Study of Venturia inaequalis sensitivity to fungicides through molecular and biological methodologies

This study was aimed to correlate the results of relative germination from in vitro tests by trifloxystrobin with those of qPCR on a wide range of V. inaequalis populations and monoconidial isolates. Samples were collected in Italian and Turkish distinct locations from orchards with different scab management. In this study, an allele-specific qPCR with primer sets designed was successfully developed to quantitatively determine the frequency of QoI-resistant allele G143A in populations and monoconidial isolates of V. inaequalis. qPCR followed a similar pattern to that obtained using in vitro conidial germination test in very sensitive and very resistant populations. However, the variability between two test results was observed in hetereogenous populations. Therefore, the results of correlations between in vitro and qPCR showed a positive but not very high correlation for Venturia inaequalis populations (R2=0.70). On the contrary, this correlation between two assessment methods was very high for monoconidial isolates (R2=0.92). Results obtained in quantitative PCR and from traditional spore germination assay differed for the same fungal population and in some cases, it is difficult to assess the resistance in the field by only qPCR. It was concluded that it is not always possible to correlate the frequency of detection of the mutation with biological assessment. In such situations, monitoring by molecular techniques must be supported by standard in vitro resistance assessments and observation of field performance in order to have correct conclusions.

Adaptive multiscale biological signal processing

Biological processes are very complex mechanisms, most of them being accompanied by or manifested as signals that reflect their essential characteristics and qualities. The development of diagnostic techniques based on signal and image acquisition from the human body is commonly retained as one of the propelling factors in the advancements in medicine and biosciences recorded in the recent past. It is a fact that the instruments used for biological signal and image recording, like any other acquisition system, are affected by non-idealities which, by different degrees, negatively impact on the accuracy of the recording. This work discusses how it is possible to attenuate, and ideally to remove, these effects, with a particular attention toward ultrasound imaging and extracellular recordings. Original algorithms developed during the Ph.D. research activity will be examined and compared to ones in literature tackling the same problems; results will be drawn on the base of comparative tests on both synthetic and in-vivo acquisitions, evaluating standard metrics in the respective field of application. All the developed algorithms share an adaptive approach to signal analysis, meaning that their behavior is not dependent only on designer choices, but driven by input signal characteristics too. Performance comparisons following the state of the art concerning image quality assessment, contrast gain estimation and resolution gain quantification as well as visual inspection highlighted very good results featured by the proposed ultrasound image deconvolution and restoring algorithms: axial resolution up to 5 times better than algorithms in literature are possible. Concerning extracellular recordings, the results of the proposed denoising technique compared to other signal processing algorithms pointed out an improvement of the state of the art of almost 4 dB.

The Integrated European Project "GEHA - GEnetics of Healthy Aging": recruitment, health status assessment and survival of the Italian 90+ sibpairs

