Thesis (Ph. D.)--West Virginia University, 2006. / Title from document title page. Document formatted into pages; contains xi, 180 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.

Thesis (Ph. D.)--West Virginia University, 2006. / Title from document title page. Document formatted into pages; contains xi, 261 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.

Thesis (Ph. D.)--University of Missouri-Columbia, 2005. / "December 2005" The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. Includes bibliographical references.

Thesis (Ph. D.)--West Virginia University, 2007. / Title from document title page. Document formatted into pages; contains ix, 103 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.

Heat shock proteins (HSP), although not considered classical defence proteins, have general cytoprotective properties, which promote survival of cells and organisms. Hsp70, in particular, provides resistance to the harmful consequences of various forms of otherwise damaging or even lethal stress including pathogen infection. Increased levels of Hsp70, due to stable transfection of cells with hsp70 genes, or elevated expression in response to stress, generally correlate with the hindrance of cell death processes triggered by a variety of noxious stimuli or toxic agents. The effect of lipopolysaccharides (LPS), the major constituent of the outer membrane of the cell wall (envelope) of almost all Gram-negative bacteria, on Hsp70/Hsc70 expression in plants is unknown. In various mammalian systems, LPS has been shown to induce Hsp70 accumulation, along with programmed (apoptotic) cell death. Contrary to the effects of LPS on animal hosts however, LPS does not elicit cell death in plants, but rather pre-treatment with LPS fraction can prevent or delay the so-called hypersensitive response (HR), thus sensitizing plant tissue to respond more rapidly, or to a greater extent, to subsequently inoculated phytopathogenic bacteria. Elevated levels of reactive oxygen species (ROS) reportedly contribute to stress sensing and hsp gene activation, and subsequent Hsp70 induction, during the stress response. Increased ROS production can also trigger cell death via either programmed cell death (PCD), an active (i.e., energy-dependent) physiological suicide mechanism that is genetically regulated, or uncontrolled necrosis, an accidental, lytic form of cell destruction passively triggered by severe trauma or injury. In plants specifically, ROS may be involved in PCD activation during the HR. As a pathogen-associated molecular pattern (PAMP) or general elicitor of defence or resistance-related responses, LPS may trigger some defence-related responses, including an oxidative burst (manifest as increased levels of reactive oxygen species or ROS) in certain plant cells as it does in animal systems. However, there is conflicting evidence that shows that LPS treatment does not elicit an oxidative burst in plants. The aim of this study was to determine the effect of LPS isolated from Ralstonia solanacearum, an extremely harmful soil-borne bacterium that causes Southern wilt in over 200 plant species by infecting the host’s roots and invading the xylem vessels, on Hsp70/Hsc70 accumulation, cell death and ROS production in suspension-cultured tobacco (Nicotiana tabacum) cells, in order to gain a better understanding of the inter-relationship between these three phenomena in plant cells responding to LPS(Ralstonia). Western (immuno)blot analysis was used to study the unknown effect of LPS(Ralstonia) on Hsp70/Hsc70 accumulation in tobacco cell suspensions. LPS(Ralstonia) (all concentrations and time periods studied) generally suppressed Hsp70/Hsc70 accumulation. However, only exposure to 100 μg LPS/ml for 3 h caused a significant reduction (P < 0.05). Therefore, there was an early suppression of Hsp70/Hsc70 accumulation in response to 100 μg LPS(Ralstonia)/ml. To determine whether the observed LPS-mediated attenuation in Hsp70/Hsc70 accumulation was due to an increase in cell death in these cells, we investigated the effect of LPS(Ralstonia) on i) the general viability of the cells, and ii) the integrity of nuclear or genomic DNA extracted from LPS-treated suspension-cultured tobacco cells. The AlamarBlue™ (AB) assay was used to investigate the general cell viability in response to LPS(Ralstonia) treatment. LPS(Ralstonia) (all concentrations and time intervals studied) did not significantly affect the overall viability of the cells. Because treatment of tobacco cell suspensions with LPS(Ralstonia) did not result in a significant decrease (P < 0.05) in AB reduction, it was presumed that LPS(Ralstonia) did not appreciably compromise metabolic activity