Land application of large, volume-based rates of municipal biosolids (MB) enhances soil physical properties and provides an alternative to disposal in landfills. Yet, topdressing or incorporation of the volume-based rates can increase non-point source losses of sediment and nutrients from excavated soils to surface waters. Research objectives were developed to evaluate the options for cycling of MB through turfgrass sod during production and after transplanting. The first objective was to compare the production of Tifway bermudagrass sod between fields grown with and without MB under increasing rates of supplemental fertilizer N. The second objective was to compare runoff losses between soils constructed with and without MB before planting to sprigs or sod transplanted from turfgrass grown in soil with and without incorporation of MB. Incorporation of 25% by volume of MB in soil enhanced (p < 0.001) turfgrass coverage of the soil surface compared to soil without MB. In addition, amending soil with MB reduced wet and dry sod weights (p < 0.001) and increased soil water content (p < 0.001) at harvest compared to sod without MB. Runoff concentrations and mass loss of total dissolved P (TDP) were significantly greater (P=0.001) for MB-amended compared to un-amended sod. In addition, a linear relationship (R2 = 0.94) was observed between water extractable soil P within the 0- to 2-cm depth and concentrations and mass loss of TDP in runoff. Similarly, runoff loss of NO3-N was greater (P = 0.05) for soil mixed with 25% by volume of MB than soil alone and variation of NO3-N loss among treatments was directly related to soil NO3-N concentration within the 0- to 5-cm depth. In contrast, runoff concentrations of NH4-N were directly related to inputs of N from turf clippings returned to soil rather than soil NH4-N concentrations. Total Kjeldahl N (TKN) concentration in runoff was unrelated to soil N concentrations, but was linearly related to mass loss of sediment in runoff. Transplanted sod reduced sediment loss compared to sprigged soil during turfgrass establishment and MB-amended soil reduced sediment loss compared to soil without MB. In addition, the MB imported in sod or incorporated in soil before sprigging increased soil organic carbon and mean soil water content compared to sod or soil without MB over a 92 day period. Incorporation of MB within soil prior to planting fertilizer grown turfgrass sod enhanced water conservation and reduced nutrient loss compared to planting MB-grown sod on un-amended soils.

Sediment Oxygen Demand (SOD) is the fluctuation of dissolved oxygen between the sediment from the overlying water. The method to acquire SOD values is inefficient and troubled by unreliable equipment. Diffusion gradients in thin film (DGT) are proposed as a potential method to collect geochemical proxy measures that can be used as SOD predictors. Field deployment of the DGTs was conducted at two locations to compare recovery and accuracy against ex-situ centrifuge methods. The results indicated DGT can be used as a statistically significant geochemical method. A principle component analysis was used to determine if reduced iron and manganese collected using DGTs clustered with SOD. Results indicated reduced iron and manganese cannot be used to predict SOD. Sulfide measurement by microelectrode from the same matrix of geochemical methods however did cluster with SOD. A stepwise multiple linear regression concluded sulfide measurement by microelectrode is a statistically significant predictor of SOD.

