Precise variant calling in the clinical settings

Identifying high quality variants in whole exome sequencing (WES) analysis can be very complex due to the different modifications that can be made in the sample sequencing preparation protocol. This can adversely affect bioinformatics analysis in the identification of variants. The evaluation and correlation of the quality parameters of each analysis stage could help to obtain a better accuracy and precision in the identification of the variants. Furthermore, after identifying high quality variants, the use of reference databases where the clinical significance and frequency of the variants can be consulted, allows for a more accurate diagnosis. During laboratory and bioinformatics analysis, it is possible to calculate many metrics to evaluate the quality of the data being processed. All this data is usually looked at separately and their history is lost over time. Besides, the process of comparing a new workflow to existing ones can be very time-consuming when done manually. In addition, for a significant diagnosis of rare variants, it is important to consider the variant frequency in the sample population. For this reason, a database that incorporates all quality metrics from the entire WES analysis over time and collects population-specific variants for accurate clinical variant identification, is needed. This thesis aims to optimise the evaluation of quality metrics and the classification of variants in the Italian population through the creation of a Structured Query Language (SQL) database directly linked to a website for more intuitive use. The thesis sets out the structure of the database and the configuration of the web page created. Furthermore, during the writing of the thesis, approximately 2,500 exomes were analysed and all quality control parameters derived from both laboratory and bioinformatics analyses were collected. All the data obtained were uploaded to the database in order to verify the usefulness of the application in monitoring data quality trends over time and in identifying possible problems. Two examples of problems identified by the implemented application and subsequently solved by modifications to the laboratory protocol are presented. Moreover, the potential of the database to simplify comparisons between existing and new laboratory protocols storing quality control parameters, is shown. All variants identified in the analysed samples were uploaded to create an accessible reference of genetic variation in Italians. The correct classification of the Italian variants is shown in relation to renowned databases that only report a broader view of the European population. This approach enables researchers to classify variants that are not observed in the most widely used databases (gnomAD Exomes, ExAC, 1KgPhase3). It also allows the identification of rare variants that are generally classified as common and might represent a disease predisposition in the Italian population. In addition, it is possible to recognize common and non-damaging variants in the Italian population that are classified as rare in the European population. In conclusion, the reported results and examples have shown how the new application (extended database with its own website) simplifies and facilitates the identification of problems in clinical WES analysis. It also makes the comparison between the various laboratory protocols easier, allowing for more precision in exome analysis aimed at identifying variants. Finally, the specific investigation of the Italian variants could improve diagnostic accuracy in the specific population

Internationalisation in the household appliances sector in Fabriano (Italy). A case study

This paper analyses the household appliances district of Jesi-Fabriano. It shows how the internationalisation strategies of many enterprises are gradually modifying the organizational structure of the local production system. The diverse goals that have led the internationalisation processes and the strategies chosen by enterprises to effectively approach far away markets are at the core of the paper that includes a broad review of the fragmentation actions of the local enterprises. Special attention is paid to the effects of the internationalisation process on the relationships among actors along the whole filire and on the models of internal enterprise management. After an analysis of the history of the district and its current transformations, the main conclusions focus on possible scenarios regarding external and endogenous factors, on the main opportunities that enterprises should seize in order to successfully complete the ongoing internationalisation process and on the important role of local and national institutions in supporting the evolution of the model of local economic development.Internationalisation, Iinternational Fragmentation, Industrial Districts, Household Appliance Industry

Jazz and Puerto Rican Traditional Music: A New Generation of Musical Exploration

Many musical styles around the world have been born from struggle and search for cultural identity. The same idea applies to Jazz and the Afro-Caribbean music created within the African diaspora and influenced by Latin and European styles in the United States. More recently, the meaning of diaspora has been used to refer to a group of people and some aspects of their culture (Rivera 2010, 104). For older and newer generations of Puerto Rican musicians two traditions have intersected to create a modern form of expression that reaffirms Puerto Rico’s musical creativity and contemporary tendency. In 1989 Warren Pinckney wrote the last known paper about jazz made by Puerto Ricans. Besides Warren Pinckney’s article, Puerto Rican Jazz and the Incorporation of Folk Music: An Analysis of New Musical Direction, (1989) I have not found another paper or essay published specifically about jazz music made by Puerto Ricans. The contribution and innovation of these musicians and rhythms has been overlooked in most cases or even completely neglected in the literature. This thesis will explore the musical influence jazz has had in traditional Puerto Rican music and vice versa. It will also discuss the modernization and revitalization of traditional Puerto Rican music by incorporating jazz, providing a new visualization of Latin American music. The field research undergirding this thesis was driven by three goals: first, to demonstrate the contribution of Puerto Rican musicians to contemporary Jazz and Latin Jazz; second, to determine how movement back and forth between the United States and the island has fueled both jazz and traditional music; and third, to describe how this movement has revitalized and modernized Puerto Rican musical traditions. What has emerged is a story that follows an outline of traditional Puerto Rican music and Jazz tradition being melded together. It is a statement of musical identity that has represented jazz musicians on the island for decades. Some of the best musicians and Jazz music have come from the exchange of Puerto Rican diaspora in the United States and the island. By merging Puerto Rico’s traditional music into the jazz format, new generations of musicians are being exposed to their roots with a contemporary approach. Puerto Rican musicians found jazz music to be a vehicle to express their musical identities. Because Latin jazz music was already sodden with Cuban influences, Puerto Rican jazz musicians decided to branch out and incorporate their own musical traditions, creating something unique