The present study is part of the EU Integrated Project “GEHA – Genetics of Healthy Aging” (Franceschi C et al., Ann N Y Acad Sci. 1100: 21-45, 2007), whose aim is to identify genes involved in healthy aging and longevity, which allow individuals to survive to advanced age in good cognitive and physical function and in the absence of major age-related diseases. Aims The major aims of this thesis were the following: 1. to outline the recruitment procedure of 90+ Italian siblings performed by the recruiting units of the University of Bologna (UNIBO) and Rome (ISS). The procedures related to the following items necessary to perform the study were described and commented: identification of the eligible area for recruitment, demographic aspects related to the need of getting census lists of 90+siblings, mail and phone contact with 90+ subjects and their families, bioethics aspects of the whole procedure, standardization of the recruitment methodology and set-up of a detailed flow chart to be followed by the European recruitment centres (obtainment of the informed consent form, anonimization of data by using a special code, how to perform the interview, how to collect the blood, how to enter data in the GEHA Phenotypic Data Base hosted at Odense). 2. to provide an overview of the phenotypic characteristics of 90+ Italian siblings recruited by the recruiting units of the University of Bologna (UNIBO) and Rome (ISS). The following items were addressed: socio-demographic characteristics, health status, cognitive assessment, physical conditions (handgrip strength test, chair-stand test, physical ability including ADL, vision and hearing ability, movement ability and doing light housework), life-style information (smoking and drinking habits) and subjective well-being (attitude towards life). Moreover, haematological parameters collected in the 90+ sibpairs as optional parameters by the Bologna and Rome recruiting units were used for a more comprehensive evaluation of the results obtained using the above mentioned phenotypic characteristics reported in the GEHA questionnaire. 3. to assess 90+ Italian siblings as far as their health/functional status is concerned on the basis of three classification methods proposed in previous studies on centenarians, which are based on: • actual functional capabilities (ADL, SMMSE, visual and hearing abilities) (Gondo et al., J Gerontol. 61A (3): 305-310, 2006); • actual functional capabilities and morbidity (ADL, ability to walk, SMMSE, presence of cancer, ictus, renal failure, anaemia, and liver diseases) (Franceschi et al., Aging Clin Exp Res, 12:77-84, 2000); • retrospectively collected data about past history of morbidity and age of disease onset (hypertension, heart disease, diabetes, stroke, cancer, osteopororis, neurological diseases, chronic obstructive pulmonary disease and ocular diseases) (Evert et al., J Gerontol A Biol Sci Med Sci. 58A (3): 232-237, 2003). Firstly these available models to define the health status of long-living subjects were applied to the sample and, since the classifications by Gondo and Franceschi are both based on the present functional status, they were compared in order to better recognize the healthy aging phenotype and to identify the best group of 90+ subjects out of the entire studied population. 4. to investigate the concordance of health and functional status among 90+ siblings in order to divide sibpairs in three categories: the best (both sibs are in good shape), the worst (both sibs are in bad shape) and an intermediate group (one sib is in good shape and the other is in bad shape). Moreover, the evaluation wanted to discover which variables are concordant among siblings; thus, concordant variables could be considered as familiar variables (determined by the environment or by genetics). 5. to perform a survival analysis by using mortality data at 1st January 2009 from the follow-up as the main outcome and selected functional and clinical parameters as explanatory variables. Methods A total of 765 90+ Italian subjects recruited by UNIBO (549 90+ siblings, belonging to 258 families) and ISS (216 90+ siblings, belonging to 106 families) recruiting units are included in the analysis. Each subject was interviewed according to a standardized questionnaire, comprising extensively utilized questions that have been validated in previous European studies on elderly subjects and covering demographic information, life style, living conditions, cognitive status (SMMSE), mood, health status and anthropometric measurements. Moreover, subjects were asked to perform some physical tests (Hand Grip Strength test and Chair Standing test) and a sample of about 24 mL of blood was collected and then processed according to a common protocol for the preparation and storage of DNA aliquots. Results From the analysis the main findings are the following: - a standardized protocol to assess cognitive status, physical performances and health status of European nonagenarian subjects was set up, in respect to ethical requirements, and it is available as a reference for other studies in this field; - GEHA families are enriched in long-living members and extreme survival, and represent an appropriate model for the identification of genes involved in healthy aging and longevity; - two simplified sets of criteria to classify 90+ sibling according to their health status were proposed, as operational tools for distinguishing healthy from non healthy subjects; - cognitive and functional parameters have a major role in categorizing 90+ siblings for the health status; - parameters such as education and good physical abilities (500 metres walking ability, going up and down the stairs ability, high scores at hand grip and chair stand tests) are associated with a good health status (defined as “cognitive unimpairment and absence of disability”); - male nonagenarians show a more homogeneous phenotype than females, and, though far fewer in number, tend to be healthier than females; - in males the good health status is not protective for survival, confirming the male-female health survival paradox; - survival after age 90 was dependent mainly on intact cognitive status and absence of functional disabilities; - haemoglobin and creatinine levels are both associated with longevity; - the most concordant items among 90+ siblings are related to the functional status, indicating that they contain a familiar component. It is still to be investigated at what level this familiar component is determined by genetics or by environment or by the interaction between genetics, environment and chance (and at what level). Conclusions In conclusion, we could state that this study, in accordance with the main objectives of the whole GEHA project, represents one of the first attempt to identify the biological and non biological determinants of successful/unsuccessful aging and longevity. Here, the analysis was performed on 90+ siblings recruited in Northern and Central Italy and it could be used as a reference for others studies in this field on Italian population. Moreover, it contributed to the definition of “successful” and “unsuccessful” aging and categorising a very large cohort of our most elderly subjects into “successful” and “unsuccessful” groups provided an unrivalled opportunity to detect some of the basic genetic/molecular mechanisms which underpin good health as opposed to chronic disability. Discoveries in the topic of the biological determinants of healthy aging represent a real possibility to identify new markers to be utilized for the identification of subgroups of old European citizens having a higher risk to develop age-related diseases and disabilities and to direct major preventive medicine strategies for the new epidemic of chronic disease in the 21st century.

