Analysis of cumulative effects of multiple stressors on saltmarshes and consideration of management options

Natural systems face pressures exerted by natural physical-chemical forcings and a myriad of co-occurring human stressors that may interact to cause larger than expected effects, thereby presenting a challenge to ecosystem management. This thesis aimed to develop new information that can contribute to reduce the existing knowledge gaps hampering the holistic management of multiple stressors. I undertook a review of the state-of-the-art methods to detect, quantify and predict stressor interactions, identifying techniques that could be applied in this thesis research. Then, I conducted a systematic review of saltmarsh multiple stressor studies in conjunction with a multiple stressor mapping exercise for the study system in order to infer potential important synergistic stressor interactions. This analysis identified key stressors that are affecting the study system, but also pointed to data gaps in terms of driver and pressure data and raised issues for potentially overlooked stressors. Using field mesocosms, I explored how a local stressor (nutrient availability) affects the responses of saltmarsh vegetation to a global stressor (increased inundation) in different soil types. Results indicate that saltmarsh vegetation would be more drastically affected by increased inundation in low than in medium organic matter soils, and especially in estuaries already under high nutrient availability. In another field experiment, I examined the challenges of managing co-occurring and potentially interacting local stressors on saltmarsh vegetation: recreational trampling and smothering by deposition of excess macroalgal wrack due to high nutrient loads. Trampling and wrack prevention had interacting effects, causing non-linear responses of the vegetation to simulated management of these stressors, such that vegetation recovered only in those treatments simulating the combined prevention of both stressors. During this research I detected, using molecular genetic methods, a widespread presence of S. anglica (and to a lesser extent S. townsendii), two previously unrecorded non-native Spartinas in the study areas.

Oscillatory mechanisms of conscious perception and attention

Although the prominent role of neural oscillations in perception and cognition has been continuously investigated, some critical questions remain unanswered. My PhD thesis was aimed at addressing some of them. First, can we dissociate oscillatory underpinnings of perceptual accuracy and subjective awareness? Current work would strongly suggest that this dissociation can be drawn. While the fluctuations in alpha-amplitude decide perceptual bias and metacognitive abilities, the speed of alpha activity (i.e., alpha-frequency) dictates sensory sampling, shaping perceptual accuracy. Second, how are these oscillatory mechanisms integrated during attention? The obtained results indicate that a top-down visuospatial mechanism modulates neural assemblies in visual areas via oscillatory re-alignment and coherence in the alpha/beta range within the fronto-parietal brain network. These perceptual predictions are reflected in the retinotopically distributed posterior alpha-amplitude, while perceptual accuracy is explained by the higher alpha-frequency at the to-be-attended location. Finally, sensory input, elaborated via fast gamma oscillations, is linked to specific phases of this slower activity via oscillatory nesting, enabling integration of the feedback-modulated oscillatory activity with sensory information. Third, how can we relate this oscillatory activity to other neural markers of behaviour (i.e., event-related potentials)? The obtained results favour the oscillatory model of ERP genesis, where alpha-frequency shapes the latency of early evoked-potentials, namely P1, with both neural indices being related to perceptual accuracy. On the other hand, alpha-amplitude dictates the amplitude of later P3 evoked-response, whereas both indices shape subjective awareness. Crucially, by combining different methodological approaches, including neurostimulation (TMS) and neuroimaging (EEG), current work identified these oscillatory-behavior links as causal and not just as co-occurring events. Current work aimed at ameliorating the use of the TMS-EEG approach by explaining inter-individual differences in the stimulation outcomes, which could be proven crucial in the way we design entrainment experiments and interpret the results in both research and clinical settings.

Molecular approach to Neurodegenerative Disorders: Role of Nociceptin/Orphanin FQ - NOP System in Parkinson and Epigenetic Mechanism in Alzheimer's Disease

With life expectancies increasing around the world, populations are getting age and neurodegenerative diseases have become a global issue. For this reason we have focused our attention on the two most important neurodegenerative diseases: Parkinson’s and Alzheimer’s. Parkinson’s disease is a chronic progressive neurodegenerative movement disorder of multi-factorial origin. Environmental toxins as well as agricultural chemicals have been associated with PD. Has been observed that N/OFQ contributes to both neurotoxicity and symptoms associated with PD and that pronociceptin gene expression is up-regulated in rat SN of 6-OHDA and MPP induced experimental parkinsonism. First, we investigated the role of N/OFQ-NOP system in the pathogenesis of PD in an animal model developed using PQ and/or MB. Then we studied Alzheimer's disease. This disorder is defined as a progressive neurologic disease of the brain leading to the irreversible loss of neurons and the loss of intellectual abilities, including memory and reasoning, which become severe enough to impede social or occupational functioning. Effective biomarker tests could prevent such devastating damage occurring. We utilized the peripheral blood cells of AD discordant monozygotic twin in the search of peripheral markers which could reflect the pathology within the brain, and also support the hypothesis that PBMC might be a useful model of epigenetic gene regulation in the brain. We investigated the mRNA levels in several genes involve in AD pathogenesis, as well DNA methylation by MSP Real-Time PCR. Finally by Western Blotting we assess the immunoreactivity levels for histone modifications. Our results support the idea that epigenetic changes assessed in PBMCs can also be useful in neurodegenerative disorders, like AD and PD, enabling identification of new biomarkers in order to develop early diagnostic programs.

