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Developing of a novel enantioselective biocatalyst acting on L-amino acids of biotechnological interest through a "semi-rational design" approach supported by computational analyses.
No full textThe synthesis of optically pure enantiomers is a challenge for traditional chemistry. Enzymes often perform better than inorganic catalysts because of their enantioselectivity, effectiveness and ecological sustainability. However, natural enzymes that evolved under the drive of natural selective pressure for their biological function, are not suitable for most industrial applications 1. For this reason, enzymes must be improved by protein engineering.
L-amino acid deaminase from Proteus myxofaciens (PmaLAAD) catalyzes the oxidative deamination of L-amino acids (AAs) to the corresponding -keto acids (KA). This biocatalyst can be used for the setup “green processes” to produce pure KA and D-AAs, which are used as raw materials for the synthesis of drugs and agrochemicals. In this project, several biocatalytic processes based on PmaLAAD have been optimized for the bioconversion, deracemization and stereoinversion of (un)natural L-AAs 2. Several natural or synthetic L-AAs have been used as substratet obtaining conversion yields close to 100%. The knowledge of the 3D structure of PmaLAAD 3 was exploited to produce several enzyme variants possessing animproved activity on L-1-naphthylalanine (L-1-Nal) through a semi-rational design approach supported by computational analysis. The most interesting variant (F318A/V412A/V438P-PmaLAAD) was able to convert D,L-1-Nal with a rate 7.5 fold higher than the wild type
Back to plants for drug discovery: from ethnomedicine to more conventional approaches
No full textOver the last years, there is rekindling of interest in drug discovery from botanical resources. This thesis proposed two different approaches (from ethnomedicine to scholarly strategies) for drug discovery from medicinal plants, identifying the areas of knowledge involved and addressing the challenges encountered, with the aim of enhancing the chance of success of the overarching process.
The first approach consists to review the literature to increase understanding of a plant of interest and generate strong hypotheses for future drug development research on this plant.
Illustrating such an approach, we focused on Tithonia diversifolia (Hemsl.) A. Gray (TD). Knowledge about TD was collected from both online databases and non-electronic resources. Overall, a total of 1,804 reports have been collected. After subsequent duplicates removal and screening for relevant titles and abstracts, a total of 119 text articles were obtained and assessed for eligibility. Finally, 168 articles were selected, of which forty-nine were added after analyses of the reference lists of the included papers.
We found that all parts of the plant are valued in several cultures for a wide scope of ailments ranging from topical issues —wounds, skeleto-muscular disorders, abscesses, dermatological conditions, and stomach pains— to systemic disorders such as diabetes, malaria, fever, hepatitis and infectious diseases. Importantly, most of the ethnomedical claims of TD have been substantiated in several studies conducted in vitro and in vivo in animals. Sometimes, findings were conflicting and thanks to this review, we were able to assess the weight of evidence for each pharmacological effect of TD. The anti-inflammatory, antimalarial, antidiabetic, antioxidant and anticancer effects do stand out but there is also a stunning array of other relevant pharmacological effects. Chemically, a hundred of chemicals, mainly terpenoids and phenols, have been isolated from various TD extracts so far. Of these, some compounds including Tagitinin C have been linked to the pharmacology of TD. About the toxicological profile, we were able to conclude based on evidence that short-term oral administration of TD is relatively well-tolerated in animals when taken at doses less than 100 mg/kg.
The second approach consists to screen the ethnomedical knowledge of indigenous people to select the best plant candidate to launch a drug discovery campaign. So, we carried out a 6-month cross-sectional questionnaire-based survey to explore the use of medicinal plants (MP) in People living with HIV (PLHIV) in the city of Dschang (West Region, Cameroon).
Of the 247 HIV-infected respondents, 54.9% reported to use plants. MP users were then kindly invited to provide photographs and/or specimens of plants for botanical identification. A total of 70 MP, chiefly the herbs, were mentioned by informants (82.2% of total MP users, mean±SEM: 2.2±0.2 MP/subject, min 1, max 11), of which forty-nine have been botanically identified. Commonly reported pathological conditions or symptoms treated with MP included malaria (n = 27, 18.4% of total citations), cough (n = 20, 13.6%) and abdominal pain (n = 16, 10.9%). The benefits of using MP reportedly ranged from moderate (n = 60, 57.7%) to complete (n = 35, 33.7%) relief, while only 8 subjects (7.7% of MP users) reported no change in their terms. Interestingly, 2 subjects (33.3 % of respondents) denounced fatigue and weight loss.