Development and performance assessment of a Plasma Focus electron beam generator for Intra-Operative Radiation Therapy

The Plasma Focus is a device designed to generate a plasma sheet between two coaxial electrodes by means of a high voltage difference. The plasma is then driven to collapse into a “pinch”, where thermonuclear conditions prevail. During the “pinch phase” charged particles are emitted, with two main components: an ion beam peaked forward and an electron beam directed backward. The electron beam emitted backward by Plasma Focus devices is being investigated as a radiation source for medical applications, using it to produce x-rays by interaction with appropriate targets (through bremsstrahlung and characteristic emission). A dedicated Plasma Focus device, named PFMA-3 (Plasma Focus for Medical Applications number 3), has been designed, put in operation and tested by the research groups of the Universities of Bologna and Ferrara. The very high dose rate (several gray per discharge, in less than 1 µs) is a peculiarity of this device that has to be investigated, as it might modify the relative biological effectiveness (RBE). Aim of this Ph.D. project was to investigate the main physical properties of the low-energy x-ray beams produced by a Plasma Focus device and their potential medical applications to IORT treatments. It was necessary to develop the optimal geometrical configuration; to evaluate the x-rays produced and their dose deposited; to estimate the energy electron spectrum produced in the “pinch phase”; to study an optimal target for the conversion of the x-rays; to conduct simulations to study the physics involved; and in order to evaluate the radio-biological features of the beam, cell holders had to be developed for both irradiations and cell growth conditions.

Development of methodologies for fish freshness assessment using metabonomics applications.

This study focuses on the use of metabonomics applications in measuring fish freshness in various biological species and in evaluating how they are stored. This metabonomic approach is innovative and is based upon molecular profiling through nuclear magnetic resonance (NMR). On one hand, the aim is to ascertain if a type of fish has maintained, within certain limits, its sensory and nutritional characteristics after being caught; and on the second, the research observes the alterations in the product’s composition. The spectroscopic data obtained through experimental nuclear magnetic resonance, 1H-NMR, of the molecular profiles of the fish extracts are compared with those obtained on the same samples through analytical and conventional methods now in practice. These second methods are used to obtain chemical indices of freshness through biochemical and microbial degradation of the proteic nitrogen compounds and not (trimethylamine, N-(CH3)3, nucleotides, amino acids, etc.). At a later time, a principal components analysis (PCA) and a linear discriminant analysis (PLS-DA) are performed through a metabonomic approach to condense the temporal evolution of freshness into a single parameter. In particular, the first principal component (PC1) under both storage conditions (4 °C and 0 °C) represents the component together with the molecular composition of the samples (through 1H-NMR spectrum) evolving during storage with a very high variance. The results of this study give scientific evidence supporting the objective elements evaluating the freshness of fish products showing those which can be labeled “fresh fish.”

