Functional Genomics and Cell Biology of the Dolphin (Tursiops runcatus): Establishment of Novel Molecular Tools to Study Marine Mammals in Changing Environments

The dolphin (Tursiops truncatus) is a mammal that is adapted to life in a totally aquatic environment. Despite the popularity and even iconic status of the dolphin, our knowledge of its physiology, its unique adaptations and the effects on it of environmental stressors are limited. One approach to improve this limited understanding is the implementation of established cellular and molecular methods to provide sensitive and insightful information for dolphin biology. We initiated our studies with the analysis of wild dolphin peripheral blood leukocytes, which have the potential to be informative of the animal’s global immune status. Transcriptomic profiles from almost 200 individual samples were analyzed using a newly developed species-specific microarray to assess its value as a prognostic and diagnostic tool. Functional genomics analyses were informative of stress-induced gene expression profiles and also of geographical location specific transcriptomic signatures, determined by the interaction of genetic, disease and environmental factors. We have developed quantitative metrics to unambiguously characterize the phenotypic properties of dolphin cells in culture. These quantitative metrics can provide identifiable characteristics and baseline data which will enable identification of changes in the cells due to time in culture. We have also developed a novel protocol to isolate primary cultures from cryopreserved tissue of stranded marine mammals, establishing a tissue (and cell) biorepository, a new approach that can provide a solution to the limited availability of samples. The work presented represents the development and application of tools for the study of the biology, health and physiology of the dolphin, and establishes their relevance for future studies of the impact on the dolphin of environmental infection and stress.

Functional components and lipid quality assessment in vegetable foods: analytical and technological approaches

Lipids are important components that contribute very significantly to nutritional and technological quality of foods because they are the least stable macro-components in foods, due to high susceptibility to oxidation. When rancidity take place, it makes food unhealthy and unacceptable for consumers. Thus, the presence of antioxidants, naturally present of added to foods, is required to enhance shelf life of foods. Moreover, antioxidant like phenolic compounds play an important role in human health enhancing the functionality of foods. The aim of this PhD project was the study of lipid quality and lipid oxidation in different vegetable foods focusing on analytical and technological aspects in order to figure out the effects of lipid composition and bioactive compounds (phenolic compounds, omega-3 fatty acids and dietary fiber) addition on their shelf life. In addition, bioavailability and antioxidant effects of phenolic compounds in human and animals, respectively, were evaluated after consumption of vegetable foods. The first section of the work was focused on the evaluation of lipid quality impact on technological behaviour of vegetable foods. Because of that, cocoa butter with different melting point were evaluated by chromatographic techniques (GC, TLC) and the sample with the higher melting point showed the presence of fatty acids, triglycerides, 2-monoglycerides and FT-IR profile different from genuine cocoa butter, meaning an adding of foreign fat (lauric-fat) not allowed by the law. Looking at lipid quality of other vegetable foods, an accelerated shelf life test (OXITEST®), was used to evaluate of lipid stability to oxidation in tarallini snacks made up using different lipid matrices (sunflower oil, extravirgin olive oil and a blend of extravirgin olive oil and lard). The results showed a good ability of OXITEST® to discriminate between lipid unsaturation and different cooking times, without any samples fat extraction. In the second section, the role of bioactive compounds on cereal based food shelf life was studied in different bakeries by GC, spectrophotometric methods and capillary electrophoresis. It was examined the relationships between phenolic compounds, added with flour, and lipid oxidation of tarallini and frollini. Both products showed an increase in lipid oxidation during storage and antioxidant effects on lipid oxidation were not as expected. Furthermore, the influence of enrichment in polyunsaturated fatty acids on lipid oxidation of pasta was evaluated. The results proved that LC n-3 PUFA were not significantly implicated in the onset of oxidation in spaghetti stored under daylight and accelerated oxidation in a laboratory heater. The importance of phenolic compounds as antioxidant in humans and rats was also studied, by HPLC/MS in the latter section. For this purpose, apigenin and apigenin glycosides excretion was investigated in six women’s urine in a 24 hours study. After a single dose of steamed artichokes, both aglicone and glucuronide metabolites were recovered in 24 h urine. Moreover, the effect of whole grain durum wheat bread and whole grain Kamut® khorasan bread in rats were evaluated. Both cereals were good sources of antioxidants but Kamut® bread fed animals had a better response to stress than wheat durum fed, especially when a sourdough bread was supplied.

