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Study of photosynthetic and stress-related enzymes: from functional characterization to physiological roles
Get PDFAs we all know, climate change poses new challenges, particularly concerning our ability to cultivate plants for food, feed, and energy in adverse environmental circumstances. In this context, a major issue regards the productivity of agricultural crops. On the one hand, plant productivity depends on plant capacity to accumulate biomass, and on the other hand, it relies on their ability to cope with both biotic and abiotic stress conditions. The thorough understanding of the molecular mechanisms underlying plant physiology is a key step in laying the foundation for future improvement.
This thesis work focuses on the study of five enzymes involved in carbon metabolism. Three of them, namely glyceraldehyde 3-phosphate dehydrogenase (GAPDH), ribulose-5-phosphate-3-epimerase (RPE) and phosphoribulokinase (PRK) participate in the Calvin-Benson-Bassham cycle, the metabolic pathway ensuring the major input of carbon into the organic world and closely influencing the planet's primary productivity. The other two enzymes, namely alcohol dehydrogenase (ADH) and nitrosoglutathione reductase (GSNOR) are involved in plant adaptation to stress conditions. Specifically, ADH is directly implicated in acclimation to oxygen deficiency, while GSNOR serves as a key regulator of intracellular levels of nitrosoglutathione, a critical signalling molecule involved in stress response.
The approach here employed integrates multidisciplinary analyses focused on the structural and biochemical characterization of recombinant enzymes, with a special emphasis on the investigation of potential cysteine-based regulatory mechanisms. Alongside, in vivo and ex vivo analyses have offered insights into the elucidation of the role of these enzymes in their physiological context
Studies on tabelliones - the authors of documents in roman law from the Republic to Justinian
Get PDFIl lavoro analizza le varie categorie di redattori susseguitasi a Roma dall’età repubblicana fino a Giustiniano, con l’intento di verificare se, prima del notaio medievale, a Roma sia esistita una figura assimilabile in qualche misura al notaio contemporaneo. Si sono esaminati in particolar modo notarii, exceptores e tabularii evidenziandone le differenze con i tabelliones, sui quali il lavoro maggiormente si concentra. Per ognuna di queste figure si sono approfondite le fonti in ordine cronologico. Infine, si è tentato di verificare se anche nell’antica Cina esistesse un soggetto paragonabile al tabellio romano e, prendendo spunto dalla necessaria presenza dei testimoni durante la redazione dei documenti tabellionici, come dimostrato dalle fonti trattate, si è verificato il ruolo di questi durante i moderni rogiti notarili in Cina.The work analyzes the various categories of documents' authors that succeeded in Rome from the Republican era to Justinian, with the aim of verifying whether, before the medieval notary, it existed a figure in Rome comparable to the contemporary notary. In particular, notarii, exceptores, and tabularii have been examined, highlighting their differences with tabelliones. For each of these figures, sources have been examined chronologically. Finally, an attempt was made to verify if there was also a subject comparable to the Roman tabellio in ancient China, and, drawing inspiration from the necessary presence of witnesses during the drafting of tabellionic documents, as demonstrated by the sources examined, the role of these witnesses during modern notarial deeds in China has been investigated
IDeS methodology applied to automotive component re-engineering focused on lightweight and high-strength by additive manufacturing
Get PDFThe project aims to gather an understanding of additive manufacturing and other manufacturing 4.0 techniques with an eyesight for industrialization. First the internal material anisotropy of elements created with the most economically feasible FEM technique was established. An understanding of the main drivers for variability for AM was portrayed, with the focus on achieving material internal isotropy. Subsequently, a technique for deposition parameter optimization was presented, further procedure testing was performed following other polymeric materials and composites. A replicability assessment by means of the use of technology 4.0 was proposed, and subsequent industry findings gathered the ultimate need of developing a process that demonstrate how to re-engineer designs in order to show the best results with AM processing. The latest study aims to apply the Industrial Design and Structure Method (IDES) and applying all the knowledge previously stacked into fully reengineer a product with focus of applying tools from 4.0 era, from product feasibility studies, until CAE – FEM analysis and CAM – DfAM. These results would help in making AM and FDM processes a viable option to be combined with composites technologies to achieve a reliable, cost-effective manufacturing method that could also be used for mass market, industry applications
Musculoskeletal disorders, research quality, and pelvic floor health in sports: exploring current evidence and new perspectives
Get PDFBackground. Pelvic floor dysfunction (PFD) is an umbrella term that includes a myriad of conditions such as urinary (UI) and anal incontinence, pelvic organ prolapse, pelvic pain, and sexual dysfunction. Literature showed high prevalence rates of PFD among athletes, especially UI, with high-impact sports have been linked with an increased risk of developing symptoms. However, comprehensive research summarising PFD prevalence across sexes, exploring treatment options, and the absence of a standardised referral screening tool are notable gaps. Misinformation is also prevalent in the sports medicine field.