"Ependymin (EPN) is a goldfish neurotrophic factor (NTF) that is one of the most abundant secreted glycoprotein components of brain extracellular fluid (ECF) and cerebrospinal fluid. This protein was first discovered due to its enhanced turnover following learning events, but has since been found to function in other important cellular events such as long-term memory formation and optic nerve elongation (Shashoua, 1976; Shashoua, 1977; Shashoua, 1985). Goldfish EPN has several demonstrated effects on mammalian cells, and immuno-reactive EPN-like proteins have been observed in a variety of organisms ranging from invertebrates (Limulus) to mice. Some NTFs have been shown to alleviate oxidative stress, one of the primary mediators of cell damage in neurodegenerative conditions. One mechanism by which they accomplish this is to increase cellular levels of anti-oxidative enzyme superoxide dismutase (SOD). In fact, our lab recently showed that a synthetic EPN fragment (CMX-8933) increases SOD mRNA and protein levels in rat primary cortical cultures (Parikh, 2003). Transgenic mice and rabbits that overexpress SOD are resistant to ischemia, while mice that lack SOD present with worse ischemic damage. Thus, due to this important SOD activating NTF-like feature of EPN may have potential therapeutic applications for treating neurodegenerative conditions such as Alzheimer’s disease, Parkinson’s or stroke. Because full-length NTFs do not efficiently cross the blood brain barrier (BBB) when administered intravenously, our lab, in collaboration with Ceremedix, Inc. (Boston, MA), is interested in designing short peptides that mimic the action of full-length NTFs, especially EPN. Due to proteases that naturally exist in ECF, EPN is partially cleaved to release the 8 aa peptide KKETLQFR. Other brain proteins, like encephalins and endorphins, also release similar short active components, so we hypothesized that this 8 aa peptide may represent an active component of EPN. Indeed, our lab demonstrated that administration of this sequence in synthetic form (termed CMX-8933) to rat primary cortical cells increases cellular titers of SOD (Parkih, 2003). This thesis was divided into three parts. The first part investigated which signal transduction pathway is responsible for CMX-8933’s ability to upregulate SOD. Because our lab also showed that CMX-8933 activates the MAPK pathway (Hasson, 1998; El-Khishin, 1999; Adams et al., 2003) we hypothesized that CMX-8933 may use this pathway to upregulate SOD. Inhibition experiments were performed to test three known components of the MAPK pathway, and a member of an unrelated pathway. Six independent SOD immunoblot experiments demonstrated that pre-treatment of rat primary cortical cultures with specific inhibitors for the protein kinase-C family (PKC), protein tyrosine kinases (PTKs), or MEK protein kinases (MEKK), completely blocked (p = 0.0001) CMX-8933’s average 15-fold upregulation of SOD. Thus, these three critical components of the MAPK pathway appear to be involved in the CMX-8933-induced upregulation of SOD. An inhibitor of transcription factor NF-êB in an unrelated pathway had no significant effect (p = 0.901). The second part of this thesis tested whether treatment of rat primary cortical cultures with CMX-8933 increases the cellular titers of mRNAs related to translation. Previous observations indicated that treatment of these cells with with CMX-8933 induces neurite sprouting (Shashoua, unpublished), and that EPN plays a role in optic nerve elongation (Schmidt and Shashoua, 1988), two processes related to growth. So we hypothesized that EPN, or CMX-8933, may stimulate the transcriptioin of mRNAs related to growth. We tested mRNAs for translation factor EF-2, and ribosomal proteins S12 and L19 based on previous observations in our lab with hybridization arrays (Parikh, 2003). RT-PCR experiments indicated that treatment of rat primary cortical cultures with 10 ng/ml CMX-8933 for 5 hrs increased the mRNAs for S12 an average of 12-fold relative to untreated cultures (N = 3, p = 0.02), L19 an average of 9-fold (N = 3, p = 0.048), and EF-2 an average of 11-fold (N = 3, p = 0.045). Levels of housekeeper polyubiquitin remained unchanged. Thus 3 gene products related to growth are indeed upregulated by CMX-8933. The third part of this thesis investigated the SOD stimulatory effects of full-length EPN versus its cleavage product CMX-8933. Previous studies showed that full-length NTFs BDNF and NGF upregulate SOD in neuronal cells. Because CMX-8933 upregulates SOD, maybe full-length EPN does too. We hypothesized that if CMX-8933 represents the receptor-binding domain of full-length EPN, that full-length EPN may show the same stimulatory effects as CMX-8933, and may upregulate SOD. Extracellular fluid (ECF) was prepared from goldfish brains, the traditional source for isolating EPN. Analysis of the ECF on protein gels demonstrated the presence of a complex protein pattern dominated by two bands at 37,000 and 31,000 daltons, the known sizes of EPN-â (glycosylated) and EPN-ã (non-glycosylated), respectively. Immunoblots performed with EPN antibody “Sheila” (directed against the C-terminal end of EPN, Shashoua and Moore, 1978) confirmed the identity of these two ECF bands as EPN. Cultured mouse Nb2a neuronal cells were treated in six independent experiments with 12 ìg/ml ECF protein for 5 hrs, and whole cell lysates were tested for levels of SOD by immunoblots. This ECF treatment of neuronal cells produced a mean 4-fold increase in SOD levels (p = 0.007), supporting our hypothesis. However, since ECF is a complex mixture, this data did not show which ECF component was resonsible for the SOD signal. Since ECF is known to contain CMX-8933 EPN cleavage product, which by itself can upregulate SOD, it is possible CMX-8933 was responsible for the signal, not full-length EPN. To address this issue, microdialysis was performed using an 8,000 dalton MWCO membrane to remove low MW components from the ECF (including CMX-8933, MW = 1149), leaving EPN-â (MW 37,000) and EPN-ã (MW 31,000) present in the dialyzed ECF. Four independent experiments indicated no significant difference (p = 0.116) between dialyzed versus non-dialyzed ECF for activating SOD. Thus, CMX-8933 does not appear to be responsible for ECF’s ability to increase SOD, but instead, high MW molecules (including full-length EPN) appear to be the active components. Altogether, the data from this thesis extends our knowledge of the mechanism of action of both full-length EPN and its cleavage product CMX-8933."

