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Integrating phenotypes and endotypes in chronic rhinosinusitis: a combined clinical and experimental approach.
No full textChronic rhinosinusitis (CRS) represents a hot and debated topic in rhinology because of its high prevalence, heterogeneity of clinical manifestations and unpredictability of disease course. The quite recent dichotomic classification of CRS with and without nasal polyps has proved to be too simplistic to fully explain CRS manifestations and the underlying pathogenetic mechanisms. Being either the same phenotype expression of substantially different pathogenic mechanisms or different phenotypes the expression of the same mechanism, a one-size-fit-all therapeutic approach turned out to be insufficient in a non-negligible proportion of patients.
Moreover, considering the attempt of giving a classification cut at a biomolecular level, a diagnostic and prognostic approach exclusively limited to subjective and objective clinical parameters is inevitably failing in many ways. However, to date no other more effective markers are available to monitor the trend of the disease.
The fact of dealing with an apparently very frequent pathology responsible for a strong discomfort on the QoL and a substantial economic impact requires a diagnostic and therapeutic appropriateness for an adequate allocation of resources within the standards of precision medicine. The development of systems for a uniform archiving and sharing of the experiences of each rhinological center would enhance the efforts of the scientific community in defining integrated and targeted care pathways.
The present thesis reports the results and the practical implications of three different experimental studies about the implementation of data storage and sharing systems, methods of analysis of therapeutic outcomes and inflammatory biomarkers in CRS
Essays in Behavioural and Experimental Economics: Emotions, uncertainty and cooperation
Full text linkIn the present doctoral thesis I apply the experimental method in the context of two lines of research in Economics, the former investigating the role of incidental emotions in decision making under risk, whereas the latter shedding light on the impact of communication on cooperation. The dissertation consists of three sections. In the first chapter I conduct a laboratory experiment in order to study the effect of incidental sadness and happiness on risky decision making. An emotion induction procedure is the treatment variable of a between-subjects design where two sessions aim at eliciting either sadness or happiness, respectively. Two further groups are characterized by neutral conditions and serve as baseline. After a manipulation check verifies the validity of the induction procedure, I use a multiple price list `a la Holt and Laury (2002) to elicit individual risk preferences in the context of a lottery-choice task. The analysis reveals that both sadness and happiness promote greater risk aversion with respect to neutral conditions, a result which might be moderated by the risk elicitation task. Therefore, as effective explanation I propose the theory of ego depletion, whereby regulating emotions so as to subsequently process information consumes a limited self-control resource, which is needed to take risks as well. The second chapter is a meta-analysis of experimental studies on the same topic, so as to explain traditional heterogeneity of outcomes in the field. After performing an advanced search in Google Scholar and filtering out studies that do not match a list of selection criteria, I include 16 studies from which 46 observations are drawn at the treatment level. At this point, I code a set of moderator variables representing experimental protocols and calculate Cohen (1988)’s d effect size as dependent variable of a weighted least squares (WLS) regression where larger studies are given more weight. Among the results, which are robust to different techniques for computing standard errors, I find that emotions induce higher risk aversion when a multiple price list `a la Holt and Laury (2002) is used in place of stated preferences methods, as well as in case the risk elicitation task is framed as an investment decision instead of an abstract choice. Given the variety of procedures employed in this type of experiments and in the absence of a tailor-made game to answer such research questions, I recommend faithful study replication as preferential path in order to investigate the influence of emotions on risky decision making and ensure comparability. The third chapter offers evidence on the impact of communication on the provision of public goods whose quality is uncertain. I run a laboratory experiment with two treatments, where the control variable is pre-play communication in the form of unrestricted text chat. A binary threshold public goods game with four-person groups and threshold of three is at the core of the design, the main novelty lying in the provision mechanism with ambiguity. Moreover, a private signal for the actual value of the public good is provided, before the contribution decision. In accordance with related literature, I find that communication significantly increases public good provision by reducing inefficiency that comes from wasteful undercontribution. Nevertheless, the players in the chat treatment seem to neglect the free-rider issue and often end up overcontributing, in contrast with previous scientific findings. After chat analysis, I propose the pursuit of symmetric payoffs within the group as original explanation of the massive overcontribution, in addition to group identity generated by the partner matching and the common fate hypothesis. Since the players prefer to minimize ambiguity than to maximize the group earnings, I finally speculate that under uncertainty satisficing is more salient than optimizing
Characterization of new synthetic or natural compounds with broad spectrum or dual antiviral activity in HIV-1 treatment
Full text linkIn recent years many advances have been made in the fight against HIV-1 infection. However, the lack of a vaccine, together with the increasing resistance to the highly active anti-retroviral therapy (HAART), make HIV-1 infection still a serious global emergency. Development of inhibitors that operate by a novel mechanism of action could expand the options for clinicians to address these unmet medical needs for the HIV-1-infected patient population or could represent novel options for an effective prevention.
