Identificazione di nuovi geni che influenzano l?espressione e la localizzazione della poli ADP ribosio polimerasi nel lievito Saccharomyces cerevisiae

Nell’uomo, la Poli (ADP-ribosio) polimerasi-1 (PARP-1) è una proteina a localizzazione prevalentemente nucleare molto abbondante nelle cellule che possiede un'attività catalitica NAD+ dipendente che sintetizza un polimero carico negativamente chiamato poli (ADP-ribosio ) o PAR e lo trasferisce sulle proteine bersaglio. Svolge una serie di attività biochimiche distinte che la rendono ben adattata per i ruoli sia strutturali e regolatori in tutto il genoma. Gioca, infatti, un ruolo chiave in un’ampia varietà di processi che avvengono all’interno del nucleo della cellula, quali la trascrizione e la riparazione del DNA; inoltre, sembra sia coinvolta nel mantenimento della corretta struttura della cromatina. PARP -1 può legarsi a diverse strutture del DNA e ai nucleosomi. Poichè PARP regola la riparazione del DNA, inibitori di PARP sono usati come coadiuvanti nelle terapie antitumorali e antinfiammatorie. In pratica, quando l’attività di PARP è inibita, non si possono più attivare quei pathway che riparano il DNA e quindi, in presenza di danno al DNA la cellula muore per apoptosi. Durante la terapia, è stato osservato che un certo numero di pazienti diventa insensibile a tali inibitori. Alcuni studi hanno proposto vari modelli per spiegare il meccanismo di questa “resistenza”, ma il meccanismo molecolare ed i geni coinvolti non sono ancora completamente chiari. Comunque, un aumentato livello proteico intracellulare o un difetto nella localizzazione nucleare di PARP1 possono essere importanti per l’efficacia degli inibitori di PARP usati in terapia. Uno screening genetico utilizzando un pool di ceppi di lievito deleti per geni non essenziali effettuato nel nostro laboratorio ha permesso di identificare 110 geni che potrebbero interferire con le funzioni di PARP e quindi avere un ruolo nella sensibilità agli inibitori. In questa tesi abbiamo determinato il livello di PARP-1 e la sua localizzazione, nel ceppo BY4743 diploide ed in 6 ceppi di lievito Saccharomyces cerevisiae identificati mediante lo screening come “resistenti” all’ effetto inibitorio sulla crescita dovuto all’espressione di PARP-1. Questi ceppi sono tutti derivati dal parentale diploide BY4743 ed ognuno di questi presenta una delezione specifica in uno dei seguenti geni: GAL3 che codifica per una proteina che regola il metabolismo del galattosio, HHO1 che codifica per l’istone H1, HUL4 che codifica per una proteina con attività ubiquitina ligasica, OTU1 per una proteasi ubiquitina specifica , NUP che codifica per una proteina del poro nulceare e SNT1 per una proteina probabilmente implicata nel legame con il DNA. Queste funzioni potrebbero in qualche modo avere un ruolo nell’ attività di PARP e potrebbero influenzare la sua localizzazione e il livello intracellulare. Inoltre, queste proteine presentano il corrispettivo omologo umano. Lo scopo di questa tesi è quello di identificare nuovi geni che possano avere un effetto sulla risposta agli inibitori di PARP. L’obiettivo finale di questo progetto è quello di individuare nuovi target terapeutici per una terapia più personalizzata.Per prima cosa abbiamo determinato il livello di PARP-1 in tutti e 6 ceppi deleti e per confronto nel ceppo parentale . Esperimenti di Western Blot dimostrano che tutti i ceppi portanti la specifica delezione esprimono PARP-1. I risultati mostrano inoltre che il livello di PARP1 nei ceppi deleti è leggermente inferiore a quello determinato nel ceppo parentale wild type. Successivamente, abbiamo trasformato i tutti i ceppi deleti presi in esame con il plasmide pYES che permette l’espressione di PARP-1 fuso con GFP, in modo tale da osservare, tramite l’utilizzo del microscopio a fluorescenza, la localizzazione di tale proteina all’interno della cellula, e soprattutto vedere se in presenza di danno al DNA varia la sua posizione intracellulare. E’ stato riscontrato soprattutto nei ceppi deleti per HH01 e HUL4 un aumento del numero di cellule rispetto al wild type, in cui PARP1 si localizza nel nucleo, confermando l’ipotesi che tali geni possano interferire con le funzioni di PARP e quindi avere un ruolo nella sensibilità agli inibitori. Tramite l’utilizzo di un anticorpo anti anti-PAR, che permette di visualizzare il polimero di ADP ribosio , sintetizzato da PARP-1 abbiamo dimostrato che i ceppi producono il polimero indicando che le funzioni geniche oggetto di studio non interferiscono sulla attività specifica di PARP.Dalla letteratura è noto che PARP-1 è implicato in modo indiretto nella riparazione dei danni al DNA tramite il pathway del Base Excision Repair (BER). Poiché il ceppo di lievito RS112 contiene un sistema di ricombinazione intercromosomica e intracromosomica, che permette di misurare la frequenza di tali eventi direttamente su piastra abbiamo determinato se l’espressione di PARP-1 influenzasse tale ricombinazione. I risultati mostrano che l’espressione di PARP non influenza il livello di ricombinazione spontanea in lievito. Per determinare l’effetto di PARP sulla ricombinazione indotta danni al DNA sono stati effettuati esperimenti irradiando in ceppo RS112 con diverse dosi di raggi UV. I risultati preliminari indicano che l’espressione di PARP riduce la frequenza di ricombinazione indotta da UV confermando i dati ottenuti su linee cellulari di mammifero. In conclusione, questo lavoro di tesi si dimostra ancora una volta che il lievito Saccharomyces cerevisiae può essere considerato un sistema genetico modello per caratterizzare il meccanismo ed i fattori coinvolti che possono influenzare la risposta a farmaci usati nella terapia antitumorale