Evolución en la distribución de malezas resistentes y tolerantes a herbicidas en el NO de la Provincia de Buenos Aires. Cuarto informe: período primavera-verano (2017-2018)

En el presente trabajo, correspondiente al período primavera-verano 2017-18, se continuó con el registro de la evolución de la distribución de malezas resistentes y tolerantes a herbicidas en el NO de la Provincia de Buenos Aires. Dentro de cada lote se realizaron inventarios de las malezas presentes por medio de transectas en forma "W" que abarcaron un área representativa del total del lote. Las malezas fueron divididas en tres categorías: especies con biotipos con resistencia comprobada a distintos principios activos de herbicidas, especies tolerantes a glifosato y especies consideradas de difícil control. Tal como en los informes anteriores, el parámetro evaluado fue la frecuencia específica (%) que surge del número de lotes en que la especie se encuentra presente respecto del total de lotes censados. A los efectos de poner de manifiesto la evolución de las especies de mayor frecuencia específica (> 10%), los valores se compararon con aquellos obtenidos en el mismo período para los años 2014-15, 2015-16 y 2016-17.EEA PergaminoFil: Acciaresi, Horacio Abel. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Pergamino. Departamento de Malezas; Argentina.Fil: Lavezzari, Daniel. Universidad Nacional del Noroeste de la Provincia de Buenos Aires; Argentina. Asesor Privado. ; ArgentinaFil: Principiano, Martín. Universidad Nacional del Noroeste de la Provincia de Buenos Aires. Comisión de Investigaciones Científicas de la Provincia de Buenos Aires (Becario CIC-UNNOBA); Argentina

Distribución y abundancia de malezas resistentes y tolerantes a herbicidas en el noroeste de la Prov. de Bs. As.

En el presente trabajo, correspondiente al período primavera-verano 2018-19, se continuó con el registro de la evolución de la distribución de malezas resistentes y tolerantes a herbicidas en el NO de la Provincia de Buenos Aires. Dentro de cada lote se realizaron inventarios de las malezas presentes por medio de transectas en forma de “W” que abarcaron un área representativa del total del lote. Las malezas fueron divididas en tres categorías: especies con biotipos con resistencia comprobada a distintos principios activos de herbicidas, especies tolerantes a glifosato y especies consideradas de difícil controlEEA PergaminoFil: Acciaresi, Horacio. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Pergamino. Malezas; ArgentinaFil: Lavezzari, Daniel. Asesor Privado; ArgentinaFil: Principiano, Martín. Universidad Nacional del Noroeste de la Provicnia de Buenos Aires (UNNOBA). Comisión de Investigaciones Científicas (CIC). Becario; Argentin

Exosomes from Plasma of Neuroblastoma Patients Contain Doublestranded DNA Reflecting the Mutational Status of Parental Tumor Cells

Neuroblastoma (NB) is an aggressive infancy tumor, leading cause of death among preschool age diseases. Here we focused on characterization of exosomal DNA (exo-DNA) isolated from plasma cell-derived exosomes of neuroblastoma patients, and its potential use for detection of somatic mutations present in the parental tumor cells. Exosomes are small extracellular membrane vesicles secreted by most cells, playing an important role in intercellular communications. Using an enzymatic method, we provided evidence for the presence of double-stranded DNA in the NB exosomes. Moreover, by whole exome sequencing, we demonstrated that NB exo-DNA represents the entire exome and that it carries tumor-specific genetic mutations, including those occurring on known oncogenes and tumor suppressor genes in neuroblastoma (ALK, CHD5, SHANK2, PHOX2B, TERT, FGFR1, and BRAF). NB exo-DNA can be useful to identify variants responsible for acquired resistance, such as mutations of ALK, TP53, and RAS/MAPK genes that appear in relapsed patients. The possibility to isolate and to enrich NB derived exosomes from plasma using surface markers, and the quick and easy extraction of exo-DNA, gives this methodology a translational potential in the clinic. Exo-DNA can be an attractive non-invasive biomarker for NB molecular diagnostic, especially when tissue biopsy cannot be easily available

