PhytoREF: a reference database of the plastidial 16S rRNA gene of photosynthetic eukaryotes with curated taxonomy

Photosynthetic eukaryotes have a critical role as the main producers in most ecosystems of the biosphere. The ongoing environmental metabarcoding revolution opens the perspective for holistic ecosystems biological studies of these organisms, in particular the unicellular microalgae that often lack distinctive morphological characters and have complex life cycles. To interpret environmental sequences, metabarcoding necessarily relies on taxonomically curated databases containing reference sequences of the targeted gene (or barcode) from identified organisms. To date, no such reference framework exists for photosynthetic eukaryotes. In this study, we built the PhytoREF database that contains 6490 plastidial 16S rDNA reference sequences that originate from a large diversity of eukaryotes representing all known major photosynthetic lineages. We compiled 3333 amplicon sequences available from public databases and 879 sequences extracted from plastidial genomes, and generated 411 novel sequences from cultured marine microalgal strains belonging to different eukaryotic lineages. A total of 1867 environmental Sanger 16S rDNA sequences were also included in the database. Stringent quality filtering and a phylogeny-based taxonomic classification were applied for each 16S rDNA sequence. The database mainly focuses on marine microalgae, but sequences from land plants (representing half of the PhytoREF sequences) and freshwater taxa were also included to broaden the applicability of PhytoREF to different aquatic and terrestrial habitats. PhytoREF, accessible via a web interface (http://phytoref.fr), is a new resource in molecular ecology to foster the discovery, assessment and monitoring of the diversity of photosynthetic eukaryotes using high-throughput sequencing.

PhytoREF: a reference database of the plastidial 16S rRNA gene of photosynthetic eukaryotes with curated taxonomy

Photosynthetic eukaryotes have a critical role as the main producers in most ecosystems of the biosphere. The ongoing environmental metabarcoding revolution opens the perspective for holistic ecosystems biological studies of these organisms, in particular the unicellular microalgae that often lack distinctive morphological characters and have complex life cycles. To interpret environmental sequences, metabarcoding necessarily relies on taxonomically curated databases containing reference sequences of the targeted gene (or barcode) from identified organisms. To date, no such reference framework exists for photosynthetic eukaryotes. In this study, we built the PhytoREF database that contains 6490 plastidial 16S rDNA reference sequences that originate from a large diversity of eukaryotes representing all known major photosynthetic lineages. We compiled 3333 amplicon sequences available from public databases and 879 sequences extracted from plastidial genomes, and generated 411 novel sequences from cultured marine microalgal strains belonging to different eukaryotic lineages. A total of 1867 environmental Sanger 16S rDNA sequences were also included in the database. Stringent quality filtering and a phylogeny-based taxonomic classification were applied for each 16S rDNA sequence. The database mainly focuses on marine microalgae, but sequences from land plants (representing half of the PhytoREF sequences) and freshwater taxa were also included to broaden the applicability of PhytoREF to different aquatic and terrestrial habitats. PhytoREF, accessible via a web interface (http://phytoref.fr), is a new resource in molecular ecology to foster the discovery, assessment and monitoring of the diversity of photosynthetic eukaryotes using high-throughput sequencing.

Applicazione di un veloce metodo di estrazione del DNA per l’identificazione di prodotti ittici marini.

