Studio in vitro dell'interazione tra DNA di TTV e Toll-like receptor 9

Nel 1997 nel siero di un paziente con epatite post-trasfusionale criptogenica fu scoperto un nuovo virus chiamato TorqueTenoVirus (TTV), prototipo del nuovo genere Anellovirus. Il virione di TTV risulta di piccole dimensioni, privo di involucro esterno, con un genoma a singolo filamento di DNA circolare a polarità negativa. Ad oggi TTV è ancora un virus orfano dato che gli studi sul suo ruolo patogeno sono stati inconcludenti anche perché resi particolarmente difficili dall’elevata prevalenza di TTV nella popolazione generale e dalla frequente cronicizzazione dell’infezione. Tuttavia, visto che TTV infetta cellule mononucleate del sangue periferico attivate e che, in bambini affetti da patologie respiratorie, i titoli del virus correlano inversamente con la percentuale di linfociti T circolanti e positivamente quella dei linfociti B, sembra plausibile che TTV sia in grado di modulare la risposta immune. Lo scopo che la tesi si propone è quello di indagare le relazioni che TTV instaura con il sistema immunitario dell’ospite. A tal fine è stata studiata l’interazione tra il DNA di TTV e il Toll-like 9 (TLR9), membro della famiglia dei TLRs, recettori fondamentali per la clearance dei patogeni attraverso l’immunità innata e acquisita. In particolare, il TLR9 riconosce specifici motivi CpG non metilati fortemente presenti a livello dei genomi virali. Per questo studio è stato utilizzato un sistema “in vitro” che permette la determinazione dell’espressione genica e della produzione di citochine proinfiammatorie (IL-12, IL-6, IL-10 e IFN-gamma) da parte di cellule di milza di topo, dopo stimolazione con il DNA di TTV. Il substrato cellulare, scelto in quanto particolarmente ricco di TLR9, è stato stimolato, oltre che col DNA virale, anche con altri induttori fra cui ODN sintetici contenenti motivi CpG e concanavalina A. I risultati hanno dimostrato come il DNA di TTV sia in grado di indurre incrementi statisticamente significativi di alcune citichine, in particolare di IL-6 e INF-gamma. Da sottolineare come l'incremento d'espressione delle citochine sia dipendente dalle quantità di DNA virale utilizzato. Ciò ha fatto ipotizzare, nell'infezione naturale, un ruolo fondamentale della carica virale. Per confermare i risultati ottenuti son state dosate le citochine prodotte nel sovranatante delle cellule (IL-6, IL-10, INF-gamma). Tale dosaggio ha mostrato un netto incremento di INF-gamma e di IL-6 ma non di IL-4 a seguito della stimolazione con DNA di TTV e/o con ODN, dimostrando come ad un'aumentata espressione del gene corrisponda la produzione della proteina. E' stato inoltre comprovato il coinvolgimento del recettore TLR9 nell'induzione alla produzione di citochine utilizzando la clorochina che opera un blocco selettivo del recettore. I risultati di tale esperimento mostrano come le cellule di milza di topo, stimolate con il DNA di TTV e con ODN, in seguito a pre-tarttamento con clorochina, riducono quasi totalmente l'espressione dell'INF-gamma. Tale effetto invece non è stato osservato quando le stesse cellule, in presenza di clorochina, sono trattate con Con A. Il passo successivo è stato quello di confermare il dato con un metodo più specifico, ossia il silenziamento genico del TLR9, ottenuto mediante la strategia degli RNA- interference. I risultati confermano il dato precedente, indicando chiaramente come la produzione di citochine, sia mediata dall'interazione del DNA di TTV con il recettore Toll-like

Forest Area Changes in Cinque Terre National Park in the Last 80 Years. Consequences on Landslides and Forest Fire Risks

Cinque Terre, one of the most important Italian cultural landscapes, has not been spared from depopulation and agricultural abandonment processes, that involved many rural areas in Europe, as a consequence of socio-economic transformations that occurred after WWII. Depopulation of rural areas, especially in mountains or in terraced areas, caused significant environmental consequences, such as the decrease of biodiversity, the landscape homogenization, the increase of hydrogeological and forest fires risks. Cinque Terre National Park (5TNP) was established in 1999, and, differently from other Italian National Parks, not just for protecting natural habitats, but mainly to preserve, restore and valorize the historical terraced landscape. Moreover, the area is a UNESCO cultural landscape site and it is partly protected by three Sites of Community Importance. The research intended to investigate the transformations that have affected forested areas inside the 5TNP in the period 1936–2018, also highlighting the connections with hydrogeological and forest fires risks, as a support for the Park planning strategies and the conservation of the UNESCO site. Results highlighted that 37% of the current forests are the consequence of dry stones terraces abandonment that occurred in the twentieth century, with negative effects on the stability of steep slopes, hydrogeological risk, forest fires and on the conservation of a unique cultural landscape. This confirms the current national trend showing no deforestation occurring, but rather a continuous increase of forests on abandoned land. While 5TNP policies and actions are effectively aimed at pursuing an equilibrium between cultivated areas and forests, the Sites of Community Importance located inside the Park mainly focuses on the conservation of “natural habitats”, even if the current vegetation is also the result of secondary successions on former cultivated land. The research highlighted the need to valorize “cultural values” in forest planning as well as the importance of forest history for an accurate planning of forest resources in protected areas

Can an Investigation of a Single Gene be Effective in Differentiating Certain Features of the Bipolar Disorder Profile?

