Identification of new candidate biomarkers for prostate cancer by affinity proteomics

Prostate cancer (PCA) is a complex malignancy that needs to be more thoroughly studied and understood at a molecular level to fill the current knowledge gap, and optimize diagnosis and to treatment. Prostate specific antigen (PSA) showed to be not specific for PCA, therefore a demand for novel specific biomarkers exists. The aim of our work was to identify new specific candidate biomarkers for PCA in tissue and plasma samples by means of affinity proteomics approaches such as reverse phase protein array and antigen arrays. Tissue samples are an invaluable source of biomarkers for cancer, but very limited in amount and requiring invasive procedures for collection. Still, they allow to directly profiling the molecular status of tumor itself. Beside tissue, a screening procedure on biological fluid such as plasma would be highly desirable, thanks to the less invasiveness and low-costs of samples collection. Among the biomarkers detectable in plasma are the autoantibodies. The first part of this thesis summarizes the current status of PCA epidemiology, treatment, and biomarkers research. Beside this, an overview of the affinity proteomics platforms available for biomarkers research, and the critical variables to consider in the biomarkers validation process are presented. The second part of the thesis reports the main results of two original studies where the author of the thesis is the main contributor. Paper I is based on the profiling of PCA tissue samples using RPPA. Our results indicate the feasibility of combining laser capture microdissection (LCM) and RPPA for evaluating the molecular architecture and cross-talking of epithelial and stromal compartments. Paper II is based on profiling the autoimmune response to PCA patients, comparing early and late stage of the disease. The authors identified and characterized the IgG reactivity toward a novel epitope for the candidate biomarker prostein. The data presented in this thesis provide two robust frameworks based on affinity proteomics platforms applied for protein profiling in tissue, and autoantibodies profiling in plasma in the context of PCA biomarkers discovery.Co-supervisors: Peter Nilsson (Biotechnology Department, KTH-Royal Institute of Technology, Stockholm, Sweden), Mariaelena Pierobon (Center for Applied Proteomics and Molecular Medicine, George Mason University, Manassas, VA, USA)openDottorato di ricerca in Medicina cellulare e molecolareopenPin, Elis

Estudio para implementar una aplicación móvil para promover servicios de trabajadores autónomos recomendados en una red de confianza

El estudio para implementar una aplicación móvil para promover servicios de trabajadores autónomos recomendados en una red de confianza; es fundamental porque con esto se pretende contribuir a la disminución de las limitaciones en la promoción de servicios de trabajadores autónomos y responder al problema de investigación. Como objetivo general se propone estudiar los aspectos esenciales para implementar la aplicación móvil. Los objetivos específicos son:1.-Analizar los aspectos teóricos, metodológicos y legales relacionados con estudio, implementación, aplicación móvil, servicios, trabajadores autónomos, red de confianza y métodos cualitativos de investigación; 2.-diagnosticar la situación actual de la funcionabilidad de los canales de promoción de servicios laborales existentes para los trabajadores autónomos ; y 3.- desarrollar del estudio para implementar una aplicación móvil para promover servicios de trabajadores autónomos recomendados en una red de confianza. Los métodos empleados para la recolección de datos son empíricos basados en la observación directa del objeto en el campo de estudio, de documentos relacionados, entrevistas estructuradas y no estructuradas y encuestas, información valiosa que a ayuda a cumplir la propuesta. Como conclusión final, se determina que el estudio para la implementación de una aplicación móvil garantizara el resultado de la premisa: “Sobre la base del estudio de los aspectos esenciales para implementar una aplicación móvil se pretende disminuir las limitaciones de promoción de servicios de los trabajadores autónomos recomendados en una red de confianza”, con esto se busca priorizar el desarrollo e innovación de transferencias tecnológicas en base a criterios de conocimientos alcanzados.The study to implement a mobile application to promote services recommended a trusted network self-employed; It is critical because this is intended to help reduce the constraints on promoting services of self-employed workers and respond to the research. The general objective we propose to study the essential aspects to implement the mobile application The specific objectives are: 1-To analyze the theoretical, methodological and legal aspects of study, implementation, mobile application services, self-employed, trusted network and qualitative methods research; 2. diagnose the current situation of the functionality of the channels existing labor promotion services for self-employed; and 3. develop the study to implement a mobile application to promote services recommended a network of trusted freelancers. The methods used to collect empirical data are based on direct observation of the object in the field of study related, structured interviews and unstructured documents and surveys, valuable information that helps fulfill the proposal. As a final conclusion, it is determined that the study for the implementation of a mobile application guarantee the result of the premise: "Based on the study of the essential aspects for the implementation of a mobile application is intended to diminish the limitations promotion services Recommended self-employed in a trusted network "this seeks to prioritize the development and innovation of technology transfer based on knowledge gained criteria