L'identificazione dei prodotti ittici è uno dei temi chiave in materia di sicurezza alimentare. L’errata etichettatura dei prodotti alimentari e la sostituzione di alcuni ingredienti rappresentano questioni emergenti in termini di qualità e sicurezza alimentare e nutrizionale. L'autenticazione e la tracciabilità dei prodotti alimentari, gli studi di tassonomia e di genetica di popolazione, così come l'analisi delle abitudini alimentari degli animali e la selezione delle prede, si basano su analisi genetiche tra cui la metodica molecolare del DNA barcoding, che consiste nell’amplificazione e nel sequenziamento di una specifica regione del gene mitocondriale chiamata COI. Questa tecnica biomolecolare è utilizzata per fronteggiare la richiesta di determinazione specifica e/o la reale provenienza dei prodotti commercializzati, nonché per smascherare errori di etichettatura e sostituzioni fraudolente, difficile da rilevare soprattutto nei prodotti ittici trasformati. Sul mercato sono disponibili differenti kit per l'estrazione del DNA da campioni freschi e conservati; l’impiego dei kit, aumenta drasticamente il costo dei progetti di caratterizzazione e di genotipizzazione dei campioni da analizzare. In questo scenario è stato messo a punto un metodo veloce di estrazione del DNA. Esso non prevede nessuna fase di purificazione per i prodotti ittici freschi e trasformati e si presta a qualsiasi analisi che preveda l’utilizzo della tecnica PCR. Il protocollo consente l'amplificazione efficiente del DNA da qualsiasi scarto industriale proveniente dalla lavorazione del pesce, indipendentemente dal metodo di conservazione del campione. L’applicazione di questo metodo di estrazione del DNA, combinato al successo e alla robustezza della amplificazione PCR (secondo protocollo barcode) ha permesso di ottenere, in tempi brevissimi e con costi minimi, il sequenziamento del DNA.

Viivakoodien ja RFID-tunnisteiden vertailu Fetek Oy:n käyttöön

Työn tavoitteena oli vähentää levyaihioiden hakuun käytettävää aikaa varastossa. Tutkimus perustuu kirjallisuuteen ja tieteellisiin lähteisiin. Fetek Oy:n levyvarastoon soveltuvaksi tunnistusjärjestelmäksi osoittautui viivakooditekniikkaan perustuva QR-koodi. QR-koodien valmistus onnistuu yrityksessä ja koodiin saadaan mahtumaan kaikki tarvittava tieto levyistä. Koodien lukeminen onnistuu työpuhelimella tai erillisellä lukijalla. Koodien käyttöönotto ja päivittäminen oli kustannustehokkain ratkaisu yrityksen tarpeisiin. Olemassa olevaa varastoa myös päätettiin selkeyttää käyttämällä väritunnistusta hyllyissä ja ottamalla käyttöön LEAN 5S. Värikoodaus nopeuttaa tunnistusta huomattavasti ja LEAN 5S puolestaan auttaa pitämään tuotantotilat aina järjestyksessä. Jatkotutkimuksessa tulisi parantaa tuotantotilojen järjestelyä ja miettiä yrityksen käytössä olevan varastonhallintaohjelmiston soveltuvuutta.

DNA Barcoding as a tool for Zoological Taxonomy: Identification of bony fish in the Mediterranean Sea

The description of all the species present in nature is a vast task to be fulfilled by using the classical approach of morphological description of the organisms. In recent years, the traditional taxonomy, based primarily on identification keys of species, has shown a number of limitations in the use of the distinctive features in many animal taxa and inconsistencies with the genetic data. Furthermore, the increasing need to get a true estimate of biodiversity has led Zoological Taxonomy to seek new approaches and methodologies to support the traditional methods. The classification procedure has added modern criteriasuch as the evolutionary relationships and the genetic, biochemical and morphological characteristics of the organisms.Until now the Linnean binomial was the only abbreviated code associated with the description of the morphology of a species. The new technologies aim to achieve a short nucleotide sequence of the DNA to be used as an unique and solely label for a particular species, a specific genetic barcode. For both morphological and genetic approaches, skills and experience are required. Taxonomy is one of zoological disciplines that has been benefited from the achievements reached by modern molecular biotechnology. Using a molecular approach it is possible to identify cryptic species, to establish a family relationship between species and their membership of taxonomic categories or to reconstruct the evolutionary history of a taxon.

