Investigating the Effect of Histone H4 Sumoylation on Chromatin Structure and Function

Thesis (Ph.D.)--University of Washington, 2016-06

Application of central composite rotatable design to modelling the effect of some operating variables on the performance of the three-product cyclone

The central composite rotatable design (CCRD) was used to design an experimental program to model the effects of inlet pressure, feed density, and length and diameter of the inner vortex finder on the operational performance of a 150-min three-product cyclone. The ranges of values of the variables used in the design were: inlet pressure: 80-130 kPa, feed density: 30 60%; length of IVF below the OVF: 50-585 mm; diameter of IVF: 35-50 mm. A total of 30 tests were conducted, which is 51 less; an that required for a three-level full factorial design. Because the model allows confident performance prediction by interpolation over the range of data in the database, it was used to construct response surface graphs to describe the effects of the variables on the performance of the three-product cyclone. To obtain a simple and yet a realistic model, it was refitted using only the variable terms that are significant at greater than or equal to 90% confidence level. Considering the selected operating variables, the resultant model is significant and predicts the experimental data well. (c) 2005 Elsevier B.V. All rights reserved.

The effect of organic mulch amendments on the physical and chemical properties and revegetation success of a saline-sodic minespoil from central Queensland, Australia

Saline-sodic clay minespoil materials excavated during open-cut coal mining in central Queensland, Australia, pose significant challenges for revegetation, particularly where suitable topsoil capping is not available. We examined the ability of sawdust or straw mulch amendments to ameliorate the adverse properties of these minespoils and improve the success of revegetation efforts. In laboratory studies, mulch application improved infiltration, increased soil moisture retention and reduced surface crust strength. In the field, mulches incorporated to a depth of 0.15 m at application rates of at least 20 t/ha straw or 80 t/ha sawdust were needed to mitigate against capillary rise of salts during drying cycles and support satisfactory vegetation cover. Further research is needed to determine whether improvements are maintained beyond the 4-year trial period reported here.

Effect of fluoromica platelet size on the properties of segmented polyurethane nanocomposites

Segmented polyurethane nanocomposites containing three different size fractions of SomasifTM ME100 (synthetic fluoromica) have been prepared via solvent casting. The platelet size was adjusted via a proprietary milling process, and average diameters of approximately 500 nm, 100 nm and 30 nm were measured via TEM. To the best of our knowledge this is the first time the effect of aspect ratio has been studied with the same t-o-t structured mineral. The mechanical properties of these nanocomposites have been found to be highly dependent upon the platelet size. Depending on the aspect ratio and surface treatment selected, significant improvements in tensile strength can be achieved with a minimal reduction in resilience: a problem encountered with elastomeric layered silicate nanocomposites.

Effect of polymorph derived oxidants on IgG in relation to rheumatoid factor binding

Immunoglobulin G from rheumatoid patients is denatured around the hinge region. This has been proposed as an explanation for the presence of circulating autoantibodies to IgG in these patients. It has previously been suggested that oxygen radicals (OR) derived from activated polymorphs may play a role in denaturation in vivo. Using sera from rheumatoid patients and age-matched controls in a modified ELISA technique, we have investigated the potential for polyclonal rheumatoid factors (RF) to bind to OR denatured IgG. Three model systems were used to generate OR in vitro: (a) purified PMN s activated by the cell surface stimulant PMA, (b) radiolysis of IgG in solution to generate specifically the superoxide radical and, in a separate system, the hydroxyl radical, (OH.), (c) purified myeloperoxide in the presence of H2O2 and halide ions. Results: 1. The binding of both IgA and IgM RF s to PMN denatured IgG increased dose dependently for seropositive sera only. 2. The OH. radical but not the superoxide radical significantly increased the binding of IgA and M RF, again only for seropositive sera. 3. The myeloperoxidase enzyme system did not increase RF binding. 4. IgG incubated with elastase was not found to be a better antigen than native IgG. These results indicate that IgG is denatured by OR released from activated PMN, thereby producing an antigen for polyclonal RF s.

