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Phenotypical and functional studies on putative cancer stem cells of human melanoma
Several lines of evidence obtained in a variety of human tumours support the theory of cancer stem cells (CSCs). The aim of this thesis was the isolation and the biomolecular characterization of the tumorigenic subpopulation of melanoma cells. Melanoma cells growing in stem cell medium as non-adherent colonies, here named 'melanospheres', were successfully isolated from melanoma specimens or from melanoma cell lines. Melanospheres displayed a self-renewal capacity and multipotency in vitro and were highly tumorigenic in vivo. Remarkably, melanosphere-derived xenografts maintained tumorigenic potential in subsequent recipients and mirrored the original melanoma of the patient. Melanospheres expressed a heterogeneous assortment of stem cell markers, and no direct and unique correlation between any of their phenotypes and in vivo tumorigenicity was found. Conversely, melanoma cells cultured in the presence of fetal calf serum displayed a lower tumorigenicity in SCID mice with limited engraftment capacity in secondary recipient animals. Moreover, adherent cell xenografts displayed a homogeneous phenotype for the expression of melanoma associated markers and contained cells with markers of full differentiation. Taken together our data provide further evidence on the heterogeneous nature of human melanomas and show that melanospheres and their xenografted tumours represent a useful model to investigate melanoma biology. The parent-to-progeny relationship between CSCs and tumour bulk does not necessarily reflect the well conserved and predictable rules operating in normal tissue development. CSCs may thus not be a static compartment but rather stemness features can be acquired by tumour cells in response to environmental signals. Thus, in this thesis, experiments have been performed to define the role of tumour environmental factors produced by melanoma cells themselves or by cells composing the tumour stroma in modulating the acquisition of sternness properties. The results provided in this thesis indicated that melanospheres display an extensive secretory capacity quantitatively and qualitatively different from that of melanoma cells growing as adherent monolayer. The roles of these immunerelated factors in shaping melanoma heterogeneity, plasticity and tumour maintenance are discussed
Fermentacija mlijeka obogaÄenog mljevenom kominom groĆŸÄa s pomoÄu bakterije Lactobacillus acidophilus
Some by-products of wine industry still contain nutrients and functional compounds that make them potential ingredients to formulate new high value-added food products. The aim of this study is to develop milk fermented with Lactobacillus acidophilus fortified with marc flour of different cultivars of Vitis vinifera from wine production and to evaluate their influence on fermentation kinetics, probiotic counts, phenolic compounds, sugar content and antioxidant activity. The acidification time was significantly shortened by these enrichments (by up to 2.7 h), and the bacterial count during cold storage resulted in stronger fortification of samples (up to 4.13 %) when compared to control tests. Fermented milk containing grape marc showed considerable amounts of phenolic compounds with notable antioxidant activity, as well as significant amounts of total sugars. The most important aspect of this paper is the feasibility of using winery by-products, rich in phenolic compounds, as natural supplements to fortify probiotic-fermented milk.Neki nusproizvodi u proizvodnji vina sadrĆŸavaju hranjive i funkcionalne tvari, pa se mogu upotrijebiti za razvoj novih prehrambenih proizvoda s dodanom vrijednoĆĄÄu. Svrha je ovoga rada bila proizvesti mlijeko obogaÄeno mljevenom kominom razliÄitih sorata groĆŸÄa i fermentirano s pomoÄu bakterije Lactobacillus acidophilus, te odrediti utjecaj dodatka komine na kinetiku fermentacije, broj probiotiÄkih mikroorganizama, udjele fenolnih spojeva i ĆĄeÄera, te antioksidativnu aktivnost fermentiranog mlijeka. Vrijeme fermentacije obogaÄenog mlijeka znatno se smanjilo (do 2,7 h), a broj bakterija tijekom skladiĆĄtenja u hladnjaku poveÄao (do 4,13 %) u usporedbi s kontrolnim uzorkom. Fermentirano je mlijeko obogaÄeno mljevenom kominom groĆŸÄa imalo veÄe udjele fenolnih spojeva i ukupnih ĆĄeÄera, te izraĆŸeniju antioksidativnu aktivnost. NajveÄi je znaÄaj ovog istraĆŸivanja u tome ĆĄto dokazuje da se nusproizvodi vinske industrije bogati fenolnim spojevima mogu upotrijebiti kao prirodni dodaci mlijeku fermentiranom s pomoÄu probiotika
Mitochondrial fitness and cancer risk
Changes in metabolism are a hallmark of cancer, but molecular signatures of altered bioenergetics to aid in clinical decision-making do not currently exist. We recently identified a group of human tumors with constitutively reduced expression of the mitochondrial structural protein, Mic60, also called mitofilin or inner membrane mitochondrial protein (IMMT). These Mic60-low tumors exhibit severe loss of mitochondrial fitness, paradoxically accompanied by increased metastatic propensity and upregulation of a unique transcriptome of Interferon (IFN) signaling and Senescence-Associated Secretory Phenotype (SASP). Here, we show that an optimized, 11-gene signature of Mic60-low tumors is differentially expressed in multiple malignancies, compared to normal tissues, and correlates with poor patient outcome. When analyzed in three independent patient cohorts of pancreatic ductal adenocarcinoma (PDAC), the Mic60-low gene signature was associated with aggressive disease variants, local inflammation, FOLFIRINOX failure and shortened survival, independently of age, gender, or stage. Therefore, the 11-gene Mic60-low signature may provide an easily accessible molecular tool to stratify patient risk in PDAC and potentially other malignancies