Development of a simple and fast “DNA extraction kit” for sea food identification and marine species

Seafood products fraud, the misrepresentation of them, have been discovered all around the world in different forms as false labeling, species substitution, short-weighting or over glazing in order to hide the correct identity, origin or weight of the seafood products. Due to the value of seafood products such as canned tuna, swordfish or grouper, these species are the subject of the commercial fraud is mainly there placement of valuable species with other little or no value species. A similar situation occurs with the shelled shrimp or shellfish that are reduced into pieces for the commercialization. Food fraud by species substitution is an emerging risk given the increasingly global food supply chain and the potential food safety issues. Economic food fraud is committed when food is deliberately placed on the market, for financial gain deceiving consumers (Woolfe, M. & Primrose, S. 2004). As a result of the increased demand and the globalization of the seafood supply, more fish species are encountered in the market. In this scenary, it becomes essential to unequivocally identify the species. The traditional taxonomy, based primarily on identification keys of species, has shown a number of limitations in the use of the distinctive features in many animal taxa, amplified when fish, crustacean or shellfish are commercially transformed. Many fish species show a similar texture, thus the certification of fish products is particularly important when fishes have undergone procedures which affect the overall anatomical structure, such as heading, slicing or filleting (Marko et al., 2004). The absence of morphological traits, a main characteristic usually used to identify animal species, represents a challenge and molecular identification methods are required. Among them, DNA-based methods are more frequently employed for food authentication (Lockley & Bardsley, 2000). In addition to food authentication and traceability, studies of taxonomy, population and conservation genetics as well as analysis of dietary habits and prey selection, also rely on genetic analyses including the DNA barcoding technology (Arroyave & Stiassny, 2014; Galimberti et al., 2013; Mafra, Ferreira, & Oliveira, 2008; Nicolé et al., 2012; Rasmussen & Morrissey, 2008), consisting in PCR amplification and sequencing of a COI mitochondrial gene specific region. The system proposed by P. Hebert et al. (2003) locates inside the mitochondrial COI gene (cytochrome oxidase subunit I) the bioidentification system useful in taxonomic identification of species (Lo Brutto et al., 2007). The COI region, used for genetic identification - DNA barcode - is short enough to allow, with the current technology, to decode sequence (the pairs of nucleotide bases) in a single step. Despite, this region only represents a tiny fraction of the mitochondrial DNA content in each cell, the COI region has sufficient variability to distinguish the majority of species among them (Biondo et al. 2016). This technique has been already employed to address the demand of assessing the actual identity and/or provenance of marketed products, as well as to unmask mislabelling and fraudulent substitutions, difficult to detect especially in manufactured seafood (Barbuto et al., 2010; Galimberti et al., 2013; Filonzi, Chiesa, Vaghi, & Nonnis Marzano, 2010). Nowadays,the research concerns the use of genetic markers to identify not only the species and/or varieties of fish, but also to identify molecular characters able to trace the origin and to provide an effective control tool forproducers and consumers as a supply chain in agreementwith local regulations.

Microfluidics-based single-cell functional proteomics microchip for portraying protein signal transduction networks within the framework of physicochemical principles, with applications in fundamental and translational cancer research

Single-cell functional proteomics assays can connect genomic information to biological function through quantitative and multiplex protein measurements. Tools for single-cell proteomics have developed rapidly over the past 5 years and are providing unique opportunities. This thesis describes an emerging microfluidics-based toolkit for single cell functional proteomics, focusing on the development of the single cell barcode chips (SCBCs) with applications in fundamental and translational cancer research.

The microchip designed to simultaneously quantify a panel of secreted, cytoplasmic and membrane proteins from single cells will be discussed at the beginning, which is the prototype for subsequent proteomic microchips with more sophisticated design in preclinical cancer research or clinical applications. The SCBCs are a highly versatile and information rich tool for single-cell functional proteomics. They are based upon isolating individual cells, or defined number of cells, within microchambers, each of which is equipped with a large antibody microarray (the barcode), with between a few hundred to ten thousand microchambers included within a single microchip. Functional proteomics assays at single-cell resolution yield unique pieces of information that significantly shape the way of thinking on cancer research. An in-depth discussion about analysis and interpretation of the unique information such as functional protein fluctuations and protein-protein correlative interactions will follow.