Molecular and functional characterization of the chemotactic genes in the PCBs-degrader Pseudomonas pseudoalcaligenes KF707

Although bacteria represent the simplest form of life on Earth, they have a great impact on all living beings. For example the degrader bacterium Pseudomonas pseudoalcaligenes KF707 is used in bioremediation procedures for the recovery of polluted sites. Indeed, KF707 strain is know for its ability to degrade biphenyl and polychlorinated biphenyls - to which is chemotactically attracted - and to tolerate the oxydative stress due to toxic metal oxyanions such as tellurite and selenite. Moreover, in bioremediation processes, target compounds can be easily accessible to KF707 through biofilm formation. All these considerations suggest that KF707 is such a unique microorganism and this Thesis work has been focused on determining the molecular nature of some of the peculiar physiological traits of this strain. The genome project provided a large set of informations: putative genes involved in the degradation of aromatic and toxic compounds and associated to stress response were identified. Notably, multiple chemotactic operons and cheA genes were also found. Deleted mutants in the cheA genes were constructed and their role in motility, chemotaxis and biofilm formation were assessed and compared to those previously attributed to a cheA1 gene in a KF707 mutant constructed by a mini-Tn5 transposon insertion and which was impaired in motility and biofilm development. The results of this present Thesis work, taken together, were interpreted to suggest that in Pseudomonas pseudoalcaligenes KF707 strain, multiple factors are involved in these networks and they might play different roles depending on the environmental conditions. The ability of KF707 strain to produce signal molecules possibly involved in cell-to-cell communication, was also investigated: lack of a lux-like QS system - which is conversely widely present in Gram negative bacteria – keeps open the question about the actual molecular nature of KF707 quorum sensing mechanism.

Functional relationship of colorectal cancer-associated glycans with malignant phenotype and transcriptome changes

Glycosyltransferases ST6GAL1 and B4GALNT2 (and their cognate antigens Sia6LacNAc and Sda, respectively) are associated with colorectal cancer (CRC) but it is not fully clear their biological and clinical significance. We explored the clinical relevance of both glycosyltransferases by interrogating The Cancer Genome Atlas (TCGA) database while the phenotypic/transcriptomic effects of ST6GAL1/B4GALNT2 overexpression were studied in genetically modified CRC cell lines. Transcriptomic data from CRC patients in TCGA database suggested a moderate impact of ST6GAL1 on CRC progression, although it was not possible to define a clear role for this glycosyltransferase. Transcriptomic analysis of ST6GAL1-transduced cell lines revealed a much deeper effect of ST6GAL1 on gene expression in SW948 than in SW48. The overexpression of ST6GAL1 induced opposite effects on soft agar growth and wound healing in both cell lines. These results indicate that the impact of a cancer-associated glycosyltransferase change on phenotype/transcriptome can be extremely variable, depending on the molecular context of the tumor cell. On the contrary, transcriptomic analysis of B4GALNT2-modified cell lines together with TCGA database survey demonstrated a strong impact of B4GALNT2 on the transcriptional activity of CRC cells, in particular its association with a better prognosis. We suggest an anti-tumoral role of B4GALNT2 in CRC. We also investigated the glycan changes related to ST6GAL1/B4GALNT2 expression in a small cohort of tissues/plasma as well as the N-glycomic profile of CRC, normal and polyp tissues. We found an increase of ST6GAL1 activity in CRC and inflammatory bowel disease plasma samples comparing with plasma from healthy donors. A different Sda protein carrier pattern was observed between healthy donors and CRC plasma samples. β-arrestin 1 is a possible candidate as Sda carrier protein in plasma samples although future validation studies are needed. The alterations found in the N-glycan pattern highlight the importance of N-glycome as a molecular signature in cancer.

