Environmental influence on the phenotype: Morphological variation in human dentition

This work is about the role that environment plays in the production of evolutionary significant variations. It starts with an historical introduction about the concept of variation and the role of environment in its production. Then, I show how a lack of attention to these topics may lead to serious mistakes in data interpretation. A statistical re-analysis of published data on the effects of malnutrition on dental eruption, shows that what has been interpreted as an increase in the mean value, is actually linked to increase of variability. In Chapter 3 I present the topic of development as a link between variability and environmental influence, giving a review of the possible mechanisms by which development influences evolutionary dynamics. Chapter 4 is the core chapter of the thesis; I investigated the role of environment in the development of dental morphology. I used dental hypoplasia as a marker of stress, characterizing two groups. Comparing the morphology of upper molars in the two groups, three major results came out: (i) there is a significant effect of environmental stressors on the overall morphology of upper molars; (ii) the developmental response increases morphological variability of the stressed population; (iii) increase of variability is directional: stressed individuals have increased cusps dimensions and number. I also hypothesized the molecular mechanisms that could be responsible of the observed effects. In Chapter 5, I present future perspectives for developing this research. The direction of dental development response is the same direction of the trend in mammalian dental evolution. Since malnutrition triggers the developmental response, and this particular kind of stressor must have been very common in our class evolutionary history, I propose the possibility that environmental stress actively influenced mammals evolution. Moreover, I discuss the possibility of reconsidering the role of natural selection in the evolution of dental morphology.

Salinity Effect on Horticultural Crops: Morphological, Physiological, and Biomolecular Elements of Salinity Stress Response

Among abiotic stresses, high salinity stress is the most severe environmental stress. High salinity exerts its negative impact mainly by disrupting the ionic and osmotic equilibrium of the cell. In saline soils, high levels of sodium ions lead to plant growth inhibition and even death. Salt tolerance in plants is a multifarious phenomenon involving a variety of changes at molecular, organelle, cellular, tissue as well as whole plant level. In addition, salt tolerant plants show a range of adaptations not only in morphological or structural features but also in metabolic and physiological processes that enable them to survive under extreme saline environments. The main objectives of my dissertation were understanding the main physiological and biomolecular features of plant responses to salinity in different genotypes of horticultural crops that are belonging to different families Solanaceae (tomato) and Cucurbitaceae (melon) and Brassicaceae (cabbage and radish). Several aspects of crop responses to salinity have been addressed with the final aim of combining elements of functional stress response in plants by using several ways for the assessment of plant stress perception that ranging from destructive measurements (eg. leaf area, relative growth rate, leaf area index, and total plant fresh and dry weight), to physiological determinations (eg. stomatal conductance, leaf gas exchanges, water use efficiency, and leaf water relation), to the determination of metabolite accumulation in plant tissue (eg. Proline and protein) as well as evaluation the role of enzymatic antioxidant capacity assay in scavenging reactive oxygen species that have been generated under salinized condition, and finally assessing the gene induction and up-down regulation upon salinization (eg. SOS pathway).

Characterization and exploiment of Microbial Strains to be used in Meat Processing and Fermentation

The principal aim of this research project has been the evaluation of the specific role of yeasts in ripening processes of dry-cured meat products, i.e. speck and in salami produced by adding Lactobacilli starter cultures, i.e. L. sakei, L. casei, L. fermentum, L. rhamnosus, L.sakei + S.xylosus. In particular the contribution of the predominant yeasts to the hydrolytic patterns of meat proteins has been studied both in model system and in real products. In fact, although several papers have been published on the microbial, enzymatic, aromatic and chemical characterization of dry-cured meat e.g. ham over ripening, the specific role of yeasts has been often underestimated. Therefore this research work has been focused on the following aspects: 1. Characterization of the yeasts and lactic acid bacteria in samples of speck produced by different farms and analyzed during the various production and ripening phases 2. Characterization of the superficial or internal yeasts population in salami produced with or without the use of lactobacilli as starter cultures 3. Molecular characterization of different strains of yeasts and detection of the dominant biotypes able to survive despite environmental stress factors (such as smoke, salt) 4. Study of the proteolytic profiles of speck and salami during the ripening process and comparison with the proteolytic profiles produced in meat model systems by a relevant number of yeasts isolated from speck and salami 5. Study of the proteolytic profiles of Lactobacilli starter cultures in meat model systems 6. Comparative statistical analysis of the proteolytic profiles to find possible relationships between specific bands and peptides and specific microorganisms 7. Evaluation of the aromatic characteristics of speck and salami to assess relationships among the metabolites released by the starter cultures or the dominant microflora

