Determinants of biological activity of nanomaterials in innate immunity cells: the role of aspect ratio, environmental contaminants and protein corona

As defined by the European Union, “ ’Nanomaterial’ (NM) means a natural, incidental or manufactured material containing particles, in an unbound state or as an aggregate or agglomerate, where, for 50 % or more of the particles in the number size distribution, one or more external dimensions is in the size range 1 nm-100 nm ” (2011/696/UE). Given their peculiar physico-chemical features, nanostructured materials are largely used in many industrial fields (e.g. cosmetics, electronics, agriculture, biomedical) and their applications have astonishingly increased in the last fifteen years. Nanostructured materials are endowed with very large specific surface area that, besides making them very useful in many industrial processes, renders them very reactive towards the biological systems and, hence, potentially endowed with significant hazard for human health. For these reasons, in recent years, many studies have been focused on the identification of toxic properties of nanostructured materials, investigating, in particular, the mechanisms behind their toxic effects as well as their determinants of toxicity. This thesis investigates two types of nanostructured TiO2 materials, TiO2 nanoparticles (NP), which are yearly produced in tonnage quantities, and TiO2 nanofibres (NF), a relatively novel nanomaterial. Moreover, several preparations of MultiWalled Carbon Nanotubes (MWCNT), another nanomaterial widely present in many products, are also investigated.- Although many in vitro and in vivo studies have characterized the toxic properties of these materials, the identification of their determinants of toxicity is still incomplete. The aim of this thesis is to identify the structural determinants of toxicity, using several in vitro models. Specific fields of investigation have been a) the role of shape and the aspect ratio in the determination of biological effects of TiO2 nanofibres of different length; b) the synergistic effect of LPS and TiO2 NP on the expression of inflammatory markers and the role played therein by TLR-4; c) the role of functionalization and agglomeration in the biological effects of MWCNT. As far as biological effects elicited by TiO2 NF are concerned, the first part of the thesis demonstrates that long TiO2 nanofibres caused frustrated phagocytosis, cytotoxicity, hemolysis, oxidative stress and epithelial barrier perturbation. All these effects were mitigated by fibre shortening through ball-milling. However, short TiO2 NF exhibited enhanced ability to activate acute pro-inflammatory effects in macrophages, an effect dependent on phagocytosis. Therefore, aspect ratio reduction mitigated toxic effects, while enhanced macrophage activation, likely rendering the NF more prone to phagocytosis. These results suggest that, under in vivo conditions, short NF will be associated with acute inflammatory reaction, but will undergo a relatively rapid clearance, while long NF, although associated with a relatively smaller acute activation of innate immunity cells, are not expected to be removed efficiently and, therefore, may be associated to chronic inflammatory responses. As far as the relationship between the effects of TiO2 NP and LPS, investigated in the second part of the thesis, are concerned, TiO2 NP markedly enhanced macrophage activation by LPS through a TLR-4-dependent intracellular pathway. The adsorption of LPS onto the surface of TiO2 NP led to the formation of a specific bio-corona, suggesting that, when bound to TiO2 NP, LPS exerts a much more powerful pro-inflammatory effect. These data suggest that the inflammatory changes observed upon exposure to TiO2 NP may be due, at least in part, to their capability to bind LPS and, possibly, other TLR agonists, thus enhancing their biological activities. Finally, the last part of the thesis demonstrates that surface functionalization of MWCNT with amino or carboxylic groups mitigates the toxic effects of MWCNT in terms of macrophage activation and capability to perturb epithelial barriers. Interestingly, surface chemistry (in particular surface charge) influenced the protein adsorption onto the MWCNT surface, allowing to the formation of different protein coronae and the tendency to form agglomerates of different size. In particular functionalization a) changed the amount and the type of proteins adsorbed to MWCNT and b) enhanced the tendency of MWCNT to form large agglomerates. These data suggest that the different biological behavior of functionalized and pristine MWCNT may be due, at least in part, to the different tendency to form large agglomerates, which is significantly influenced by their different capability to interact with proteins contained in biological fluids. All together, these data demonstrate that the interaction between physico-chemical properties of nanostructured materials and the environment (cells + biological fluids) in which these materials are present is of pivotal importance for the understanding of the biological effects of NM. In particular, bio-persistence and the capability to elicit an effective inflammatory response are attributable to the interaction between NM and macrophages. However, the interaction NM-cells is heavily influenced by the formation at the nano-bio interface of specific bio-coronae that confer a novel biological identity to the nanostructured materials, setting the basis for their specific biological activities.

