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Scienze Veterinarie Alma Mater Studiorum – Università di Bologna DOTTORATO DI RICERCA IN SCIENZE VETERINARIE Ciclo XXXII Settore Concorsuale: 07/H1 Settore Scientifico Disciplinare: VET/01 Aspetti Morfo-Fisiologici dell’Apparato Digerente nei Mammiferi Presentata da: Dott.ssa Agnese Stanzani Coordinatore Dottorato Supervisore Prof. Arcangelo Gentile Prof. Paolo Clavenzani Esame finale anno 2020 Abstract Tesi La tesi verte sullo studio morfo-fisiologico e/o patologico del tratto gastrointestinale (GI) di mammiferi. La prima ricerca è basata sull’identificazione, attraverso metodiche di next generation sequencing, di geni coinvolti nell’insorgenza di acalasia sporadica nell’uomo. Tale patologia risulta caratterizzata dalla presenza di anomalie a carico di neuroni del plesso mienterico e cellule interstiziali di Cajal (ICC), che esita in una compromissione della peristalsi del tratto esofageo. É emerso che nei pazienti con acalasia vs controlli il gene C-KIT è down-regolato, mentre INPP4B è up-regolato. Questi geni risultano essere importanti per lo sviluppo e differenziamento neuronale e delle ICC. Il secondo argomento verte allo studio dei processi di de- e fosforilazione della proteina Tau in condizioni fisiologiche e neurodegenerative. Nell’Alzheimer si osserva un accumulo irreversibile di phospho-Tau che induce neurotossicità. Nell’encefalo di animali letargici la proteina Tau viene iperfosforilata in ipotermia, ma defosforila al rientro della normotermia. Inducendo torpore sintetico nel ratto si è osservato che nel sistema nervoso centrale e nel tratto gastrointestinale la phospho-Tau si accumula durante lo stato ipotermico, ma defosforila al ripristino delle temperature fisiologiche. Comprendere le modificazioni associate alla Tau può rappresentare un valido supporto per meglio comprendere quali meccanismi sono alterati in condizioni patologiche. L’ultima parte delle ricerche è stata finalizzata allo studio della distribuzione dei recettori per cannabinoidi quali CB1R, CB2R, GPR55, PPARα, PPARγ, TRPA1 e 5-HT1aR, nel tratto GI di gatto attraverso metodiche immunoistochimiche. Molti autori riportano come l’attivazione di questi recettori svolga un ruolo chiave nei processi infiammatorie ed in generale sulla sensibilità viscerale. La conoscenza della presenza e distribuzione di questi recettori può rappresentare un efficace contributo per future applicazioni in campo farmaceutico/terapeutico. La parte finale dell’elaborato contiene progetti ai quali ho partecipato che esulano dalla tematica principale della tesi ma che hanno esitato in pubblicazioni o partecipazioni a Congressi. INPP4B overexpression and c-KIT downregulation in human achalasia 1 Introduzione .............................................................................................................................. 1 1.1 Il Sistema Nervoso Enterico: organizzazione morfo-funzionale ......................................... 3 1.2 Organizzazione dei Plessi ................................................................................................... 6 1.3 Classificazione dei neuroni del SNE ................................................................................... 9 1.4 Cellule Interstiziali di Cajal (ICC) .................................................................................... 14 1.5 Alterato funzionamento del SNE e conseguenti anomalie della motilità del tGI .............. 15 1.5.1 Gastroparesi ................................................................................................................ 15 1.5.2 Malattia di Hirschsprung (HSCR) .............................................................................. 16 1.6 Acalasia ............................................................................................................................. 17 1.6.1 Eziopatogenesi ........................................................................................................... 18 2 Scopo........................................................................................................................................ 26 3 Materiali e Metodi .................................................................................................................. 27 3.1 Reclutamento dei pazienti e dei tessuti ............................................................................. 27 3.2 Estrazione dell’RNA, allestimento della libreria e sequenziamento dell’RNA (RNAseq) . 27 3.3 Analisi dei dati di RNAseq ................................................................................................ 29 3.4 Validazione dei dati ........................................................................................................... 30 3.4.1 Estrazione DNA ......................................................................................................... 30 3.4.2 Retrotrascrizione ........................................................................................................ 31 3.4.3 PCR Real time (RT-PCR) .......................................................................................... 32 3.4.4 Western Blot ............................................................................................................... 34 3.4.5 Immunoistochimica (IHC) ......................................................................................... 35 3.4.6 Analisi Statistica ......................................................................................................... 36 4 Risultati ................................................................................................................................... 37 4.1 Analisi dell’RNA totale tramite RNAseq ........................................................................... 37 4.2 Analisi dei geni tramite software PANTHER .................................................................... 39 4.3 Analisi dei network molecolari in cui sono coinvolti i geni alterati nell’acalasia............ 40 4.4 Conferma dei risultati di RNAseq ..................................................................................... 42 4.4.1 PCR real-time quantitativa (rt-qPCR) su 9 geni selezionati ....................................... 42 4.4.2 Western Blot su proteine estratte da tessuto esofageo ................................................ 45 4.4.3 Immunoistochimica (IHC) sviluppata con tecnica DAB su biopsie a tutto spessore . 47 4.4.4 INPP4B: importanza nel signaling di Akt? ................................................................ 48 5 Discussione .............................................................................................................................. 50 6 Bibliografia ............................................................................................................................. 52 Phosphorylation and Dephosphorylation of Tau Protein During Synthetic Torpor 1 Introduzione ............................................................................................................................ 61 1.1 Espressione e Struttura della Proteina Tau ...................................................................... 61 1.2 Ruolo Fisiologico della Proteina Tau ............................................................................... 65 1.3 Ruolo Patologico della Proteina Tau ................................................................................ 66 1.4 Ruolo della proteina Tau nel torpore ................................................................................ 71 1.5 Sistema Nervoso Centrale e Controllo della Termoregolazione ....................................... 75 1.6 Torpore Sintetico ............................................................................................................... 78 2 Scopo........................................................................................................................................ 82 3 Materiali e Metodi .................................................................................................................. 83 3.1 Animali .............................................................................................................................. 83 3.2 Procedura Chirurgica ....................................................................................................... 83 3.3 Torpore Sintetico ............................................................................................................... 84 3.4 Immonoistochimica ........................................................................................................... 86 3.5 Acquisizione Immagini e Analisi ....................................................................................... 88 3.6 Analisi Statistica ................................................................................................................ 91 4 Risultati ................................................................................................................................... 93 4.1 Analisi complessiva delle aree encefaliche AT8-pTau-IR ................................................. 93 4.2 Analisi delle singole aree encefaliche AT8-pTau-IR ......................................................... 93 4.2.1 N30 (campioni ottenuti durante l’induzione del TS) .................................................. 93 4.2.2 N (campioni ottenuti durante il TS, al nadir ipotermico) ........................................... 94 4.2.3 ER (campioni ottenuti durante early recovery da TS, quando la TB raggiunge 35.5°C) ............................................................................................................................................
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