Ex vivo culture of patient tissue and examination of gene delivery

Gene therapy has emerged as a realistic prospect for the treatment of cancer due to its potential for selective tumour cell targeting. The greatest challenge gene delivery vectors face is the ability to safely and efficiently deliver genes into target cells. The overall objectives of this thesis are to evaluate the efficacy of various gene delivery methods in a clinically relevant tumour model and to also investigate potential strategies for tumour selective delivery. We began with the development of a tumour slice model system using patient waste tissue. This model involves the use of fresh human tumour tissue, cut into thin slices and maintained ex vivo and is universally applicable to gene delivery methods, using a real-time luminescence detection method to assess gene delivery. The nature of the ex vivo culture system permitted examination of specific physiological variables, the influence of intratumoural factors and tissue specific effects on vector expression. Adenoviral vectors under the control of the human CXCR4 promoter demonstrated a 'tumour on' and 'normal off' expression profile when compared with the ubiquitously active CMV promoter when tested in patient tumour tissue. In addition, we developed an ex vivo system of changing oxygenation using the hypoxia inducer, cobalt, to mimic the transient hypoxic conditions found in solid tumours. We found that Adenoviral transgene expression was robust in the cycling hypoxic conditions relevant to solid tumours and re-oxygenation of chronically hypoxic tissue enhanced transgene expression. Finally, we demonstrated an AAV-based tumour targeting strategy using a tumour-selective promoter allowing for the efficient targeting of AAV vectors to cancer cells and the sparing of normal tissue in both murine metastatic liver tumours models and patient tissue. The thesis highlights the importance of indepth preclinical assessment of novel therapeutics and may serve as a platform for further testing of novel gene delivery approaches.

Fabrication and characterization of doped and porous silicon nanowires as anodes for lithium ion batteries

By using Si(100) with different dopant type (n++-type (As) or p-type (B)), it is shown how metal-assisted chemically (MAC) etched silicon nanowires (Si NWs) can form with rough outer surfaces around a solid NW core for p-type NWs, and a unique, defined mesoporous structure for highly doped n-type NWs. High resolution electron microscopy techniques were used to define the characteristic roughening and mesoporous structure within the NWs and how such structures can form due to a judicious choice of carrier concentration and dopant type. Control of roughness and internal mesoporosity is demonstrated during the formation of Si NWs from highly doped n-type Si(100) during electroless etching through a systematic investigation of etching parameters (etching time, AgNO3 concentration, %HF and temperature). Raman scattering measurements of the transverse optical phonon confirm quantum size effects and phonon scattering in mesoporous wires associated with the etching condition, including quantum confinement effects for the nanocrystallites of Si comprising the internal structure of the mesoporous NWs. Laser power heating of NWs confirms phonon confinement and scattering from internal mesoporosity causing reduced thermal conductivity. The Li+ insertion and extraction characteristics at n-type and p-type Si(100) electrodes with different carrier density and doping type are investigated by cyclic voltammetry and constant current measurements. The insertion and extraction potentials are demonstrated to vary with cycling and the occurrence of an activation effect is shown in n-type electrodes where the charge capacity and voltammetric currents are found to be much higher than p-type electrodes. X-ray photo-electron spectroscopy (XPS) and Raman scattering demonstrate that highly doped n-type Si(100) retains Li as a silicide and converts to an amorphous phase as a two-step phase conversion process. The findings show the succinct dependence of Li insertion and extraction processes for uniformly doped Si(100) single crystals and how the doping type and its effect on the semiconductor-solution interface dominate Li insertion and extraction, composition, crystallinity changes and charge capacity. The effect of dopant, doping density and porosity of MAC etched Si NWs are investigated. The CV response is shown to change in area (current density) with increasing NW length and in profile shape with a changing porosity of the Si NWs. The CV response also changes with scan rate indicative of a transition from intercalation or alloying reactions, to pseudocapactive charge storage at higher scan rates and for p-type NWs. SEM and TEM show a change in structure of the NWs after Li insertion and extraction due to expansion and contraction of the Si NWs. Galvanostatic measurements show the cycling behavior and the Coulombic efficiency of the Si NWs in comparison to their bulk counterparts.

In situ synthesis of DNA microarray on functionalized cyclic olefin copolymer substrate.

