objectives. Goal reasoning differs from frameworks in which agents are told what goals to achieve, and possibly how goals can be decomposed into subgoals, but not how to dynamically and autonomously decide what goals they should pursue. This constraint can be limiting for agents that solve tasks in complex environments when it is not feasible to manually engineer/encode complete knowledge of what goal(s) should be pursued for every conceivable state. Yet, in such environments, states can be reached in which actions can fail, opportunities can arise, and events can otherwise take place that strongly motivate changing the goal(s) that the agent is currently trying to achieve. This topic is not new; researchers in several areas have studied goal reasoning (e.g., in the context of cognitive architectures, automated planning, game AI, and robotics). However, it has infrequently been the focus of intensive study, and (to our knowledge) no other series of meetings has focused specifically on goal reasoning. As shown in these papers, providing an agent with the ability to reason about its goals can increase performance measures for some tasks. Recent advances in hardware and software platforms (involving the availability of interesting/complex simulators or databases) have increasingly permitted the application of intelligent agents to tasks that involve partially observable and dynamically-updated states (e.g., due to unpredictable exogenous events), stochastic actions, multiple (cooperating, neutral, or adversarial) agents, and other complexities. Thus, this is an appropriate time to foster dialogue among researchers with interests in goal reasoning. Research on goal reasoning is still in its early stages; no mature application of it yet exists (e.g., for controlling autonomous unmanned vehicles or in a deployed decision aid). However, it appears to have a bright future. For example, leaders in the automated planning community have specifically acknowledged that goal reasoning has a prominent role among intelligent agents that act on their own plans, and it is gathering increasing attention from roboticists and cognitive systems researchers. In addition to a survey, the papers in this workshop relate to, among other topics, cognitive architectures and models, environment modeling, game AI, machine learning, meta-reasoning, planning, selfmotivated systems, simulation, and vehicle control. The authors discuss a wide range of issues pertaining to goal reasoning, including representations and reasoning methods for dynamically revising goal priorities. We hope that readers will find that this theme for enhancing agent autonomy to be appealing and relevant to their own interests, and that these papers will spur further investigations on this important yet (mostly) understudied topic

Goal Reasoning: Papers from the ACS Workshop

This technical report contains the 14 accepted papers presented at the Workshop on Goal Reasoning, which was held as part of the 2015 Conference on Advances in Cognitive Systems (ACS-15) in Atlanta, Georgia on 28 May 2015. This is the fourth in a series of workshops related to this topic, the first of which was the AAAI-10 Workshop on Goal-Directed Autonomy; the second was the Self-Motivated Agents (SeMoA) Workshop, held at Lehigh University in November 2012; and the third was the Goal Reasoning Workshop at ACS-13 in Baltimore, Maryland in December 2013