Effect of a liposomal spray on the pre-ocular tear film

Purpose: With the potential to address evaporative dry eye, a novel spray has been developed in which phospholipid liposomes are delivered to the tear film via the surface of the closed eyelid. This study evaluated the short-term effects of liposomal spray application on the lipid and stability characteristics of the pre-ocular tear film in normal eyes. Methods: Twenty-two subjects (12M, 10F) aged 35.1 ± 7.1 years participated in this prospective, randomised, double-masked investigation in which the liposomal spray was applied to one eye, and an equal volume of saline spray (control) applied to the contralateral eye. Lipid layer grade (LLG), non-invasive tear film stability (NIBUT) and tear meniscus height (TMH) were evaluated at baseline, and at 30, 60, 90 and 135 minutes post-application. Subjective reports of comfort were also compared. Results: Treated and control eyes were not significantly different at baseline (p>0.05). Post-application, LLG increased significantly, at 30 and 60 minutes, only in the treated eyes (p=0.005). NIBUT also increased significantly in the treated eyes only (p<0.001), at 30, 60 and 90 minutes. TMH did not alter significantly (p>0.05). Comfort improved relative to baseline in 46% of treated and 18% of control eyes, respectively, at 30 minutes post-application. Of those expressing a preference in comfort between the eyes, 68% preferred the liposomal spray. Conclusions: Consistent with subjective reports of improved comfort, statistically and clinically significant improvements in lipid layer thickness and tear film stability are observed in normal eyes for at least an hour after a single application of a phospholipid liposomal spray.

The postantibiotic effect of the carbapenem antibiotics on gram-negative bacteria

Postantibiotic effect (PAE) describes the suppression of microbial growth occurring after a short exposure to an antimicrobial agent. PAE appears to be a property of the majority of antimicrobial agents and is demonstrated by a wide variety of microorganisms. At present, carbapenems and penems are the only members of the -lactam group of antimicrobial agents that exhibit a significant PAE on Gram-negative bacilli. A standardised method was developed to evaluate the in vitro PAE of three carbapenems; imipenem, meropenem and biapenem on Gram-negative bacteria under reproducible laboratory conditions that partially mimicked those occurring in vivo. The effects on carbapenem PAE of the method of antimicrobial removal, concentration, exposure duration, inoculum size, inoculum growth phase, multiple exposures and pooled human serum were determined. Additionally, the reproducibility, susceptibility prior to and after PAE determination and inter-strain variation of carbapenem PAE were evaluated. The method developed determined PAE by utilising viable counts and demonstrated carbapenem PAE to be reproducible, constant over successive exposures, dependent on genera, concentration, duration of exposure, inoculum size and growth phase. In addition, carbapenem PAE was not significantly effected either by agitation, the antimicrobial removal method or the viable count diluent. At present, the mechanism underlying PAE is undetermined. It is thought to be due to either the prolonged persistence of the antimicrobial at the cellular site of action or the true recovery period from non-lethal damage. Increasing the L-lysine concentration and salinity at recovery decreased and increased the carbapenem and imipenem PAE of Pseudomonas aeruginosa, respectively. In addition, no apparent change was observed in the production of virulence factors by P.aeruginosa in PAE phase. However, alterations in cell morphology were observed throughout PAE phase, and the reappearance of normal cell morphology corresponded to the duration of PAE determined by viable count. Thus, the recovery of the penicillin binding protein target enzymes appears to be the mechanism behind carbapenem PAE in P. aeruginosa.