Functional heterogeneity of medial posterior parietal cortex of macaque monkey

The posterior parietal cortex (PPC) of primates represents a remarkable platform that has evolved over time to solve some of the computational challenges that we face in the everyday life, such as sensorimotor integration, spatial attention, and motor planning. With the aim of further investigating the multifaceted functional characteristics of medial PPC, we conducted three studies to explore the visuomotor, somatic, visual, and attention-related properties of two PPC areas: V6A, a visuomotor area part of the dorsomedial visual stream, and PE, an area strongly dominated by somatomotor input, residing mainly on the exposed surface of the superior parietal lobule. In the first study, we tested the impact of visual feedback on V6A grasp-related activity during arm movements towards objects of different shapes. Our results demonstrate that V6A is modulated by both grip type and visual information during grasping preparation and execution, with a predominance of cells influenced by grip type. In the second study, we explored the influence of depth and direction information on reach-related activity of neurons in the so far largely neglected medial part of area PE. We observed a remarkable trend in medial PPC, going from the joint coding of depth and direction signals caudally, in area V6A, to a largely segregated processing of the two signals rostrally, in area PE. In the third study, we used a combined fMRI-electrophysiology experiment to investigate the neuronal mechanisms underlying covert shift of attention processes in area V6A. Our preliminary results reveal that half of the cells showed shift-selective activity when the monkey covertly shifted its attention towards the receptive field. All together these findings highlight the role of the medial PPC in integrating information coming from different sources (vision, somatosensory and motor) and emphasize the involvement of action-related regions of the dorsomedial visual stream in higher level cognitive functions.

Emerging plant-based proteins: comparison from extraction to functional properties and food application

The increasing demand for alternatives to meat food products, which is linked to ethical and environmental reasons, highlights the necessity of using different protein sources. Plant proteins provide a valid option, thanks to the relative low costs, high availability and wide supply sources. The current process used to produce plant concentrates and isolates is the alkaline extraction followed by isoelectric precipitation. However, despite the high purity of the proteins, it presents some drawbacks. Innovative protein extraction processes are emerging, with the aim of reducing the environmental impact and the costs, as well as improving the functional properties. In this study, the traditional wet protein extraction and another simplified wet process were used to obtain protein-rich extracts out of different plants. The sources considered in the project were de-oiled sunflower and canola, chickpea, lentils, and the camelina meal, an emerging oleaginous seed interesting for its high content of omega 3. The extracts obtained from the two processes were then analysed for their capacities to hold water and fat, to form gel and a stable foam. Results highlighted strong differences concerning the protein content, yield and functionalities. The extracts obtained with the alkaline process confirmed the literature data about the four plant sources (sunflower, canola, chickpea and lentils) and allow to obtain a camelina concentrate with a protein content of 63 % and a protein recovery of 41 %. The second easiest process was not effective to obtain a protein enrichment in oleaginous sources, whereas an enrichment of 10 and 15 % was obtained in chickpea and lentils, respectively. The functional properties were also completely different: the easiest process produced protein ingredients completely water-soluble at pH 7, with a discrete foaming capacity compared to the extracts obtained with alkaline process. These characteristics could make these extracts suitable for the plant milk-analogue products.

Impact of anthropogenic stressors on the microbial communities in marine holobionts and ecosystems