Thermoplastic materials such as cyclic-olefin copolymers (COC) provide a versatile and cost-effective alternative to the traditional glass or silicon substrate for rapid prototyping and industrial scale fabrication of microdevices. To extend the utility of COC as an effective microarray substrate, we developed a new method that enabled for the first time in situ synthesis of DNA oligonucleotide microarrays on the COC substrate. To achieve high-quality DNA synthesis, a SiO(2) thin film array was prepatterned on the inert and hydrophobic COC surface using RF sputtering technique. The subsequent in situ DNA synthesis was confined to the surface of the prepatterned hydrophilic SiO(2) thin film features by precision delivery of the phosphoramidite chemistry using an inkjet DNA synthesizer. The in situ SiO(2)-COC DNA microarray demonstrated superior quality and stability in hybridization assays and thermal cycling reactions. Furthermore, we demonstrate that pools of high-quality mixed-oligos could be cleaved off the SiO(2)-COC microarrays and used directly for construction of DNA origami nanostructures. It is believed that this method will not only enable synthesis of high-quality and low-cost COC DNA microarrays but also provide a basis for further development of integrated microfluidics microarrays for a broad range of bioanalytical and biofabrication applications.

Phosphorus export from a restored wetland ecosystem in response to natural and experimental hydrologic fluctuations

Wetland restoration is a commonly used approach to reduce nutrient loading to freshwater and coastal ecosystems, with many wetland restoration efforts occurring in former agricultural fields. Restored wetlands are expected to be effective at retaining or removing both nitrogen and phosphorus (P), yet restoring wetland hydrology to former agricultural fields can lead to the release of legacy fertilizer P. Here, we examined P cycling and export following rewetting of the Timberlake Restoration Project, a 440 ha restored riverine wetland complex in the coastal plain of North Carolina. We also compared P cycling within the restored wetland to two minimally disturbed nearby wetlands and an adjacent active agricultural field. In the restored wetland we observed increased soluble reactive phosphorus (SRP) concentrations following initial flooding, consistent with our expectations that P bound to iron would be released under reducing conditions. SRP concentrations in spring were 2.5 times higher leaving the restored wetland than a forested wetland and an agricultural field. During two large-scale drawdown and rewetting experiments we decreased the water depth by 1 m in ∼10 ha of inundated wetland for 2 weeks, followed by reflooding. Rewetting following experimental drainage had no effect on SRP concentrations in winter, but SRP concentrations did increase when the experiment was repeated during summer. Our best estimates suggest that this restored wetland could release legacy fertilizer P for up to a decade following hydrologic restoration. The time lag between restoration and biogeochemical recovery should be incorporated into management strategies of restored wetlands. Copyright 2010 by the American Geophysical Union.

Design and synthesis of hierarchical MnO2 nanospheres/carbon nanotubes/conducting polymer ternary composite for high performance electrochemical electrodes.

For efficient use of metal oxides, such as MnO(2) and RuO(2), in pseudocapacitors and other electrochemical applications, the poor conductivity of the metal oxide is a major problem. To tackle the problem, we have designed a ternary nanocomposite film composed of metal oxide (MnO(2)), carbon nanotube (CNT), and conducting polymer (CP). Each component in the MnO(2)/CNT/CP film provides unique and critical function to achieve optimized electrochemical properties. The electrochemical performance of the film is evaluated by cyclic voltammetry, and constant-current charge/discharge cycling techniques. Specific capacitance (SC) of the ternary composite electrode can reach 427 F/g. Even at high mass loading and high concentration of MnO(2) (60%), the film still showed SC value as high as 200 F/g. The electrode also exhibited excellent charge/discharge rate and good cycling stability, retaining over 99% of its initial charge after 1000 cycles. The results demonstrated that MnO(2) is effectively utilized with assistance of other components (fFWNTs and poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) in the electrode. Such ternary composite is very promising for the next generation high performance electrochemical supercapacitors.

The role of tectonic uplift, climate, and vegetation in the long-term terrestrial phosphorous cycle

Phosphorus (P) is a crucial element for life and therefore for maintaining ecosystem productivity. Its local availability to the terrestrial biosphere results from the interaction between climate, tectonic uplift, atmospheric transport, and biotic cycling. Here we present a mathematical model that describes the terrestrial P-cycle in a simple but comprehensive way. The resulting dynamical system can be solved analytically for steady-state conditions, allowing us to test the sensitivity of the P-availability to the key parameters and processes. Given constant inputs, we find that humid ecosystems exhibit lower P availability due to higher runoff and losses, and that tectonic uplift is a fundamental constraint. In particular, we find that in humid ecosystems the biotic cycling seem essential to maintain long-term P-availability. The time-dependent P dynamics for the Franz Josef and Hawaii chronosequences show how tectonic uplift is an important constraint on ecosystem productivity, while hydroclimatic conditions control the P-losses and speed towards steady-state. The model also helps describe how, with limited uplift and atmospheric input, as in the case of the Amazon Basin, ecosystems must rely on mechanisms that enhance P-availability and retention. Our novel model has a limited number of parameters and can be easily integrated into global climate models to provide a representation of the response of the terrestrial biosphere to global change. © 2010 Author(s).