Brain-derived neurotrophic factor and neurotrophin receptors modulate glutamate-induced phase shifts of the suprachiasmatic nucleus

Light information reaches the suprachiasmatic nucleus (SCN) through a subpopulation of retinal ganglion cells. Previous work raised the possibility that brain-derived neurotrophic factor (BDNF) and its high-affinity tropomyosin-related receptor kinase may be important as modulators of this excitatory input into the SCN. In order to test this possibility, we used whole-cell patch-clamp methods to measure spontaneous excitatory currents in mouse SCN neurons. We found that the amplitude and frequency of these currents were increased by BDNF and decreased by the neurotrophin receptor inhibitor K252a. The neurotrophin also increased the magnitude of currents evoked by application of N-methyl-D-aspartate and amino-methyl proprionic acid. Next, we measured the rhythms in action potential discharge from the SCN brain slice preparation. We found that application of K252a dramatically reduced the magnitude of phase shifts of the electrical activity rhythm generated by the application of glutamate. By itself, BDNF caused phase shifts that resembled those produced by glutamate and were blocked by K252a. The results demonstrate that BDNF and neurotrophin receptors can enhance glutamatergic synaptic transmission within a subset of SCN neurons and potentiate glutamate-induced phase shifts of the circadian rhythm of neural activity in the SCN. Originally published European Journal of Neuroscience, Vol. 24, No. 4, Aug 200

c-Myc expression in human anagen hair follicles

The hair follicle represents a very attractive organ system for studying the precise balance between cell proliferation, growth, differentiation, and death of cells, because it periodically and regularly regenerates, retaining its morphogenetic signals throughout its life. One of the most intriguing oncogenes which is able to induce both cell growth and apoptosis, depending upon the environmental conditions, is c-Myc. The aim of the present study was to investigate its presence and localization in human hair follicles by immunohistochemistry and immunofluorescence. Our observations demonstrated the consistent presence of two clusters of c-Myc-expressing cells in anagen follicles, located in two annular regions of the inner root sheath, at the border between cells characterized by putative trichohyalin granules and cells which are keratinized. The lower group belongs to Henle's layer, while the upper group belongs to Huxley's layer. c-Myc oncoprotein seems to favour apoptosis/differentiation and may be a marker for terminal differentiation of trichocytes, at least in the inner root sheath. Our findings agree with the interpretation that the complex morphology of the hair follicle reflects its complex function; the extrusion of a highly organized multicellular structure, the hair shaft, driven by another highly organized multicellular structure, the inner root sheath

The developmental shift of NMDA receptor composition proceeds independently of GluN2 subunit-specific GluN2 C-terminal sequences

The GluN2 subtype (2A versus 2B) determines biophysical properties and signaling of forebrain NMDA receptors (NMDARs). During development, GluN2A becomes incorporated into previously GluN2B-dominated NMDARs. This “switch” is proposed to be driven by distinct features of GluN2 cytoplasmic C-terminal domains (CTDs), including a unique CaMKII interaction site in GluN2B that drives removal from the synapse. However, these models remain untested in the context of endogenous NMDARs. We show that, although mutating the endogenous GluN2B CaMKII site has secondary effects on GluN2B CTD phosphorylation, the developmental changes in NMDAR composition occur normally and measures of plasticity and synaptogenesis are unaffected. Moreover, the switch proceeds normally in mice that have the GluN2A CTD replaced by that of GluN2B and commences without an observable decline in GluN2B levels but is impaired by GluN2A haploinsufficiency. Thus, GluN2A expression levels, and not GluN2 subtype-specific CTD-driven events, are the overriding factor in the developmental switch in NMDAR composition

Mitochondrial ROS control neuronal excitability and cell fate in frontotemporal dementia.

INTRODUCTION: The second most common form of early-onset dementia-frontotemporal dementia (FTD)-is often characterized by the aggregation of the microtubule-associated protein tau. Here we studied the mechanism of tau-induced neuronal dysfunction in neurons with the FTD-related 10+16 MAPT mutation. METHODS: Live imaging, electrophysiology, and redox proteomics were used in 10+16 induced pluripotent stem cell-derived neurons and a model of tau spreading in primary cultures. RESULTS: Overproduction of mitochondrial reactive oxygen species (ROS) in 10+16 neurons alters the trafficking of specific glutamate receptor subunits via redox regulation. Increased surface expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptors containing GluA1 and NR2B subunits leads to impaired glutamatergic signaling, calcium overload, and excitotoxicity. Mitochondrial antioxidants restore the altered response and prevent neuronal death. Importantly, extracellular 4R tau induces the same pathological response in healthy neurons, thus proposing a mechanism for disease propagation. DISCUSSION: These results demonstrate mitochondrial ROS modulate glutamatergic signaling in FTD, and suggest a new therapeutic strategy