The effect of R-plasmid RP1 on the properties of Proteus mirabilis

A clinical isolate of Proteus mirabilis containing R-plasmid RP1 (R+ cells), grown in both iron- and carbon- limited chemically defined media in mixed culture with plasmid-free (R- cells), did not disappear as expected, due to adherence of R+ cells to the wall of the chemostat vessel. Plasmid RP1 promoted adherence to glass and to medical prostheses. The hydrophobicity and surface charge of R+ cells were different from those of R- cells and both factors may contribute to the adherence of R+ cells to surfaces. The mode of cultivation of the cells, whether batch or continuous culture, were also found to affect the result. Antibodies raised against homologous cells increased the surface hydrophobicity of both R+ and R- cells and eliminated the differences between them. Results for surface hydrophobicity varied with the method used for measuring it. R+ cells were more sensitive than R- cells to tbe bacteridical action of normal serum and whole blood and to phagocytosis as measured by chemiluminescence. No clear differences were revealed in the protein antigens of R+ and R- cells by both SDS PAGE gels and immunoblots reacted with homologous antibodies. However, lectins revealed differences in the sugars exposed on the cell surfaces. Chemical analysis of R&43 and R- cells also revealed differences in the content of 2-keto-3-deoxy-D-manno-2-octulosonate, lipopolysaccharide and total fatty acids, when cells were grown in media containing added iron; however, no qualitative differences in the lipopolysaccharide were found. Removal of iron from the medium was found to have considerable effects on the chemical structure of R+ cells but not of R- ones. Adhesion to prostheses and to leucocytes is discussed in the light of the results and the clinical relevance outlined with respect to the initiation of infection and the association of virulence with antibiotic resistance.

The effect of iron oxide nanoparticles on the fate and transformation of arsenic in aquatic environments

Iron oxides and arsenic are prevalent in the environment. With the increase interest in the use of iron oxide nanoparticles (IONPs) for contaminant remediation and the high toxicity of arsenic, it is crucial that we evaluate the interactions between IONPs and arsenic. The goal was to understand the environmental behavior of IONPs in regards to their particle size, aggregation and stability, and to determine how this behavior influences IONPs-arsenic interactions. ^ A variety of dispersion techniques were investigated to disperse bare commercial IONPs. Vortex was able to disperse commercial hematite nanoparticles into unstable dispersions with particles in the micrometer size range while probe ultrasonication dispersed the particles into stable dispersions of nanometer size ranges for a prolonged period of time. Using probe ultrasonication and vortex to prepare IONPs suspensions of different particle sizes, the adsorption of arsenite and arsenate to bare hematite nanoparticles and hematite aggregates were investigated. To understand the difference in the adsorptive behavior, adsorption kinetics and isotherm parameters were determined. Both arsenite and arsenate were capable of adsorbing to hematite nanoparticles and hematite aggregates but the rate and capacity of adsorption is dependent upon the hematite particle size, the stability of the dispersion and the type of sorbed arsenic species. Once arsenic was adsorbed onto the hematite surface, both iron and arsenic can undergo redox transformation both microbially and photochemically and these processes can be intertwined. Arsenic speciation studies in the presence of hematite particles were performed and the effect of light on the redox process was preliminary quantified. The redox behavior of arsenite and arsenate were different depending on the hematite particle size, the stability of the suspension and the presence of environmental factors such as microbes and light. The results from this study are important and have significant environmental implications as arsenic mobility and bioavailability can be affected by its adsorption to hematite particles and by its surface mediated redox transformation. Moreover, this study furthers our understanding on how the particle size influences the interactions between IONPs and arsenic thereby clarifying the role of IONPs in the biogeochemical cycling of arsenic.^

Effect of enhanced pCO2 levels on the production of dissolved organic carbon and transparent exopolymer particles in short-term bioassay experiments

