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Piazzesi, Antonia Felicia (2014) Drosophila melanogaster as a model for mitochondrial biology, mitochondrial disease and neurological disorders. [Tesi di dottorato]

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Abstract (inglese)

Drosophila melanogaster has a long history of being used as an animal model for a wide variety of human diseases, including genetic diseases, neurodegeneration and alcoholism. Despite the fact that Drosophila was first used as an animal model over 100 years ago, it still remains an extremely relevant model today, thanks to its short life cycle, its low cost ease to rear in laboratory conditions and the sophistication of the molecular tools available for genetic manipulation in Drosophila melanogaster. This model also has far less genetic redundancy with respect to mammals, making the study of the role of certain genes far more straightforward, and yet despite this, still possesses an ortholog for 75% of human disease-causing genes. All of these properties contribute to the relevance of this model and were taken advantage of during this project.
In the first part of this project, Drosophila melanogaster was used as a model for mitochondrial deoxynucleotide transport. The Drosophila homolog CG18317 of the yeast gene RIM2, which was previously reported to be a pyrimidine dNTP transporter, was characterized. Knock-out (K.O.) flies for gene CG18317, here referred to as drim2, were characterized for mitochondrial function and mtDNA integrity. The two human homologs for this gene, PNC-1 and SLC25A36 were also expressed in this mutant background, in order to investigate the functional homology of these genes and confirm the validity of this model for human mitochondrial dNTP transport.
This project also focuses on further characterizing a K.O. fly line for dTTC19, a gene whose human homolog has already been tied to mitochondrial encephalopathy and psychosis in humans. This characterization was also accompanied by the generation of three K.O. lines which express the dTTC19 gene in a mutant background, in order to finally confirm that the entirety of the mutant phenotype is due to the absence of transcription of the dTTC19 gene.
Finally, this project attempts to propose a new protocol which will enable researchers to use Drosophila melanogaster as a model for neurological disorders which present with antisocial symptoms. A protocol was developed to investigate social behaviour in Drosophila melanogaster and to demonstrate that subtle changes in either dopamine levels or previous social contact can have dramatic effects on their social interactions. We therefore propose that Drosophila can also be a useful model for the investigation of the genetic factors involved in diseases which present with antisocial behaviour such as autism, obsessive compulsive disorder, depression and so forth.
In conclusion, this project takes full advantage of Drosophila melanogaster as an animal model for mitochondrial biology and disease. Furthermore, it proposes yet another way in which Drosophila can be used as a model which has not yet been done.

Abstract (italiano)

Drosophila melanogaster ha una lunga storia come animale modello per tante malattie umane, incluse le malattie genetiche, la neurodegenerazione e l’alcolismo. Anche se Drosophila fu inizialmente utilizzata come animale modello più di 100 anni fa, rimane comunque un modello rilevante oggi grazie al suo ciclo vitale breve, il suo basso costo e la sofisticazione degli attrezzi molecolari disponibili per la sua manipolazione genetica. Questo modello ha anche meno ridondanza genetica rispetto ai mammiferi, rendendo lo studio della funzione di questi geni molto più diretto, ma malgrado questo possiede un ortologo per 75% dei geni legati a malattie umane. Tutte queste proprietà contribuiscono alla sua rilevanza come modello e sono state sfruttate durante questo progetto.
In primis, Drosophila melanogaster è stata usata come modello per il trasporto mitocondriale di deossinucleotidi. Il gene RIM2 in lievito, che è stato precedentemente caratterizzato come trasportatore mitocondriale di deossinucleotidi pirimidinici, ha un omologo in Drosophila: CG18317, qui chiamato drim2, che è stato caratterizzato in questo progetto. Questo gene è stato rimosso in vivo e la funzione mitocondriale e l’integrità del mtDNA sono state caratterizzate. I due omologhi umani per questo gene, PNC-1 e SLC25A36, sono stati espressi nel mutante, per determinare l’omologia funzionale di questi geni e per confermare la validità di questo mutante come modello per il trasporto mitocondriale umano di deossinucleotidi.
Questo progetto si è anche focalizzato su una caratterizzazione più approfondita di una linea mutante per dTTC19, un omologo di un gene umano che è già stato collegato alla encefalopatia mitocondriale e la psicosi. Questa caratterizzazione è stata accompagnata dalla generazione di tre linee mutanti che esprimono dTTC19, per confermare che il fenotipo mutante osservato sia dovuto alla mancanta trascrizione di dTTC19.
In fine, questo progetto propone un nuovo protocollo che, nella nostra opinione, permetterà di utilizzare Drosophila melanogaster come modello per disturbi neurologici che presentano con sintomi asociali. Un protocollo è stato sviluppato per studiare il comportamento sociale in Drosophila melanogaster e per dimostrare che piccole differenze nei livelli di dopamina o nel contatto sociale dopo l’eclosione possono avere effetti drammatici sulle interazioni sociali in Drosophila. Proponiamo che Drosophila può essere un modello utile per lo studio dei fattori genici coinvolti nelle malattie che presentano con comportamento asociale come l’autismo, il disturbo ossessivo compulsivo, la depressione eccetera.
In conclusione, questo progetto sfrutta interamente Drosophila melanogaster come animale modello per la biologia e le malattie mitocondriali. In più, propone un nuovo modo per utilizzare Drosophila come modello che non è stato finora sfruttato.

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Tipo di EPrint:Tesi di dottorato
Relatore:Zordan, Mauro A.
Dottorato (corsi e scuole):Ciclo 26 > Scuole 26 > BIOSCIENZE E BIOTECNOLOGIE > GENETICA E BIOLOGIA MOLECOLARE DELLO SVILUPPO
Data di deposito della tesi:30 Gennaio 2014
Anno di Pubblicazione:30 Gennaio 2014
Parole chiave (italiano / inglese):Drosophila melanogaster, mitochondria, dNTP, transporter, drim2, ttc19, dopamine, sociality, social behaviour, snap-25.
Settori scientifico-disciplinari MIUR:Area 05 - Scienze biologiche > BIO/18 Genetica
Struttura di riferimento:Dipartimenti > Dipartimento di Biologia
Codice ID:6681
Depositato il:28 Apr 2015 17:28
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