An Easy and Reliable Strategy for Making Type I Interferon Signature Analysis Comparable among Research Centers

Interferon-stimulated genes (ISGs) are a set of genes whose transcription is induced by interferon (IFN). The measure of the expression of ISGs enables calculating an IFN score, which gives an indirect estimate of the exposition of cells to IFN-mediated inflammation. The measure of the IFN score is proposed for the screening of monogenic interferonopathies, like the Aicardi-Gouti\ue8res syndrome, or to stratify subjects with systemic lupus erythematosus to receive IFN-targeted treatments. Apart from these scenarios, there is no agreement on the diagnostic value of the score in distinguishing IFN-related disorders from diseases dominated by other types of cytokines. Since the IFN score is currently measured in several research hospitals, merging experiences could help define the potential of scoring IFN inflammation in clinical practice. However, the IFN score calculated at different laboratories may be hardly comparable due to the distinct sets of IFN-stimulated genes assessed and to different controls used for data normalization. We developed a reliable approach to minimize the inter-laboratory variability, thereby providing shared strategies for the IFN signature analysis and allowing different centers to compare data and merge their experiences

Altered pattern of tumor necrosis factor-Alpha production in peripheral blood monocytes from Crohn's disease basic study

AIM: To evaluate the inflammatory state in Crohn's disease (CD) patients and correlate it with genetic background and microbial spreading. METHODS: By means of flow cytometry, production of tumor necrosis factor-alpha (TNF-\u3b1) was measured in peripheral blood monocytes from patients suffering from CD, ulcerative colitis (UC) and in healthy subjects after stimulation of the NOD2 and TLR pathways. CD patients were genotyped for the three most common NOD2 variants (R702W, G908R and L1007Pfs*2) and basal production of TNF-\u3b1 was correlated to NOD2 genotype. Also, production of TNF-\u3b1 was correlated to plasmatic levels of LPS Binding Protein (LBP), soluble (s) CD14 and to the activity state of the disease. RESULTS: The patients with CD were characterized by a significantly higher monocyte basal expression of TNF-\u3b1 compared with healthy subjects and UC patients, and after stimulation with Pam3CSK4 (ligand of TLR2/1) and MDP-L18 (ligand of NOD2) this difference was maintained, while other microbial stimuli (LPS, ligand of TLR4 and PolyI:C, ligand of TLR3) induced massive activation in CD monocytes as well as in UC and in healthy control cells. There was no significant difference in the production of TNF-\u3b1 between patients who carried CD-associated heterozygous or homozygous variants in NOD2 and patients with wild type NOD2 genotype. Although serum LBP levels have been shown to correlate positively with the state of activity of the disease, TNF-\u3b1 production did not show a clear correlation with either LBP or sCD14 levels in plasma. Moreover, no clear correlation was seen between TNF-\u3b1 production and activity indices in either CD or UC. CONCLUSION: Peripheral monocytes from CD express higher basal and stimulated TNF-\u3b1 than controls, regardless of NOD2 genotype and without a clear correlation with disease activity

Epigenetic targeting of bromodomain protein BRD4 counteracts cancer cachexia and prolongs survival

Cancer cachexia is a devastating metabolic syndrome characterized by systemic inflammation and massive muscle and adipose tissue wasting. Although it is responsible for approximately one-third of cancer deaths, no effective therapies are available and the underlying mechanisms have not been fully elucidated. We previously identified the bromodomain and extra-terminal domain (BET) protein BRD4 as an epigenetic regulator of muscle mass. Here we show that the pan-BET inhibitor (+)-JQ1 protects tumor-bearing mice from body weight loss and muscle and adipose tissue wasting. Remarkably, in C26-tumor-bearing mice (+)-JQ1 administration dramatically prolongs survival, without directly affecting tumor growth. By ChIP-seq and ChIP analyses, we unveil that BET proteins directly promote the muscle atrophy program during cachexia. In addition, BET proteins are required to coordinate an IL6-dependent AMPK nuclear signaling pathway converging on FoxO3 transcription factor. Overall, these findings indicate that BET proteins may represent a promising therapeutic target in the management of cancer cachexia

Loss of MUTYH function in human cells leads to accumulation of oxidative damage and genetic instability

