Cable Internet Unbundling: Local Leadership in the Deployment High Speed Access

With the pending merger of TCI and AT&T and their promise of one-stop television, Internet, and telephone service, the cable Internet issues move to the forefront. The desire of traditional Internet Service Providers to gain access to new high-speed technologies for Internet access led to requests for unbundling or open access to cable systems. Despite the heated debate on the need for unbundling that has occurred at the federal level, local authorities have taken the lead in requiring open access to cable for competing ISPs. General anticompetitive concerns with cable Internet dominated by the cable company could be alleviated in large part by requiring open access to cable for Internet Service Providers

Cable Internet Unbundling: Local Leadership in the Deployment High Speed Access

With the pending merger of TCI and AT&T and their promise of one-stop television, Internet, and telephone service, the cable Internet issues move to the forefront. The desire of traditional Internet Service Providers to gain access to new high-speed technologies for Internet access led to requests for unbundling or open access to cable systems. Despite the heated debate on the need for unbundling that has occurred at the federal level, local authorities have taken the lead in requiring open access to cable for competing ISPs. General anticompetitive concerns with cable Internet dominated by the cable company could be alleviated in large part by requiring open access to cable for Internet Service Providers

Structure-Activity Relationships Governing the Interaction of Nanoparticles with Mammalian Cells – Predictive Models for Toxicology and Medical Applications.

Nanoscience is seen as one of the key enabling technologies of the 20th century and as its range of applications increases it is important to look at how nanomaterials interact with biological environments. Some of these interactions have given rise to toxic effects and thus, the creation of the field of nanotoxicology, it has also been noted that current methods of evaluating toxicity may not be sufficient to keep up with the rapidly emerging range of nanomaterials becoming available. It is clear that alternatives are necessary. In this thesis, a phenomenological rate equation model is constructed to simulate nanoparticle uptake and subsequent cellular response. Poly (amido amine) (PAMAM) dendrimers, generations 4 – 6 were employed to model the response due to their well defined physico-chemical properties. Upon particle uptake, the model simulates the temporal evolution of: increased reactive oxygen species (ROS) generation, decrease in cellular anti-oxidants, caspase activation, mitochondrial membrane potential decay (MMPD), Tumour Necrosis Factor – alpha (TNF-α) and Interleukin 8 (IL-8) activation, based on previously obtained experimental data. The simulated results match well the experimental observations over a range of different doses and time points, and over a range of different cell lines: including HaCaT (human keratinocytes), SW480 (human colon andenocarcinoma cells) and J774A.1 (mouse macrophages). Furthermore, differences in the cytotoxic responses of each cell line is understood via variation of intracellular antioxidant levels and differences in the responses to several assays is understood in terms of their sensitivity to different cellular events. The model is able to simultaneously visualise the dose and time dependence of the toxic response of these cells to nanoparticle uptake and describes this interaction using a system of rates, which decouple the cell and particle based parameters. This rate based approach may form the basis of a valid alternative to effective concentrations for the classification of nanotoxicity and can lay the foundation for Quantitative Structure Activity Relationships (QSARs) and a better understanding of nanoparticle Adverse Outcome Pathways (AOPs). To further the range of applicability, the model was extended to simulate the toxicity of Polystyrene Nanoparticles with amine modified surface groups (PSNP-NH2). PSNP-NH2 did not share the same well-defined physico-chemical properties as PAMAM dendrimers, therefore, information on the size and extent of surface functionalisation was obtained via Scanning Electron Microscopy (SEM) and Nuclear Magnetic Resonance (NMR), respectively. The toxicity was evaluated using Alamar Blue (AB) and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) at various time points from 6 – 72 hours. Again, the model results were seen to faithfully simulate the experimental results, with identical model parameters, with the exception of the endocytosis rate (a particle size dependant property, which was modified to account for the huge disparity in size between PAMAM dendrimers and PSNP-NH2) and rate of generation of ROS (determined by the number of surface functional groups). The consistancy of the model over a range of differnt nanopaticle types adds validity to such an approach to evaluate nanoparticle toxic potential. Nanoparticle uptake is a critical process in the induction of toxicity. Therefore, to bypass this process could eliminate the subsequent toxic response. PAMAM dendrimers (G4 and 6) and HaCaT cells were again employed. Cells were treated with DL-Buthionine-(S,R)-sulfoximine (BSO) to enduce membrane permeability and endosomal activity was tracked using confocal laser scanning microscopy (CLSM), ROS generation tracked with the dye Carboxy-H2DCFDA and viability measued with AB and MTT. A dramatic change compared to untreated cells occured: reduced uptake of both generations of PAMAM dendrimer, reduced ROS generation and huge increases in mitochondrial activity were observed. These results indicated that dendrimer uptake appeared to be occuring via passive diffusion into the cell, thereby avoiding the endocytosis associated ROS production. Toxicity was still noted for the higher dose regime. BSO is also known to reduce cellular Glutathione and the study highlights the importance of GSH in maintaining the redox balance of the mitochondria, but also points to the potential of controling nanoparticle toxicity via control of the uptake mechanism employed by the cell. Finally, an acellular assay was designed to quantify the potential for a nanoparticle to generate ROS. The enzyme Monoamine Oxidase – A (MAO-A) was used to generate ROS (Hydrogen Peroxide – H2O2) and the effect of PAMAM and PPI dendrimer poylmers on this production were examined. The acellular reaction rate (ARR) was found to be dependant on the particle physico-chemical properties, and within a series of nanoparticles can also be related to the toxicity. The difference in relating the acellular data to experimental results between different series of particles points to the importance of cellular parameters such as uptake rate in determining toxicity. The acellular results were then modelled based on a similar series of equations as used in the cellular studies. The cellular and acellular modelling approaches developed within this thesis, indicate that such a method may form an efficient and cost effective alternative for the evaluation of nanoparticle toxicity and furthermore, this toxicity is primarily associated with two parameters: the cellular dependant uptake rate and the particle dependant number of surface groups