In this contest, one possibility is the development of single drug with multiple targets binding, such as the natural compound kuwanon-L. Derived from the computational identification of novel potential allosteric inhibitors of HIV integrase, in this thesis kuwanon-L has been further characterised in enzymatic assays to evaluate its activity in vitro. Moreover, kuwanon-L was tested in cellular-based assays to confirm its antiviral activity also on viral strains. Finally, its mechanism of action was further investigated through a time-of-addition (TOA) experiment and its innovative dual mode of action on both HIV integrase and reverse transcriptase was confirmed in enzymatic assays.
On the other hand, compounds with novel mechanisms of action and targeting very early steps of viral life cycle, as the novel series of rhodanine derivatives investigated in the second part of this work, may represent potent anti-HIV-1 agents suitable for prevention approaches. Their activity not only on HIV but also on other sexually transmitted infections such as HSV-1/2 viruses, and their favourable pharmacokinetic profile further emphasize their usage as topical microbicides in pre-exposure prophylaxis (PrEP) approaches
Barred galaxies in cosmological simulations. Tidal perturbations and feedback.
Full text linkBars are truly common objects among disc galaxies. Even though it is now commonly accepted that non-axisymmetric structures could deeply affect the life of isolated disc galaxies, by shaping their stellar and gaseous distribution, some fundamental aspects of their formation and growth are still debated. The following manuscript is divided in two parts, in which I contribute to the study of bar formation and evolution through the use of state-of-the-art hydrodynamical and cosmological simulations. In the first part of my work, I investigate the influence of tidal encounters on either unbarred, or already barred systems. Here, I take advantage of two specific simulations belonging to the Eris suite – ErisBH and Eris2k – which evolve two Milky Way-sized barred galaxies in their cosmological volume. I both analyse the direct output of these simulations, and run a sample of new cosmological zoom-in simulations, by altering the original history of galaxy-satellite interactions in the ErisBH run. In the second part, I focus on the analysis of the effect of the different feedback prescriptions implemented in the ErisBH and Eris2k runs. My conclusions depict a scenario where, once the disc has grown to a mass large enough to sustain global non-axisymmetric modes, bar formation is inevitable and tidal encounters have, a destructive effect on the two-fold overdensity. In addition, the self-gravity of the disc and its interplay with various internal processes, seem to be the main drivers of bar formation and growth. I confirm the importance of accurate feedback prescriptions in cosmological zoom-in simulations on the investigated processes, and propose a possible use of a statistical sample of barred galaxies to quantitatively assess this dependence
Improving ecosystem exposure assessment of organic chemicals: evaluating their mobility and bioavailability with novel chemical measurements and modelling approaches.
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Semantics for Homotopy Type Theory
Full text linkThe main aim of my PhD thesis is to define a semantics for Homotopy type theory based on elementary categorical tools. This led us to extend the study of this system in other directions: we proved a Normalisation theorem, and defined a generic syntax. All those results are obtained for a subset of the whole Homotopy type theory, which we called 1-HoTT theories.