Identification of Altered miRNAs in Cerumen of Dogs Affected by Otitis Externa
Otitis externa is one of the most common diseases in dogs. It is associated with bacteria and yeast, which are regarded as secondary causes. Cerumen is a biological substance playing an important role in the protection of ear skin. The involvement of cerumen in immune defense is poorly understood. MicroRNAs can modulate the host immune response and can provide promising biomarkers for several inflammatory and infectious disorder diagnosis. The aims of this study were to profile the cerumen miRNA signature associated with otitis externa in dogs, integrate miRNAs to their target genes related to immune functions, and investigate their potential use as biomarkers. Cerumen was collected from healthy and otitis affected dogs and the expression of miRNAs was profiled by Next Generation Sequencing; the validation of the altered miRNAs was performed using RT-qPCR. The potential ability of miRNAs to modulate immune-related genes was investigated using bioinformatics tools. The results pointed out that 32 miRNAs, of which 14 were up- and 18 down-regulated, were differentially expressed in healthy vs. otitis-affected dogs. These results were verified by RT-qPCR. To assess the diagnostic value of miRNAs, ROC analysis was carried out, highlighting that 4 miRNAs are potential biomarkers to discriminate otitis-affected dogs. Bioinformatics showed that cerumen miRNAs may be involved in the modulation of host immune response. In conclusion, we have demonstrated for the first time that miRNAs can be efficiently extracted and quantified from cerumen, that their profile changes between healthy and otitis affected dogs, and that they may serve as potential biomarkers. Further studies are necessary to confirm their diagnostic value and to investigate their interaction with immune-related genes
De novo UBE2A mutations are recurrently acquired during chronic myeloid leukemia progression and interfere with myeloid differentiation pathways
Despite the advent of tyrosine kinase inhibitors, a proportion of chronic myeloid leukemia patients in chronic phase fails to respond to Imatinib or to second generation inhibitors and progress to blast crisis. Limited improvements in the understanding of the molecular mechanisms responsible for chronic myeloid leukemia transformation from chronic phase to the aggressive blast crisis were achieved until now. We present here a massive parallel sequencing analysis of 10 blast crisis samples and of the corresponding autologous chronic phase controls which reveals, for the first time, recurrent mutations affecting the ubiquitin-conjugating enzyme E2A gene (UBE2A, formerly RAD6A). Additional analyses on a cohort of 24 blast crisis, 41 chronic phase as well as 40 acute myeloid leukemia and 38 atypical chronic myeloid leukemia patients at onset confirmed that UBE2A mutations are specifically acquired during chronic myeloid leukemia progression with a frequency of 16.7% in advanced phases. In vitro studies show that the mutations here described cause a decrease in UBE2A activity, leading to an impairment of myeloid differentiation in chronic myeloid leukemia cells
A Comprehensive Patient-Derived Xenograft Collection Representing the Heterogeneity of Melanoma
Therapy of advanced melanoma is changing dramatically. Following mutational and biological subclassification of this heterogeneous cancer, several targeted and immune therapies were approved and increased survival significantly. To facilitate further advancements through pre-clinical in vivo modeling, we have established 459 patient-derived xenografts (PDX) and live tissue samples from 384 patients representing the full spectrum of clinical, therapeutic, mutational, and biological heterogeneity of melanoma. PDX have been characterized using targeted sequencing and protein arrays and are clinically annotated. This exhaustive live tissue resource includes PDX from 57 samples resistant to targeted therapy, 61 samples from responders and non-responders to immune checkpoint blockade, and 31 samples from brain metastasis. Uveal, mucosal, and acral subtypes are represented as well. We show examples of pre-clinical trials that highlight how the PDX collection can be used to develop and optimize precision therapies, biomarkers of response, and the targeting of rare genetic subgroups
Ghost mitochondria drive metastasis through adaptive GCN2/Akt therapeutic vulnerability
