Development of sensitive proteomic approaches for protein tyrosine phosphorylation detection.

The elucidation of the complex array of cell signalling cascades is imperative for a deeper understanding of cell biology in both physiological and patho-physiological states. Extensive biochemical characterisation of signalling networks has revealed the importance of post-translational modifications (PTMs), particularly phosphorylation. Signalling via protein phosphorylation occurs across homeostatic proliferative, differentiative and anti-apoptotic events. Dysregulation of the kinase signalling pathways as well as mutations in kinases involved in phosphorylation have been implicated in a number of pathologies such as cancer or immune deficiencies. While it is estimated that 50% of all proteins are phosphorylated during their lifetime, phosphorylated proteins are present in relatively low abundance compared to their non-phosphorylated counterparts. The rarity of phosphorylation, which occurs on serine, threonine and tyrosine residues, has prompted the development of sensitive approaches to improve phosphorylation characterisation. Proteomic-based strategies offer novel approaches to overcome the limitations of currently available strategies for phosphoprotein analysis. The research presented within describes the development of proteomic-based methodologies for phosphotyrosine identification, quantitation and characterisation. These methods utilise the antiphosphotyrosine. Antibody 4G10 along with other MS-compatible approaches for phosphotyrosine enrichment prior to MS analysis. Methods for more targeted phosphoprotein analyses involved coupling of 4G10 covalently to super para-magnetic beads or by affinity to super para-magnetic beads with protein G covalently attached. These 4G10-coupled beads successfully enriched tyrosine phosphopeptides derived from tryptic phosphoprotein digests for identification and characterisation of phosphopeptides using MALDI-TOF/TOF MS analysis. The limited capacity of the magnetic bead approach for analysis of more complex samples necessetated the development of a more global proteomic strategy for tyrosine phosphorylation analysis. A global strategy that provides not only qualitative pTyr information but also shows quantitative changes that occur with pTyr signalling is imperative for detailed signalling cascade analyses. The global approach presented here utilised the 4G10 Ab/bead approach as well as Hydrophilic interaction chromatography (HILIC) for the enrichment of pTyr peptides from complex samples isotopically-labelled to quantify tyrosine phosphorylation after LC-MALDI-TOF/TOF MS analysis. Aspects of this approach were modified to improve phosphopeptide detection and characterisation, including the development of a novel optimised matrix-deposition strategy for LCMALDI-TOF/TOF MS. The strategy, termed EZYprep LC, allowed the effective use of the atypical 2,5-DHB matrix with phosphoric acid to improve phosphopeptide ionisation and subsequently identify and characterise more phosphorylation sites on phosphoprotein samples compared with LC-ESI-IT-MS/MS. Another aspect of the global strategy was the development of a modified isotope protein coded label strategy (modified ICPL). The optimised ICPL approach ensured quantitative information from a larger sub-set of peptides after tryptic digest of complex samples. The improved ability to quantify using this approach was highlighted by a comparative analysis of complex cell lysates labelled using the conventional ICPL strategy and the modified ICPL strategy. The modified ICPL labelling strategy identified more proteins and provided more quantitative information that the conventional ICPL methodology. As such, the global phospho-tyrosine strategy, combined the modified ICPL labelling and 4G10 Ab/bead enrichment with peptide fractionation and MALDI-TOF/TOF MS analysis, was subsequently utilised to identify and quantify tyrosine phosphorylation occurring in insulin-stimulated insulin receptor A- and B-subtypes.Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 201

Comparini, Alberto. La poetica dei Dialoghi con Leucò di Cesare Pavese. Sesto San Giovanni (MI): Mimesis, 2017, 224 p

Obra ressenyada: Alberto COMPARINI, La poetica dei Dialoghi con Leucò di Cesare Pavese. Sesto San Giovanni (MI): Mimesis, 2017

Increased phospho-keratin 8 isoforms in colorectal tumors associated with EGFR pathway activation and reduced apoptosis

Extent: 8p.Hyperphosphorylated keratin (K) 8 acts as a phosphate “sponge” for stress-activated protein kinases thereby inhibiting pro-apoptotic molecules and thus apoptosis. MAP kinase/ERK1 has increased activity in colorectal cancer (CRC) and is known to phosphorylate K8. The aims were to identify the K8 isoforms abundantly present in colon tumors, using 2D difference gel electrophoresis (DIGE), to identify the modifications using mass spectrometry, and to validate the differential abundance of these isoforms in tumors relative to matched normal mucosae. 2D DIGE showed 3 isoforms of K8 significantly increased in tumor ≥2-fold in 6/8 pairs. Metal oxide affinity chromatography mass spectrometry and bioinformatics were used to identify phosphorylated serine residues. Levels of PS24, PS432, and PS74 by western blotting were found to be significantly increased in tumor versus matched normal. Blocking of EGFR signaling in Caco2 cells showed a significant decrease (P < 0.0001) in K8 PS74 and PS432 levels by 59% and 66%, respectively, resulting in increased apoptosis.Georgia Arentz, Tim Chataway, Mark R. Condina, Timothy J. Price, Peter Hoffmann, and Jennifer E. Hardingha