Comparing different approach and avoidance models of learning and personality in the prediction of work, university, and leadership outcomes

Jackson (2005) developed a hybrid model of personality and learning, known as the learning styles profiler (LSP) which was designed to span biological, socio-cognitive, and experiential research foci of personality and learning research. The hybrid model argues that functional and dysfunctional learning outcomes can be best understood in terms of how cognitions and experiences control, discipline, and re-express the biologically based scale of sensation-seeking. In two studies with part-time workers undertaking tertiary education (N=137 and 58), established models of approach and avoidance from each of the three different research foci were compared with Jackson's hybrid model in their predictiveness of leadership, work, and university outcomes using self-report and supervisor ratings. Results showed that the hybrid model was generally optimal and, as hypothesized, that goal orientation was a mediator of sensation-seeking on outcomes (work performance, university performance, leader behaviours, and counterproductive work behaviour). Our studies suggest that the hybrid model has considerable promise as a predictor of work and educational outcomes as well as dysfunctional outcomes.

Added luminance ramp alters perceived edge blur and contrast:A critical test for derivative-based models of edge coding

In many models of edge analysis in biological vision, the initial stage is a linear 2nd derivative operation. Such models predict that adding a linear luminance ramp to an edge will have no effect on the edge's appearance, since the ramp has no effect on the 2nd derivative. Our experiments did not support this prediction: adding a negative-going ramp to a positive-going edge (or vice-versa) greatly reduced the perceived blur and contrast of the edge. The effects on a fairly sharp edge were accurately predicted by a nonlinear multi-scale model of edge processing [Georgeson, M. A., May, K. A., Freeman, T. C. A., & Hesse, G. S. (in press). From filters to features: Scale-space analysis of edge and blur coding in human vision. Journal of Vision], in which a half-wave rectifier comes after the 1st derivative filter. But we also found that the ramp affected perceived blur more profoundly when the edge blur was large, and this greater effect was not predicted by the existing model. The model's fit to these data was much improved when the simple half-wave rectifier was replaced by a threshold-like transducer [May, K. A. & Georgeson, M. A. (2007). Blurred edges look faint, and faint edges look sharp: The effect of a gradient threshold in a multi-scale edge coding model. Vision Research, 47, 1705-1720.]. This modified model correctly predicted that the interaction between ramp gradient and edge scale would be much larger for blur perception than for contrast perception. In our model, the ramp narrows an internal representation of the gradient profile, leading to a reduction in perceived blur. This in turn reduces perceived contrast because estimated blur plays a role in the model's estimation of contrast. Interestingly, the model predicts that analogous effects should occur when the width of the window containing the edge is made narrower. This has already been confirmed for blur perception; here, we further support the model by showing a similar effect for contrast perception. © 2007 Elsevier Ltd. All rights reserved.

Role of fluctuations in membrane models:thermal versus nonthermal

We study the comparative importance of thermal to nonthermal fluctuations for membrane-based models in the linear regime. Our results, both in 1+1 and 2+1 dimensions, suggest that nonthermal fluctuations dominate thermal ones only when the relaxation time τ is large. For moderate to small values of τ, the dynamics is defined by a competition between these two forces. The results are expected to act as a quantitative benchmark for biological modeling in systems involving cytoskeletal and other nonthermal fluctuations. © 2011 American Physical Society.