Eco-evolutionary feedbacks in community and ecosystem ecology: interactions between the ecological theatre and the evolutionary play.

Interactions between natural selection and environmental change are well recognized and sit at the core of ecology and evolutionary biology. Reciprocal interactions between ecology and evolution, eco-evolutionary feedbacks, are less well studied, even though they may be critical for understanding the evolution of biological diversity, the structure of communities and the function of ecosystems. Eco-evolutionary feedbacks require that populations alter their environment (niche construction) and that those changes in the environment feed back to influence the subsequent evolution of the population. There is strong evidence that organisms influence their environment through predation, nutrient excretion and habitat modification, and that populations evolve in response to changes in their environment at time-scales congruent with ecological change (contemporary evolution). Here, we outline how the niche construction and contemporary evolution interact to alter the direction of evolution and the structure and function of communities and ecosystems. We then present five empirical systems that highlight important characteristics of eco-evolutionary feedbacks: rotifer-algae chemostats; alewife-zooplankton interactions in lakes; guppy life-history evolution and nutrient cycling in streams; avian seed predators and plants; and tree leaf chemistry and soil processes. The alewife-zooplankton system provides the most complete evidence for eco-evolutionary feedbacks, but other systems highlight the potential for eco-evolutionary feedbacks in a wide variety of natural systems.

The toxicology of climate change: environmental contaminants in a warming world.

Climate change induced by anthropogenic warming of the earth's atmosphere is a daunting problem. This review examines one of the consequences of climate change that has only recently attracted attention: namely, the effects of climate change on the environmental distribution and toxicity of chemical pollutants. A review was undertaken of the scientific literature (original research articles, reviews, government and intergovernmental reports) focusing on the interactions of toxicants with the environmental parameters, temperature, precipitation, and salinity, as altered by climate change. Three broad classes of chemical toxicants of global significance were the focus: air pollutants, persistent organic pollutants (POPs), including some organochlorine pesticides, and other classes of pesticides. Generally, increases in temperature will enhance the toxicity of contaminants and increase concentrations of tropospheric ozone regionally, but will also likely increase rates of chemical degradation. While further research is needed, climate change coupled with air pollutant exposures may have potentially serious adverse consequences for human health in urban and polluted regions. Climate change producing alterations in: food webs, lipid dynamics, ice and snow melt, and organic carbon cycling could result in increased POP levels in water, soil, and biota. There is also compelling evidence that increasing temperatures could be deleterious to pollutant-exposed wildlife. For example, elevated water temperatures may alter the biotransformation of contaminants to more bioactive metabolites and impair homeostasis. The complex interactions between climate change and pollutants may be particularly problematic for species living at the edge of their physiological tolerance range where acclimation capacity may be limited. In addition to temperature increases, regional precipitation patterns are projected to be altered with climate change. Regions subject to decreases in precipitation may experience enhanced volatilization of POPs and pesticides to the atmosphere. Reduced precipitation will also increase air pollution in urbanized regions resulting in negative health effects, which may be exacerbated by temperature increases. Regions subject to increased precipitation will have lower levels of air pollution, but will likely experience enhanced surface deposition of airborne POPs and increased run-off of pesticides. Moreover, increases in the intensity and frequency of storm events linked to climate change could lead to more severe episodes of chemical contamination of water bodies and surrounding watersheds. Changes in salinity may affect aquatic organisms as an independent stressor as well as by altering the bioavailability and in some instances increasing the toxicity of chemicals. A paramount issue will be to identify species and populations especially vulnerable to climate-pollutant interactions, in the context of the many other physical, chemical, and biological stressors that will be altered with climate change. Moreover, it will be important to predict tipping points that might trigger or accelerate synergistic interactions between climate change and contaminant exposures.

Four-decade responses of soil trace elements to an aggrading old-field forest: B, Mn, Zn, Cu, and Fe.