A 1-HoTT theory is composed by Martin-Löf type theory with generic inductive types, the axioms of function extensionality and univalence, truncation and generic 1-higher inductive types, which are a subset of the higher inductive types in which the higher constructor of a type T is limited to the type =T .
For those theories we obtained some proof theoretic results; the main one is a Normalisation theorem, following Girard's reducibility candidates technique.
The semantics is sound and complete, with the completeness result following from the existence of a canonical model, which is also classifying.
Our conjecture is that our proof theory and semantics can be extended to every single higher inductive type. The dissertation shows that a very large amount of higher inductive types can be analysed inside our framework: what prevents to extend the results is the lack of a systematic treatment of the syntax of the higher inductive types, which is still an open issue in Homotopy type theory
Lo sviluppo rurale nel contesto pedemontano: la dimensione territoriale della 'nuova' agricoltura varesina
No full textThe thesis analyzes the role of the agricultural system in the Lombardy foothills context, in particular deepening the phenomena of growth and diversification of farms, referring to the paradigms of rural and local development. The aim of the research is to identify, starting from these peculiarities, the potential and the possible evolution of the role of agriculture in the development of the foothills territories, and in the Varese context in particular, investigating the relationship between the agricultural system and the territory, reframing the role of the territorial dimension of the economic development. Therefore, the research started with the review of the theoretical economic models that, by focusing on the territorial dimension of development, appear able to integrate new paradigms of rural development, recognizing, in the comparison between the different disciplinary approaches, the elements of convergence towards a 'territorialist' approach. The thesis then investigated the different methods of analysis of agricultural systems and rural areas and their outcomes in terms of classification of rural areas in Lombardy, comparing them with an analysis of the structural variables of the regional agricultural system. The case study in the Varese area was studied in greater depth, focusing on its specificity and in particular: deepening the dynamic aspects of change and evolution of the agricultural system, interpreting the 'return' to agriculture given by the increase of firms as a potential perspective; examining the role of multifunctionality both from the point of view of producing services related to agriculture and therefore of firm diversification, income integration and relations with other economic sectors, and with a view to producing common goods and ecosystem services, or rather considering the role of the territory in agricultural activity, but, on the contrary, the territory produced by agricultural activity; overcome the dichotomic approach between city and country, not only by identifying the peri-urban areas, but rather by recognizing the complex and integrated links between different places and landscapes that constitute, by virtue of their relationships, a single territorial system. In the end, the interpretative elements that emerged from the case study has been systematized and made it possible to recognize the possible roles of the territorial dimension in the Varese agricultural system and the potential for integrated development of the various “local farming systems
Preliminar evaluation of epigenetic modifications followingi vitro hydroquinone exposure in models of human acute promyelocytic leukemia cells (HL-60) and of human umbilical cord mesenchimal stem cell (hUCMSC)
No full textBenzene, a volatile aromatic hydrocarbon, is extensively used in industry even though it is recognized as a myelotoxin with leukemogenic activity, representing a significant occupational risk. Metabolism of benzene plays a fundamental role in its toxicity, and among the different metabolites, hydroquinone (HQ) is one of the most important, as it accumulates in the bone marrow where it can induce several genetic and epigenetic changes. Nevertheless, the actual mechanisms behind the carcinogenetic effects of benzene and HQ and the role of epigenetic alterations in the process of tumorigenesis are not fully clarified yet.
Aberrant patterns of DNA methylation, including loss of imprinting (LOI), gene-specific hyper- or hypomethylation and global hypo-methylation are all common in several tumor types, including AML, and are important for transcriptional repression or activation of cancer-associated genes. Most of the studies on the epigenetic effects of benzene conducted so far have been centered on DNA methylation (measured on repeated elements LINE-1 and Alu, used as a surrogate for the entire genome), whereas only very recently a few investigations have analyzed the effects of environmental chemicals on histone modifications.