Structure-Activity Relationships Governing the Interaction of Nanoparticles With Mammalian Cells- Predictive Models for Toxicology and Medical Appliances

Nanoscience is seen as one of the key enabling technologies of the 20th century and as its range of applications increases it is important to look at how nanomaterials interact with biological environments. Some of these interactions have given rise to toxic effects and thus, the creation of the field of nanotoxicology, it has also been noted that current methods of evaluating toxicity may not be sufficient to keep up with the rapidly emerging range of nanomaterials becoming available. It is clear that alternatives are necessary. In this thesis, a phenomenological rate equation model is constructed to simulate nanoparticle uptake and subsequent cellular response. Poly (amido amine) (PAMAM) dendrimers, generations 4 – 6 were employed to model the response due to their well defined physico-chemical properties. Upon particle uptake, the model simulates the temporal evolution of: increased reactive oxygen species (ROS) generation, decrease in cellular anti-oxidants, caspase activation, mitochondrial membrane potential decay (MMPD), Tumour Necrosis Factor – alpha (TNF-α) and Interleukin 8 (IL-8) activation, based on previously obtained experimental data. The simulated results match well the experimental observations over a range of different doses and time points, and over a range of different cell lines: including HaCaT (human keratinocytes), SW480 (human colon andenocarcinoma cells) and J774A.1 (mouse macrophages). Furthermore, differences in the cytotoxic responses of each cell line is understood via variation of intracellular antioxidant levels and differences in the responses to several assays is understood in terms of their sensitivity to different cellular events. The model is able to simultaneously visualise the dose and time dependence of the toxic response of these cells to nanoparticle uptake and describes this interaction using a system of rates, which decouple the cell and particle based parameters. This rate based approach may form the basis of a valid alternative to effective concentrations for the classification of nanotoxicity and can lay the foundation for Quantitative Structure Activity Relationships (QSARs) and a better understanding of nanoparticle Adverse Outcome Pathways (AOPs). To further the range of applicability, the model was extended to simulate the toxicity of Polystyrene Nanoparticles with amine modified surface groups (PSNP-NH2). PSNP-NH2 did not share the same well-defined physico-chemical properties as PAMAM dendrimers, therefore, information on the size and extent of surface functionalisation was obtained via Scanning Electron Microscopy (SEM) and Nuclear Magnetic Resonance (NMR), respectively. The toxicity was evaluated using Alamar Blue (AB) and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) at various time points from 6 – 72 hours. Again, the model results were seen to faithfully simulate the experimental results, with identical model parameters, with the exception of the endocytosis rate (a particle size dependant property, which was modified to account for the huge disparity in size between PAMAM dendrimers and PSNP-NH2) and rate of generation of ROS (determined by the number of surface functional groups). The consistancy of the model over a range of differnt nanopaticle types adds validity to such an approach to evaluate nanoparticle toxic potential. Nanoparticle uptake is a critical process in the induction of toxicity. Therefore, to bypass this process could eliminate the subsequent toxic response. PAMAM dendrimers (G4 and 6) and HaCaT cells were again employed. Cells were treated with DL-Buthionine-(S,R)-sulfoximine (BSO) to enduce membrane permeability and endosomal activity was tracked using confocal laser scanning microscopy (CLSM), ROS generation tracked with the dye Carboxy-H2DCFDA and viability measued with AB and MTT. A dramatic change compared