The DNA glycosylase MUTYH (mutY homolog (Escherichia coli)) counteracts the mutagenic effects of 8-oxo-7,8-dihydroguanine (8-oxodG) by removing adenine (A) misincorporated opposite the oxidized purine. Biallelic germline mutations in MUTYH cause the autosomal recessive MUTYH-associated adenomatous polyposis (MAP). Here we designed new tools to investigate the biochemical defects and biological consequences associated with different MUTYH mutations in human cells. To identify phenotype(s) associated with MUTYH mutations, lymphoblastoid cell lines (LCLs) were derived from seven MAP patients harboring missense as well as truncating mutations in MUTYH. These included homozygous p.Arg245His, p.Gly264TrpfsX7 or compound heterozygous variants (p.Gly396Asp/Arg245Cys, p.Gly396Asp/Tyr179Cys, p.Gly396Asp/Glu410GlyfsX43, p.Gly264TrpfsX7/Ala385ProfsX23 and p.Gly264TrpfsX7/Glu480del). DNA glycosylase assays of MAP LCL extracts confirmed that all these variants were defective in removing A from an 8-oxoG:A DNA substrate, but retained wild-type OGG1 activity. As a consequence of this defect, MAP LCLs accumulated DNA 8-oxodG in their genome and exhibited a fourfold increase in spontaneous mutagenesis at the PIG-A gene compared with LCLs from healthy donors. They were also hypermutable by KBrO3--a source of DNA 8-oxodG--indicating that the relatively modest spontaneous mutator phenotype associated with MUTYH loss can be significantly enhanced by conditions of oxidative stress. These observations identify accumulation of DNA 8-oxodG and a mutator phenotype as likely contributors to the pathogenesis of MUTYH variants

Autoantibody Profiling and Anti-Kinesin Reactivity in ANCA-Associated Vasculitis

ANCA-associated vasculitides (AAV) are rare autoimmune diseases causing inflammation and damage to small blood vessels. New autoantibody biomarkers are needed to improve the diagnosis and treatment of AAV patients. In this study, we aimed to profile the autoantibody repertoire of AAV patients using in-house developed antigen arrays to identify previously unreported antibodies linked to the disease per se, clinical subgroups, or clinical activity. A total of 1743 protein fragments representing 1561 unique proteins were screened in 229 serum samples collected from 137 AAV patients at presentation, remission, and relapse. Additionally, serum samples from healthy individuals and patients with other type of vasculitis and autoimmune-inflammatory conditions were included to evaluate the specificity of the autoantibodies identified in AAV. Autoreactivity against members of the kinesin protein family were identified in AAV patients, healthy volunteers, and disease controls. Anti-KIF4A antibodies were significantly more prevalent in AAV. We also observed possible associations between anti-kinesin antibodies and clinically relevant features within AAV patients. Further verification studies will be needed to confirm these findings.</p

Autoantibody Profiling and Anti-Kinesin Reactivity in ANCA-Associated Vasculitis

ANCA-associated vasculitides (AAV) are rare autoimmune diseases causing inflammation and damage to small blood vessels. New autoantibody biomarkers are needed to improve the diagnosis and treatment of AAV patients. In this study, we aimed to profile the autoantibody repertoire of AAV patients using in-house developed antigen arrays to identify previously unreported antibodies linked to the disease per se, clinical subgroups, or clinical activity. A total of 1743 protein fragments representing 1561 unique proteins were screened in 229 serum samples collected from 137 AAV patients at presentation, remission, and relapse. Additionally, serum samples from healthy individuals and patients with other type of vasculitis and autoimmune-inflammatory conditions were included to evaluate the specificity of the autoantibodies identified in AAV. Autoreactivity against members of the kinesin protein family were identified in AAV patients, healthy volunteers, and disease controls. Anti-KIF4A antibodies were significantly more prevalent in AAV. We also observed possible associations between anti-kinesin antibodies and clinically relevant features within AAV patients. Further verification studies will be needed to confirm these findings.</p

Autoantibody Profiling and Anti-Kinesin Reactivity in ANCA-Associated Vasculitis

ANCA-associated vasculitides (AAV) are rare autoimmune diseases causing inflammation and damage to small blood vessels. New autoantibody biomarkers are needed to improve the diagnosis and treatment of AAV patients. In this study, we aimed to profile the autoantibody repertoire of AAV patients using in-house developed antigen arrays to identify previously unreported antibodies linked to the disease per se, clinical subgroups, or clinical activity. A total of 1743 protein fragments representing 1561 unique proteins were screened in 229 serum samples collected from 137 AAV patients at presentation, remission, and relapse. Additionally, serum samples from healthy individuals and patients with other type of vasculitis and autoimmune-inflammatory conditions were included to evaluate the specificity of the autoantibodies identified in AAV. Autoreactivity against members of the kinesin protein family were identified in AAV patients, healthy volunteers, and disease controls. Anti-KIF4A antibodies were significantly more prevalent in AAV. We also observed possible associations between anti-kinesin antibodies and clinically relevant features within AAV patients. Further verification studies will be needed to confirm these findings.</p