We also observed that THPs were the main advisors of PLHIV on the use of MP. Thus, in the rest of our study, their knowledge and attitudes towards HIV/AIDS were surveyed aiming at understanding whether they may be an appropriate resource to assist in the scaling up of HIV prevention and treatment delivery services in Cameroon. 16 THPs were recruited by the chairperson of the Cooperative Society of Producers of Medicinal Plants of West Region based on their good reputation in traditional healing practice. Three of them acknowledged the use of MP to manage HIV diseases in their clients. All THPs who agreed to participate in the survey were also evaluated for their knowledge of HIV transmission, prevention and diagnosis. We found that their knowledge related to HIV was relatively low raising concern about their aptitude to effectively assist conventional health practitioners in fighting against HIV/AIDS.
Resulting from literature mining and ethnomedical claims is the adoption of a relevant pharmacological testing system. In any case, the testing systems should represent the biological activities that best match the ethnomedical uses of the selected plant species. In addition, it is important to bear in mind that plant extracts are complex mixtures containing various components and, therefore, their overall activity results from interactions between their naturally occurring ingredients.
It is with this background that we conducted a study of comparison of the effects of a whole extract of a particular strain of Cannabis sativa L. to that of cannabidiol (CBD). We knew cannabis is endowed with a potent anti-inflammatory effect attributable mainly to CBD, but also to its entourage. This mechanism by which other compounds occurring in cannabis may contribute to its clinical effects has been espoused as an “entourage effect”. The concept of entourage effect was first introduced in 1998 by Ben-Shabat and Raphael Mechoulam but still, there was no hard evidence that the entourage effect is real. So, thanks to a collaboration with a pharmaceutical company, we grew a particular strain of cannabis deprived of THC and standardized in 5% CBD (CM5). Then, we tested the effects of an extract of CM5 in parallel to that of pure CBD at equimolar concentrations on neutrophil functions including oxidative metabolism, migration and production of proinflammatory cytokines.
Results show that CM5 0.05-50 μg/mL and CBD 10-8-10-5 M inhibit the neutrophil functions including ROS production, cell migration, mRNA levels of proinflammatory cytokines (but at the protein level, only TNF-a was inhibited) to a comparable extent, indicating that CBD may be the main responsible of the anti-inflammatory effects of Cannabis. The effects of CBD and CM5 show however remarkable differences in terms of potency and efficacy, suggesting that beyond CBD, other components of cannabis may contribute to its biological effects. As a whole, such results support the use of cannabis and CBD to stem inflammation, however also warrant in-depth investigation of the underlying cellular and molecular mechanisms to better exploit their therapeutic potential
RUNX2 associated long non-coding RNA characterization
No full textRUNX2 is a lineage-specific transcription factor (TF) known to promote cancer progression. However, the molecular mechanisms that control RUNX2 expression in cancer remain widely unknown. Long non-coding RNAs (lncRNAs) are a novel class of transcripts that do not code for proteins and are often engaged in gene expression regulation.
Using the ENCODE annotation data, we identified a previously uncharacterized family of lncRNAs within the RUNX2 locus, that we named RAIN (RUNX2 Associated Intergenic Non-coding RNA). We showed that RAIN comprises 4 major variants that share a common central region but differ at the 5'- and 3'- ends. The longest isoform (l-RAIN) is nuclear and strongly associated with chromatin, suggesting a role of RAIN in gene expression regulation. Expression analysis in cancer cell lines and patient samples demonstrated that RAIN correlates with RUNX2. Furthermore, RAIN silencing resulted in a significant RUNX2 repression demonstrating that this lncRNA is required for the expression of this TF in cancer. We showed that RAIN promotes RUNX2 expression at least through two distinct mechanisms. Interacting with WDR5 and directing its recruitment to the P2 promoter, RAIN modifies its transcriptional activation status, bursting transcription initiation. In parallel, RAIN sequesters NELFe preventing the binding of the NELF complex to the RUNX2 P2 promoter and restraining its inhibitory function on nascent transcripts elongation. Finally, we investigated the RAIN associated transcriptional profile in thyroid cancer showing that beside RUNX2, this lncRNA controls a panel of cancer associated TFs. Overall, our data characterize the function of novel lncRNA and identify an additional layer in the complex RUNX2 regulation in cancer
Boring bacteria: a morphological research on bone diagenesis.
