The study of dermal wound healing has long been used to elucidate the cellular and molecular processes guiding the connective tissue response to injury. Of particular interest are the mechanisms by which soluble mediators, including inflammatory signals, guide fibroblast activity within the wound bed. This thesis addresses the role of prostaglandin E2 (PGE2) in the regulation of fibroblast activities relevant to restoration of tissue structure and function. Although PGE2 has been previously shown to play an important role in various wound healing steps, its precise contribution to the overall outcome of dermal repair is unclear. Using three well defined human dermal fibroblast phenotypes this study demonstrates that while PGE2 signaling during dermal repair triggers pro-inflammatory cascades, its effects on fibroblast activities are putatively anti-fibrotic. Specifically, exogenous PGE2 decreases the migratory and contractile potential of dermal fibroblasts through destabilization of the actin cytoskeleton and inhibits endogenous collagen synthesis. While PGE2 effects on fibroblast activity are largely conserved across phenotypes, fetal fibroblasts maintain a quantitatively diminished response to PGE2-induced alterations of cytoskeletal dynamics. Upon further analysis, this effect was shown to be representative of a larger intrinsic fibroblast phenotype. Fetal dermal fibroblasts were shown to maintain elevated rates of migration and contraction, as part of a generalized hyperactive dynamic state. Surprisingly, this phenotype was found to be sufficiently robust so as to persist despite changes in substrate and environmental constraints. In light of this finding, one additional approach was used to ascertain the robustness of the fetal fibroblast. Transplantation of fetal dermal fibroblasts into an adult wound environment was used to assess whether the intrinsic fetal fibroblast phenotype can survive the multitude of events comprising adult wound healing. While results are preliminary, this approach does present a useful tool for future studies aimed at elucidating the precise fetal fibroblast phenotype and its contribution to overall wound healing response.

Cell migration is a complex biophysical event that is dysregulated in a variety of human diseases including cancer. The ability of tumor cells to migrate enables cancer dissemination causing significant mortality thus making it an important therapeutic target. Motility is exhibited epigenetically by activation of numerous signaling pathways that transmit extracellular cues to the final effectors of cell movement. Such signaling switches are a part of larger and highly complex signaling (proteomic) networks that are under the control of numerous activators or inhibitors. Although majority of the proteins that are 'required' during cell motility have been identified, it is yet unclear wherein they fit within the signaling network to govern motility. Thus, a 'systems biology' approach is needed to understand the complex interplay of signaling cascades in mediating cell motility so that better therapeutic targets can be defined. We utilized a mathematical modeling approach, called decision tree analysis to map the interplay between five key signaling proteins known to regulate vital biophysical processes of fibroblast motility downstream of EGF receptor activation. Interestingly, our model identified myosin light chain (MLC) mediated cell contractility as a crucial node for maximal motility. Even more non-intuitively the decision tree model predicted that subtotal inhibition of MLC can actually increase motility. Confirmatory experiments with fibroblasts and cancer cells have shown that to be the case. Since the model proposed that total abrogation of contractility can limit cell migration, we asked if such an intervention can limit tumor invasion. Since PKCδ is implicated in EGF receptor mediated transcellular contractility, we abrogated PKCδ using pharmacological (Rottlerin) and molecular (RNAi) interventions. Such depletion of PKCδ reduced migration as well as invasiveness of prostate carcinoma cells predominantly by decreasing their contractility through myosin light chain (MLC). Additionally, activation of PKCδ correlated with human prostate cancer progression as assessed by immunohistochemistry of prostate tissue sections. In summation our studies illustrate the importance of quantitative (total versus subtotal) disruption of key signaling nodes in mediating a desired cell response. Novel computational modeling approaches are needed to identify newer molecular switches from existing proteomic networks that can be explored, using classical experimental methods, as therapeutic targets.