Free-living or host-associated marine microbiomes play a determinant role in supporting the functioning and biodiversity of marine ecosystems, providing essential ecological services, and promoting the health of the entire biosphere. Currently, the fast and restless increase of World’s human population strongly impacts life on Earth in the forms of ocean pollution, coastal zone destruction, overexploitation of marine resources, and climate change. Thanks to their phylogenetic, metabolic, and functional diversity, marine microbiomes represent the Earth’s biggest reservoir of solutions against the major threats that are now impacting marine ecosystems, possibly providing valuable insights for biotechnological applications to preserve the health of the ocean ecosystems. Microbial-based mitigation strategies heavily rely on the available knowledge on the specific role and composition of holobionts associated microbial communities, thus highlighting the importance of pioneer studies on microbial-mediated adaptive mechanisms in the marine habitats. In this context, we propose different models representing ecologically important, widely distributed, and habitat-forming organisms, to further investigate the ability of marine holobionts to dynamically adapt to natural environmental variations, as well as to anthropogenic stress factors. In this PhD thesis, we were able to supply the characterization of the microbial community associated with the model anthozoan cnidaria Corynactis viridis throughout a seasonal gradient, to provide critical insights into microbiome-host interactions in a biomonitoring perspective. We also dissected in details the microbial-derived mitigation strategies implemented by the benthonic anthozoan Anemonia viridis and the gastropod Patella caerulea as models of adaptation to anthropogenic stressors, in the context of bioremediation of human-impacted habitats and for the monitoring and preservation of coastal marine ecosystems, respectively. Finally, we provided a functional model of adaptation to future ocean acidification conditions by characterizing the microbial community associated with the temperate coral Balanophyllia europaea naturally living at low pH conditions, to implement microbial based actions to mitigate climate change.

Nanostructured smart materials as functional components of medical devices

The field of medical devices has experienced, more than others, technological advances, developments and innovations, thanks to the rapidly expanding scientific knowledge and collaboration between different disciplines such as biology, engineering and materials science. The design of functional components can be achieved by exploiting composite materials based on nanostructured smart materials, that due to the inherent characteristics of single constituents develop unique properties that make them suitable for different applications preserving excellent mechanical proprieties. For instance, recent developments have focused on the fabrication of piezoelectric devices with multiple biomedical functions, as actuation and sensing functions in one component for monitoring pressure signals. The present Ph.D. Thesis aims at investigating nanostructured smart materials embedded into a polymeric matrix to obtain a composite material that can be used as a functional component for medical devices. (i) Nanostructured piezoelectric material with self-sensing capability was successfully manufactured by using ceramic (i.e. lead zirconate titanate (PZT)) and (ii) polymeric (i.e. poly(vinylidene fluoride-trifluoro ethylene (PVDF-TRFE)) piezoelectric materials. PZT nanofibers were obtained by sol-gel electrospinning starting from synthetized PZT precursor solution. Synthesis, sol-gel electrospinning process, and thermal treatment were accurately controlled to obtain PZT nanofibers dimensionally stable with densely packed grains in the perovskite phase. To guarantee the impact resistance of the laminate, the morphology and size of the hosting filler were accurately designed by increasing the surface area to volume ratio. Moreover, to solve the issue relative to the mechanical discrepancy between rigid electronic materials/soft human tissues/different material of the device (iii) a nanostructured flexible composite material based on a network of Poly-L-lactic acid (PLLA) made of curled nanofibers that present a tuneable mechanical response as a function of the applied stress was successful fabricated.

Development of sexing systems functional to mass production of Aedes albopictus sterile males

Aedes albopictus is a vector able to transmit several arboviruses. Due to its high impact on human health, it is important to develop an efficient control strategy for this pest. Nowadays, control based on chemical insecticides is limited by the number of available active principles and the occurrence of resistance. A valuable alternative to the conventional control strategies is the sterile insect technique (SIT) which relies on releasing sterile males of the target insect. Mating between wild females and sterile males results in no viable offspring. A crucial aspect of SIT is the production of a large number of sterile males with a low presence of females that can bite and transmit viruses. The present thesis aimed to find, implement and study the most reliable mechanical sex sorter and protocol to implement male productivity and reduce female contamination. In addition, I evaluated different variables and sorting protocols to enable female recovery for breeding purposes. Furthermore, I studied the creation of a hyper-protandric strain potentially able to produce only males. I also assessed the integration of artificial intelligence with an optical unit to identify sexes at the adult stage. All these applications helped to realise a mass production model in Italy with a potential weekly production of 1 million males. Moreover, I studied and applied for aerial sterile male release in an urban environment. This technology could allow the release of males in a wide area, overcoming environmental and urban obstacles. However, the development and application of drone technologies in a metropolitan area close to airports, such as in Bologna area, must fit specific requirements. Lastly, at Réunion Island, during a Short Term Scientific Mission France (AIM-COST Action), Indian Ocean, I studied the Boosted SIT application. Coating sterile males with Pyriproxyfen may help spread the insecticide into the larval breeding sites.