No full textPost mortem interval (PMI) estimation is a crucial issue in forensic medicine. To date, little is known about factors affecting post-mortem changes in hard tissues and it is still unclear who between exogenous bacteria from the environment and endogenous microbiota is the cause of microscopical alterations observed in human bone after death. Recent research highlighted an important role of endogenous bacteria in the earlier stages of the process. The aim of this study was to probe a potential endogenous model of human bone biodeterioration, based on the action of oral cavity endogenous microorganisms.
A total of seventy-four fragments of human bone samples were incubated with six bacterial strains, isolated from human tartar specimens. In a forty-eight months long prospective study, the onset and development of bone tissue alterations were serially analysed by scanning electron microscope.
The research furnished evidence that endogenous bacteria are able to bore into human dead bone, giving rise to microstructural changes morphologically indistinguishable from those observed in archaeological and forensic bone
Aspetti etici del trattamento delle malformazioni cranio-facciali.
No full textWhen treating dento-facial deformities, the attention has always been put on the technical aspects of the various interventions instead of the ethical aspects, given the fact that dealing with children the primary worry is providing them with the best cure and the fastest path to a normalization of their deformities.
Nonetheless, surgical and dental procedures do modify the aesthetics of the patient, thus you need to take into account also the ethical aspects of such treatments.
Our aim has been to identify the outstanding ethical issues regarding the treatment of dento-facial deformities and to evaluate the perception of some peculiar aspects of the treatment by submitting a questionnaire to both the children and the parents.
Facial aesthetics represent a psychological issue for most people, and our research confirms it; in particular parents are concerned about the social consequences related to an uncertain facial aesthetic.
The crucial point, as always when treating children, is to pursue the best interest for them, leaving aside those concerns typical of the adults
Storia ambientale dell’energia nucleare. Gli anni della contestazione.
No full textFor more than half a century, nuclear energy, both for military and civil uses, has outlined one of the most controversial and debated political, scientific as well as cultural issues: A long-lasting and often violent dispute, tragically opened by the atomic bombs on Hiroshima and Nagasaki, which has weighed heavily upon all the different phases of the Cold War, up to the dawn of the new century and beyond. Making use of a wide variety of archive sources, the book aims at offering one of the first accounts about nuclear energy from the perspective of the environmental history studies. It focuses mainly on that particular historical period which began with the social protests of 1968 and was followed by the oil crisis (1973) and economic turmoil of the Seventies. However, it should not be ignored this “age” was also distinguished by the spread of ecological sciences as well as the emergence of massive environmental public pressures. Laying emphasis on the Italian background, the monograph considers those geological and environmental concerns which involved debates about industrial applications of nuclear technology in the post-oil-crisis backdrop. The work focuses on a period during which the public controversy on nuclear energy was not yet affected by the catastrophic accidents of Černobyl’ and Fukushima, nevertheless it reveals how conflicts, contradictions and divisions were already subverting both science and society
Towards the Extremely Large Telescopes era in exoplanetary science: simulation tools, instrumental optimization and design for high resolution spectroscopy. The cases of ESPRESSO and ELT-HIRES.
No full textIn this thesis I present my PhD activities concerning the development of simulation tools both for ground-based high resolution spectrograph design, ESPRESSO and ELT-HIRES, and for scientific investigations in the field of exoplanetary high-dispersion transmission spectroscopy. In the ESPRESSO (the Echelle SPectrograph for Rocky Exoplanets Super Stable Observations) project, the instrument modeling through ray-tracing bsoftware and ad-hoc developed sensivity analysis tool were fruitfully used for component optimization and alignment verification. In the framework of the ELT-HIRES (the HIgh REsolution Spectrograph for the ELT) project, an End-to-End simulator and a parametric paraxial model of the spectrograph were developed with the purpose to evaluate the different effects which can affect the final instrument performances, since they directly influence the scientific data observational performances. The synthetic echellogram (raw frame) produced and successfully processed by the CRIRES+ instrument data reduction pipeline are presented, thus probing the full chain feasibility and consistency of the system. Large telescopes allowing very high contrast, could also imply the possibility to detect the light emitted from background sources and/or from gravitationally bounded companion of exoplanetary system, which could act as signal contamination. A transmission spactra simulator, a tool aimed at generating synthetic spectra, was developed and used to show that the maximum contamination occurs for background stars of G-to-M type, even though for high resolution spectra with a 4m class telescope this contamination seems to not introduce obvious shifts or line broadening in the exoplanet atmospheric features
A new methodological approach in the diagnosis of extra-medullary lymphomas: the role of classical and molecular cytogenetics.