Thiobencarb (TB), widely used for the control of broadleaf weeds, grasses, and sedges in rice fields, is considered safe for rice plants when used at recommended rates. TB's reductive dechlorination product, dechlorinated thiobencarb (DTB), is highly toxic to rice. TB is naturally transformed into DTB in field soils in certain areas in Japan and the United States. The resultant syndrome is called delayed phytotoxicity syndrome (DPS). This research was conducted to characterize DPS in Louisiana, to compare the toxicity of TB and DTB to rice, to determine uptake and retention rates of TB and DTB by rice, to confirm that soil microorganisms convert TB to DTB, to determine factors affecting the dechlorination of TB, and to develop methods for isolating dechlorinating microorganisms. An in vitro bioassay developed in this study showed that seedling heights were reduced as concentrations of TB and DTB in soil increased. The effective dosage for 50% reduction in height, using Lafitte rice, was 6.6 μg/ml for TB and 0.3 μg/ml for DTB. By developing and using a gas chromatography/mass spectrometry (GC/MS) method, it was shown that DTB was not taken up preferentially by rice plants. Rice plants absorbed and accumulated more TB than DTB when exposed at equal concentrations. The toxic effects of TB and DTB to rice seedlings was additive. When rice cultivars were evaluated for sensitivity to DTB, M201 was more tolerant than Bengal, Cocodrie, and Lafitte. The conditions affecting the transformation of TB into DTB in soil were studied using a special apparatus developed to measure the redox potential of soil columns at different depths. Reductive dechlorination of TB peaked after 14 days incubation, at a position in the soil column corresponding to an Eh of -230 mV. TB was converted to DTB in vitro in a conducive soil, but not after the soil was autoclaved. Bacterial and fungal isolates from conducive soil inoculated into sterile soil suspensions, or the soil column, all failed to dechlorinate TB. Repeated attempts to isolate the organisms responsible for dechlorination of TB in Louisiana rice field soils failed.

Parasympodiella

Clinical information was collected from 283 randomly chosen ovarian cancer cases from at the Saskatoon Cancer Centre between the years 1983-1995. The data was evaluated for its significance in predicting survival and relapse free survival (RFS) using univariate and multivariate analysis. Several clinical prognostic factors were identified by univariate analysis. Additionally, using Cox's regression model the independent markers of survival and RFS were FIGO stage, and residual disease in 173 and 178 patients respectively. Data on CA 125 serum level, (available in 89 patients) was a marker of prognostic significance in the patients treated with platinum based chemotherapy. CA 125 and CEA antigen expression were also evaluated in seventy one cases. It was found that mucinous neoplasms exclusively expressed CEA antigen. This study indicates that the evaluation of serum level CEA may be a complementary tool for patients with cancers not expressing CA 125. In this retrospective study, DNA from paraffin embedded tissue (PET) in patients with ovarian carcinoma was examined to identify gene abnormalities in p53, p16INK4A, RB-1, p21WAF1/CIP1, Cyclin D1, Erb-B2, and MSH2. Adverse outcome was also examined in addition to survival and RFS, to identify novel molecular prognostic markers. P53 overexpression in 44 of 112 (39%) was associated with reduced survival and RFS (' p' = '0.04' and 'p' = '0.008 '). Aneuploid DNA content, found in 34 of 112 (30%) cases, was associated with shorter survival and RFS ('p' = '0.03' and 'p' = '0.01'). Dot blot hybridization of G1-S control genes (p16INK4A, Cyclin D1, RB-1, and CDK4) did not identify amplification or deletion events to be associated with adverse outcome. A number of gene alterations in 59 of 63 (94%) ovarian cancer cases were detected by dot blot hybridization; the lack of association with clinical outcome indicated that there may be some other genes in addition to those examined that are of prognostic significance. For eighteen cases, microsatellite instability (MSI) was evaluated by using fluorescently labeled primers at nine loci. LOH was a common event in ovarian carcinoma but MSI was infrequent. Molecular and clinical marker multivariate analysis indicated: (a) residual disease for survival, (b) stage and residual disease for RFS, were independent markers of prognosis.