No description available.

For trace elements, such as copper (Cu) and zinc (Zn), the bioavailability of these elements, Cu and Zn, in biosolids is important because both are essential elements and both are potential contaminants when biosolids are land applied. A greenhouse study was conducted in factorial experiment based on a completely randomized design (CRD) with four replications on a soil treated with four rates of Cu (0, 50, 100, and 150 mg/kg) and four rates of Zn (0, 150, 300, and 450 mg/kg) on celery plants to investigate the distribution and mobility of these elements as well as growth and antioxidant changes of celery. The results of antioxidant changes were inconclusive due to irregular changes with Zn and Cu applications. However, generally the results show that Cu did not affect superoxide dismutase (SOD) or peroxidase (POD) activities in most of the treatments. On the other hand, Zn stimulated SOD and POD activities in most of the treatments. The photosynthesis rate decreased with the applications of Cu and Zn at the rates above 100 and 300 mg/kg and increased in low Cu concentration (50 mg/kg) compared to S (soil without biosolid).

TAT peptide is one of the best-characterised cell penetrating peptides (CPPs) derived from the transactivator of transcription protein from the human immunodeficiency virus 1 (HIV-1). TAT peptide is able to cross the cell membrane and deliver various biomolecules into cells with low immunogenicity and no toxicity. However, the exact mechanism of internalization still remains a subject of controversy. Lamellar neutron scattering was used to determine the location of TAT peptide in the negativelycharged phospholipids bilayers. The results reveal two locations, one in the peripheral aqueous phase between the adjacent bilayers and the second one below the glycerol backbone region of the lipid bilayer. A concentrationindependent membrane thinning above a peptide concentration threshold (1mol%) and a contiguous transbilayer water channel at the largest peptide concentration (10mol%) were also found. This evidence led to the suggestion that the toroidal pore model might be involved in the transmembrane mechanism at high peptide concentration. Another set of neutron diffraction experiments examined the interaction between the TAT peptide and neutral phospholipids showed that TAT peptide preferentially intercalated into the hydrophobic core and the glycerol backbone region of the neutral lipid bilayer at the lowest peptide concentration investigated (0.1mol%), indicating that the insertion did not require negatively-charged phospholipids. There was also clear evidence for the concentration-dependent reorientation of TAT peptide. A plasmid containing the human copper-zinc SOD gene linked with the coding sequence for a 11-aa HIV-TAT peptide (pGEX-TAT-SOD, 513bp) was constructed and used to express a recombinant fusion protein in Escherichia coli strain BL21 (DE3). High-level expression of TAT-SOD soluble protein with a GST tag (44-kDa) was achieved under optimal expression conditions and a small-scale glutathione affinity column or large-scale ion-exchange chromatography used for its purification. The potential protective effect of TAT-SOD against UV-induced cell damage was studied on UVC-irradiated MDCK epithelial cells. Before any further clinical study, the UV full-length absorption of TAT-SOD protein was measured. The results showed the potential UV protective effect of TAT-SOD was not due to the physical absorption of UV irradiation. In a preclinical study with five healthy volunteers, the penetration of TAT-SOD through human stratum corneum on the inner upper arm was identified by the tape stripping and specific SOD activity analysis. Significant increases on SOD activity were found on the outer layers of stratum corneum in TAT-SOD treated group, compared to placebo treated control, indicating that the TAT peptide assisted SOD to penetrate into the human stratum corneum . In a clinical study with ten healthy volunteers, eight showed a significant increase of minimal erythema dose (MED) with TAT-SOD pre-treatment. The median blood flow value of ten subjects at the UVB-irradiated site decreased with TAT-SOD pretreatment. Taken together, this evidence showed that TATvi SOD did have a marked protective effect against UVB induced skin damage. In a second clinical study, five healthy volunteers were challenged with a series of UVB doses. Skin punch biopsies were taken from four test sites on the lower back for H&E and immunohistochemical staining analysis. UVB-induced apoptotic sunburn cell (SBC) formation, p53 up-regulation and thymine dimer formation in epidermis were not attenuated by pretreatment with TAT-SOD. These data suggest that transdermal superoxide scavenger TAT-SOD reduced the UVB-induced inflammation, but did not abrogate the direct DNA damage of UVB irradiation on the skin. However, the hope of TAT-SOD could reduce UVA indirect DNA damage remains.

