Baculovirus as a gene delivery and expression vector in mammalian cells

The interior homeostasis of a solid organ, such as a kidney, is greatly altered during the surgical removal, storage and transplantation process from donor to recipient. Consequently, normal organ function may be delayed or prevented following transplantation. The main injury that organs have to withstand often occurs subsequent to the restoration of blood flow; this injury is known as ischaemia reperfusion-injury (I/R injury) and is associated with hypoxia, free radical formation and organ failure. Viral gene therapy, such as BacMam gene transfer technology (Baculovirus-based expression in mammalian cells) is an attractive option for reducing I/R injury due to its high safety profile compared to mammalian viruses. However, limiting factors, including the requirement of high-doses of viral vector and low transgene expression levels are still a hurdle for the successful use of this technology. Therefore, the initial objectives of this study were to develop and validate a number of methods for concentrating BacMam along with the use of a number of chemical compounds to improve BacMam gene delivery in kidney cells. Amongst all the approaches screened, high-speed (for small volumes) and low-speed (for large volumes) centrifugation methods were the most effective for concentrating BacMam virus (4x109 and 1x1010 pfu/ml, respectively). Transduction of HEK cells was improved between two- to 10-fold in the presence of sodium butyrate or hydroxyurea alone and significantly improved egfp (enhanced green fluorescence protein) expression (~40-fold) when both chemicals were used in combination. Subsequently, in the second part of this study an in vitro culture model was developed and evaluated for assessing ischaemic injury in kidney cells using antimycin A in combination with 2-deoxyglucose. It was shown that sod-2 and bcl-2 overexpression in injured kidney cells improved the effect of I/R injury by enhancing the recovery of ATP post-reperfusion Furthermore, to examine the possibility of reducing injury associated with cold preservation, an ex vivo hypothermic perfusion system of porcine kidney was established and validated using reporter and protective genes. Importantly, for the first time, BacMam has been used to transfer ex vivo a protective gene (sod-2), into kidneys which appeared to provide protection against hypothermic preservation-associated injury by improving the recovery of cellular ATP

Memorizzare "in campo aperto": neumi, canto gregoriano, tropi liturgici e tecnologia della memoria

Il saggio si sofferma su un momento posto all’inizio della parabola storica delineata dalla tecnologia della scrittura musicale nella cultura occidentale: l’introduzione, verso l’inizio del ix secolo, delle prime notazioni neumatiche, il cui principale scopo consistette nel registrare il repertorio di canti liturgici noto come canto gregoriano. Partendo dal presupposto che nella cultura medioevale la scrittura costituisse una tecnologia della memoria, il saggio tenta di interpretare in quest’ottica sia le caratteristiche della notazione neumatica, sia alcune caratteristiche formali dei canti liturgici trasmessi mediante essa.Si propone una nuova interpretazione della tipica costruzione formulaica del canto gregoriano, intesa come conseguenza di un uso sofisticato della memoria improntato al modello concettuale della scrittura. L’analisi di alcuni brani melismatici tramandati in notazione neumatica, a volte accompagnati da testi composti ex novo (prosulae), mostra come la scrittura neumatica corrisponda a strategie di analisi e creazione fondate sulla memorizzazione. Un’idea che emerge al termine dalla trattazione riguarda il nesso tra la tecnologia della memoria e la concezione sostanzialmente aperta dei canti liturgici – e in particolar modo dei tropi liturgici –, il fatto cioè che questi non fossero pensati come unità autonome e conchiuse, ma piuttosto come differenti configurazioni di un certo numero di elementi fissi (mnemonici) che caratterizzano un vasto complesso di brani differenti. The essay focuses on a historical moment placed at the beginning of the parable outlined by the technology of music writing in Western culture: the introduction, at the beginning of the ninth century, of the first neumatic notations, whose main aim consisted in recording the repertoire of liturgical songs known as Gregorian chant. Assuming that writing in medieval culture constituted a technology of memory, the essay tries to interpret in this light the characteristics of neumatic notation, as well as some formal characteristics of liturgical chants transmitted through it. A new interpretation of the typical formulaic construction of Gregorian chant is suggested: formularity is understood as a result of a sophisticated use of memory based on the conceptual model of writing. The analysis of some pieces handed down in melismatic neumatic notation, sometimes accompanied by texts composed ex novo (prosulae), shows how neumatic writing corresponds to analytical and creative strategies based on memorization. An idea that emerges at the end of the discussion concerns the link between the technology of memory and the essentially open vision of the liturgical chants – especially the liturgical tropes – namely the fact that they were not conceived as autonomous and concluded units, but rather as different configurations of a number of  fixed (mnemonic) elements that feature a wide range of different songs