Field studies conducted over two growing seasons evaluated the effect of 2,4-D applied at 1.6 kg ai/ha to LCP 85-384 sugarcane (Saccharum interspecific hybrid) 7, 5, 3, and 1 wk before planting (WBP). Sugarcane was planted in mid-September using both whole stalk and billet (45 cm) seed pieces. When 2,4-D was applied 5 wk or closer to planting, sugarcane shoot emergence and population averaged across planting methods was reduced 5, 7, and 28 wk after planting (WAP) when compared to the nontreated control. Sugarcane height in one of two years was reduced when 2,4-D was applied 5 wk or closer to harvest of sugarcane for seed and sugarcane and sugar yield were reduced around 11% when compared with the nontreated control. For LCP 85-384 a 7 wk period should be allowed between 2,4-D application and harvest for seed when planted using whole stalks or billets. In field studies complete control of red morningglory (Ipomoea coccinea L.) 30 and 60 cm in height was obtained 14 or 21 days after treatment (DAT) over two years with 2,4-D at 0.53 kg/ha, 2,4-D at 0.4 kg/ha or more plus dicamba, atrazine at 2.23 kg ai/ha, flumioxazin at 0.10 kg ai/ha, sulfentrazone at 0.35 kg ai/ha, and V10064 at 1.75 kg ai/ha. Red morningglory 1.8 m tall was controlled 100% 28 DAT the first year with 2,4-D at 1.06 kg/ha and 78% the second year. In the second year when herbicides were applied three weeks earlier than the previous year and when weed growth was more vigorous, the 2,4-D plus the 2,4-D and dicamba premix at 0.79 + 0.1 / 0.04 kg/ha provided control greater than that of 2,4-D alone at 1.06 kg/ha, but was the only treatment that included dicamba to control red morningglory equal to that of 2,4-D at 1.59 kg/ha (87%). Directed applications to the lower 45 cm of 1.8 m red morningglory plants with atrazine at 4.47 kg/ha, sulfentrazone at 0.35 kg/ha, and V10064 at 1.75 kg/ha the first year controlled weeds at least 96%, but control was 23 to 30 percentage points less the second year.

Field studies were conducted over two years in drill- and water-seeded rice to evaluate weed control and crop response with imazethapyr programs. Barnyardgrass [Echinochloa crus-galli (L.) Beauv.] was evaluated with imazethapyr applied alone at various rates and timings. Imazethapyr controlled barnyardgrass 95 to 97% with a soil application at 87 or 70 g/ha fb 53 or 70 g/ha EPOST or LPOST. A single EPOST application of imazethapyr at 140 g/ha controlled barnyardgrass and rice yield was equal to or above those treatments receiving two applications of imazethapyr. Research was also conducted at Crowley, LA and Rayne, LA to evaluate the addition of a herbicide with broadleaf activity into imazethapyr programs applied alone early postemergence (EPOST) and in combination with imazethapyr postemergence (POST). Weeds evaluated included barnyardgrass, red rice (Oryza sativa L.), alligatorweed [Alternanthera philoxeroides (Mart.) Griseb.], and hemp sesbania [Sesbania exaltata (Raf.) Rydb. ex A.W. Hill]. Treatments consisting of imazethapyr applied at 87 g/ha alone to the soil fb a POST imazethapyr application at 53 g/ha controlled barnyardgrass above 90% late season in all studies, expect in water-seeded rice when an imazethapyr application was made at the three- to four-leaf rice stage. Red rice control with a total imazethapyr program was equivalent to, or higher, than other treatments in both drill- and water-seeded studies. Single imazethapyr applications resulted in reduced control of red rice indicating two applications of imazethapyr are required to obtain adequate control. Alligatorweed control increased with soil applications of imazethapyr. However, alligatorweed control with imazethapyr as the only herbicide in a weed control program was inconsistent and suggested only suppression. Treatments receiving a broadleaf herbicide application increased hemp sesbania control as compared with the total imazethapyr program; however, treatments with bensulfuron and triclopyr were inconsistent at controlling hemp sesbania. In drill-seeded studies, hemp sesbania was less of a problem and red rice had a greater impact on rice yield. Rice yields with total imazethapyr programs were equal to, or higher than, other treatments. However, in water-seeded studies, hemp sesbania growth was favored and yields from rice treated with broadleaf herbicides were higher than total imazethapyr programs.

This research was conducted to enhance utilization of the Liberty herbicide resistance transgene in rice. Non-lethal methods to determine the sensitivity of transgenic rice plants to hygromycin B and Liberty were developed, tested and used in this research. Four homozygous transformed plants were selected to make reciprocal crosses with their non-transformed parent cultivars Taipei 309 and Nipponbare. Their resistances to Liberty and hygromycin B were controlled by the closely linked single dominant genes bar and hpt. Some non-resistant phenotypes in the F2 populations were due to gene silencing. The bar gene in some of these plants were allelic and some were non-allelic. When seven independently transformed homozygous transgenic plants with bar and hygromycin genes from Taipei 309 and Nipponbare were crossed reciprocally, progeny evaluations showed five allelic locations among the seven transgenic plants. Twenty additional homozygous transgenic plants from independent transformation events were crossed reciprocally with the previous seven transgenic plants. Evaluation of F1, F2, and F3 populations showed that some of the genes were allelic, but most of them were non-allelic with two or more pairs of genes being expressed. The functional foreign gene (bar) appeared to be restrictively inserted into the rice genome in some cases and was not randomly inserted and expressed. Three to five repeated backcrosses were made using transgenic plants as the donor and current cultivars as the recurrent parents. The results from selected progeny rows, and two-years of yield tests with selected lines, indicated that the target bar gene could be transferred to lines similar to commercial cultivars from homozygous transformants in 4-5 years of backcrossing, giving lines similar to the recurrent parents based on phenotype and yield potential. Liberty herbicide has antibiotic characteristics and suppressed growth of several rice fungal pathogens and Burkholderia glumae in in vitro tests. Liberty had a short residual activity against Rhizoctonia solani in field tests, but single applications of Liberty after disease development had started in the field significantly reduced sheath blight ratings and yield loss. Control of sheath blight by Liberty was equal to or better than that given by the registered fungicide Quadris.