Oxidative stress has a degenerative effect on neuronal health. Mutations in the copper zinc superoxide dismutase (SOD1), an important antioxidant, have been found in patients suffering from amyotrophic lateral sclerosis (ALS). Classical EMS induced mutations to SOD1 in Drosophila show similar loss of motor coordination and shortened lifespan seen in humans. A study of newly created human ALS point mutants along with the classic alleles show similar phenotypes in their neurodegeneration. I examined markers of oxidative stress, neuronal health and behavioral phenotypes throughout the lifecycle of aging flies. Larvae were largely found to be unaffected by mutations in SOD1, with no measured increase in ROS level over wild type (WT) flies. Mutant pupae were found to have two major defects in their circadian eclosion rhythm and their fundamental ability to eclose from the pupal casing. Adults showed the classic reduced lifespan and motor abilities. To further examine the health on non-glutamatergic synapses electroretinograms (ERGs) were recorded at different levels of survivorship indicated by Kaplan-Meier Survival curves. These ERGs show that the histaminergic synapses they record have greater degeneration in aging SOD1 mutants than in WT flies. This is true for their chronological age as well as their biological age. There was coinciding disruption of the photo transduction pathway of the photoreceptors that coincided with degeneration at the synapse. This demonstrates the separate degenerative effect of high levels of oxidative stress impart separate for the normal aging process.

Concerns over water quality have led to required removal of 50 % of dairy manure phosphorus (P) from the impaired Bosque River Watershed. Application of composted dairy manure (CDM) to sod and moving P off the watershed with sod has prompted a study using box lysimeters to determine NO3--N and P leaching from transplanted sod grown with CDM and inorganic fertilizer as well as sprigs top-dressed with CDM. Treatments were applied to lysimeters filled with a silica sand medium. Three leaching events were imposed, leaching 0.07 to 0.09 % of the total P applied and 0.09 to 1.43 % of total N applied. Concentrations of P in leachate averaged 0.04 to 0.25 mg L-1. Top-dressed CDM on sprigs leached statistically greater amounts of NO3--N than both transplanted sod treatments and greater P than the fertilizer grown sod. After the third leaching event, all treatments received an additional application of P, 100 kg ha-1 as CDM for manure-grown sod and sprigs, 50 kg ha-1 as triple superphosphate for fertilizer-grown sod. An additional three leachings were imposed. Top-dressed sprigs and transplanted sod leached similar amounts of P following the additional P application. Applied nutrients appeared to stay mainly in the sod layer and in the sand medium just below the sod layer. Top-dressed CDM appears to exhibit greater leaching losses of NO3--N than transplanted manure-grown sod and greater N and P losses than transplanted fertilizer grown sod.