It has been proposed that increasing levels of pCO2 in the surface ocean will lead to more partitioning of the organic carbon fixed by marine primary production into the dissolved rather than the particulate fraction. This process may result in enhanced accumulation of dissolved organic carbon (DOC) in the surface ocean and/or concurrent accumulation of transparent exopolymer particles (TEPs), with important implications for the functioning of the marine carbon cycle. We investigated this in shipboard bioassay experiments that considered the effect of four different pCO2 scenarios (ambient, 550, 750 and 1000 µatm) on unamended natural phytoplankton communities from a range of locations in the northwest European shelf seas. The environmental settings, in terms of nutrient availability, phytoplankton community structure and growth conditions, varied considerably between locations. We did not observe any strong or consistent effect of pCO2 on DOC production. There was a significant but highly variable effect of pCO2 on the production of TEPs. In three of the five experiments, variation of TEP production between pCO2 treatments was caused by the effect of pCO2 on phytoplankton growth rather than a direct effect on TEP production. In one of the five experiments, there was evidence of enhanced TEP production at high pCO2 (twice as much production over the 96 h incubation period in the 750 ?atm treatment compared with the ambient treatment) independent of indirect effects, as hypothesised by previous studies. Our results suggest that the environmental setting of experiments (community structure, nutrient availability and occurrence of phytoplankton growth) is a key factor determining the TEP response to pCO2 perturbations.

Effect of the processing conditions on the microstructure and properties of rotational molded polyamide 11

Rotomolded containers for solvents and hydrocarbons require the use of high-permeability resins such as polyamide (PA). The published studies with this material are very scarce. In this work, a commercial grade of PA11 was rotational-molded using different processing temperatures and characterized with a range of techniques. The study aims at investigating the influence of the processing conditions on the microstructure and properties of molded parts. The results showed that the spherulitic morphology and the mechanical properties are affected by the processing temperature, the optimum processing range being between 220°C and 240°C. Overheating causes a decrease of the impact strength and a severe increase in the formation of pinholes at the outer surface due to polymer degradation and formation of volatile products. The thermo-oxidation reactions occurring at the inner surface of the samples result in the formation of products that absorb in the UV and visible light regions and cause the microhardness and the melt viscosity of the material to increase. The extent and severity of the degradation at the inner surface may be easily assessed by fluorescence microscopy. © 2008 Wiley Periodicals, Inc.

The effect of ionic Co presence on the structural, optical and photocatalytic properties of modified cobalt–titanate nanotubes

With the aim of producing materials with enhanced optical and photocatalytic properties, titanate nanotubes (TNTs) modified by cobalt doping (Co-TNT) and by Na+ -> Co ion-exchange (TNT/Co) were successfully prepared by a hydrothermal method. The influence of the doping level and of the cobalt position in the TNT crystalline structure was studied. Although no perceptible influence of the cobalt ion position on the morphology of the prepared titanate nanotubes was observed, the optical behaviour of the cobalt modified samples is clearly dependent on the cobalt ions either substituting the Ti4+ ions in the TiO6 octahedra building blocks of the TNT structure (doped samples) or replacing the Na+ ions between the TiO6 interlayers (ion-exchange samples). The catalytic ability of these materials on pollutant photodegradation was investigated. First, the evaluation of hydroxyl radical formation using the terephthalic acid as a probe was performed. Afterwards, phenol, naphthol yellow S and brilliant green were used as model pollutants. Anticipating real world situations, photocatalytic experiments were performed using solutions combining these pollutants. The results show that the Co modified TNT materials (Co-TNT and TNT/Co) are good catalysts, the photocatalytic performance being dependent on the Co/Ti ratio and on the structural metal location. The Co(1%)-TNT doped sample was the best photocatalyst for all the degradation processes studied.

