Sintesi di inibitori dell'isoforma 5 della lattato deidrogenasi umana

Many studies have demonstrated the presence of regions at very low oxygen concentration (hypoxia) in solid tumors; so during the tumoural progression only cells that change their metabolism and switch to glycolytic phenotype survive. Although the upregulation of glycolysis is a successful adaptation to hypoxia, it also has significant negative consequences because of increased acid production, which provokes considerable decreases in local extracellular pH. The acidification of the microenvironment facilitates tumour invasion. As a consequence, the development of hypoxic areas is very closely linked to the development of a malignant and metastatic phenotype. The molecular mechanisms that underlie metabolic reprogramming of cancer cells are complex, but the activation of hypoxia-inducible factor (HIF) represents one of the principal causes of the metabolic switch. HIF-1 is a transcription factor that is activated by hypoxic stress, causing a up-regulation of GLUT1, HK1 and HK2, and lactate dehydrogenase A (LDH-A). In my thesis work I tried to synthesize molecules able to inhibit LDH-A, in fact, by inhibiting the lactic fermentation, the lack of NAD+ cofactors causes the block of glycolysis and, consequently, the interruption of energy production

Cytokine secretion responsiveness of lymphomonocytes following cortisol cell exposure: Sex differences

The stress hormone cortisol has been recognized as a coordinator of immune response. However, its different ability to modulate the release of inflammatory mediators in males and females has not been clarified yet. Indeed, the dissection of cortisol specific actions may be difficult due to the complex hormonal and physio-pathological individual status. Herein, the release of inflammatory mediators following increasing cortisol concentrations was investigated in an in vitro model of primary human male and female lymphomonocytes. The use of a defined cellular model to assess sex differences in inflammatory cytokine secretion could be useful to exclude the effects of divergent and fluctuating sex hormone levels occurring in vivo. Herein, the cells were challenged with cortisol concentrations resembling the plasma levels achieving in physiological and stressful conditions. The production of cytokines and other molecules involved in inflammatory process was determined. In basal conditions, male cells presented higher levels of some pro-inflammatory molecules (NF-kB and IDO-1 mRNAs, IL-6 and kynurenine) than female cells. Following cortisol exposure, the levels of the pro-inflammatory cytokines, IL-6 and IL-8, were increased in male cells. Conversely, in female cells IL-6 release was unchanged and IL-8 levels were decreased. Anti-inflammatory cytokines, IL-4 and IL-10, did not change in male cells and increased in female cells. Interestingly, kynurenine levels were higher in female cells than in male cells following cortisol stimulus. These results highlighted that cortisol differently affects male and female lymphomonocytes, shifting the cytokine release in favour of a pro-inflammatory pattern in male cells and an anti-inflammatory secretion profile in female cells, opening the way to study the influences of other stressful factors involved in the neurohumoral changes occurring in the response to stress conditions

Potential biomarkers and novel pharmacological targets in protein aggregation-related neurodegenerative diseases

The aggregation of specific proteins plays a pivotal role in the etiopathogenesis of several neurodegenerative diseases (NDs). β-Amyloid (Aβ) peptide-containing plaques and intraneuronal neurofibrillary tangles composed of hyperphosphorylated protein tau are the two main neuropathological lesions in Alzheimer's disease. Meanwhile, Parkinson's disease is defined by the presence of intraneuronal inclusions (Lewy bodies), in which α-synuclein (α-syn) has been identified as a major protein component. The current literature provides considerable insights into the mechanisms underlying oligomeric-related neurodegeneration, as well as the relationship between protein aggregation and ND, thus facilitating the development of novel putative biomarkers and/or pharmacological targets. Recently, α-syn, tau and Aβ have been shown to interact each other or with other "pathological proteins" to form toxic heteroaggregates. These latest findings are overcoming the concept that each neurodegenerative disease is related to the misfolding of a single specific protein. In this review, potential opportunities and pharmacological approaches targeting α-syn, tau and Aβ and their oligomeric forms are highlighted with examples from recent studies. Protein aggregation as a biomarker of NDs, in both the brain and peripheral fluids, is deeply explored. Finally, the relationship between biomarker establishment and assessment and their use as diagnostics or therapeutic targets are discussed

Dissecting the miRNome impact on metastatic pathways in Triple Negative Breast Cancer