Sugarcane (inter-specific hybrids of Saccharum) is grown largely under long-term monoculture production in Louisiana. This can lead to a complex problem termed "yield decline" that results in poor root health and reduced productive capacity of sugarcane. This problem has been documented to be a limiting factor for sugarcane production in diverse regions, including Louisiana, Hawaii, Jamaica, and Australia. Previous work showed that biological factors affect root health and contribute to yield decline. The objectives of this study were to increase our understanding of microbial communities in sugarcane soils, to determine if there are differences in microbial communities associated with sugarcane roots in soil with and without a sugarcane cropping history, and to provide information on possible changes in the microbial communities resulting from monoculture that may contribute to yield decline. <p> To achieve these objectives, two approaches were used for comparing culturable organisms in soil microbial communities from soil with and without a sugarcane cropping history, and methods were adapted to reliably obtain DNA from soil microbial communities for molecular comparisons. In one approach, colonies grown on different types of culture media were quantified and characterized. In the other approach, sole carbon source utilization profiles (SCSUP) of soil communities grown in Biolog(tm) GN2 microplates were compared. Comparisons of the numbers and types of microorganisms that grew on various culture media demonstrated that differences exist between microbial communities associated with sugarcane roots in Louisiana soils with and without a recent sugarcane cropping history. The differences in community functional diversity detected by SCSUP supported the differences found in types of microorganisms isolated on selective media. The SCSUP results showed that differences in community functional diversity exist between sites in soils with a long-term sugarcane cropping history in common. <p> Methods for DNA extraction and polymerase chain reaction (PCR) amplification were optimized for sugarcane soil microbial community samples from Louisiana. This will allow molecular characterization of sugarcane rhizosphere microbial communities in the future.

Many signals involved in pathophysiology are controlled by hypoxia-inducible factors (HIFs), transcription factors that induce expression of hypoxia-responsive genes. HIFs are highly conserved master regulators of oxygen homeostasis. These factors are post-translationally regulated by a family of oxygen-dependent HIF hydroxylases, whose members include four prolyl 4-hydroxylases (PHD1-4) and an asparaginyl hydroxylase (FIH-1). All HIF hydroxylases require molecular oxygen, Fe(II), ascorbate, and 2-oxoglutarate as cofactors. We hypothesized that alterations in subcellular localization may provide an additional point of regulation for the HIF pathway in response to hypoxia. Most of these enzymes are abundant in resting liver, an organ which is itself unique due to its physiologic oxygen gradient, and they can exist in both nuclear and cytoplasmic pools. In this study, we analyzed the localization of endogenous HIFs and their regulatory hydroxylases in primary rat hepatocytes cultured under hypoxia-reoxygenation conditions. We observed an absence of nuclear HIF-1á activation in hypoxic hepatocytes, even though several known HIF target genes were upregulated, suggesting that HIF-2á and HIF-3á are the predominant isoforms in liver. We show that in hepatocytes, hypoxia-reoxygenation targets HIF-1á to the peroxisome rather than the nucleus, where it co-localizes with the von Hippel Lindau protein (VHL) and the HIF hydroxylases. Confocal immunofluorescence microscopy demonstrated that the HIF hydroxylases can translocate from the nucleus to the cytoplasm in response to hypoxia, with increased accumulation in peroxisomes upon reoxygenation. These results were confirmed via immuno-transmission electron microscopy and Western blotting. Surprisingly, in resting liver tissue, peri-venous localization of the HIF hydroxylases was detected, consistent with areas of low oxygenation. This was in contrast to nuclear HIF-1á, which was undetectable in a number of liver injury models. In conclusion, these studies establish the peroxisome as a highly relevant site of subcellular localization and function for the endogenous HIF pathway in hepatocytes.