Satisfaction with online teaching of medical statistics during the COVID-19 pandemic: A survey by the Education Committee of the Italian Society of Medical Statistics and Clinical Epidemiology

: On May 2020, after 2 months of online teaching with no face-to-face lectures, the Education Committee of the Italian scientific Society of Medical Statistics and Clinical Epidemiology conceived an online survey to assess satisfaction of Italian academics of medical statistics with online teaching and remote exams. This survey highlighted teachers' perceptions as well as opportunities and limitations of online teaching of medical statistics, biostatistics, and epidemiology. Although 61% of Italian academics of medical statistics declared to be favorable to provide online teaching of medical statistics, biostatistics, and epidemiology in the future, we recognize that distance education cannot substitute the unique value of teaching and knowledge exchange that could only be transmitted through a personal interaction between students and teachers. These indications may be useful to improve the quality of the teaching process in the future

Changes in cerebellar output abnormally modulates cortical myoclonus sensorimotor hyperexcitability

Cortical myoclonus is produced by abnormal neuronal discharges within the sensorimotor cortex, as demonstrated by electrophysiology. Our hypothesis is that the loss of cerebellar inhibitory control over the motor cortex, via cerebello-thalamo-cortical connections, could induce the increased sensorimotor cortical excitability that eventually causes cortical myoclonus. To explore this hypothesis, in the present study we applied anodal transcranial direct current stimulation over the cerebellum of patients affected by cortical myoclonus and healthy controls and assessed its effect on sensorimotor cortex excitability. We expected that anodal cerebellar transcranial direct current stimulation would increase the inhibitory cerebellar drive to the motor cortex and therefore reduce the sensorimotor cortex hyperexcitability observed in cortical myoclonus. Ten patients affected by cortical myoclonus of various aetiology and 10 aged-matched healthy controls were included in the study. All participants underwent somatosensory evoked potentials, long-latency reflexes, and short-interval intracortical inhibition recording at baseline and immediately after 20 min session of cerebellar anodal transcranial direct current stimulation. In patients, myoclonus was recorded by the means of surface electromyography before and after the cerebellar stimulation. Anodal cerebellar transcranial direct current stimulation did not change the above variables in healthy controls, while it significantly increased the amplitude of somatosensory evoked potential cortical components, long-latency reflexes and decreased short-interval intracortical inhibition in patients; alongside, a trend towards worsening of the myoclonus after the cerebellar stimulation was observed. Interestingly, when dividing patients in those with and without giant somatosensory evoked potentials, the increment of the somatosensory evoked potential cortical components was observed mainly in those with giant potentials. Our data showed that anodal cerebellar transcranial direct current stimulation facilitates, and does not inhibit, sensorimotor cortex excitability in cortical myoclonus syndromes. This paradoxical response might be due to an abnormal homeostatic plasticity within the sensorimotor cortex, driven by dysfunctional cerebello-thalamo-cortical input to the motor cortex. We suggest that the cerebellum is implicated in the pathophysiology of cortical myoclonus and that these results could open the way to new forms of treatment or treatment targets