The use of erythrocytic and animal models in the study of protein phosphorylation

Phosphorylation processes are common post-transductional mechanisms, by which it is possible to modulate a number of metabolic pathways. Proteins are highly sensitive to phosphorylation, which governs many protein-protein interactions. The enzymatic activity of some protein tyrosine-kinases is under tyrosine-phosphorylation control, as well as several transmembrane anion-fluxes and cation exchanges. In addition, phosphorylation reactions are involved in intra and extra-cellular 'cross-talk' processes. Early studies adopted laboratory animals to study these little known phosphorylation processes. The main difficulty encountered with these animal techniques was obtaining sufficient kinase or phosphatase activity suitable for studying the enzymatic process. Large amounts of biological material from organs, such as the liver and spleen were necessary to conduct such work with protein kinases. Subsequent studies revealed the ubiquity and complexity of phosphorylation processes and techniques evolved from early rat studies to the adaptation of more rewarding in vitro models. These involved human erythrocytes, which are a convenient source both for the enzymes, we investigated and for their substrates. This preliminary work facilitated the development of more advanced phosphorylative models that are based on cell lines. © 2005 Elsevier B.V. All rights reserved.

Detection and quantification of protein oxidation in sarcopenic models:a mass spectrometry study

Oxidised biomolecules in aged tissue could potentially be used as biomarkers for age-related diseases; however, it is still unclear whether they causatively contribute to ageing or are consequences of the ageing process. To assess the potential of using protein oxidation as markers of ageing, mass spectrometry (MS) was employed for the identification and quantification of oxidative modifications in obese (ob/ob) mice. Lean muscle mass and strength is reduced in obesity, representing a sarcopenic model in which the levels of oxidation can be evaluated for different muscular systems including calcium homeostasis, metabolism and contractility. Several oxidised residues were identified by tandem MS (MS/MS) in both muscle homogenate and isolated sarcoplasmic reticulum (SR), an organelle that regulates intracellular calcium levels in muscle. These modifications include oxidation of methionine, cysteine, tyrosine, and tryptophan in several proteins such as sarcoplasmic reticulum calcium ATPase (SERCA), glycogen phosphorylase, and myosin. Once modifications had been identified, multiple reaction monitoring MS (MRM) was used to quantify the percentage modification of oxidised residues within the samples. Preliminary data suggests proteins in ob/ob mice are more oxidised than the controls. For example SERCA, which constitutes 60-70% of the SR, had approximately a 2-fold increase in cysteine trioxidation of Cys561 in the obese model when compared to the control. Other obese muscle proteins have also shown a similar increase in oxidation for various residues. Further analysis with complex protein mixtures will determine the potential diagnostic use of MRM experiments for analysing protein oxidation in small biological samples such as muscle needle biopsies.

The development of attenuation compensation models of fluorescence spectroscopy signals

This study examines the effect of blood absorption on the endogenous fluorescence signal intensity of biological tissues. Experimental studies were conducted to identify these effects. To register the fluorescence intensity, the fluorescence spectroscopy method was employed. The intensity of the blood flow was measured by laser Doppler flowmetry. We proposed one possible implementation of the Monte Carlo method for the theoretical analysis of the effect of blood on the fluorescence signals. The simulation is constructed as a four-layer skin optical model based on the known optical parameters of the skin with different levels of blood supply. With the help of the simulation, we demonstrate how the level of blood supply can affect the appearance of the fluorescence spectra. In addition, to describe the properties of biological tissue, which may affect the fluorescence spectra, we turned to the method of diffuse reflectance spectroscopy (DRS). Using the spectral data provided by the DRS, the tissue attenuation effect can be extracted and used to correct the fluorescence spectra.

Potential for Cell-Transplant Therapy with Human Neuronal Precursors to Treat Neuropathic Pain in Models of PNS and CNS Injury: Comparison of hNT2.17 and hNT2.19 Cell Lines

Effective treatment of sensory neuropathies in peripheral neuropathies and spinal cord injury (SCI) is one of the most difficult problems in modern clinical practice. Cell therapy to release antinociceptive agents near the injured spinal cord is a logical next step in the development of treatment modalities. But few clinical trials, especially for chronic pain, have tested the potential of transplant of cells to treat chronic pain. Cell lines derived from the human neuronal NT2 cell line parentage, the hNT2.17 and hNT2.19 lines, which synthesize and release the neurotransmitters gamma-aminobutyric acid (GABA) and serotonin (5HT), respectively, have been used to evaluate the potential of cell-based release of antinociceptive agents near the lumbar dorsal (horn) spinal sensory cell centers to relieve neuropathic pain after PNS (partial nerve and diabetes-related injury) and CNS (spinal cord injury) damage in rat models. Both cell lines transplants potently and permanently reverse behavioral hypersensitivity without inducing tumors or other complications after grafting. Functioning as cellular minipumps for antinociception, human neuronal precursors, like these NT2-derived cell lines, would likely provide a useful adjuvant or replacement for current pharmacological treatments for neuropathic pain.