The SCBC is a powerful tool to resolve the functional heterogeneity of cancer cells. It has the capacity to extract a comprehensive picture of the signal transduction network from single tumor cells and thus provides insight into the effect of targeted therapies on protein signaling networks. We will demonstrate this point through applying the SCBCs to investigate three isogenic cell lines of glioblastoma multiforme (GBM).

The cancer cell population is highly heterogeneous with high-amplitude fluctuation at the single cell level, which in turn grants the robustness of the entire population. The concept that a stable population existing in the presence of random fluctuations is reminiscent of many physical systems that are successfully understood using statistical physics. Thus, tools derived from that field can probably be applied to using fluctuations to determine the nature of signaling networks. In the second part of the thesis, we will focus on such a case to use thermodynamics-motivated principles to understand cancer cell hypoxia, where single cell proteomics assays coupled with a quantitative version of Le Chatelier's principle derived from statistical mechanics yield detailed and surprising predictions, which were found to be correct in both cell line and primary tumor model.

The third part of the thesis demonstrates the application of this technology in the preclinical cancer research to study the GBM cancer cell resistance to molecular targeted therapy. Physical approaches to anticipate therapy resistance and to identify effective therapy combinations will be discussed in detail. Our approach is based upon elucidating the signaling coordination within the phosphoprotein signaling pathways that are hyperactivated in human GBMs, and interrogating how that coordination responds to the perturbation of targeted inhibitor. Strongly coupled protein-protein interactions constitute most signaling cascades. A physical analogy of such a system is the strongly coupled atom-atom interactions in a crystal lattice. Similar to decomposing the atomic interactions into a series of independent normal vibrational modes, a simplified picture of signaling network coordination can also be achieved by diagonalizing protein-protein correlation or covariance matrices to decompose the pairwise correlative interactions into a set of distinct linear combinations of signaling proteins (i.e. independent signaling modes). By doing so, two independent signaling modes – one associated with mTOR signaling and a second associated with ERK/Src signaling have been resolved, which in turn allow us to anticipate resistance, and to design combination therapies that are effective, as well as identify those therapies and therapy combinations that will be ineffective. We validated our predictions in mouse tumor models and all predictions were borne out.

In the last part, some preliminary results about the clinical translation of single-cell proteomics chips will be presented. The successful demonstration of our work on human-derived xenografts provides the rationale to extend our current work into the clinic. It will enable us to interrogate GBM tumor samples in a way that could potentially yield a straightforward, rapid interpretation so that we can give therapeutic guidance to the attending physicians within a clinical relevant time scale. The technical challenges of the clinical translation will be presented and our solutions to address the challenges will be discussed as well. A clinical case study will then follow, where some preliminary data collected from a pediatric GBM patient bearing an EGFR amplified tumor will be presented to demonstrate the general protocol and the workflow of the proposed clinical studies.

DETEÇÃO DE FUNGOS ENDOFÍTICOS EM FOLHAS DE OLIVEIRA INFETADAS COM OLHO-DE-PAVÃO

Os fungos endofíticos, que colonizam naturalmente os tecidos de plantas, têm vindo a crescer de importância em termos de pesquisa e aplicações no âmbito da proteção das plantas. Todavia, a sua presença nos tecidos da oliveira ainda permanece bastante desconhecida. Dessa forma, com o objetivo de identificar e relacionar a presença de fungos endofíticos com o fungo patogénico Spilocaea oleaginea, agente causal da doença olho-de-pavão na oliveira, durante a primavera e verão de 2013 colheram-se folhas das cultivares Cobrançosa, Picual e Galega, que apresentavam lesões características da presença da doença, em quatro olivais localizados em diferentes regiões do Alentejo. Após a extração do DNA total e subsequente PCR com primers universais para a região ITS (internal transcribed spacer), foram sequenciados e analisados dez clones de cada cultivar. De acordo com as bases de dados Fungal Barcode e NCBI foi identificada a presença de fungos endofíticos pertencentes a oito famílias, nomeadamente, Botryosphaeriaceae, Dothioraceae, Leptosphaeriaceae, Mycosphaerellaceae, Phaeosphaeriaceae, Pleosporaceae, Sclerotiniaceae e Venturiaceae.