El trabajo (labour) digital en la economía de plataforma: el caso de Facebook

El objetivo del trabajo es analizar las características del trabajo (labour) digital conectado con la llamada economía de plataforma. Muchos modelos de negocios basados en plataformas se basan en una nueva composición del capital capaz de capturar información personal y transformarla en big data. Comenzando con el ejemplo del modelo de negocio de Facebook, explicamos el proceso de valorización en el núcleo de capitalismo de plataforma, haciendo hincapié en la relevancia del trabajo digital, para aclarar la distinción crucial entre el trabajo como labour y el mismo como work. Nuestro análisis difiere de la tesis de Fuchs y Sevignani sobre el trabajo digital como labour y el trabajo digital como work y resulta consistente con la idea de que Facebook extrae una renta de la información generada por el trabajo (labour) libre de sus usuarios.The aim of the paper is to analyse the features of the digital labour connected with the so-called platform economy. Many platform-based business models rely on a new composition of capital capable of capturing personal information and transforming it into big data. Starting with the example of the Facebook business model, we explain the valorisation process at the core of platform capitalism, stressing the relevance of digital labour, to clarify the crucial distinction between labour and work. Our analysis differs from Fuchs and Sevignani’s thesis about digital work and digital labour and seems consistent with the idea that Facebook extracts a rent from the information produced by the free labour of its users.O objetivo do trabalho é analisar as características do trabalho digital (trabalho) ligado à chamada economia de plataforma. Muitos modelos de negócios baseados em plataformas são baseados em uma nova composição de capital capaz de capturar informações pessoais e transformá-las em big data. Começando com o exemplo do modelo de negócios do Facebook, explicamos o processo de valorização no núcleo do capitalismo de plataforma, enfatizando a relevância do trabalho digital, para esclarecer a distinção crucial entre trabalho como labour e o mesmo como work. Nossa análise difere da tese de Fuchs e Sevignani sobre trabalho digital como work e trabalho digital como labour e é consistente com a idéia de que o Facebook extrai uma renda da informação gerada pelo trabalho (labour) livre de seus usuários.El presente artículo es una traducción de "Digital Labour in the Platform Economy: The Case of Facebook", publicado por Fumagalli, A.; Lucarelli, S.; Musolino, E; y Rocchi, G., en Sustainability, 2018, 10, 1757. La traducción, a cargo del consejo editorial, fue realizada por Emilio Cafassi, Martina Lasalle, Santiago Liaudat e Ignacio Rocca.Facultad de Trabajo Socia