In-House Packed Porous Graphitic Carbon Columns for Liquid Chromatography-Mass Spectrometry Analysis of N-Glycans

Protein glycosylation is a common post-translational modification that modulates biological processes such as the immune response and protein trafficking. Altered glycosylation profiles are associated with cancer and inflammatory diseases, as well as impacting the efficacy of therapeutic monoclonal antibodies. Consisting of oligosaccharides attached to asparagine residues, enzymatically released N-linked glycans are analytically challenging due to the diversity of isomeric structures that exist. A commonly used technique for quantitative N-glycan analysis is liquid chromatography-mass spectrometry (LC-MS), which performs glycan separation and characterization. Although many reversed and normal stationary phases have been utilized for the separation of N-glycans, porous graphitic carbon (PGC) chromatography has become desirable because of its higher resolving capability, but is difficult to implement in a robust and reproducible manner. Herein, we demonstrate the analytical properties of a 15 cm fused silica capillary (75 µm i.d., 360 µm o.d.) packed in-house with Hypercarb PGC (3 µm) coupled to an Agilent 6550 Q-TOF mass spectrometer for N-glycan analysis in positive ion mode. In repeatability and intermediate precision measurements conducted on released N-glycans from a glycoprotein standard mixture, the majority of N-glycans reported low coefficients of variation with respect to retention times (≤4.2%) and peak areas (≤14.4%). N-glycans released from complex samples were also examined by PGC LC-MS. A total of 120 N-glycan structural and compositional isomers were obtained from formalin-fixed paraffin-embedded ovarian cancer tissue sections. Finally, a comparison between early- and late-stage formalin-fixed paraffin-embedded ovarian cancer tissues revealed qualitative changes in the α2,3- and α2,6-sialic acid linkage of a fucosylated bi-antennary complex N-glycan. Although the α2,3-linkage was predominant in late-stage ovarian cancer, the alternate α2,6-linkage was more prevalent in early-stage ovarian cancer. This study establishes the utility of in-house packed PGC columns for the robust and reproducible LC-MS analysis of N-glycans

Cancer tissue classification using supervised machine learning applied to maldi mass spectrometry imaging

Matrix assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) can determine the spatial distribution of analytes such as protein distributions in a tissue section according to their mass-to-charge ratio. Here, we explored the clinical potential of machine learning (ML) applied to MALDI MSI data for cancer diagnostic classification using tissue microarrays (TMAs) on 302 colorectal (CRC) and 257 endometrial cancer (EC)) patients. ML based on deep neural networks discriminated colorectal tumour from normal tissue with an overall accuracy of 98% in balanced cross-validation (98.2% sensitivity and 98.6% specificity). Moreover, our machine learning approach predicted the presence of lymph node metastasis (LNM) for primary tumours of EC with an accuracy of 80% (90% sensitivity and 69% specificity). Our results demonstrate the capability of MALDI MSI for complementing classic histopathological examination for cancer diagnostic applications.Paul Mittal, Mark R. Condina, Manuela Klingler-Hoffmann, Gurjeet Kaur, Martin K. Oehler, Oliver M. Siebe

Chemoresistant Cancer Cell Lines Are Characterized by Migratory, Amino Acid Metabolism, Protein Catabolism and IFN1 Signalling Perturbations

Chemoresistance remains the major barrier to effective ovarian cancer treatment. The molecular features and associated biological functions of this phenotype remain poorly understood. We developed carboplatin-resistant cell line models using OVCAR5 and CaOV3 cell lines with the aim of identifying chemoresistance-specific molecular features. Chemotaxis and CAM invasion assays revealed enhanced migratory and invasive potential in OVCAR5-resistant, compared to parental cell lines. Mass spectrometry analysis was used to analyse the metabolome and proteome of these cell lines, and was able to separate these populations based on their molecular features. It revealed signalling and metabolic perturbations in the chemoresistant cell lines. A comparison with the proteome of patient-derived primary ovarian cancer cells grown in culture showed a shared dysregulation of cytokine and type 1 interferon signalling, potentially revealing a common molecular feature of chemoresistance. A comprehensive analysis of a larger patient cohort, including advanced in vitro and in vivo models, promises to assist with better understanding the molecular mechanisms of chemoresistance and the associated enhancement of migration and invasion.Mitchell Acland, Noor A. Lokman, Clifford Young, Dovile Anderson, Mark Condina, Chris Desire, Tannith M. Noye, Wanqi Wang, Carmela Ricciardelli, Darren J. Creek, Martin K. Oehler, Peter Hoffmann, and Manuela Klingler-Hoffman

Comparini, Alberto. La poetica dei Dialoghi con Leucò di Cesare Pavese. Sesto San Giovanni (MI): Mimesis, 2017, 224 p