Analisi e standardizzazione del ciclo commessa dei materiali in conto lavoro: il caso HP Composites S.p.A.

HP Composites, azienda leader nella produzione di componenti in fibra di carbonio, a seguito della crescita di ordini commissionati dai suoi clienti, ha deciso di affidare a dei fornitori la produzione di alcune commesse, in alcune o in tutte le fasi del loro ciclo. L’elaborato di tesi nasce da un’esperienza di tirocinio nell’ufficio pianificazione di HP Composites, e si pone l’obiettivo di analizzare tutte le attività che, evitando di dedicare risorse alle lavorazioni selezionate, deve compiere per supportare il fornitore; l’analisi è stata effettuata tramite la costruzione di un diagramma di flusso e fornendo un indicazione del tempo richiesto da ogni attività, per poi poi avanzare delle proposte di ottimizzazione. Il ciclo commessa può essere suddiviso in quattro fasi: scelta della commessa e selezione del fornitore, attivazione del fornitore, gestione settimanale dell’evasione degli ordini e gestione del fine commessa. L’ufficio acquisti nel tempo si è specializzato individuando i temi da chiarire prima di attivare la fornitura per evitare l’insorgere di problematiche a produzione già attivata. Per quanto riguarda la gestione settimanale del fornitore, è stato istituito il ruolo del responsabile delle esternalizzazioni, una figura che funga da intermediario con il fornitore e monitori la produzione esterna: questa figura apporta un'alta quantità di interazioni, ma HP valuta maggiori i vantaggi di questa gestione centralizzata. Inoltre nel tempo le attività pianificazione sono state ottimizzate grazie alla stesura di file precompilati da aggiornare. Un’attività fonte di inefficienza è quella dell’emissione dei documenti di trasporto, soprattutto nel caso in cui il compito di consegnare il prodotto al cliente è deputato al fornitore di conto lavoro: HP Composites ha intenzione di implementare un lettore barcode per l’emissione del documento che ridurrebbe i tempi richiesti dall’attività e permetterebbe una gestione del conto lavoro a sistema più corretta.

PickingList App: progettazione ed implementazione di un’applicazione mobile per magazzini ospedalieri

Questo documento illustra la progettazione e lo sviluppo di un’applicazione Android in ambito aziendale. L’idea è stata concepita dall’azienda Vettore Rinascimento, la quale richiesta era d’implementare un’applicazione per la gestione di liste di prelievo in magazzini di strutture mediche. Lo scopo è di rendere il lavoro manuale più efficiente e affidabile aumentando così le prestazioni dei dipendenti. La tesi analizza le fasi tipiche di sviluppo di un software ovvero: l’analisi dei requisiti, la progettazione, la realizzazione e il collaudo. Durante ciascuna fase saranno analizzate anche le scelte progettuali e implementative. La discussione non si limiterà al solo sviluppo di un’applicazione mobile: siccome il prodotto sviluppato non sarà un software stand-alone, verrà anche spiegato il funzionamento del server preesistente e dello sviluppo di un’api per caricare e salvare i dati. Si presenterà inoltre il prodotto finale con tutte le sue features, alcune delle più interessanti sono la sua capacità di funzionare sia online che offline, di adattarsi a ogni tipo di magazzino, previa configurazione, e di poter scannerizzare i codici a barre degli articoli da prelevare con la fotocamera del dispositivo.