El trabajo (labour) digital en la economía de plataforma: el caso de Facebook

The aim of the paper is to analyse the features of the digital labour connected with the so-called platform economy. Many platform-based business models rely on a new composition of capital capable of capturing personal information and transforming it into big data. Starting with the example of the Facebook business model, we explain the valorisation process at the core of platform capitalism, stressing the relevance of digital labour, to clarify the crucial distinction between labour and work. Our analysis differs from Fuchs and Sevignani’s thesis about digital work and digital labour and seems consistent with the idea that Facebook extracts a rent from the information produced by the free labour of its users.El objetivo del trabajo es analizar las características del trabajo (labour) digital conectado con la llamada economía de plataforma. Muchos modelos de negocios basados en plataformas se basan en una nueva composición del capital capaz de capturar información personal y transformarla en big data. Comenzando con el ejemplo del modelo de negocio de Facebook, explicamos el proceso de valorización en el núcleo de capitalismo de plataforma, haciendo hincapié en la relevancia del trabajo digital, para aclarar la distinción crucial entre el trabajo como labour y el mismo como work[1]. Nuestro análisis difiere de la tesis de Fuchs y Sevignani sobre el trabajo digital como labour y el trabajo digital como work y resulta consistente con la idea de que Facebook extrae una renta de la información generada por el trabajo (labour) libre de sus usuarios.   [1] [N. de los Traductores] El equipo de traducción de Hipertextos se confronta permanentemente con dificultades relativas a la distinción anglosajona entre work y labour en inglés o werk y arbeit en alemán. Una precisión indispensable que recorre buena parte de los debates de la filosofía, la sociología, la economía política, impregnando además a la historia y la antropología. Distinción que, en suma, tiene un impacto decisivo en las llamadas ciencias sociales, sin que en lenguas latinas se reconozcan inmediatas equivalencias. En efecto, a pesar de que la palabra labour tiene una raíz latina (laborem), cuando se introduce en la lengua inglesa en el siglo XIV, queda ya asociada inequívocamente al sufrimiento, al padecimiento y la aspereza. Como reconoce Raymond Williams, labour designa formas de organización del trabajo bajo relaciones de clase, o en otros términos, relaciones de poder en las que el trabajador no domina y dirige su fuerza de trabajo o es compelido a trabajar por otros. Aquello que Marx, apelando a Hegel, designa con el término “alienación” y entiende la unidad de estas formas de alienación como explotación de la fuerza de trabajo. Como reconoce uno de los traductores de Williams, la traducción como labor resulta insatisfactoria. Otro tanto sucede con la expresión germana arbeit, que deriva del término arba que signfica esclavo. Inversamente tanto Work como werk aluden a la creatividad, a la ejecutividad y si se nos permite la hipótesis antropológica, a la realización humana. Ante la recurrencia nada casual de esta díada contradictoria entre los artículos que la nuestra revista pone a disposición de los lectores de lengua española, el equipo de traducción de Hipertextos, seguirá intentando utilizar un significante en español que mejor se adapte al espíritu del texto, pero indicando entre paréntesis el término originalmente utilizado por los autores.O objetivo do trabalho é analisar as características do trabalho digital (trabalho) ligado à chamada economia de plataforma. Muitos modelos de negócios baseados em plataformas são baseados em uma nova composição de capital capaz de capturar informações pessoais e transformá-las em big data. Começando com o exemplo do modelo de negócios do Facebook, explicamos o processo de valorização no núcleo do capitalismo de plataforma, enfatizando a relevância do trabalho digital, para esclarecer a distinção crucial entre trabalho como labour e o mesmo como work. Nossa análise difere da tese de Fuchs e Sevignani sobre trabalho digital como work e trabalho digital como labour e é consistente com a idéia de que o Facebook extrai uma renda da informação gerada pelo trabalho (labour) livre de seus usuários

Safety at high altitude: the importance of emotional dysregulation on pilots’ risk attitudes during flight

IntroductionAviation psychology is very interested in understanding how personological and psychological variables influence flight performances. Indeed, risk attitudes have been considered as a risk factor for aviation accidents. In this context, emotions and coping style are key variables which could influence concentration by affecting cognition and attention. In addition, the specific training backgrounds seemed to be associated with differences in in-flight accident rates. The aim of the present study was to investigate the association between age, sex, flight experience, emotional dysregulation, coping styles, flight licenses, and pilots’ risk attitudes.MethodsEighty pilots completed an online survey composed of ad hoc questionnaire for sociodemographic and work-related information’s and self-report questionnaires that assessed emotional dysregulation, coping styles, and risk attitudes.ResultsResults showed that older age and emotional dysregulation were associated with higher risk attitudes in pilots. Moreover, emotional dysregulation seemed to promote worse self-confidence. Ultralight pilots appeared to be more risk-oriented and less self-confident than civil pilots, while more flight experience appeared to favorite greater self-confidence.DiscussionIn conclusion, the study suggests the importance of promoting interventions based on sharing pilots’ difficulties and emotions and promoting safe attitudes with special attention to ultralight pilots, age, and sex differences