Cancer and aging: a multidisciplinary medicinal chemistry approach on relevant biological targets such as proteasome, sirtuins and interleukin 6

It is well known that ageing and cancer have common origins due to internal and environmental stress and share some common hallmarks such as genomic instability, epigenetic alteration, aberrant telomeres, inflammation and immune injury. Moreover, ageing is involved in a number of events responsible for carcinogenesis and cancer development at the molecular, cellular, and tissue levels. Ageing could represent a “blockbuster” market because the target patient group includes potentially every person; at the same time, oncology has become the largest therapeutic area in the pharmaceutical industry in terms of the number of projects, clinical trials and research and development (R&D) spending, but cancer remains one of the leading causes of mortality worldwide. The overall aim of the work presented in this thesis was the rational design of new compounds able to modulate activity of relevant targets involved in cancer and aging-related pathologies, namely proteasome and immunoproteasome, sirtuins and interleukin 6. These three targets play different roles in human cells, but the modulation of its activity using small molecules could have beneficial effects on one or more aging-related diseases and cancer. We identified new moderately active and selective non-peptidic compounds able to inhibit the activity of both standard and immunoproteasome, as well as novel and selective scaffolds that would bind and inhibit SIRT6 selectively and can be used to sensitize tumor cells to commonly used anticancer agents such gemcitabine and olaparib. Moreover, our virtual screening approach led us also to the discovery of new putative modulators of SIRT3 with interesting in-vitro and cellular activity. Although the selectivity and potency of the identified chemical scaffolds are susceptible to be further improved, these compounds can be considered as highly promising leads for the development of future therapeutics.

Effects of environmental complexity on neurogenesis in the hippocampal dentate gyrus

