Longitudinal Bottom-Up Proteomics of Serum, Serum Extracellular Vesicles, and Cerebrospinal Fluid Reveals Candidate Biomarkers for Early Detection of Glioblastoma in a Murine Model

Glioblastoma Multiforme (GBM) is a brain tumor with a poor prognosis and low survival rates. GBM is diagnosed at an advanced stage, so little information is available on the early stage of the disease and few improvements have been made for earlier diagnosis. Longitudinal murine models are a promising platform for biomarker discovery as they allow access to the early stages of the disease. Nevertheless, their use in proteomics has been limited owing to the low sample amount that can be collected at each longitudinal time point. Here we used optimized microproteomics workflows to investigate longitudinal changes in the protein profile of serum, serum small extracellular vesicles (sEVs), and cerebrospinal fluid (CSF) in a GBM murine model. Baseline, pre-symptomatic, and symptomatic tumor stages were determined using non-invasive motor tests. Forty-four proteins displayed significant differences in signal intensities during GBM progression. Dysregulated proteins are involved in cell motility, cell growth, and angiogenesis. Most of the dysregulated proteins already exhibited a difference from baseline at the pre-symptomatic stage of the disease, suggesting that early effects of GBM might be detectable before symptom onset

Reazioni tipo Sonogashira su alogenuri olefinici alifatici: un primo studio sistematico sulla fattibilita in presenza di semplici catalizzatori supportati

Sono state effettuate diverse serie di prove esplorative di alchinilazioni tipo Sonogashira, in presenza di semplici catalizzatori supportati, commerciali e non. Sono stati scelti come substrati alogenuri alchenilici non arilsostituiti di vario tipo: 1-bromo-1-alcheni, 2-bromo-1-alcheni, 1-iodo-1-alcheni e bromoacrilati, in vista di una possibile applicazione delle migliori condizioni trovate nella sintesi di composti naturali di interesse applicativo

Neuroproteomics: method development and applications

With this Thesis I have exploited several aspects of proteomics and phosphoproteomics related to neurologic diseases, such as glioblastoma, Alzheimer disease and globoid cell leukodystrophy (GLD or Krabbe disease). The work was focused both on methodological and applicative aspects. I worked mostly with murine cerebrospinal fluid and cell pellets and experimented several digestion methods for different starting protein amount, such as SP3, Urea and SDC digestion protocols. The longitudinal analysis of murine cerebrospinal fluid allowed to identify some molecular changes between the baseline, the asymptomatic stage and the symptomatic stage of the glioblastoma, such as macrophage capping protein, measured using a linear mixed effect model. I also focused on the relevance of phosphorylation as post-translational modification in different contexts, such as Alzheimer or Krabbe disease. As potential biomarker for Alzheimer disease I identified the phosphorylation of centrin-2, indicating a role of phosphocentrin-2 in the early phases of Alzheimer disease, never fully investigated before. From a methodological point of view, I also implemented an automated on-column TMT labeling procedure for both peptides and phosphopeptides, using AssayMAP BRAVO liquid handling robot, and I applied it for the analysis of differentiated neurons belonging to treated and untreated GLD cell lines, to explain better the multimodal action of psychosine in the pathogenesis of Krabbe disease

Low-melting point agarose as embedding medium for MALDI mass spectrometry imaging and laser-capture microdissection-based proteomics

Abstract The combination of MALDI mass spectrometry imaging, laser-capture microdissection, and quantitative proteomics allows the identification and characterization of molecularly distinct tissue compartments. Such workflows are typically performed using consecutive tissue sections, and so reliable sectioning and mounting of high-quality tissue sections is a prerequisite of such investigations. Embedding media facilitate the sectioning process but can introduce contaminants which may adversely affect either the mass spectrometry imaging or proteomics analyses. Seven low-temperature embedding media were tested in terms of embedding temperature and cutting performance. The two media that provided the best results (5% gelatin and 2% low-melting point agarose) were compared with non-embedded tissue by both MALDI mass spectrometry imaging of lipids and laser-capture microdissection followed by bottom-up proteomics. Two out of the seven tested media (5% gelatin and 2% low-melting point agarose) provided the best performances on terms of mechanical properties. These media allowed for low-temperature embedding and for the collection of high-quality consecutive sections. Comparisons with non-embedded tissues revealed that both embedding media had no discernable effect on proteomics analysis; 5% gelatin showed a light ion suppression effect in the MALDI mass spectrometry imaging experiments, 2% agarose performed similarly to the non-embedded tissue. 2% low-melting point agarose is proposed for tissue embedding in experiments involving MALDI mass spectrometry imaging of lipids and laser-capture microdissection, proteomics of consecutive tissue sections