The experiments in this thesis tested <i>in vitro</i> and <i>in vivo</i> the proposal that zinc-deficient superoxide dismutase, resulting from mutations or oxidative damage to the enzyme, gains ascorbate oxidase activity that contributes to the pathology of amyotrophic lateral sclerosis (ALS). They also tested whether flavonoids can help protect against this activity.<p>The <i>in vitro</i> experiments showed that zinc-extracted Cu/Zn-SOD (Cu-SOD) as well as SOD treated with H2O2 or H2O2 plus ascorbate accelerated ascorbate oxidation 100 to 300 %, while native SOD had no effect. With Cu-SOD, the activity was unaffected by EDTA, EGTA, or catalase, showing that the catalytic copper was firmly bound and that the H2O2 product of SOD activity was not responsible. Catechin and uric acid slowed ascorbate oxidation by Cu-SOD by 72% and 67%, respectively.<p>The <i>in vivo</i> study investigated tissue levels of ascorbate and biomarkers of oxidative stress in a transgenic mice bearing a mutation in Cu/Zn-SOD as a model of familial ALS (FALS mice), and the effects of dietary ascorbate and quercetin. In FALS mice on control modified AIN93G diet for 10 weeks compared to the wild-type, liver thiobarbituric acid reactive substances (TBARS) were 47% higher and liver oxidized vitamin C was 2800% higher. These results support, in liver, that mutant SOD acquired ascorbate oxidase activity and increased oxidative stress. The only difference in other tissues was a 136% increase in GSH/GSSG ratio in thigh muscle of FALS mice.<p>In dietary treatments of FALS mice, spinal cord TBARS was 93 % higher with ascorbate-supplemented diet compared to control diet, suggesting that dietary ascorbate increased oxidative stress. Also in spinal cord, oxidized-vitamin C was 250% higher in ascorbate + quercetin-fed FALS mice, which suggests there is no protection by quercetin against ascorbate oxidation. In brain, protein thiols were 56% and 58% lower in quercetin-fed and ascorbate + quercetin-fed FALS mice, suggesting that quercetin worsened oxidative damage. In liver, quercetin feeding produced a 40% decrease in vitamin C, total vitamin C and oxidized-vitamin C, perhaps by down-regulating ascorbate biosynthesis. Overall the results support a gain of ascorbate oxidase activity of mutant SOD in ALS, but do not support protection by dietary treatment with ascorbate or quercetin.

The experiments in this thesis tested <i>in vitro</i> and <i>in vivo</i> the proposal that zinc-deficient superoxide dismutase, resulting from mutations or oxidative damage to the enzyme, gains ascorbate oxidase activity that contributes to the pathology of amyotrophic lateral sclerosis (ALS). They also tested whether flavonoids can help protect against this activity.<p>The <i>in vitro</i> experiments showed that zinc-extracted Cu/Zn-SOD (Cu-SOD) as well as SOD treated with H2O2 or H2O2 plus ascorbate accelerated ascorbate oxidation 100 to 300 %, while native SOD had no effect. With Cu-SOD, the activity was unaffected by EDTA, EGTA, or catalase, showing that the catalytic copper was firmly bound and that the H2O2 product of SOD activity was not responsible. Catechin and uric acid slowed ascorbate oxidation by Cu-SOD by 72% and 67%, respectively.<p>The <i>in vivo</i> study investigated tissue levels of ascorbate and biomarkers of oxidative stress in a transgenic mice bearing a mutation in Cu/Zn-SOD as a model of familial ALS (FALS mice), and the effects of dietary ascorbate and quercetin. In FALS mice on control modified AIN93G diet for 10 weeks compared to the wild-type, liver thiobarbituric acid reactive substances (TBARS) were 47% higher and liver oxidized vitamin C was 2800% higher. These results support, in liver, that mutant SOD acquired ascorbate oxidase activity and increased oxidative stress. The only difference in other tissues was a 136% increase in GSH/GSSG ratio in thigh muscle of FALS mice.<p>In dietary treatments of FALS mice, spinal cord TBARS was 93 % higher with ascorbate-supplemented diet compared to control diet, suggesting that dietary ascorbate increased oxidative stress. Also in spinal cord, oxidized-vitamin C was 250% higher in ascorbate + quercetin-fed FALS mice, which suggests there is no protection by quercetin against ascorbate oxidation. In brain, protein thiols were 56% and 58% lower in quercetin-fed and ascorbate + quercetin-fed FALS mice, suggesting that quercetin worsened oxidative damage. In liver, quercetin feeding produced a 40% decrease in vitamin C, total vitamin C and oxidized-vitamin C, perhaps by down-regulating ascorbate biosynthesis. Overall the results support a gain of ascorbate oxidase activity of mutant SOD in ALS, but do not support protection by dietary treatment with ascorbate or quercetin.