Metastatic disease at distant sites is the main cause of death for patients of breast cancer. Among them, patients diagnosed with the subtype termed Triple Negative (TNBC) are affected the most by both residual disease after treatment and distant metastases. It is therefore of utmost importance to investigate more in depth the role of pathways related to TNBC development and metastasization, possibly identifying novel therapeutic or diagnostic targets. In this PhD project, the effect of the miRNome (i.e. known miRNAs encoded in the genome) was analysed on three pathways relevant for TNBC metastatic processes. A high-throughput proteomic approach termed Reverse Phase Protein Arrays (RPPA) was exploited to quantify the effect of miRNAs on the expression of 62 proteins belonging to c-Met, Integrin, and WNT/β-catenin signalling pathways. Then, a novel network analysis method was developed, taking into consideration the role of the proteins within the pathway of interest, and therefore contextualising the miRNA:target interactions. miRNAs identified as modulators of WNT/β-catenin signalling were tested functionally by means of a reporter gene assay, characterising miR-193b, -409, -494-, and -92b as strong repressors of the pathway. Their ability to regulate proliferation was assessed in a context of pathway overactivation, revealing that miR-193b, -409, and -494 reduce it. Analysis of stem-associated surface markers CD44 and CD24 showed that miR-193b is able to decrease the CD44+ population. Additionally, direct molecular interactions with predicted target sequences in 3’UTRs of mRNAs corresponding to the proteins probed were validated for miR-193b and miR-494. In summary, the findings of this dissertation describe the role of miRNAs as putative repressors or activators of metastatic pathways in TNBC, validating miR-193b, miR-409, and miR-494 as highly integrated within the network of WNT/β-catenin signalling. Furthermore, the RPPA results provide a useful resource available to the scientific community for further studies related to miRNA function in TNBC

TUMOR NECROSIS FACTOR ALPHA TRIGGERS OSTEOGENESIS THROUGH THE INVOVLVEMENT OF Gs-COUPLED RECEPTOR SIGNALS

Tumor Necrosis Factor alpha (TNF-α) plays a role in several chronic immune and inflammatory diseases, where inhibition of TNF has led to significant clinical improvement. Actually, this cytokine is involved in bone healing by affecting mesenchymal stem cell (MSC) behaviour in a dose- and time-dependent manner1,2. Indeed, in the early inflammatory phase after fracture, low doses of TNF-α are required to favour MSC migration, survival and differentiation, thus initiating bone repair. At high dose, in the chronic uncontrolled phase of inflammation, the same cytokine has destructive effects on bone and contribute to bone loss1,2. As other soluble factors released in cell microenvironment, the cytokine modulates expression and functioning of different G protein coupled receptors (GPCRs) and of their regulatory proteins (GPCR regulated kinases, GRKs)3, thus dictating the final biological outcome of these receptor proteins in controlling bone anabolic processes. Herein, we investigated the effects of TNF-α low doses on the expression and functional responsiveness of A2B adenosine receptor (A2B AR), a Gs-coupled puringergic receptor that controls mesenchymal stem cell (MSC) differentiation to osteoblasts4,5. In our hands, TNF-α exerted a pro-differentiating action on MSCs, pushing towards an osteoblast phenotype, and without any effects on cell proliferation. The cytokine increased the A2B AR-mediated pro-osteogenic effects, through the A2B AR desensitization impairment mediated by GRK2 inhibition. These data i) support the anabolic effect of sub-massimal concentration of TNF-α in bone reparative processes and ii) demonstrate that the cytokine regulates GPCR responses by interfering with desensitization machinery and potentiating in turn the anabolic responses evoked by Gs-GPCRs. Overall these results indicated that manipulating MSC local environment by lregulates membrane receptors favouring bone remodelling

The A2B adenosine receptor modulates the epithelial- mesenchymal transition through the balance of cAMP/PKA and MAPK/ERK pathway activation in human epithelial lung cells