Cancer metabolism, including in mitochondria, is a disease hallmark and therapeutic target, but its regulation is poorly understood. Here, we show that many human tumors have heterogeneous and often reduced levels of Mic60, or Mitofilin, an essential scaffold of mitochondrial structure. Despite a catastrophic collapse of mitochondrial integrity, loss of bioenergetics, and oxidative damage, tumors with Mic60 depletion slow down cell proliferation, evade cell death, and activate a nuclear gene expression program of innate immunity and cytokine/chemokine signaling. In turn, this induces epithelial-mesenchymal transition (EMT), activates tumor cell movements through exaggerated mitochondrial dynamics, and promotes metastatic dissemination in vivo. In a small-molecule drug screen, compensatory activation of stress response (GCN2) and survival (Akt) signaling maintains the viability of Mic60-low tumors and provides a selective therapeutic vulnerability. These data demonstrate that acutely damaged, ghost mitochondria drive tumor progression and expose an actionable therapeutic target in metastasis-prone cancers
Science with the Einstein Telescope: a comparison of different designs
The Einstein Telescope (ET), the European project for a third-generation
gravitational-wave detector, has a reference configuration based on a
triangular shape consisting of three nested detectors with 10 km arms, where in
each arm there is a `xylophone' configuration made of an interferometer tuned
toward high frequencies, and an interferometer tuned toward low frequencies and
working at cryogenic temperature. Here, we examine the scientific perspectives
under possible variations of this reference design. We perform a detailed
evaluation of the science case for a single triangular geometry observatory,
and we compare it with the results obtained for a network of two L-shaped
detectors (either parallel or misaligned) located in Europe, considering
different choices of arm-length for both the triangle and the 2L geometries. We
also study how the science output changes in the absence of the low-frequency
instrument, both for the triangle and the 2L configurations. We examine a broad
class of simple `metrics' that quantify the science output, related to compact
binary coalescences, multi-messenger astronomy and stochastic backgrounds, and
we then examine the impact of different detector designs on a more specific set
of scientific objectives.Comment: 197 pages, 72 figure
Serum Albumin Is Inversely Associated With Portal Vein Thrombosis in Cirrhosis
We analyzed whether serum albumin is independently associated with portal vein thrombosis (PVT) in liver cirrhosis (LC) and if a biologic plausibility exists. This study was divided into three parts. In part 1 (retrospective analysis), 753 consecutive patients with LC with ultrasound-detected PVT were retrospectively analyzed. In part 2, 112 patients with LC and 56 matched controls were entered in the cross-sectional study. In part 3, 5 patients with cirrhosis were entered in the in vivo study and 4 healthy subjects (HSs) were entered in the in vitro study to explore if albumin may affect platelet activation by modulating oxidative stress. In the 753 patients with LC, the prevalence of PVT was 16.7%; logistic analysis showed that only age (odds ratio [OR], 1.024; P = 0.012) and serum albumin (OR, -0.422; P = 0.0001) significantly predicted patients with PVT. Analyzing the 112 patients with LC and controls, soluble clusters of differentiation (CD)40-ligand (P = 0.0238), soluble Nox2-derived peptide (sNox2-dp; P < 0.0001), and urinary excretion of isoprostanes (P = 0.0078) were higher in patients with LC. In LC, albumin was correlated with sCD4OL (Spearman's rank correlation coefficient [r(s)], -0.33; P < 0.001), sNox2-dp (r(s), -0.57; P < 0.0001), and urinary excretion of isoprostanes (r(s), -0.48; P < 0.0001) levels. The in vivo study showed a progressive decrease in platelet aggregation, sNox2-dp, and urinary 8-iso prostaglandin F2 alpha-III formation 2 hours and 3 days after albumin infusion. Finally, platelet aggregation, sNox2-dp, and isoprostane formation significantly decreased in platelets from HSs incubated with scalar concentrations of albumin. Conclusion: Low serum albumin in LC is associated with PVT, suggesting that albumin could be a modulator of the hemostatic system through interference with mechanisms regulating platelet activation
Search for dark matter produced in association with bottom or top quarks in âs = 13 TeV pp collisions with the ATLAS detector
A search for weakly interacting massive particle dark matter produced in association with bottom or top quarks is presented. Final states containing third-generation quarks and miss- ing transverse momentum are considered. The analysis uses 36.1 fbâ1 of protonâproton collision data recorded by the ATLAS experiment at âs = 13 TeV in 2015 and 2016. No significant excess of events above the estimated backgrounds is observed. The results are in- terpreted in the framework of simplified models of spin-0 dark-matter mediators. For colour- neutral spin-0 mediators produced in association with top quarks and decaying into a pair of dark-matter particles, mediator masses below 50 GeV are excluded assuming a dark-matter candidate mass of 1 GeV and unitary couplings. For scalar and pseudoscalar mediators produced in association with bottom quarks, the search sets limits on the production cross- section of 300 times the predicted rate for mediators with masses between 10 and 50 GeV and assuming a dark-matter mass of 1 GeV and unitary coupling. Constraints on colour- charged scalar simplified models are also presented. Assuming a dark-matter particle mass of 35 GeV, mediator particles with mass below 1.1 TeV are excluded for couplings yielding a dark-matter relic density consistent with measurements
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