Identification and characterisation of Gamma-herpesviruses in zoo artiodactyla

Background: Viruses within the γ-herpesviruses subfamily include the causative agents of Malignant Catarrhal Fever (MCF) in several species of the order Artiodactyla. MCF is a usually fatal lymphoproliferative disease affecting non-adapted host species. In adapted host species these viruses become latent and recrudesce and transmit during times of stress or immunosuppression. The undetected presence of MCF-causing viruses (MCFVs) is a risk to non-adapted hosts, especially within non-sympatric zoological collections. This study investigated the presence of MCFVs in six different zoological collections in the UK, to evaluate the presence of subclinical/latent MCFVs in carrier animals. Methods: One-hundred and thirty eight samples belonging to 54 different species of Artiodactyla were tested by Consensus Pan-herpes PCR. The positive samples were sequenced and subjected to phylogenetic analyses to understand their own evolutionary relationships and those with their hosts. Results: Twenty-five samples from 18 different species tested positive. All viruses but one clustered in the γ-herpesvirus family and within the Macavirus as well as the non-Macavirus groups (caprinae and alcelaphinae/hippotraginae clusters, respectively). A strong association between virus and host species was evident in the Macavirus group and clustering within the caprinae group indicated potential pathogenicity. Conclusion: This study shows the presence of pathogenic and non-pathogenic MCFVs, as well as other γ-herpesviruses, in Artiodactyla species of conservation importance and allowed the identification of new herpesviruses in some non-adapted species

How socio-institutional contexts and cultural worldviews relate to COVID-19 acceptance rates: A representative study in Italy

Rationale Despite its importance to counter the COVID-19 pandemic, vaccination has raised hesitation in large segments of the population. This hesitation makes it important to understand the mechanisms underlying vaccine acceptance. To this end, the study adopts the Semiotic Cultural Psychology Theory, holding that social behaviors – and therefore, vaccination acceptance – depend on the cultural meanings in terms of which people interpret the social world. Objective The study aims at estimating the impact a) of the way people interpret the socio-institutional context of the pandemic and b) of the underlying cultural worldviews on vaccine acceptance. More particularly, the study tested the three following hypotheses. a) The meanings grounding the interpretation of the socio-institutional framework – that is, trust in institutions and political values – are an antecedent of vaccination acceptance. b) The impact of these meanings is moderated by the cultural worldviews (operationalized as symbolic universes). And c), the magnitude of the symbolic universes’ moderator effect depends on the uncertainty to which the respondent is exposed. The exposure to uncertainty was estimated in terms of socioeconomic status – the lower the status, the high the exposure to uncertainty. Methods An Italian representative sample (N = 3020) completed a questionnaire, measuring vaccination acceptance, the meanings attributed to the socio-institutional context – that is, political values and trust in institutions – and symbolic universes. Results The findings were consistent with the hypotheses. a) Structural equation modelling proved that vaccine acceptance was predicted by trust in institutions. b) Multigroup analysis revealed that symbolic universes moderated the correlation between trust in institutions and vaccine acceptance. And c), the moderation effect of symbolic universes proved to occur only in the segment of lower socio-economic status (i.e., the group exposed to higher uncertainty). Conclusions Vaccination acceptance is not only a medical issue; it is also dependent upon the rationalization of the socio-institutional context. Implications for the promotion of vaccination acceptance are discussed

Refinement of Bos taurus sequence assembly based on BAC-FISH experiments

<p>Abstract</p> <p>Background</p> <p>The sequencing of the cow genome was recently published (Btau_4.0 assembly). A second, alternate cow genome assembly (UMD2), based on the same raw sequence data, was also published. The two assemblies have been subsequently updated to Btau_4.2 and UMD3.1, respectively.</p> <p>Results</p> <p>We compared the Btau_4.2 and UMD3.1 alternate assemblies. Inconsistencies were grouped into three main categories: (i) DNA segments showing almost coincidental chromosomal mapping but discordant orientation (inversions); (ii) DNA segments showing a discordant map position along the same chromosome; and (iii) sequences present in one chromosomal assembly but absent in the corresponding chromosome of the other assembly. The latter category mainly consisted of large amounts of scaffolds that were unassigned in Btau_4.2 but successfully mapped in UMD3.1. We sampled 70 inconsistencies and identified appropriate cow BACs for each of them. These clones were then utilized in FISH experiments on cow metaphase or interphase nuclei in order to disambiguate the discrepancies. In almost all instances the FISH results agreed with the UMD3.1 assembly. Occasionally, however, the mapping data of both assemblies were discordant with the FISH results.</p> <p>Conclusions</p> <p>Our work demonstrates how FISH, which is assembly independent, can be efficiently used to solve assembly problems frequently encountered using the shotgun approach.</p