Introduction. Postnatal neurogenesis in the hippocampal dentate gyrus, can be modulated by numerous determinants, such as hormones, transmitters and stress. Among the factors positively interfering with neurogenesis, the complexity of the environment appears to play a particularly striking role. Adult mice reared in an enriched environment produce more neurons and exhibit better performance in hippocampus-specific learning tasks. While the effects of complex environments on hippocampal neurogenesis are well documented, there is a lack of information on the effects of living under socio-sensory deprivation conditions. Due to the immaturity of rats and mice at birth, studies dealing with the effects of environmental enrichment on hippocampal neurogenesis were carried out in adult animals, i.e. during a period of relatively low rate of neurogenesis. The impact of environment is likely to be more dramatic during the first postnatal weeks, because at this time granule cell production is remarkably higher than at later phases of development. The aim of the present research was to clarify whether and to what extent isolated or enriched rearing conditions affect hippocampal neurogenesis during the early postnatal period, a time window characterized by a high rate of precursor proliferation and to elucidate the mechanisms underlying these effects. The experimental model chosen for this research was the guinea pig, a precocious rodent, which, at 4-5 days of age can be independent from maternal care. Experimental design. Animals were assigned to a standard (control), an isolated, or an enriched environment a few days after birth (P5-P6). On P14-P17 animals received one daily bromodeoxyuridine (BrdU) injection, to label dividing cells, and were sacrificed either on P18, to evaluate cell proliferation or on P45, to evaluate cell survival and differentiation. Methods. Brain sections were processed for BrdU immunhistochemistry, to quantify the new born and surviving cells. The phenotype of the surviving cells was examined by means of confocal microscopy and immunofluorescent double-labeling for BrdU and either a marker of neurons (NeuN) or a marker of astrocytes (GFAP). Apoptotic cell death was examined with the TUNEL method. Serial sections were processed for immunohistochemistry for i) vimentin, a marker of radial glial cells, ii) BDNF (brain-derived neurotrofic factor), a neurotrophin involved in neuron proliferation/survival, iii) PSA-NCAM (the polysialylated form of the neural cell adhesion molecule), a molecule associated with neuronal migration. Total granule cell number in the dentate gyrus was evaluated by stereological methods, in Nissl-stained sections. Results. Effects of isolation. In P18 isolated animals we found a reduced cell proliferation (-35%) compared to controls and a lower expression of BDNF. Though in absolute terms P45 isolated animals had less surviving cells than controls, they showed no differences in survival rate and phenotype percent distribution compared to controls. Evaluation of the absolute number of surviving cells of each phenotype showed that isolated animals had a reduced number of cells with neuronal phenotype than controls. Looking at the location of the new neurons, we found that while in control animals 76% of them had migrated to the granule cell layer, in isolated animals only 55% of the new neurons had reached this layer. Examination of radial glia cells of P18 and P45 animals by vimentin immunohistochemistry showed that in isolated animals radial glia cells were reduced in density and had less and shorter processes. Granule cell count revealed that isolated animals had less granule cells than controls (-32% at P18 and -42% at P45). Effects of enrichment. In P18 enriched animals there was an increase in cell proliferation (+26%) compared to controls and a higher expression of BDNF. Though in both groups there was a decline in the number of BrdU-positive cells by P45, enriched animals had more surviving cells (+63) and a higher survival rate than controls. No differences were found between control and enriched animals in phenotype percent distribution. Evaluation of the absolute number of cells of each phenotype showed that enriched animals had a larger number of cells of each phenotype than controls. Looking at the location of cells of each phenotype we found that enriched animals had more new neurons in the granule cell layer and more astrocytes and cells with undetermined phenotype in the hilus. Enriched animals had a higher expression of PSA-NCAM in the granule cell layer and hilus Vimentin immunohistochemistry showed that in enriched animals radial glia cells were more numerous and had more processes.. Granule cell count revealed that enriched animals had more granule cells than controls (+37% at P18 and +31% at P45). Discussion. Results show that isolation rearing reduces hippocampal cell proliferation but does not affect cell survival, while enriched rearing increases both cell proliferation and cell survival. Changes in the expression of BDNF are likely to contribute to he effects of environment on precursor cell proliferation. The reduction and increase in final number of granule neurons in isolated and enriched animals, respectively, are attributable to the effects of environment on cell proliferation and survival and not to changes in the differentiation program. As radial glia cells play a pivotal role in neuron guidance to the granule cell layer, the reduced number of radial glia cells in isolated animals and the increased number in enriched animals suggests that the size of radial glia population may change dynamically, in order to match changes in neuron production. The high PSA-NCAM expression in enriched animals may concur to favor the survival of the new neurons by facilitating their migration to the granule cell layer. Conclusions. By using a precocious rodent we could demonstrate that isolated/enriched rearing conditions, at a time window during which intense granule cell proliferation takes place, lead to a notable decrease/increase of total granule cell number. The time-course and magnitude of postnatal granule cell production in guinea pigs are more similar to the human and non-human primate condition than in rats and mice. Translation of current data to humans would imply that exposure of children to environments poor/rich of stimuli may have a notably large impact on dentate neurogenesis and, very likely, on hippocampus dependent memory functions.

Studio di caratteri correlati a stress idrico in una collezione di cultivar di frumento duro (Triticum durum Desf.)

L’agricoltura si trova ad affrontare una diminuzione della disponibilità d’acqua ed una crescente domanda della produzione di cereali per scopi alimentari. Sono perciò necessarie strategie di coltivazione innovative per migliorare la produttività e nuovi genotipi migliorati nell'efficienza dell’uso delle risorse in condizioni di siccità. Questi rappresentano gli obietti principali del progetto “DROPS” (Drought tolerant yielding Plants) all’interno del quale ha avuto luogo il mio progetto di Dottorato. La mia attività di ricerca è stata svolta come segue: 1. Caratterizzazione molecolare di un panel di188 accessioni di frumento duro con marcatori SSR e DaRT; 2. Esperimenti in serra su 100 accessioni del panel per valutare la Water-Use Efficiency (WUE) in sei repliche secondo un Alpha Lattice design; 3. Prove sul campo, effettuate secondo un Alpha Lattice design, in due stagioni di crescita: a. 2010/11, valutazione di 100 accessioni presso l’Azienda sperimentale dell'Università di Cadriano (BO); b. 2011/12, valutazione del panel completo in 3 ambienti, con due diversi regimi irrigui In entrambi gli anni, abbiamo valutato caratteri agronomici correlati con il ciclo di sviluppo, la resa di granella e sue componenti, nonché diversi fattori ambientali e del suolo. Per quanto riguarda WUE, abbiamo trovato differenze altamente significative tra accessioni; inoltre, cinque accessioni hanno mostrato elevati valori di WUE e cinque accessioni valori molto bassi di WUE in tutte e sei le repliche. Gli esperimenti di campo nelle stagioni 2011 e 2012 hanno evidenziato differenze altamente significative tra le accessioni del panel per la maggior parte dei caratteri analizzati, confermando inoltre che il panel di fiorisce entro una settimana. L'esperimento del secondo anno ci ha permesso osservare un significativa interazione Genotipo X Ambiente. Questi risultati saranno integrati con ulteriori analisi QTL, per identificare regioni cromosomiche coinvolte nel controllo genetico dei caratteri di interesse e verificare la stabilità dei QTL in diversi ambienti.