Induction of Hebbian associative plasticity through paired non-invasive brain stimulation of premotor-motor areas to elucidate the network's functional role

The ventral premotor cortex (PMv) is believed to play a pivotal role in a multitude of visuomotor behaviors, such as sensory-guided goal-directed visuomotor transformations, arbitrary visuomotor mapping, and hyper-learnt visuomotor associations underlying automatic imitative tendencies. All these functions are likely carried out through the copious projections connecting PMv to the primary motor cortex (M1). Yet, causal evidence investigating the functional relevance of the PMv-M1 network remains elusive and scarce. In the studies reported in this thesis we addressed this issue using a transcranial magnetic stimulation (TMS) protocol called cortico-cortical paired associative stimulation (ccPAS), which relies on multisite stimulation to induce Hebbian spike-timing dependent plasticity (STDP) by repeatedly stimulating the pathway connecting two target areas to manipulate their connectivity. Firstly, we show that ccPAS protocols informed by both short- and long-latency PMv-M1 interactions effectively modulate connectivity between the two nodes. Then, by pre-activating the network to apply ccPAS in a state-dependent manner, we were able to selectively target specific functional visuo-motor pathways, demonstrating the relevance of PMv-M1 connectivity to arbitrary visuomotor mapping. Subsequently, we addressed the PMv-to-M1 role in automatic imitation, and demonstrated that its connectivity manipulation has a corresponding impact on automatic imitative tendencies. Finally, by combining dual-coil TMS connectivity assessments and ccPAS in young and elderly individuals, we traced effective connectivity of premotor-motor networks and tested their plasticity and relevance to manual dexterity and force in healthy ageing. Our findings provide unprecedent causal evidence of the functional role of the PMv-to-M1 network in young and elderly individuals. The studies presented in this thesis suggest that ccPAS can effectively modulate the strength of connectivity between targeted areas, and coherently manipulate a networks’ behavioral output. Results open new research prospects into the causal role of cortico-cortical connectivity, and provide necessary information to the development of clinical interventions based on connectivity manipulation.

Towards a more sustainable aquaculture: diets effects and environmental impact of aquaculture from a microbiome perspective

There is a lot of interest to optimize aquaculture production due to its overexploitation of marine resources, ocean pollution and habitat destruction. Since feed production is one of the greatest issues in aquaculture, feeding strategy optimization is important. The study of several different feed additives or supplementation is important to secure optimal growth, gut health, and function in farmed fish. Feed additives are typically supplied to ensure good health and to help the animal ward off pathogens during both normal and challenging conditions, which could stress animals and promote insurgence of pathologies or pathogens invasions. In this context has an increasing interest the study of host associated microbiome to understand the influence of novel functional feed on the health and physiology of animals. To achieve a more sustainable aquaculture sector, show a great importance the understanding of the environmental impact of this human activity in terms of habitat destruction, ocean pollution and reduction marine environments biodiversity. Marine microbiomes, either free-living or associated with multicellular hosts, is acquiring an increasing interest because their role in supporting the functioning and biodiversity of marine ecosystems, providing essential ecological services. Becoming extremely important to understand how these activities can affect marine microbiomes by altering their function and diversity. In this thesis work, we were able to present a comprehensive evaluation of different functional feeds assessing their effects in terms of growth and gut health of three fish species, Rainbow Trout (Oncorhynchus mykiss), Gilthead seabream (Sparus aurata) and Zebrafish (Danio rerio). We also explored the impact of Aquaculture on the surrounding marine microbiomes, using Patella caerulea as a model holobionts. Finally, we provided a synoptical study on the microbiomes of the water column and surface sediments in North-Western Adriatic Sea (Italy), providing the finest-scale mapping of marine microbiomes in the Mediterranean Sea.