Space-for-Time Substitution Works in Everglades Ecological Forecasting Models

Space-for-time substitution is often used in predictive models because long-term time-series data are not available. Critics of this method suggest factors other than the target driver may affect ecosystem response and could vary spatially, producing misleading results. Monitoring data from the Florida Everglades were used to test whether spatial data can be substituted for temporal data in forecasting models. Spatial models that predicted bluefin killifish (Lucania goodei) population response to a drying event performed comparably and sometimes better than temporal models. Models worked best when results were not extrapolated beyond the range of variation encompassed by the original dataset. These results were compared to other studies to determine whether ecosystem features influence whether space-for-time substitution is feasible. Taken in the context of other studies, these results suggest space-for-time substitution may work best in ecosystems with low beta-diversity, high connectivity between sites, and small lag in organismal response to the driver variable.

Diatom-based Models for Inferring Hydrology and Periphyton Abundance in a Subtropical Karstic Wetland: Implications for Ecosystem-Scale Bioassessment

We developed diatom-based prediction models of hydrology and periphyton abundance to inform assessment tools for a hydrologically managed wetland. Because hydrology is an important driver of ecosystem change, hydrologic alterations by restoration efforts could modify biological responses, such as periphyton characteristics. In karstic wetlands, diatoms are particularly important components of mat-forming calcareous periphyton assemblages that both respond and contribute to the structural organization and function of the periphyton matrix. We examined the distribution of diatoms across the Florida Everglades landscape and found hydroperiod and periphyton biovolume were strongly correlated with assemblage composition. We present species optima and tolerances for hydroperiod and periphyton biovolume, for use in interpreting the directionality of change in these important variables. Predictions of these variables were mapped to visualize landscape-scale spatial patterns in a dominant driver of change in this ecosystem (hydroperiod) and an ecosystem-level response metric of hydrologic change (periphyton biovolume). Specific diatom assemblages inhabiting periphyton mats of differing abundance can be used to infer past conditions and inform management decisions based on how assemblages are changing. This study captures diatom responses to wide gradients of hydrology and periphyton characteristics to inform ecosystem-scale bioassessment efforts in a large wetland.

The importance of realistic dispersal models in planning for conservation : application of a novel modelling platform to evaluate management scenarios in an Afrotropical biodiversity hotspot

Funded by European Union's Horizon 2020 Marie Sklodowska-Curie. Grant Number: 661211 Research Foundation Flanders (FWO). Grant Numbers: G.0055.08, G.0149.09, G.0308.13 FWO Research Network on Eco-Evolutionary dynamics French Ministère de l'Energie de l'Ecologie du Développement Durable et de la Mer through the EU FP6 BiodivERsA Eranet NERC. Grant Number: NE/J008001/1

Using individual tracking data to validate the predictions of species distribution models

The authors would like to thank the College of Life Sciences of Aberdeen University and Marine Scotland Science which funded CP's PhD project. Skate tagging experiments were undertaken as part of Scottish Government project SP004. We thank Ian Burrett for help in catching the fish and the other fishermen and anglers who returned tags. We thank José Manuel Gonzalez-Irusta for extracting and making available the environmental layers used as environmental covariates in the environmental suitability modelling procedure. We also thank Jason Matthiopoulos for insightful suggestions on habitat utilization metrics as well as Stephen C.F. Palmer, and three anonymous reviewers for useful suggestions to improve the clarity and quality of the manuscript.

Strong inference from transect sign surveys : combining spatial autocorrelation and misclassification occupancy models to quantify the detectability of a recovering carnivore

Funding — Forest Enterprise Scotland and the University of Aberdeen provided funding for the project. The Carnegie Trust supported the lead author, E. McHenry, in this research through the award of a tuition fees bursary.