Methods. This doctoral project comprises four studies addressing different aspects of pelvic health in athletes. The first two studies were scoping reviews of epidemiological PFD data in male and female athletes, as well as available interventions. Study 3 concerned the development of a new screening tool for PFD in female athletes, aiming to guide sports medicine clinicians in referring patients to PFD specialists through a worldwide Delphi consensus. Study 4 summarised all previous findings, integrating data into an infographic.
Results and conclusions. In Study 1, the findings of 100 articles on PFD in both sexes have been collected, highlighting a higher prevalence of studies on female athletes evaluating UI across multiple sports. Other conditions remain rarely investigated. Study 2 found a diverse range of interventions for female PFD, with a notable emphasis on conservative approaches. Recommendations for clinical practice often relied on the transferability of results from the nonathlete population or expert opinions. In Study 3, 41 international experts took part in the consensus development of the Pelvic Floor Dysfunction-ScrEeNing Tool IN fEmale athLetes (PFD-SENTINEL). It incorporates a cluster of PFD symptoms, items (risk factors, clinical, and sports-related characteristics), and a clinical algorithm. Lastly, Study 4 included ten evidence-based information with a relative description concerning pelvic floor health in athletes
Chemiluminescence – based bioanalytical devices for astrobiological, food safety and clinical applications
Get PDFAt the intersection of biology, chemistry, and engineering, biosensors are a multidisciplinary innovation that provide a cost-effective alternative to traditional laboratory techniques. Due to their advantages, biosensors are used in medical diagnostics, environmental monitoring, food safety and many other fields. The first part of the thesis is concerned with learning the state of the art of paper-based immunosensors with bioluminescent (BL) and chemiluminescent (CL) detection. The use of biospecific assays combined with CL detection and paper-based technology offers an optimal approach to creating analytical tools for on-site applications and we have focused on the specific areas that need to be considered more in order to ensure a future practical implementation of these methods in routine analyses. The subsequent part of the thesis addresses the development of an autonomous lab-on-chip platform for performing chemiluminescent-based bioassays in space environment, exploiting a CubeSat platform for astrobiological investigations. An origami-inspired microfluidic paper-based analytical device has been developed with the purpose of assesses its performance in space and to evaluate its functionality and the resilience of the (bio)molecules when exposed to a radiation-rich environment. Subsequently, we designed a paper-based assay to detect traces of ovalbumin in food samples, creating a user-friendly immunosensing platform. To this purpose, we developed an origami device that exploits a competitive immunoassay coupled with chemiluminescence detection and magnetic microbeads used to immobilize ovalbumin on paper. Finally, with the aim of exploring the use of biomimetic materials, an hydrogel-based chemiluminescence biosensor for the detection of H2O2 and glucose was developed. A guanosine hydrogel was prepared and loaded with luminol and hemin, miming a DNAzyme activity. Subsequently, the hydrogel was modified by incorporating glucose oxidase enzyme to enable glucose biosensing. The emitted photons were detected using a portable device equipped with a smartphone's CMOS (complementary metal oxide semiconductor) camera for CL emission detection
Advancing digital healthcare research and machine learning-aided knowledge extraction amidst the COVID-19 pandemic
Get PDFThe rapid progression of biomedical research coupled with the explosion of scientific literature has generated an exigent need for efficient and reliable systems of knowledge extraction. This dissertation contends with this challenge through a concentrated investigation of digital health, Artificial Intelligence, and specifically Machine Learning and Natural Language Processing's (NLP) potential to expedite systematic literature reviews and refine the knowledge extraction process.
The surge of COVID-19 complicated the efforts of scientists, policymakers, and medical professionals in identifying pertinent articles and assessing their scientific validity. This thesis presents a substantial solution in the form of the COKE Project, an initiative that interlaces machine reading with the rigorous protocols of Evidence-Based Medicine to streamline knowledge extraction.