Influence of plant structure, cultural pratices and environmental conditions on the development of the bacterial canker of kiwifruits

Italy has a preeminent rank in kiwifruit industry, being the first exporter and the second largest producer after China. However, in the last years kiwifruit yields and the total cultivated area considerably decreased, due to the pandemic spread of the bacterial canker caused by Pseudomonas syringae pv. actinidiae (Psa). Several climatic conditions and cultural practices affect the development of the bacterial canker. This research work focused on the impact of agricultural practices and microclimate conditions on the incidence and epidemiology of Psa in the orchard. Therefore, the effect of fertilization, irrigation, use of bio-regulators, rootstock, training system and pruning were examined. The effect of different tunnel systems was analyzed as well, to study the plant-pathogen interaction. Considering the importance of insects as vectors in other pathosystems, the role of Metcalfa pruinosa in the spread of the bacterial canker was investigated in controlled conditions. In addition, quality and storage properties of fruits from infected plants were assessed. The study of all these aspects of the agronomic practices is useful to define a strategy to limit the bacterial diffusion in the orchard. Overall, excess nitrogen fertilization, water stress, stagnant water supplies, pruning before summer and the high number of Metcalfa pruinosa increased the Psa incidence. In contrast, tunnel covers may be useful for the control of the disease, with special attention to the kind of material.

Stress abiotici e trattamenti ultra-diluiti: effetti fisiologici e molecolari sulla crescita in vitro di frumento

Gli stress abiotici determinando modificazioni a livello fisiologico, biochimico e molecolare delle piante, costituiscono una delle principali limitazioni per la produzione agricola mondiale. Nel 2007 la FAO ha stimato come solamente il 3,5% della superficie mondiale non sia sottoposta a stress abiotici. Il modello agro-industriale degli ultimi cinquant'anni, oltre ad avere contribuito allo sviluppo economico dell'Europa, è stato anche causa di inquinamento di acqua, aria e suolo, mediante uno sfruttamento indiscriminato delle risorse naturali. L'arsenico in particolare, naturalmente presente nell'ambiente e rilasciato dalle attività antropiche, desta particolare preoccupazione a causa dell'ampia distribuzione come contaminante ambientale e per gli effetti di fitotossicità provocati. In tale contesto, la diffusione di sistemi agricoli a basso impatto rappresenta una importante risorsa per rispondere all'emergenza del cambiamento climatico che negli anni a venire sottoporrà una superficie agricola sempre maggiore a stress di natura abiotica. Nello studio condotto è stato utilizzato uno stabile modello di crescita in vitro per valutare l'efficacia di preparati ultra diluiti (PUD), che non contenendo molecole chimiche di sintesi ben si adattano a sistemi agricoli sostenibili, su semi di frumento preventivamente sottoposti a stress sub-letale da arsenico. Sono state quindi condotte valutazioni sia a livello morfometrico (germinazione, lunghezza di germogli e radici) che molecolare (espressione genica valutata mediante analisi microarray, con validazione tramite Real-Time PCR) arricchendo la letteratura esistente di interessanti risultati. In particolare è stato osservato come lo stress da arsenico, determini una minore vigoria di coleptile e radici e a livello molecolare induca l'attivazione di pathways metabolici per proteggere e difendere le cellule vegetali dai danni derivanti dallo stress; mentre il PUD in esame (As 45x), nel sistema stressato ha indotto un recupero nella vigoria di germoglio e radici e livelli di espressione genica simili a quelli riscontrati nel controllo suggerendo un effetto "riequilibrante" del metabolismo vegetale.