In the ancient and acidic Ultisol soils of the Southern Piedmont, USA, we studied changes in trace element biogeochemistry over four decades, a period during which formerly cultivated cotton fields were planted with pine seedlings that grew into mature forest stands. In 16 permanent plots, we estimated 40-year accumulations of trace elements in forest biomass and O horizons (between 1957 and 1997), and changes in bioavailable soil fractions indexed by extractions of 0.05 mol/L HCl and 0.2 mol/L acid ammonium oxalate (AAO). Element accumulations in 40-year tree biomass plus O horizons totaled 0.9, 2.9, 4.8, 49.6, and 501.3 kg/ha for Cu, B, Zn, Mn, and Fe, respectively. In response to this forest development, samples of the upper 0.6-m of mineral soil archived in 1962 and 1997 followed one of three patterns. (1) Extractable B and Mn were significantly depleted, by -4.1 and -57.7 kg/ha with AAO, depletions comparable to accumulations in biomass plus O horizons, 2.9 and 49.6 kg/ha, respectively. Tree uptake of B and Mn from mineral soil greatly outpaced resupplies from atmospheric deposition, mineral weathering, and deep-root uptake. (2) Extractable Zn and Cu changed little during forest growth, indicating that nutrient resupplies kept pace with accumulations by the aggrading forest. (3) Oxalate-extractable Fe increased substantially during forest growth, by 275.8 kg/ha, about 10-fold more than accumulations in tree biomass (28.7 kg/ha). The large increases in AAO-extractable Fe in surficial 0.35-m mineral soils were accompanied by substantial accretions of Fe in the forest's O horizon, by 473 kg/ha, amounts that dwarfed inputs via litterfall and canopy throughfall, indicating that forest Fe cycling is qualitatively different from that of other macro- and micronutrients. Bioturbation of surficial forest soil layers cannot account for these fractions and transformations of Fe, and we hypothesize that the secondary forest's large inputs of organic additions over four decades has fundamentally altered soil Fe oxides, potentially altering the bioavailability and retention of macro- and micronutrients, contaminants, and organic matter itself. The wide range of responses among the ecosystem's trace elements illustrates the great dynamics of the soil system over time scales of decades.

Sexually coercive male chimpanzees sire more offspring.

In sexually reproducing animals, male and female reproductive strategies often conflict. In some species, males use aggression to overcome female choice, but debate persists over the extent to which this strategy is successful. Previous studies of male aggression toward females among wild chimpanzees have yielded contradictory results about the relationship between aggression and mating behavior. Critically, however, copulation frequency in primates is not always predictive of reproductive success. We analyzed a 17-year sample of behavioral and genetic data from the Kasekela chimpanzee (Pan troglodytes schweinfurthii) community in Gombe National Park, Tanzania, to test the hypothesis that male aggression toward females increases male reproductive success. We examined the effect of male aggression toward females during ovarian cycling, including periods when the females were sexually receptive (swollen) and periods when they were not. We found that, after controlling for confounding factors, male aggression during a female's swollen periods was positively correlated with copulation frequency. However, aggression toward swollen females was not predictive of paternity. Instead, aggression by high-ranking males toward females during their nonswollen periods was positively associated with likelihood of paternity. This indicates that long-term patterns of intimidation allow high-ranking males to increase their reproductive success, supporting the sexual coercion hypothesis. To our knowledge, this is the first study to present genetic evidence of sexual coercion as an adaptive strategy in a social mammal.

The body language: The spontaneous influence of congruent bodily arousal on the awareness of emotional words

Nowadays, the idea of a reciprocal influence of physiological and psychological processes seems to be widely accepted. For instance, current theories of embodied emotion suggest that knowledge about an emotion concept involves simulations of bodily experienced emotional states relevant to the concept. In line with this framework, the present study investigated whether actual levels of physiological arousal interact with the processing of emotional words. Participants performed 2 blocks of an attentional blink task, once after a cycling session (increased arousal) and once after a relaxation session (reduced arousal). Concretely, participants were instructed to detect and report 2 target words (T1 and T2) presented among a series of nonword distractors. T1 and T2 were either neutral, high arousal, or low arousal words. Results revealed that increased physiological arousal led to improved reports of high arousal T2 words, whereas reduced physiological arousal led to improved reports of low arousal T2 words. Neutral T2 remained unaffected by the arousing conditions. These findings emphasize that actual levels of physiological arousal modulate the cognitive access to arousal (in-)congruent emotional concepts and suggest a direct grounding of emotion knowledge in our bodily systems of arousal.

Characterization of autonomous thyroid adenoma: metabolism, gene expression, and pathology.

