Diletta Ami

Publications (25) View all

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  Available from: Diletta Ami

  Chapter: Multivariate Analysis for Fourier Transform Infrared Spectra of Complex Biological Systems and Processes

  Diletta Ami, Paolo Mereghetti, Silvia Maria, Doglia
  01/2013;
• Article: Fourier transform infrared microspectroscopy of complex biological systems: from intact cells to whole organisms.

  Diletta Ami, Antonino Natalello, Silvia Maria Doglia
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  ABSTRACT: Fourier transform infrared (FTIR) microspectroscopy is a powerful tool for the study of complex biological systems. Indeed, it is employed to characterize intact cells, tissues, and whole model organisms such as nematodes, since it allows to obtain a chemical fingerprint of the sample under investigation, giving information on the molecular composition and structures. The successful application of this technique for the in situ study of biological processes requires specific sample preparations, in order to obtain reliable and reproducible results. In the present work, we illustrate the optimized procedures to prepare biological samples for IR measurements and the method to collect and analyze their FTIR spectra. In particular, we describe here the investigations on bacterial cells, intact eukaryotic cells, and whole intact nematode specimens.
  Methods in molecular biology (Clifton, N.J.) 01/2012; 895:85-100.
• Article: Fourier transform infrared spectroscopy of intrinsically disordered proteins: measurement procedures and data analyses.

  Antonino Natalello, Diletta Ami, Silvia Maria Doglia
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  ABSTRACT: A peculiar property of intrinsically disordered proteins (IDPs), or of intrinsically disordered domains, is the absence of a well-defined three dimensional structure under native conditions. Moreover, IDPs usually acquire a specific structure in the presence of different interactors. In this framework, Fourier transform infrared (FTIR) spectroscopy is a powerful tool to assess the disordered character of a protein and to study its induced folding. In this chapter, we will show the detailed experimental procedures to measure the FTIR spectra of protein samples and the spectral analyses required to obtain information on the protein secondary structures and aggregation.
  Methods in molecular biology (Clifton, N.J.) 01/2012; 895:229-44.
• Article: Biophysical characterization of Met-G-CSF: effects of different site-specific mono-pegylations on protein stability and aggregation.

  Antonino Natalello, Diletta Ami, Maddalena Collini, Laura D'Alfonso, Giuseppe Chirico, Giancarlo Tonon, Silvia Scaramuzza, Rodolfo Schrepfer, Silvia Maria Doglia
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  ABSTRACT: The limited stability of proteins in vitro and in vivo reduces their conversion into effective biopharmaceuticals. To overcome this problem several strategies can be exploited, as the conjugation of the protein of interest with polyethylene glycol, in most cases, improves its stability and pharmacokinetics. In this work, we report a biophysical characterization of the non-pegylated and of two different site-specific mono-pegylated forms of recombinant human methionyl-granulocyte colony stimulating factor (Met-G-CSF), a protein used in chemotherapy and bone marrow transplantation. In particular, we found that the two mono-pegylations of Met-G-CSF at the N-terminal methionine and at glutamine 135 increase the protein thermal stability, reduce the aggregation propensity, preventing also protein precipitation, as revealed by circular dichroism (CD), Fourier transform infrared (FTIR), intrinsic fluorescence spectroscopies and dynamic light scattering (DLS). Interestingly, the two pegylation strategies were found to drastically reduce the polydispersity of Met-G-CSF, when incubated under conditions favouring protein aggregation, as indicated by DLS measurements. Our in vitro results are in agreement with preclinical studies, underlining that preliminary biophysical analyses, performed in the early stages of the development of new biopharmaceutical variants, might offer a useful tool for the identification of protein variants with improved therapeutic values.
  PLoS ONE 01/2012; 7(8):e42511. · 4.09 Impact Factor
• Article: Effects of recombinant protein misfolding and aggregation on bacterial membranes.

  D Ami, A Natalello, T Schultz, P Gatti-Lafranconi, M Lotti, S M Doglia, A de Marco
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  ABSTRACT: The expression of recombinant proteins is known to induce a metabolic rearrangement in the host cell. We used aggregation-sensitive model systems to study the effects elicited in Escherichia coli cells by the aggregation of recombinant glutathione-S-transferase and its fusion with the green fluorescent protein that, according to the expression conditions, accumulate intracellularly as soluble protein, or soluble and insoluble aggregates. We show that the folding state of the recombinant protein and the complexity of the intracellular aggregates critically affect the cell response. Specifically, protein misfolding and aggregation induce changes in specific host proteins involved in lipid metabolism and oxidative stress, a reduction in the membrane permeability, as well as a rearrangement of its lipid composition. The temporal evolution of the host cell response and that of the aggregation process pointed out that the misfolded protein and soluble aggregates are responsible for the membrane modifications and the changes in the host protein levels. Interestingly, native recombinant protein and large insoluble aggregates do not seem to activate stress markers and membrane rearrangements.
  Biochimica et Biophysica Acta 12/2008; 1794(2):263-9. · 4.66 Impact Factor