Bipolar disorder (BD) is amongst the most common heritable mental disorders, but the clarification of its genetic roots has proven to be very challenging. Many single nucleotide polymorphisms (SNPs) have been identified to be associated with BD. SNPs in the CACNA1C gene have emerged as the most significantly associated with the disease. The aim of the present study is to provide a concise description of SNP 1006737 variants identified by Real Time PCR and confirm sequencing analysis with the Sanger method in order to estimate the association with BD. The molecular method was tested on 47 Sardinian subjects of whom 23 were found to not be mutated, 1 was found to be a carrier of the homozygous A allele and 23 were found to be carriers of the heterozygous G allele. Moreover, the positive results of the preliminary application suggest that the development of the screener could be extended to the other 5 genetic variables identified as associated with BD

Assessment of Tuscany Landscape Structure According to the Regional Landscape Plan Partition

The landscape is considered a strategic asset by the Tuscan regional government, also for its economic role, meaning that a specific Landscape Plan has been developed, dividing the region into 20 Landscape Units and representing the main planning instrument at the regional level. Following the aims of the Landscape Plan and the guidelines of the European Landscape Convention, it is necessary to develop an adequate assessment of the landscape, evaluating the main typologies and their characteristics. The aim of this research is to carry out an assessment of the landscape diversity in Tuscany based on 20 study areas, analyzing land uses and landscape mosaic structures through the application of landscape metrics: number of land uses, mean patch size (MPS), Hill’s diversity number, edge density (ED), patch density (PD), land use diversity (LUD). The results highlight a correlation between the landscape typologies (forest, agricultural, mixed, periurban) and the complexity of the landscape structure, especially in relation to MPS and PD, while the combination of PD and LUD calculated on the basis of a hexagonal grid allows obtaining landscape complexity maps. Despite the phenomena of reforestation and urban sprawl of recent decades, Tuscany still preserves different landscape typologies characterized by a good level of complexity. This is particularly evident in mixed landscapes, while agricultural landscapes have a larger variability because of different historical land organization forms. The methodology applied in this study provided a large amount of data about land uses and the landscape mosaic structure and complexity and proved to be effective in assessing the landscape structure and in creating a database that can represent a baseline for future monitoring

Usefulness of salivary sampling for the molecular detection of a genetic variant associated with bipolar disorders

Under certain conditions, the hyperthymic temperament traits associated with an increased risk of developing bipolar disorders may in fact produce adaptive responses. The purpose of this study is to see if the type of biological material used for genetic analysis (saliva or blood) affects the detection of mutations in the CACNA1C (RS1006737) gene. The first experimental group consisted of Sardinian migrants (“volunteers”) in South American and European megacities. The second experimental group consisted of older healthy subjects with hyperactivity and novelty-seeking characteristics from Cagliari, Italy. The genetic procedure included DNA extraction, real-time PCR, and the Sanger method. Nonetheless, the authors believe that saliva is the most appropriate biological material, given its many advantages. In contrast to blood, saliva can be collected by any type of healthcare provider after following a few simple instructions

Glutamine Starvation Affects Cell Cycle, Oxidative Homeostasis and Metabolism in Colorectal Cancer Cells

Cancer cells adjust their metabolism to meet energy demands. In particular, glutamine addiction represents a distinctive feature of several types of tumors, including colorectal cancer. In this study, four colorectal cancer cell lines (Caco-2, HCT116, HT29 and SW480) were cultured with or without glutamine. The growth and proliferation rate, colony-forming capacity, apoptosis, cell cycle, redox homeostasis and metabolomic analysis were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test (MTT), flow cytometry, high-performance liquid chromatography and gas chromatography/mass spectrometry techniques. The results show that glutamine represents an important metabolite for cell growth and that its deprivation reduces the proliferation of colorectal cancer cells. Glutamine depletion induces cell death and cell cycle arrest in the GO/G1 phase by modulating energy metabolism, the amino acid content and antioxidant defenses. Moreover, the combined glutamine starvation with the glycolysis inhibitor 2-deoxy-D-glucose exerted a stronger cytotoxic effect. This study offers a strong rationale for targeting glutamine metabolism alone or in combination with glucose metabolism to achieve a therapeutic benefit in the treatment of colon cancer