Fifty-one in vivo characterized autonomous single adenomas have been studied for functional parameters in vitro, for gene and protein expression and for pathology, and have been systematically compared to the corresponding extratumoral quiescent tissue. The adenomas were characterized by a high level of iodide trapping that corresponds to a high level of Na+ /iodide symporter gene expression, a high thyroperoxidase mRNA and protein content, and a low H2O2 generation. This explains the iodide metabolism characteristics demonstrated before, ie, the main cause of the "hot" character of the adenomas is their increased iodide transport. The adenomas spontaneously secreted higher amounts of thyroid hormone than the quiescent tissue and in agreement with previous in vivo data, this secretion could be further enhanced by thyrotropin (TSH). Inositol uptake was also increased but there was no spontaneous increase of the generation of inositol phosphates and this metabolism could be further activated by TSH. These positive responses to TSH are in agreement with the properties of TSH-stimulated thyroid cells in vitro and in vivo. They are compatible with the characteristics of mutated TSH receptors whose constitutive activation accounts for the majority of autonomous thyroid adenomas in Europe. The number of cycling cells, as evaluated by MIB-1 immunolabeling was low but increased in comparison with the corresponding quiescent tissue or normal tissue. The cycling cells are observed mainly at the periphery; there was very little apoptosis. Both findings account for the slow growth of these established adenomas. On the other hand, by thyroperoxidase immunohistochemistry, the whole lesion appeared hyperfunctional, which demonstrates a dissociation of mitogenic and functional stimulations. Thyroglobulin, TSH receptor, and E-cadherin mRNA accumulations were not modified in a consistent way, which confirms the near-constitutive expression of the corresponding genes in normal differentiated tissue. On the contrary, early immediate genes expressions (c-myc, NGF1B, egr 1, genes of the fos and jun families) were decreased. This may be explained by the proliferative heterogeneity of the lesion and the previously described short, biphasic expression of these genes when induced by mitogenic agents. All the characteristics of the autonomous adenomas can therefore be explained by the effect of the known activating mutations of genes coding for proteins of the TSH cyclic adenosine monophosphate (cAMP) cascade, all cells being functionally activated while only those at the periphery multiply. The reason of this heterogeneity is unknown.

Characteristics of the biphasic action of androgens and of the potent antiproliferative effects of the new pure antiestrogen EM-139 on cell cycle kinetic parameters in LNCaP human prostatic cancer cells

The most potent steroid in human prostatic carcinoma LNCaP cells, i.e. dihydrotestosterone (DHT), has a biphasic stimulatory effect on cell proliferation. At the maximal stimulatory concentration of 0.1 nM DHT, analysis of cell kinetic parameters shows a decrease of the G0-G1 fraction with a corresponding increase of the S and G2 + M fractions. In contrast, concentrations of 1 nM DHT or higher induce a return of cell proliferation to control levels, reflected by an increase in the G0-G1 fraction at the expense of the S and especially the G2 + M fractions. Continuous labeling for 144 h with the nucleotide analogue 5'-bromodeoxyuridine shows that the percentage of cycling LNCaP cells rises more than 90% after treatment with stimulatory concentrations of DHT, whereas in control cells as well as in cells treated with high concentrations of the androgen, this value remains below 50%. Although LNCaP cells do not contain detectable estrogen receptors, the new pure steroidal antiestrogen EM-139 not only reversed the stimulation of cell proliferation and cell kinetics induced by stimulatory doses of DHT but also inhibited basal cell proliferation.

Optimization and finite element analysis for reliable electronic packaging

This paper demonstrates a modeling and design approach that couples computational mechanics techniques with numerical optimisation and statistical models for virtual prototyping and testing in different application areas concerning reliability of eletronic packages. The integrated software modules provide a design engineer in the electronic manufacturing sector with fast design and process solutions by optimizing key parameters and taking into account complexity of certain operational conditions. The integrated modeling framework is obtained by coupling the multi-phsyics finite element framework - PHYSICA - with the numerical optimisation tool - VisualDOC into a fully automated design tool for solutions of electronic packaging problems. Response Surface Modeling Methodolgy and Design of Experiments statistical tools plus numerical optimisaiton techniques are demonstrated as a part of the modeling framework. Two different problems are discussed and solved using the integrated numerical FEM-Optimisation tool. First, an example of thermal management of an electronic package on a board is illustrated. Location of the device is optimized to ensure reduced junction temperature and stress in the die subject to certain cooling air profile and other heat dissipating active components. In the second example thermo-mechanical simulations of solder creep deformations are presented to predict flip-chip reliability and subsequently used to optimise the life-time of solder interconnects under thermal cycling.

Optimisation modelling for flip-chip solder joint reliability

This paper details and demonstrates integrated optimisation-reliability modelling for predicting the performance of solder joints in electronic packaging. This integrated modelling approach is used to identify efficiently and quickly the most suitable design parameters for solder joint performance during thermal cycling and is demonstrated on flip-chip components using “no-flow” underfills. To implement “optimisation in reliability” approach, the finite element simulation tool – PHYSICA, is coupled with optimisation and statistical tools. This resulting framework is capable of performing design optimisation procedures in an entirely automated and systematic manner.