In the framework of the COKE (“COVID-19 Knowledge Extraction framework for next-generation discovery science”) Project, this thesis aims to underscore the capacity of machine reading to create knowledge graphs from scientific texts. The project is remarkable for its innovative use of NLP techniques such as a BERT + bi-LSTM language model. This combination is employed to detect and categorize elements within medical abstracts, thereby enhancing the systematic literature review process.
The COKE project's outcomes show that NLP, when used in a judiciously structured manner, can significantly reduce the time and effort required to produce medical guidelines. These findings are particularly salient during times of medical emergency, like the COVID-19 pandemic, when quick and accurate research results are critical
Contribution of cytokines to the etiology and progression of Primary Myelofibrosis
Get PDFPrimary Myelofibrosis (PMF) is the end-stage of Philadelphia-negative myeloproliferative neoplasms (MPN) and is characterized by fibrosis and hematopoietic failure in bone marrow, with a consequential migration of the malignant hematopoietic stem cells (HSC) in the spleen where they induce ineffective haematopoiesis. To date, available therapies for PMF are still palliative and do not halt the progression of this neoplasm.
During my PhD years, our laboratory investigated the factors promoting the onset and progression of PMF. In our PMF mice model, Gata1low mouse, we studied the role of the interaction of HSC niche with megakaryocytes and HSC localization in the bone marrow during their division and cycle. We observed the inflammation and the main protagonists (LNC-2, CXCL1, and TGF-β) of this process and how their level changes before and after the onset of the disease. We investigated the different megakaryocyte populations in the fibrotic environment in different organs (lung and bone marrow) to define the megakaryocytes implicated in this process. In human samples, we described different ultrastructural abnormalities of megakaryocytes from the bone marrow and the spleen, identifying a possible different metabolism in those two populations.
In conclusion, we highlighted the intricated crosstalk between the megakaryocytes, the niche and HSC in PMF. We identified megakaryocytes-dependent cytokines altering the homeostasis of the niche and HSC. Those cytokines could be used as alternative therapeutic targets. Furthermore, we observed different megakaryocytic populations in different organs, providing new prospective on the role of megakaryocytes in different microenvironments
Omission of axillary clearance in patients with early breast cancer and positive sentinel node biopsy treated with conservative surgery: application of the ACOSOG Z011 trial in clinical practice
Get PDFBackground: Axillary lymph node dissection (ALND) in presence of sentinel lymph node (SLN) metastases has been the standard in breast cancer (BC) patients for many years. Today, after the publication of the ACOSOG Z0011 trial, ALND is a procedure restricted to a dwindling group of patients with a clearly metastatic axilla.
Material and methods: This was a prospective observational trial involving two Italian Breast Units: Policlinico di Sant’Orsola and San Raffaele hospital. Objective was to evaluate that the omission of ALND in patients with cT1-2 cN0 BC undergoing breast conserving surgery (BCS) and histological finding of metastases in 1 or 2 SLN is not associated with a worse prognostic outcome. Primary endpoint was overall survival (OS). Secondary endpoints were disease free survival (DFS) and locoregional recurrence. All BC patients treated between the 1st of November 2020 and 31st of July 2023 with cT1-2 cN0 BC, preoperative negative axillary ultrasound and 1 or 2 metastatic SLN treated with sentinel node biopsy (SLNB) alone entered the study.
Results: 795 cT1-2 cN0 BC patients underwent BCS and SLNB. Ninety patients were included. Median age was 60 (52-68) years. Seventy-five patients (83%) had T1 tumor and 15 (17%) T2. Median tumor size was 16 mm (11-19). The median SLN removed was 2 (1-3). Eighty-one patients had 1 positive SLN (90%), while 9 had 2 SLN metastasis (10%). 39 (43%) micrometastases were identified and 51 macrometastasis (57%). All patients underwent radiotherapy. Seventeen (19%) performed adjuvant chemotherapy. Two received immunotherapy with trastuzumab and pertuzumab. Endocrine therapy was given to 84 (93%). At a median follow-up of 19 months (IQR 13-23) OS and DFS were 100%. No loco-regional recurrence was seen.