A step forwards in immunomodulation and immunetolerance knowledge. HLA-G expression in co-cultures of peripheral blood mononuclear cells and stem cells after in vitro NGAL stimulation

NGAL (Neutrophil Gelatinase-associated Lipocalin ) is a protein of lipocalin superfamily. Recent literature focused on its biomarkers function in several pathological condition (acute and chronic kidney damage, autoimmune disease, malignancy). NGAL biological role is not well elucidated. Several are the demonstration of its bacteriostatic role. Recent papers have indeed highlight NGAL role in NFkB modulation. The aim of this study is to understand whether NGAL may exert a role in the activation (modulation) of T cell response through the regulation of HLA-G complex, a mediator of tolerance. From 8 healthy donors we obtained peripheral blood mononuclear cells (PBMCs) and we isolated by centrifugation on a Ficoll gradient. Cells were then treated with four concentrations of NGAL (40-320 ng/ml) with or without iron. We performed flow cytometry analysis and ELISA test. NGAL increased the HLA-G expression on CD4+ T cells, with an increasing corresponding to the dose. Iron effect is not of unique interpretation. NGAL adiction affects regulatory T cells increasing in vitro expansion of CD4+ CD25+ FoxP3+ cells. Neutralizing antibody against NGAL decreased HLA-G expression and reduced significantly CD4+ CD25+ FoxP3+ cells percentage. In conclusion, we provided in vitro evidence of NGAL involvement in cellular immunity. The potential role of NGAL as an immunomodulatory molecule has been evaluated: it has been shown that NGAL plays a pivotal role in the induction of immune tolerance up regulating HLA-G and T regulatory cells expression in healthy donors. As potential future scenario we highlight the in vivo role of NGAL in immunology and immunomodulation, and its possible relationship with immunosuppressive therapy efficacy, tolerance induction in transplant patients, and/or in other immunological disorders.

Involvement of the opioid system and BDNF in neuroplastic alterations occurring in neuropathic pain conditions

Chronic pain affects one in five adults, reducing quality of life and increasing risk of developing co-morbidities such as depression. Neuropathic pain results by lesions to the nervous system that alter its structure and function leading to spontaneous pain and amplified responses to noxious and innocuous stimuli. The Opioid System is probably the most important system involved in control of nociceptive transmission. Dynorphin and nociceptin systems have been suggested key mediators of some neuropathic pain aspects. An important role also for BDNF has been recently suggested since its involvement in the peripheral and central sensitization phenomena is known. We studied neuroplastic alterations occurring in chronic pain in mice subjected to the chronic constriction injury (CCI). We investigated gene expression alterations of both BDNF and Opioid System at spinal level at different intervals of time. A transient upregulation of pBDNF and pDYN was observed in spinal cord, while increasing upregulation of ppN/OFQ was found in the DRGs of injured mice. Development of neuropathic behavioral signs has been observed in ICR/CD-1 and BDNF+/+ mice, subjected to CCI. A different development of these signs was observed in BDNF+/-. We also studied gene expression changes of investigated systems in different brain areas fourteen days after surgery. We found pBDNF, pDYN, pKOP, ppN/OFQ and pNOP gene expression alterations in several areas of CCI mice. In the same brain regions we also determined bioactive nociceptin peptide levels, and elevated N/OFQ levels were observed in the amygdala area. Histone modifications studies have been performed in BDNF and DYN gene promoters of CCI animal spinal cord showing selected alterations in pDYN gene promoter. In addition, a preliminary characterization of the innovative NOP-EGFP mice was performed. Overall, our results could be useful to understand which and how neuropeptidergic systems are involved in neuroplastic mechanism occurring in neuropathic pain.

Disorders of arousal: a physiopathological window to explore the mechanisms regulating sleep arousal

Disorders of Arousal (DoA) belong to NREM parasomnias and are characterized by motor and emotional episodes arising from incomplete awakenings from NREM sleep. DoA episodes embody at the same time the double nature of the arousal process, that is preserving sleep as well as respond to sleep perturbations, thus being an ideal model to study sleep arousal. In the first part of this work, we performed a spectral whole scalp EEG analysis exploring the neurophysiologic correlates of the pre-motor onset of the episodes in a large sample of patients with DoA, disclosing the co-existence of both slow and fast EEG frequencies over overlapping areas before DoA episodes, suggesting an alteration of local sleep mechanisms. Episodes of different complexity were preceded by a similar EEG activation, implying that they possibly share a similar pathophysiology. In the second part of this work, we performed a spectral whole scalp EEG analysis comparing the pre-motor onset of the episodes and normal arousals from healthy sleepers, disclosing the persistence of slow frequencies as well as sigma band (expression of sleep spindles) in DoA episodes. Overall, these results might subtend a higher tendence to preserve sleep and a more defective mechanism toward developing a complete arousal in patients with DoA. In the last part of our work, we evaluated 15 patients with DoA with 15 controls in a functional MRI study during wakefulness in addition to a proton magnetic resonance spectroscopy (1H-MRS) focused on cingulate cortex. We disclosed subtle alterations on posterior cingulate cortex as well as an increased connectivity in sensory-motor network, possibly representing a trait-functional feature responsible for the dysfunctional arousal process in DoA patients