Several studies have shown a strong correlation between DNA methylation and histone modifications, indicating a cross-talk between DNMTs and histone modifications. For example, trimethylation of H3K27 (H3K27me3) can be associated with increased DNA methylation while de novo methylation does not occur in regions where bi-or trimethylation of the histone 3 lysine 4 (H3K4me2 or H3K4me3) is observed. Conversely, the simultaneous presence of H3K27me3 and H3K4me3 is recognized as a bivalent mark; it was firstly described during development and it appears to play a role in poisoning non-active genes for aberrant transcription in cancer.
Most notably, epidemiological studies on healthy subjects have shown a difference in the pattern of methylation between subjects exposed to benzene (gasoline station attendants) compared to office workers, with a significant reduction in the methylation of LINE-1 and Alu. This profile of global hypomethylation following exposure to low doses of benzene appears to be qualitatively comparable to what observed in AML and other neoplasms.
Thus, in the present work we attempted to reproduce in vitro the conditions of chronic exposure to benzene found in the peripheral blood of subjects exposed. We started from “low” doses of hydroquinone in vitro (below 15 μM) and we proceeded by progressively lowering the concentration to explore the cellular response to different types of exposure and concentrations, to evaluate if HQ might be able to alter the epigenetic signature in two different biological systems, thereby describing a poorly explored
step in the mechanism of toxicity associated with benzene exposure.
We finally set up a chronic treatment 4 weeks-long with HQ 1 μM, corresponding to 110 ng/mL, a concentration within the amount of total HQ (between 20 and 120 ng/mL, corresponding to 2–16 ng/mL of free HQ) found in the blood of subjects exposed to airborne benzene ranging from 1 mg/m3 (around 0,3 ppm) to 80mg/m3 (around 25 ppm). This concentration was used on a stabilized cell line of human acute promyelocytic leukemia (hAPML), HL60, analyzed as a model of hematopoietic cells; exploring the epigenetic events occurring in chromatin, we found the instauration of the distinctive signature combining the repressive H3K27me3 and the activating H3K4me3, with the gradual increase in H3K4me3 levels, on LINE-1 promoter region. We observed the absence of statistically significant variations in DNA methylation and expression levels of LINE-1, despite a decrease in protein levels of UHRF1, DNA methyl-transferases and histone methyl-transferases.
Moving onto a model of normal stem cells, we had to further lower the concentration to avoid cytotoxicity. Thus, human umbilical cord mesenchymal stem cells (hUCMSC) were treated for 4 weeks with HQ 0,1 μM to verify if long-term exposure to low doses of HQ could be able to alter the epigenetic signature in staminal cells outside the hematopoietic compartment. Surprisingly, we found a progressive increase of H3K4me3 in our control cells, with the instauration of the bivalent mark at the third week; the fourth week was non-evaluable, since the cytotoxic effect was prevalent despite the reduced concentration. Most interestingly, at the third week we observed the peculiar inversion in the levels of H3K27me3 and H3K4me3 on LINE-1 promoter region, with higher H3K27me3 in HQ treated cells as compared to the control, and opposite to what observed in HL60. On the other hand, as for HL60 cells no statistically significant variations in DNA methylation was appreciable. These differences were seen not only on chromatin but also on the profile of mRNA expression: preliminary Principal Component Analysis showed a significant distance between controls in the three biological replicates under consideration.
In conclusion, in vitro treatment with low-dose HQ determined the instauration of a poisoned state of chromatin in LINE-1 sequences in both the models considered, suggesting that prolonged exposure could cause persistent epigenetic alterations
DNA hypomethylation and hypermethylation in colorectal cancer initiation.