No full textLymphomas are a group of heterogeneous malignant neoplasms involving B, T and NK lymphocytes. Their localization is predominantly lymph node, but frequently they can diffuse to the bone marrow. Cytogenetic analysis, required for a correct diagnosis, prognosis and therapy is harder when the lymphoma is extra-medullary: in these cases Fluorescence In Situ Hybridization (FISH) analysis is commonly applied, performed on interphase nuclei coming from sections of lymph node tissue. However, this method has some limitations: 1) it is based on the use of locus specific probes and it provides targeted information; 2) the outcome of FISH investigations is strongly conditioned by the correct processing of the biopsy sample and by the presence of a sufficient amount of neoplastic cells.
The aim of this work is trying to optimize the tissue taken by lymph node biopsy in a patient with suspected extra-medullary lymphoma, producing cell cultures added with a specific combination of B-cell mitogen named DSP30 with Interleukin 2, the same used in the BM cultures. On this "enriched" and correctly processed sample, we performed a complete analysis of the karyotype that, in some cases, allowed to identify additional chromosomal alterations comparing to those highlighted by FISH analysis.
Based on the obtained results, we believe that we have developed an ideal protocol to improve the cytogenetic analysis of lymphomas, including the different types of cell culture, the use of an appropriate mitogen and the analyses made by conventional and molecular cytogenetics. Our methodological approach, moreover, allows storing cells suspension, bioptical and histological samples, that can be used also later to perform molecular genetic investigations
Indagini antropologiche e paleopatologiche di un sito archeologico: San Biagio in Citiglio.
No full textThe present PhD thesis focuses on the site of the church of San Biagio in Cittiglio (Va). This work is part of a project started in 2006 in collaboration between the Research Center of Osteoarchaeology and Paleopathology of the University of Insubria and the Superintendence for Archaeological Heritage of Lombardy.
This site is particularly interesting because it covers a very wide chronological range from the eleventh to the seventeenth century and due to the presence of numerous infantile remains. The study is based on a work of systematic integration between anthropological investigation and archaeological investigation to arrive at a definition of the socio-demographic dynamics of the area. In fact, anthropological analysis has been useful not only to reconstruct the biological history of the individual, but also to contribute to the reconstruction of the past of an entire community. The skeletal material was analyzed according to standard methods for identification, paying attention to the taphonomic aspects with reference to the study of burials and to the paleopathological and traumatological aspects important for defining the internal organization of the community
Selection, cultivation and biochemical analysis of fungi with pharmacological properties.
No full textThe importance of medicinal mushrooms has rapidly increased in the last decades thanks to the numerous studies carried out on their beneficial properties and the isolation of bioactive compounds able to bring significant benefits to human health. Among the most studied are found in particular, the saprotrophic fungi Lentinula edodes and Pleurotus ostreatus, belonging to the phylum Basidiomycota. Their economic and commercial interest has rapidly grown thanks to new methods of cultivation on substrates made ad hoc that allow to obtain high yields and high-quality mushrooms. The main purpose of this PhD thesis is to characterize the shiitake and oyster strains collected by different world banks, both commercial and research, comparing their activities and evaluating their beneficial potentials to create high-potential food products for the market of Italian and international functional food. In order to achieve this goal, different properties were evaluated, and two cultivation methods were compared, one on a commercial substrate based on sawdust and straw and the second on hardwood logs using a traditional method. This research project allows to select specific strains according to the type of activity considered, also evaluating production costs, yields and timing in order to create food products for the functional food market