to untreated cells occured: reduced uptake of both generations of PAMAM dendrimer, reduced ROS generation and huge increases in mitochondrial activity were observed. These results indicated that dendrimer uptake appeared to be occuring via passive diffusion into the cell, thereby avoiding the endocytosis associated ROS production. Toxicity was still noted for the higher dose regime. BSO is also known to reduce cellular Glutathione and the study highlights the importance of GSH in maintaining the redox balance of the mitochondria, but also points to the potential of controling nanoparticle toxicity via control of the uptake mechanism employed by the cell. Finally, an acellular assay was designed to quantify the potential for a nanoparticle to generate ROS. The enzyme Monoamine Oxidase – A (MAO-A) was used to generate ROS (Hydrogen Peroxide – H2O2) and the effect of PAMAM and PPI dendrimer poylmers on this production were examined. The acellular reaction rate (ARR) was found to be dependant on the particle physico-chemical properties, and within a series of nanoparticles can also be related to the toxicity. The difference in relating the acellular data to experimental results between different series of particles points to the importance of cellular parameters such as uptake rate in determining toxicity. The acellular results were then modelled based on a similar series of equations as used in the cellular studies. The cellular and acellular modelling approaches developed within this thesis, indicate that such a method may form an efficient and cost effective alternative for the evaluation of nanoparticle toxicity and furthermore, this toxicity is primarily associated with two parameters: the cellular dependant uptake rate and the particle dependant number of surface groups

Planar lipid bilayers in recombinant ion channel research

There are a number of methods of investigating the function of recombinant proteins such as ion channels. However, after channel purification there are few methods to guarantee that the protein still functions. For ion channels, reconstituting back into planar lipid bilayers and demonstrating preserved function is a convenient and trusted method. It is cell free and even inaccessible, intracellular ion channels can be studied. We have used this method to study the function of recombinant channels of known subunit composition and have found it convenient for investigating the mode of action of ion channel modulators

Numerically Modelling Time and Dose Dependent Cytotoxicity

Dose-response curves are fundamental tools of in vitro toxicology, extensively employed in toxicant or drug screening. They are expressed by a variety of end-points and assays, measured at different time-points in a choice of cell-lines, but are typically quantified only using the mean concentration for 50% response (e.g. drug efficacy or pathway inhibition) as an indicator of overall effect. However, the response is the result of a complex and dynamic cascade of events which occur between the initial exposure and the measured end-point, and the characteristic rates of the contributing stages govern the dose response and ultimately the measured characteristic concentration. A better understanding of the effects and interdependencies of these can help in interpreting the response curves. The system can be modelled according to a phenomenological rate equation approach, in which each stage of the process is characterised by a rate constant, and causal relationships between different processes are incorporated. The current study utilises such an approach to simulate some common response cascades of cell populations to exogenous agents and explores the dependences of the dose dependent response on, for example, number of steps in a cascade, time-point, and scenarios such as additive, synergistic and antagonistic response of multiple exogenous agents

Acellular Reactivity of Polymeric Dendrimer Nanoparticles as an Indicator of Oxidative Stress in Vitro