No full textThis project was focused on identification of new valuable molecular risk factors for the onset of colorectal cancer (CRC), studying both KRAS/NRAS/BRAF/PIK3CA mutations and DNA global hypomethylation in the early events of colorectal carcinogenesis. We analyzed a cohort of 52 colorectal adenomas and 11 carcinomas derived from MAP subjects, 80 sporadic adenomas and 15 carcinomas and a control set of 36 FAP/AFAP adenomas. Moreover, we characterized the L1-MET transcript induced by L1 hypomethylation. We observed that the early steps of oxidative DNA damage in MAP carcinogenesis are characterized by a specific pattern of somatic mutations. We also found that MAP adenomas and carcinomas show a decreased DNA global methylation and specific L1-MET hypomethylation. Finally, we hypothesized that DNA hypomethylation and expression of L1-MET chimeric transcript may play an early role in colorectal carcinogenesis characterizing a subset of more aggressive precursor lesions and cancers. In the second part of the thesis, we studied the promoter of MutL homolog 1 (MLH1) in order to elucidate the relationship between methylation and risk/protective allele at rs1800734 during CRC progression. We confirmed the association of rs1800734 with microsatellite instability (MSI) in our own data. In 33 normal colon biopsies, small allele-specific differences exist only in methylation, but not gene expression. In contrast, allele-specific differences in both MLH1 methylation and expression are present in 35 MSI cancers. We showed that MLH1 transcriptional repression is dependent on DNA methylation and can be reversed by a methylation inhibitor. The rs1800734 allele influences the rate of methylation loss and amount of re-expression
Genomic molecular markers to monitor minimal residual disease with a non invasive liquid biopsy in breast cancer patients
Full text linkBackground
Circulating cell free DNA (cfDNA) is one of the most intriguing and developing topic in the field of precision and personalized medicine.
From 1977, when Leon and colleagues reported an increase in cfDNA in cancer patients, several studies have tried to explain and understand deeper how cfDNA is produced, lasts into the bloodstream, and what kind of information it contains and can be useful to detect and/or manage the disease.
Liquid biopsy has already been approved to determine those patients that will benefit from an epidermal growth factor receptor (EGFR)-targeted therapy in non-small cell lung cancer (NSCLC).
Nowadays, several studies on clinical applicability and clinical trials are on-going, in order to determine if information obtained from cfDNA can drive clinical decisions.
Homologous recombination (HR) is a high-fidelity DNA repair mechanism involved in double-strand DNA (dsDNA) break repair. Recent studies have highlighted a broader involvement of HR status in BRCA wild type breast cancers (BC), in particular in triple negative BC (TNBC).
Identification of mutations in the HR pathway can suggest the administration of a specific therapy, such as platinum agents or PARP inhibitors (PARPi).
Material & Methods
In this work we have collected fresh tissue and blood from 6 BC patients.
Plasma was separated with two consecutive centrifugations to completely remove cell and cellular debris.
We have extracted genomic DNA (gDNA) from fresh tissue and cfDNA from plasma using commercial kits.
We have analysed in parallel both DNAs with the Homologous Recombination Solution™ kit (Sophia™ Genetics) that allows the identification of mutations in exonic regions of 16 genes involved in HR.
Results
All samples passed target enrichment and sequencing quality controls (QC).
In 3 out of 6 patients no mutations were detected in both gDNA and cfDNA. Patients 1 and 6 had mutations in gDNA not detected in cfDNA. Patient 2 had 2 mutations in gDNA and only one of the two was detected in cfDNA.
Conclusions
This kit can be used to analyse cfDNA, as confirmed by QC reports.
Interestingly, the identification of the mutation in TP53 in patient 2’s cfDNA supports the possibility to detect mutations with this kit. The higher variant fraction (VF) in cfDNA compared to gDNA can sustain the possibility to have a broader detection of heterogeneity.
The non-recognition of mutations in cfDNA in patients carrying gDNA mutations can be ascribed to the reduced amount of cfDNA analysed and to the fact that tumoral DNA is only a fraction of the total cfDNA. Thus, the sample analysed can be unrepresentative of the circulating DNA pool.
The analysis of a higher number of samples will give us a clearer idea of the applicability of this kit to monitor HR mutational status in cfDNA of BC patients, giving us statistical significance of detection