Concussion: Examining the Effect of Neuronal Oxidative Stress on the Pathophysiology of Brain and Blood Brain Barrier Cells

Neuronal stretching during concussion alters glucose transport and reduces neuronal viability, also affecting other cells in the brain and the Blood Brain Barrier (BBB). Our hypothesis is that oxidative stress (OS) generated in neurons during concussions contributes to this outcome. To validate this, we investigated: (1) whether OS independently causes alterations in brain and BBB cells, namely human neuron-like, neuroblastoma cells (NCs), astrocyte cells (ACs) and brain microvascular endothelial cells (ECs), and (2) whether OS originated in NCs (as in concussion) is responsible for causing the subsequent alterations observed in ACs and ECs. We used H2O2 treatment to mimic OS, validated by examining the resulting reactive oxygen species, and evaluated alterations in cell morphology, expression and localization of the glucose transporter GLUT1, and the overall cell viability. Our results showed that OS, either directly affecting each cell type or originally affecting NCs, caused changes in several morphological parameters (surface area, Feret diameter, circularity, inter-cellular distance), slightly varied GLUT1 expression and lowered the overall cell viability of all NCs, ACs, and ECs. Therefore, we can conclude that oxidative stress, which is known to be generated during concussion, caused alterations in NCs, ACs, and ECs whether independently originated in each cell or when originated in the NCs and could further propagate the ACs and ECs.

Effect of vitamin D supplementation on bone status, glucose homeostasis and immune function in children with vitamin D deficiency

Background: Between 1961-1971 vitamin D deficiency was recognized as a public health issue in the UK, because of the lack of effective sunlight and the population mix [1, 2]. In recent years, health care professionals have cited evidence suggesting a re-emergence of the vitamin D deficiency linked to a number of health consequences as a concern [3-6]. Evidence from observational studies has linked low vitamin D status with impairment in glucose homeostasis and immune dysfunction [7-9]. However, interventional studies, particularly those focused on paediatric populations, have been limited and inconsistent. There is a need for detailed studies, to clarify the therapeutic benefits of vitamin D in these important clinical areas. Objective: The aims of this PhD thesis were two-fold. Firstly, to perform preliminary work assessing the association between vitamin D deficiency and bone status, glucose homeostasis and immune function, and to explore any changes in these parameters following short term vitamin D3 replacement therapy. Secondly, to assess the effectiveness of an electronic surveillance system (ScotPSU) as a tool to determine the current incidence of hospital-based presentation of childhood vitamin D deficiency in Scotland. Methods: Active surveillance was performed for a period of two years as a part of an electronic web-based surveillance programme performed by the Scottish Paediatric Surveillance Unit (ScotPSU). The validity of the system was assessed by identifying cases with profound vitamin D deficiency (in Glasgow and Edinburgh) from the regional laboratory. All clinical details were checked against those identified using the surveillance system. Thirty-seven children aged 3 months to 10 years, who had been diagnosed with vitamin D deficiency, were recruited for the bone, glucose and immunity studies over a period of 24 months. Twenty-five samples were analysed for the glucose and bone studies; of these, 18 samples were further analysed for immune study. Treatment consisted of six weeks taking 5000 IU units cholecalciferol orally once a day. At baseline and after completion of treatment, 25 hydroxyvitamin D (25(OH)D), parathyroid hormone (PTH), alkaline phosphatase (ALP), collagen type 1 cross-linked C-telopeptide (CTX), osteocalcin (OCN), calcium, phosphate, insulin, glucose, homeostasis model assessment index, estimated insulin resistance (HOMA IR), glycated hemoglobin (HbA1c), sex hormone binding globulin (SHBG), lipids profiles, T helper 1 (Th1) cytokines (interleukin-2 ( IL-2), tumor necrosis factors-alpha (TNF-α), interferon-gamma (INF-γ)), T helper 2 (Th2) cytokines (interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6)), T helper 17 (Th17) cytokine (interleukin-17 (IL-17)), Regulatory T (Treg) cytokine (interleukin-10 (IL-10)) and chemokines/cytokines, linked with Th1/Th2 subset balance and/or differentiation (interleukin-8 (IL-8), interleukin-12 (IL-12), eosinophil chemotactic protein ( EOTAXIN), macrophage inflammatory proteins-1beta (MIP-1β), interferon-gamma-induced protein-10 (IP-10), regulated on activation, normal T cell expressed and secreted (RANTES), monocyte chemoattractant protein-1(MCP-1)) were measured. Leukoocyte subset analysis was performed for T cells, B cells and T regulatory cells and a luminex assay was used to measure the cytokiens. Results: Between September 2009 and August 2011, 163 cases of vitamin D deficiency were brought to the attention of the ScotPSU, and the majority of cases (n = 82) were reported in Glasgow. The cross-validation checking in Glasgow and Edinburgh over a one-year period revealed only 3 (11%) cases of clearly symptomatic vitamin D deficiency, which had been missed by the ScotPSU survey in Glasgow. While 16 (67%) symptomatic cases had failed to be reported through the ScotPSU survey in Edinburgh. For the 23 children who are included in bone and glucose studies, 22 (96%) children had basal serum 25(OH)D in the deficiency range (< 50 nmol/l) and one (4%) child had serum 25(OH)D in the insufficiency range (51-75 nmol/l). Following vitamin D3 treatment, 2 (9%) children had final serum 25(OH)D lower than 50 nmol/l, 6 (26%) children had final serum 25(OH)D between >50-75 nmol/l, 12 (52%) children reached a final serum 25(OH)D >75-150 nmol/l and finally 3 (13%) exceeded the normal reference range with a final 25(OH)D >150 nmol/l. Markers for remodelling ALP and PTH had significantly decreased (p = 0.001 and <0.0001 for ALP and PTH respectively). In 17 patients for whom insulin and HOMA IR data were available and enrolled in glucose study, significant improvements in insulin resistance (p = 0.04) with a trend toward a reduction in serum insulin (p = 0.05) was observed. Of those 14 children who had their cytokines profile data analysed and enrolled in the immunity study, insulin and HOMA IR data were missed in one child. A significant increase in the main Th2 secreted cytokine IL-4 (p = 0.001) and a tendency for significant increases in other Th2 secreted cytokines IL-5 (p = 0.05) and IL-6 (p = 0.05) was observed following vitamin D3 supplementation. Conclusion: An electronic surveillance system can provide data for studying the epidemiology of vitamin D deficiency. However, it may underestimate the number of positive cases. Improving vitamin D status in vitamin D deficient otherwise healthy children significantly improved their vitamin D deficient status, and was associated with an improvement in bone profile, improvements in insulin resistance and an alteration in main Th2 secreting cytokines.

