Molecular basis oh herpes simplex virus entry into the cell and retargeting of the viral tropism for the design of oncolytic herpesviruses

Herpes simplex virus 1 (HSV-1) infects oral epitelial cells, then spreads to the nerve endings and estabilishes latency in sensory ganglia, from where it may, or may not reactivate. Diseases caused by virus reactivation include mild diseases such as muco-cutaneous lesions, and more severe, and even life-threatening encephalitis, or systemic infections affecting diverse organs. Herpes simplex virus represents the most comprehensive example of virus receptor interaction in Herpesviridae family, and the prototype virus encoding multipartite entry genes. In fact, it encodes 11-12 glycoproteins and a number of additional membrane proteins: five of these proteins play key roles in virus entry into subsceptible cells. Thus, glycoprotein B (gB) and glycoprotein C (gC) interact with heparan sulfate proteoglycan to enable initial attachment to cell surfaces. In the next step, in the entry cascade, gD binds a specific surface receptor such as nectin1 or HVEM. The interaction of glycoprotein D with the receptor alters the conformation of gD to enable the activation of gB, glycoprotein H, and glycoprotein L, a trio of glycoproteins that execute the fusion of the viral envelope with the plasma membrane. In this thesis, I described two distinct projects: I. The retargeting of viral tropism for the design of oncolytic Herpesviruses: • capable of infecting cells through the human epitelial growth factor receptor 2 (HER2), overexpressed in highly malignant mammary and ovarian tumors and correlates with a poor prognosis; • detargeted from its natural receptors, HVEM and nectin1. To this end, we inserted a ligand to HER2 in gD. Because HER2 has no natural ligand, the selected ligand was a single chain antibody (scFv) derived from MAb4D5 (monoclonal antibody to HER2), herein designated scHER2. All recombinant viruses were targeted to HER2 receptor, but only two viruses (R-LM113 and R-LM249) were completely detargeted from HVEM and nectin1. To engineer R-LM113, we removed a large portion at the N-terminus of gD (from aa 6 to aa 38) and inserted scHER2 sequence plus 9-aa serine-glycine flexible linker at position 39. On the other hand, to engineer R-LM249, we replaced the Ig-folded core of gD (from aa 61 to aa 218) with scHER2 flanked by Ser-Gly linkers. In summary, these results provide evidence that: i. gD can tolerate an insert almost as big as gD itself; ii. the Ig-like domain of gD can be removed; iii. the large portion at the N-terminus of gD (from aa 6 to aa 38) can be removed without loss of key function; iv. R-LM113 and R-LM249 recombinants are ready to be assayed in animal models of mammary and ovary tumour. This finding and the avaibility of a large number of scFv greatly increase the collection of potential receptors to which HSV can be redirected. II. The production and purification of recombinant truncated form of the heterodimer gHgL. We cloned a stable insect cell line expressing a soluble form of gH in complex with gL under the control of a metalloprotein inducible promoter and purified the heterodimer by means of ONE-STrEP-tag system by IBA. With respect to biological function, the purified heterodimer is capable: • of reacting to antibodies that recognize conformation dependent epitopes and neutralize virion infectivity; • of binding a variety cells at cell surface. No doubt, the availability of biological active purified gHgL heterodimer, in sufficient quantities, will speed up the efforts to solve its crystal structure and makes it feasible to identify more clearly whether gHgL has a cellular partner, and what is the role of this interaction on virus entry.

Il risparmio delle risorse alla scala abitativa e di insediamento. Tecnologie non convenzionali e valutazione ambientale attraverso metodologia LCA.

Nel corso del mio lavoro di ricerca mi sono occupata di identificare strategie che permettano il risparmio delle risorse a livello edilizio e di approfondire un metodo per la valutazione ambientale di tali strategie. La convinzione di fondo è che bisogna uscire da una visione antropocentrica in cui tutto ciò che ci circonda è merce e materiale a disposizione dell’uomo, per entrare in una nuova era di equilibrio tra le risorse della terra e le attività che l’uomo esercita sul pianeta. Ho quindi affrontato il tema dell’edilizia responsabile approfondendo l’ambito delle costruzioni in balle di paglia e terra. Sono convinta che l’edilizia industriale abbia un futuro molto breve davanti a sé e lascerà inevitabilmente spazio a tecniche non convenzionali che coinvolgono materiali di semplice reperimento e posa in opera. Sono altresì convinta che il solo utilizzo di materiali naturali non sia garanzia di danni ridotti sull’ecosistema. Allo stesso tempo ritengo che una mera certificazione energetica non sia sinonimo di sostenibilità. Per questo motivo ho valutato le tecnologie non convenzionali con approccio LCA (Life Cycle Assessment), approfondendo gli impatti legati alla produzione, ai trasporti degli stessi, alla tipologia di messa in opera, e ai loro possibili scenari di fine vita. Inoltre ho approfondito il metodo di calcolo dei danni IMPACT, identificando una carenza nel sistema, che non prevede una categoria di danno legata alle modifiche delle condizioni idrogeologiche del terreno. La ricerca si è svolta attraverso attività pratiche e sperimentali in cantieri di edilizia non convenzionale e attività di ricerca e studio sull’LCA presso l’Enea di Bologna (Ing. Paolo Neri).