The epithelial-mesenchymal transition (EMT) is a complex process in which cell phenotype switches from the epithelial to mesenchymal one. The deregulations of this process have been related with the occurrence of different diseases such as lung cancer and fibrosis. In the last decade, several efforts have been devoted in understanding the mechanisms that trigger and sustain this transition process. Adenosine is a purinergic signaling molecule that has been involved in the onset and progression of chronic lung diseases and cancer through the A2Badenosine receptor subtype activation, too. However, the relationship between A2BAR and EMT has not been investigated, yet. Herein, the A2BAR characterization was carried out in human epithelial lung cells. Moreover, the effects of receptor activation on EMT were investigated in the absence and presence of transforming growth factor-beta (TGF-β1), which has been known to promote the transition. The A2BAR activation alone decreased and increased the expression of epithelial markers (E-cadherin) and the mesenchymal one (Vimentin, N-cadherin), respectively, nevertheless a complete EMT was not observed. Surprisingly, the receptor activation counteracted the EMT induced by TGF-β1. Several intracellular pathways regulate the EMT: high levels of cAMP and ERK1/2 phosphorylation has been demonstrated to counteract and promote the transition, respectively. The A2BAR stimulation was able to modulated these two pathways, cAMP/PKA and MAPK/ERK, shifting the fine balance toward activation or inhibition of EMT. In fact, using a selective PKA inhibitor, which blocks the cAMP pathway, the A2BAR-mediated EMT promotion were exacerbated, and conversely the selective inhibition of MAPK/ERK counteracted the receptor-induced transition. These results highlighted the A2BAR as one of the receptors involved in the modulation of EMT process. Nevertheless, its activation is not enough to trigger a complete transition, its ability to affect different intracellular pathways could represent a mechanism at the basis of EMT maintenance/inhibition based on the extracellular microenvironment. Despite further investigations are needed, herein for the first time the A2BAR has been related to the EMT process, and therefore to the different EMT-related pathologies

Synthesis, chemical characterization, and biological evaluation of a novel auranofin derivative as an anticancer agent

A novel gold(I) complex inspired by the known medicinal inorganic compounds auranofin and thimerosal, namely ethylthiosalicylate(triethylphosphine)gold(I) (AFETT hereafter), was synthesized and characterised and its structure was resolved through X-ray diffraction. The solution behavior of AFETT and its interactions with two biologically relevant proteins (i.e. human serum albumin and haemoglobin) and with a synthetic dodecapeptide reproducing the C-terminal portion of thioredoxin reductase were comparatively analyzed through 31P NMR and ESI-MS. Remarkable binding properties toward these biomolecules were disclosed. Moreover, the cytotoxic effects produced by AFETT on two ovarian cancer cell lines (A2780 and A2780 R) and one colorectal cancer cell line (HCT116) were analyzed and found to be strong and nearly superimposable to those of auranofin. Interestingly, for both compounds, the ability to induce downregulation of vimentin expression in A2780 R cells was evidenced. Despite its close similarity to auranofin, AFETT is reported to exhibit some peculiar and distinctive features such as a lower lipophilicity, an increased water solubility and a faster reactivity towards the selected target biomolecules. These differences might confer to AFETT significant pharmaceutical and therapeutic advantages over auranofin itself

Gross Cystic Disease Fluid Protein-15(GCDFP-15)/Prolactin-Inducible Protein (PIP) as Functional Salivary Biomarker for Primary Sjögren's Syndrome

Gross cystic disease fluid protein-15(GCDFP-15)/prolactin-inducible protein (PIP) is a secretory acinar glycoprotein of 14 KDa which we have recently described as significantly lower in salivary samples of patients with primary Sjögren's syndrome (pSS) in comparison to healthy volunteers by proteomic analysis

Education of clinical reasoning in patients with multimorbidity: a scoping review and perspectives for technology-enhanced learning

Multimorbidity is defined as the co-existence of two or more chronic diseases in a patient, and it is increasing in prevalence. This condition poses new problems for clinical reasoning. Few studies inquire regarding the construct of reasoning in multimorbidity and the teaching/learning methods. The objectives of this scoping review were searching for a definition of the construct of clinical reasoning in multimorbidity and the related learning methods, and special ways in which information technology can help. We searched PubMed, Scopus, ERIC and CORE databases. After an iterative process of selection and thematic analysis, we selected 30 articles, that were thematized in three classes: the multimorbid patient as a teacher (8 articles), defining a framework of competence (11 articles), representing multimorbidity and related clinical reasoning (11 articles). In this last theme were also grouped studies using technology to enhance learning. The construct of clinical reasoning in multimorbidity expands over three domains: clinical (including managing uncertainty, anticipating, and detecting evolutions and conflicting guidelines, and setting priorities); relational (concerning communicating uncertainty and developing a feasible, shared plan of care with the patient; organizational) (managing the wide system of resources needed to take care of a multimorbid patient). The preferred teaching methods are based on the encounter with real or expert patients, technology enhanced case-based learning and graphical representations of clinical cases. Perspectives of research should be addressed to permit the learner to experience a patient's life-long experience by moving forward and back over time while exploring interactions among diseases and social determinants with respect to possibly conflicting treatments. Perspectives on rich, technology-enhanced simulations should be researched