Conclusion: The preliminary results of our study confirm that omitting ALND in patients meeting Z011 criteria is oncologically safe and should be the standard of care
Pharmacological targeting of P-selectin to halt the progression of myelofibrosis in the Gata1low mouse model
Get PDFPrimary myelofibrosis is a clonal hematopoietic disorder characterized by marked degrees of systemic inflammation. The release of pro-inflammatory factors by clonal hematopoietic cell populations cause the remodeling of a specialized microenvironment, defined niche, in which the hematopoietic stem cells reside. The main source of pro-inflammatory cytokines is represented by malignant megakaryocytes. The bone marrow and spleen from myelofibrosis patients, as well as those from the Gata1low mouse model of the disease, contain increased number of abnormal megakaryocytes. These cells express on their surface high levels of the adhesion receptor P-selectin that, by triggering a pathological megakaryocyte-neutrophil emperipolesis, lead to increased bioavailability of TGF-β1 in the microenvironment and disease progression. Gata1low mice develop with age a phenotype similar to that of patients with myelofibrosis. We previously demonstrated that deletion of the P-selectin gene in Gata1low mice prevented the development of the myelofibrotic phenotype in these mice. In the current study, we tested the hypothesis that pharmacological inhibition of P-selectin may rescue the fibrotic phenotype of Gata1low mice. To test this hypothesis, we have investigated the phenotype expressed by old Gata1low mice treated with the anti-mouse monoclonal antibody against P-selectin RB40.34, alone or in combination with the JAK2 inhibitor Ruxolitinib. The results showed that the combined therapy normalized the phenotype of Gata1low mice with limited toxicity by reducing fibrosis, TGF-β1 and CXCL1 content in the BM and spleen and by restoring hematopoiesis in the bone marrow and the normal architecture of the spleen. In conclusion, pharmacological inhibition of P-selectin was effective in targeting malignant megakaryocytes and the microenvironmental abnormalities that affect the hematopoietic stem cell compartment in this model. These results suggest that P-selectin and JAK1/2 inhibitors in combination may represent a valid therapeutic option for patients with myelofibrosis
A prospective study on the early evaluation of response to androgen receptor-targeted agents with 11C-Choline, 68Ga-PSMA and 18F-FACBC PET in metastatic castration-resistant prostate cancer: a single center experience.
Get PDFBackground: The early identification of responsive and resistant patients to androgen-receptor targeting agents (ARTA) in metastatic castration resistant-prostate cancer (CRPC) is not completely possible with PSA assessment and conventional imaging. Considering its ability to determine metabolic activity of lesions, PET assessment might be a promising tool.
Materials and methods: We performed a monocentric prospective study in patients with metastatic CRPC under treatment with ARTA to evaluate the role of different PET radiotracers: 49 patients were randomized to receive 11C-Choline, 18F-FACBC or 68Ga-PSMA PET, one scan before therapy onset and one two months later.
The primary aim was to investigate the performance of three different novel PET radiotracers for the early evaluation of response to ARTA in metastatic CRPC patients; with regards to this aim, the outcome evaluated was biochemical response (PSA reduction ≥50%). The secondary aim was to investigate the prognostic role of several semiquantitative PET parameters and their variations with the different radiotracers in terms of biochemical PFS (bPFS) and overall survival (OS).
The study was promoted by the Italian Department of Health (code RF-2016-02364809).
Results: With regards to the primary endpoint, at univariate analysis a statistically significant correlation was found between MTV_VARIATION% (p=0.018) and TLA_VARIATION% (p=0.025) with 68Ga-PSMA PET and biochemical response. As for the secondary endpoints, significant correlations with bPFS were found for 68Ga-PSMA PET MTV_TOT_PET1 (p=0.001), TLA_TOT_PET1 (p=0.025), MTV_VARIATION% (p=0.031). For OS, statistically significant correlations were found for: MAJ_SUV_MAX_PET1 with 11C-Choline PET (p=0.007); MTV_TOT_PET1 (p=0.004), MAJ_SUV_MAX_PET1 (p=0.029), SUVMAX_VARIATION% (p=0.04), MTV_VARIATION% (p=0.015), TLA_VARIATION% (p=0.03) with 68Ga-PSMA PET,; MTV_TOT_PET1 (p=0.011), TLA_TOT_PET1 (p=0.009), MAJ_SUV_MAX_PET1 (p=0.027), MTV_VARIATION% (p=0.048) with 18F-FACBC.
Conclusions: Our prospective study highlighted that several 68Ga-PSMA and 18F-FACBC semiquantitative PET parameters and their variations present a prognostic value in terms of OS and bPFS and a correlation with biochemical response, that could help to assess response to ARTA