The need for rapid and cost effective pre-screening protocols of the toxicological response of the vast array of emerging nanoparticle types is apparent and the emerging consensus on the paradigm of oxidative stress by generation of intracellular reactive oxygen species as a primary source of the toxic response suggests the development of acellular assays to screen for nanoparticle surface reactivity. This study explores the potential of the monoamine oxidase A (MAO-A) enzyme based assay with polymeric dendrimers as cofactors and serotonin as substrate, which generates H2O2, quantified by the conversion of the Carboxy-H2DCFDA dye to its fluorescent form. A range of generations of both PAMAM (poly(amido amine)) (G4-G7) and PPI (poly(propylene imine)) (G0-G4) dendritic polymer nanoparticles are used as test particles to validate the quantitative nature of the assay response as a function of nanoparticle physico-chemical properties. The assay is well behaved as a function of dose, over low dose ranges and the acellular reaction rate (ARR) is well correlated with the number of surface amino groups for the combined dendrimer series. For each series, the ARR is also well correlated with the previously documented cytotoxicity, although the correlation is substantially different for each series of dendrimers, pointing to the additional importance of cellular uptake rates in the determination of toxicity

Measuring the electrical impedance of mouse brain tissue

We report on an experimental method to measure conductivity of cortical tissue. We use a pair of 5mm diameter Ag/AgCl electrodes in a Perspex sandwich device that can be brought to a distance of 400 microns apart. The apparatus is brought to uniform temperature before use. Electrical impedance of a sample is measured across the frequency range 20 Hz-2.0 MHz with an Agilent 4980A four-point impedance monitor in a shielded room. The equipment has been used to measure the conductivity of mature mouse brain cortex in vitro. Slices 400 microns in thickness are prepared on a vibratome. Slices are bathed in artificial cerebrospinal fluid (ACSF) to keep them alive. Slices are removed from the ACSF and sections of cortical tissue approximately 2 mm times 2 mm are cut with a razor blade. The sections are photographed through a calibrated microscope to allow identification of their cross-sectional areas. Excess ACSF is removed from the sample and the sections places between the electrodes. The impedance is measured across the frequency range and electrical conductivity calculated. Results show two regions of dispersion. A low frequency region is evident below approximately 10 kHz, and a high frequency dispersion above this. Results at the higher frequencies show a good fit to the Cole-Cole model of impedance of biological tissue; this model consists of resistive and non-linear capacitive elements. Physically, these elements are likely to arise due to membrane polarization and migration of ions both intra- and extra-cellularly.http://www.iupab2014.org/assets/IUPAB/NewFolder/iupab-abstracts.pd

Toxicological Assessment of Nanomaterials: The Role of In Vitro Raman Microspectroscopic Analysis

The acceleration of nanomaterials research has brought about increased demands for rapid analysis of their bioactivity, in a multi-parametric fashion, to minimise the gap between potential applications and knowledge of their toxicological properties. The potential of Raman microspectroscopy for the analysis of biological systems with the aid of multivariate analysis techniques has been demonstrated. In this study, an overview of recent efforts towards establishing a ‘label-free high content nanotoxicological assessment technique’ using Raman microspectroscopy is presented. The current state of the art for cellular toxicity assessment and the potential of Raman microspectroscopy are discussed, and the spectral markers of the cellular toxic responses upon exposure to nanoparticles, changes on the identified spectral markers upon exposure to different nanoparticles, cell death mechanisms and the effects of nanoparticles on different cell lines are summarised. Moreover, 3D toxicity plots of spectral markers, as a function of time and dose, are introduced as new methodology for toxicological analysis based on the intrinsic properties of the biomolecular changes, such as cytoplasmic RNA aberrations, protein and lipid damage associated with the toxic response. The 3D evolution of the spectral markers are correlated with the results obtained by commonly-used cytotoxicity assays and significant similarities are observed between band intensity and percentage viability obtained by the Alamar Blue assay, as an example. Therefore, the developed 3D plots can be used to identify toxicological properties of a nanomaterial and can potentially be used to predict toxicity which can provide rapid advances in nanomedicine