Effect of Melatonin and Analogues on Corneal Wound Healing: Involvement of Mt2 Melatonin Receptor

Purpose: We have investigated the effect of melatonin and its analogues on rabbit corneal epithelial wound healing. Methods: New Zealand rabbits were anaesthetised and wounds were made by placing Whatman paper discs soaked in n-heptanol on the cornea. Melatonin and analogues (all 10 nmol) were instilled. Wound diameter was measured every 2 hours by means of fluorescein application with a Topcon SL-8Z slit lamp. Melatonin antagonists (all 10 nmol) were applied 2 hours before the application of the n-heptanol-soaked disc and then every 6 hours together with melatonin. To confirm the presence of MT2 receptors in corneal epithelial cells immunohistochemistry, Western blot and RT-PCR assays in native tissue and in rabbit corneal epithelial cells were performed. The tear components were extracted then processed by HPLC to quantify melatonin in tears. Results: Migration assays revealed that melatonin and particularly the treatment with the MT2 agonist IIK7, accelerated the rate of healing (p < 0.001). The application of the non-selective melatonin receptor antagonist luzindole and the MT2 antagonist DH97 (but not prazosin), prevented the effect of melatonin on wound healing (both p < 0.001). Immunohistochemistry, Western blot and RT-PCR assays showed the presence of MT2 melatonin receptor in corneal epithelial cells. In addition, we have identified melatonin in tears and determined its daily variations. Conclusions: These data suggest that MT2 receptors are implicated in the effect of melatonin on corneal wound healing regulating migration rate. This suggests the potential use of melatonin and its analogues to enhance epithelial wound healing in ocular surface disease.