Nature and relevance of inherited blueprints on business models choices and innovation: an assessment

One important metaphor, referred to biological theories, used to investigate on organizational and business strategy issues is the metaphor about heredity; an area requiring further investigation is the extent to which the characteristics of blueprints inherited from the parent, helps in explaining subsequent development of the spawned ventures. In order to shed a light on the tension between inherited patterns and the new trajectory that may characterize spawned ventures’ development we propose a model aimed at investigating which blueprints elements might exert an effect on business model design choices and to which extent their persistence (or abandonment) determines subsequent business model innovation. Under the assumption that academic and corporate institutions transmit different genes to their spin-offs, we hence expect to have heterogeneity in elements that affect business model design choices and its subsequent evolution. This is the reason why we carry on a twofold analysis in the biotech (meta)industry: under a multiple-case research design, business model and especially its fundamental design elements and themes scholars individuated to decompose the construct, have been thoroughly analysed. Our purpose is to isolate the dimensions of business model that may have been the object of legacy and the ones along which an experimentation and learning process is more likely to happen, bearing in mind that differences between academic and corporate might not be that evident as expected, especially considering that business model innovation may occur.

Effects of pesticides on honey bees (Apis mellifera L.): study of a specific route of exposure and evaluation of biochemical-physiological changes in the assessment of the pesticides toxicity

In this study, some important aspects of the relationship between honey bees (Apis mellifera L.) and pesticides have been investigated. In the first part of the research, the effects of the exposure of honey bees to neonicotinoids and fipronil contaminated dusts were analyzed. In fact, considerable amounts of these pesticides, employed for maize seed dressing treatments, may be dispersed during the sowing operations, thus representing a way of intoxication for honey bees. In particular, a specific way of exposure to this pesticides formulation, the indirect contact, was taken into account. To this aim, we conducted different experimentations, in laboratory, in semi-field and in open field conditions in order to assess the effects on mortality, foraging behaviour, colony development and capacity of orientation. The real dispersal of contaminated dusts was previously assessed in specific filed trials. In the second part, the impact of various pesticides (chemical and biological) on honey bee biochemical-physiological changes, was evaluated. Different ways and durations of exposure to the tested products were also employed. Three experimentations were performed, combining Bt spores and deltamethrin, Bt spores and fipronil, difenoconazole and deltamethrin. Several important enzymes (GST, ALP, SOD, CAT, G6PDH, GAPDH) were selected in order to test the pesticides induced variations in their activity. In particular, these enzymes are involved in different pathways of detoxification, oxidative stress defence and energetic metabolism. The results showed a significant effect on mortality of neonicotinoids and fipronil contaminated dusts, both in laboratory and in semi-field trials. However, no effects were evidenced in honey bees orientation capacity. The analysis of different biochemical indicators highlighted some interesting physiological variations that can be linked to the pesticide exposure. We therefore stress the attention on the possibility of using such a methodology as a novel toxicity endpoint in environmental risk assessment.

Influence of Electromagnetic Fields On Biological Signalling: An Experimental and Theoretical Approach

The primary goals of this study were to develop a cell-free in vitro assay for the assessment of nonthermal electromagnetic (EMF) bioeffects and to develop theoretical models in accord with current experimental observations. Based upon the hypothesis that EMF effects operate by modulating Ca2+/CaM binding, an in vitro nitric oxide (NO) synthesis assay was developed to assess the effects of a pulsed radiofrequency (PRF) signal used for treatment of postoperative pain and edema. No effects of PRF on NO synthesis were observed. Effects of PRF on Ca2+/CaM binding were also assessed using a Ca2+-selective electrode, also yielding no EMF Ca2+/CaM binding. However, a PRF effect was observed on the interaction of hemoglobin (Hb) with tetrahydrobiopterin, leading to the development of an in vitro Hb deoxygenation assay, showing a reduction in the rate of Hb deoxygenation for exposures to both PRF and a static magnetic field (SMF). Structural studies using pyranine fluorescence, Gd3+ vibronic sideband luminescence and attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy were conducted in order to ascertain the mechanism of this EMF effect on Hb. Also, the effect of SMF on Hb oxygen saturation (SO2) was assessed under gas-controlled conditions. These studies showed no definitive changes in protein/solvation structure or SO2 under equilibrium conditions, suggesting the need for real-time instrumentation or other means of observing out-of-equilibrium Hb dynamics. Theoretical models were developed for EMF transduction, effects on ion binding, neuronal spike timing, and dynamics of Hb deoxygenation. The EMF sensitivity and simplicity of the Hb deoxygenation assay suggest a new tool to further establish basic biophysical EMF transduction mechanisms. If an EMF-induced increase in the rate of deoxygenation can be demonstrated in vivo, then enhancement of oxygen delivery may be a new therapeutic method by which clinically relevant EMF-mediated enhancement of growth and repair processes can occur.