Obra ressenyada: Alberto COMPARINI, La poetica dei Dialoghi con Leucò di Cesare Pavese. Sesto San Giovanni (MI): Mimesis, 2017

Più donne che cavalieri. Orlando furioso: il poema di Bradamante

[spa] Este trabajo aborda por primera vez de forma sistemática y monográfica el papel desempeñado por los personajes femeninos en el Orlando furioso (1532) de Ludovico Ariosto (1474-1533), evidenciando su carácter profundamente innovador respecto a la tradición, incluida la caballeresca ferraresa, y en buena medida también respecto al contexto cultural de las cortes renacentistas en Italia. Con tal fin se conjuga el enfoque comparatista y de fuentes con el análisis descriptivo e interpretativo para elaborar una tipología de las distintas figuras definiendo sus funciones tanto individualmente como en su interacción recíproca y respecto al conjunto de la trama. Desde este último punto de vista se demuestra también el papel de auténtica protagonista desempeñado por Bradamante, la figura femenina a la que el poema de Ariosto dedica no solo más espacio, sino también una mayor continuidad en la acción, y a la que asigna una riqueza superior de intervenciones en el plano privado y público. La tesis se estructura en tres partes, precedidas por una introducción metodológica que plantea el problema y delinea el status quaestionis, que muestra el carácter parcial de los estudios realizados sobre el tema hasta el momento. En la primera parte se analiza la presencia de los personajes femeninos en la tradición épica occidental – desde los poemas homéricos hasta la Chanson de geste, pasando por la Eneida y la Tebaida – y en las principales obras que mantienen una relación de intertextualidad con el Furioso, como las Metamorfosis y las Heroidas de Ovidio; paralelamente se toman en consideración algunos lugares fundamentales de la representación femenina en otros géneros y épocas de la literatura occidental, para llegar, a través de la poesía provenzal y las obras de Dante, Petrarca y Boccaccio, a la querelle des femmes, que especialmente desde la segunda mitad del siglo XV involucró a intelectuales y escritores. Este recorrido permite delinear los precedentes y el contexto cutural del que partió Ariosto, eligiendo una vía eminentemente original. La segunda parte está dedicada al análisis detallado de los personajes femeninos del poema, vistos individualmente y clasificados en grupos y subgrupos temáticos: en cada uno de los cuales se ponen de manifiesto tipologías, relaciones intertextuales y características siguiendo un orden que culmina con las figuras más significativas: las de Angelica y Bradamante. Por último, en la tercera parte se estudian las funciones de los distintos personajes dentro de los episodios en los que intervienen, teniendo en cuenta su relación con otras figuras – masculinas y femeninas – y la perspectiva de la trama en su conjunto así como del telos del poema. Un espacio se dedica asimismo a las figuras femeninas históricas citadadas en el Furioso y a las damas de corte que configuran el público elegido por el poeta para su obra. En las conclusiones se evidencia, en fin, la complejidad de la óptica ariostesca sobre la mujer, encaminada a desenmascarar cualquier postura unívoca y cualquier prejuicio, mostrando las dos caras de los falsos contrarios (de aquí también la presencia subterránea de duplicaciones y figuras desdobladas). Los resultados de este análisis hacen aflorar, pues, aspectos hasta ahora descuidados de la obra maestra del Renacimiento literario italiano y despejan el campo de los intentos de encuadrar a Ariosto en los estrechos márgenes de la filoginia o la misoginia.[eng] This dissertation studies, for the first time in a systematic and monographic way, the role of female characters in Ludovico Ariosto’s Orlando Furioso (1532), highlighting its extremely innovative characteristics compared to tradition, included the chivalric tradition of Ferrara, and in part also compared to the cultural context of the Italian courts of Renaissance. The fundamental role of Bradamante, to whom Ariosto reserves a more constant presence and richer interventions both on private and on public level, is proved as well. The dissertation is divided in three parts, each preceded by a methodological introduction delineating the status quaestionis, from which the incomplete nature of the existing studies on the subject emerges. The first part of the work analyses the presence of female characters in the Western epic tradition and in the main works which are in a relationship of intertextuality with the Furioso. Simultaneously, some fundamental passages of the representation of women in Western literature are retraced up to the fifteenth/sixteenth century querelle des femmes. This path allows to delineate the precedents and the cultural context from which Ariosto moved. The second part of the dissertation is dedicated to a detailed analysis of the female characters of the poem, which are classified in groups and subgroups: for each of them the typologies, the relationship of intertextuality and the characteristics are analysed. Finally, in the third part, the functions of the different characters in the single episodes are faced, in relation with the other – male and female – figures both from the plot perspective and in the epic telos of the poem. The conclusions underline the complexity of Ariosto’s approach, aimed at unmasking every univocal position and every prejudice, and to show the two sides of false opposites (from which duplications and double figures originate). The results of this analysis show hitherto neglected aspects of the masterpiece of Italian literary Renaissance and clear the air of any attempt to enlist Ariosto among the misogynists or the philogynists