The aim of this thesis is to study the microbiota in subjects with celiac disease, and to compare the data obtained. There are several scientific studies that correlate the immune processes with a modification of the communities that colonize the intestine. The microbiota itself is considered a real organism that coexists with a symbiotic relationship with the host organism. It is also known that dysbiosis is associated with an inflammatory or pathological state, or alterations in the relationships between the different microbial communities, including alterations in the permeability of the intestinal barrier which, in genetically predisposed subjects, are at the base of the onset of celiac disease and have been also found in individuals with nerve or neurodegenerative diseases. According to some data from the literature, in particular the gram-negative bacteria are the ones most present in celiac patients.The approach followed in this thesis work was of a metagenomic type. DNA extracted from faecal samples was amplified using universal primers, specific for the V3 and V4 sequences of the 16S gene of the bacterial rRNA and subsequently the clustered sequences of the pre-constituted library were sequenced by the Illumina platform. Finally the sequence readings are filtered and analyzed through specific bioinformatics tools that allow to classify and cluster the sequences obtained in OTU (Operational Tassonomic Units), through comparison with reference databases. The abundance of OTUs allows to carry out evaluations of the different composition of a given sample or of several samples compared simultaneously and provides significant biostatistic parameters that allow to associate the microbiota-host interactions with the onset of celiac disease.
Obiettivo di questo lavoro di tesi è di studiare il microbiota in soggetti con celiachia, e di confrontare i dati ottenuti. Sono diversi gli studi scientifici che correlano i processi immunitari con una modificazione delle comunità che colonizzano l’intestino. Il microbiota stesso è considerato un vero e proprio organismo che convive con un rapporto di tipo simbiontico con l’organismo ospite. E’ anche noto come disbiosi siano associate a stati infiammatori o patologici, ovvero alterazioni dei rapporti tra le diverse comunità microbiche, tra cui alterazioni della permeabilità della barriera intestinale che, in soggetti geneticamente predisposti, sono alla base dell’insorgenza della celiachia e sono state riscontrate anche in individui affetti da malattie nervose o neurodegenerative. Secondo alcuni dati della letteratura, sono in particolare i batteri gram-negativi quelli maggiormente presenti nei pazienti celiaci. L’approccio seguito in questo lavoro di tesi è stato di tipo metagenomico. Il DNA estratto da campioni fecali è stato amplificato mediante primers universali, specifici per le sequenze V3 e V4 del gene 16S dell’rRNA batterico e successivamente le sequenze clusterizzate della library pre-costituita sono state sequenziate mediante piattaforma Illumina. Infine le letture di sequenza sono state filtrate ed analizzate tramite specifici tools bioinformatici che permettono di classificare e clusterizzare le sequenze ottenute in OTUs (Unità Tassonomiche Operazionali), attraverso il confronto con databases di riferimento. L’abbondanza delle OTUs permette di effettuare valutazioni della differente composizione di un determinato campione o di più campioni confrontati contemporaneamente e fornisce significativi parametri biostatistici che permettono di associare le interazioni microbiota-ospite con l’insorgenza della malattia celiaca.
Alterazioni del microbiota intestinale nella malattia celiaca
SPALTRO, ANGELA
2016/2017
Abstract
The aim of this thesis is to study the microbiota in subjects with celiac disease, and to compare the data obtained. There are several scientific studies that correlate the immune processes with a modification of the communities that colonize the intestine. The microbiota itself is considered a real organism that coexists with a symbiotic relationship with the host organism. It is also known that dysbiosis is associated with an inflammatory or pathological state, or alterations in the relationships between the different microbial communities, including alterations in the permeability of the intestinal barrier which, in genetically predisposed subjects, are at the base of the onset of celiac disease and have been also found in individuals with nerve or neurodegenerative diseases. According to some data from the literature, in particular the gram-negative bacteria are the ones most present in celiac patients.The approach followed in this thesis work was of a metagenomic type. DNA extracted from faecal samples was amplified using universal primers, specific for the V3 and V4 sequences of the 16S gene of the bacterial rRNA and subsequently the clustered sequences of the pre-constituted library were sequenced by the Illumina platform. Finally the sequence readings are filtered and analyzed through specific bioinformatics tools that allow to classify and cluster the sequences obtained in OTU (Operational Tassonomic Units), through comparison with reference databases. The abundance of OTUs allows to carry out evaluations of the different composition of a given sample or of several samples compared simultaneously and provides significant biostatistic parameters that allow to associate the microbiota-host interactions with the onset of celiac disease.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/19247