Contaminants of emerging concern in drinking water: assessment of the effectiveness of removal by drinking water treatments and possible risks to human health

Contaminants of emerging concern are increasingly detected in the water cycle, with endocrine-disrupting chemicals (EDCs) receiving attention due to their potential to cause adverse health effects even at low concentrations. Although the EU has recently introduced some EDCs into drinking water legislation, most drinking water treatment plants (DWTPs) are not designed to remove EDCs, making their detection and removal in DWTPs an important challenge. The aim of this doctoral project was to investigate hormones and phenolic compounds as suspected EDCs in drinking waters across the Romagna area (Italy). The main objectives were to assess the occurrence of considered contaminants in source and drinking water from three DWTPs, characterize the effectiveness of removal by different water treatment processes, and evaluate the potential biological impact on drinking water and human health. Specifically, a complementary approach of target chemical analysis and effect-based methods was adopted to explore drinking water quality, treatment efficacy, and biological potential. This study found that nonylphenol (NP) was prevalent in all samples, followed by BPA. Sporadic contamination of hormones was found only in source waters. Although the measured EDC concentrations in drinking water did not exceed threshold guideline values, the potential role of DWTPs as an additional source of EDC contamination should be considered. Significant increases in BPA and NP levels were observed during water treatment steps, which were also reflected in estrogenic and mutagenic responses in water samples after the ultrafiltration. This highlights the need to monitor water quality during various treatment processes to improve the efficiency of DWTPs. Biological assessments on finished water did not reveal any bioactivity, except for few treated water samples that exhibited estrogenic responses. Overall, the data emphasize the high quality of produced drinking water and the value of applying integrated chemical analysis and in vitro bioassays for water quality assessment.

Network analysis and machine learning assist drug repurposing and safety assessment in neurological diseases

In recent decades, two prominent trends have influenced the data modeling field, namely network analysis and machine learning. This thesis explores the practical applications of these techniques within the domain of drug research, unveiling their multifaceted potential for advancing our comprehension of complex biological systems. The research undertaken during this PhD program is situated at the intersection of network theory, computational methods, and drug research. Across six projects presented herein, there is a gradual increase in model complexity. These projects traverse a diverse range of topics, with a specific emphasis on drug repurposing and safety in the context of neurological diseases. The aim of these projects is to leverage existing biomedical knowledge to develop innovative approaches that bolster drug research. The investigations have produced practical solutions, not only providing insights into the intricacies of biological systems, but also allowing the creation of valuable tools for their analysis. In short, the achievements are: • A novel computational algorithm to identify adverse events specific to fixed-dose drug combinations. • A web application that tracks the clinical drug research response to SARS-CoV-2. • A Python package for differential gene expression analysis and the identification of key regulatory "switch genes". • The identification of pivotal events causing drug-induced impulse control disorders linked to specific medications. • An automated pipeline for discovering potential drug repurposing opportunities. • The creation of a comprehensive knowledge graph and development of a graph machine learning model for predictions. Collectively, these projects illustrate diverse applications of data science and network-based methodologies, highlighting the profound impact they can have in supporting drug research activities.