and was therefore not particularly toxic to these cells. Genomic DNA from cells undergoing PCD-associated internucleosomal DNA fragmentation (IDF) typically runs as a ladder of internucleosomal-sized DNA fragments corresponding to multimers of ca. 180 bp in agarose gels. In contrast, random DNA cleavage, usually manifest as smearing of nuclear DNA following agarose gel electrophoresis, is a token of uncontrolled necrosis. Therefore, if so-called “DNA laddering” is observed following agarose gel electrophoresis of genomic DNA extracted from suspension-cultured tobacco cells exposed to LPS(Ralstonia) then it can be assumed that LPS(Ralstonia) induced PCD. Alternatively, if a long, continuous “necrotic smear” is evident after electrophoretic separation of nuclear DNA from LPS-treated cells then LPS(Ralstonia) clearly induced uncontrolled necrosis. Whether or not LPS(Ralstonia) induced PCD-associated IDF or necrotic smearing was determined by investigating genomic DNA fragmentation (or DNA integrity) in response to LPS(Ralstonia) iii treatment. Although no typical DNA ladders were detected following electrophoresis of DNA isolated from LPS-treated cells, PCD may still have transpired. However, this is highly unlikely. No necrotic smearing was evident in LPS-treated samples either, which verifies the hypothesis that LPS(Ralstonia) (25–100 μg/ml) did not induce uncontrolled necrosis in suspension-cultured tobacco cells. In fact, these concentrations of LPS(Ralstonia) did not seem to significantly compromise DNA integrity given that LPS(Ralstonia) (25–100 μg/ml) generally had no appreciable effect on genomic DNA fragmentation (compared to untreated control samples). Incidentally, 24-h exposure of tobacco cell suspensions to higher concentrations of LPS(Ralstonia) (500 and 1000 μg/ml) may have resulted in partial DNA cleavage and/or degradation. Exposure of tobacco cell suspensions to 400 μg LPS(Burkholderia)/ml for 7 days may also have evoked partial DNA cleavage and/or degradation. Whether this cleavage and/or degradation occurred deliberately by means of a fixed or predetermined mechanism or randomly by an uncontrolled mechanism remains uncertain. Finally, the H2DCF-DA (2′, 7′-dihydrodichlorofluorescein-diacetate) fluorescence assay was used to investigate the effect of LPS(Ralstonia) on ROS production, a common factor in the regulation of HSP expression and cell death activation. LPS(Ralstonia) treatment (25–100 μg/ml) generally increased ROS levels in suspension-cultured tobacco cells (compared to untreated control cells). Exposure to 75 μg LPS(Ralstonia)/ml resulted in a particularly prominent elevation in ROS levels almost instantaneously. Incidentally, higher concentrations of LPS(Ralstonia) (500 and 1000 μg/ml) resulted in decreased ROS levels at some point during the assay. Although LPS(Ralstonia) (100 μg/ml for 3 h) significantly decreased Hsp70/Hsc70 accumulation in tobacco cell suspensions, cell death did not appear to be induced. In fact, LPS(Ralstonia) had no effect on general cell viability and appeared to be ineffective at causing PCD-associated IDF (DNA laddering) or necrotic smearing regardless of concentration or time of exposure. Despite these findings, treatment of suspension-cultured tobacco cells with LPS(Ralstonia) (≤ 100 μg/ml) resulted in a mild increase in ROS production. Although the exact mechanism(s) by which LPS(Ralstonia) suppressed Hsp70/Hsc70 accumulation is elusive, our results suggest that the suppression is not related to excessive LPS-mediated injury caused by excessively high ROS levels or increased cell death. We speculate that the prevention of HR-related PCD often observed in plants that are pre-treated with LPS and subsequently inoculated with phytopathogenic bacteria may be dependent on the LPS-mediated suppression of cytosolic Hsp70 expression. / Dr. M.J. Cronje

Forensic biology encompasses the examination of evidentiary items from crime scenes for biological fluids, often identifying the specific biological fluid present and developing a DNA profile that can be used to link a suspect to a crime. Blood identification consists of visual examination, presumptive tests based on the catalytic activity of hemoglobin, and confirmatory tests based on antigen-antibody interactions. Issues encountered in blood identification include the occurrence of false positive and false negative results. Many causes of these results are well-known but more recently three substances resulting in false negatives with catalytic color tests, chemiluminescent reagents, and immunoassays have been explored. Quebracho extract (a common leather tannin), sodium percarbonate (the main component of detergents containing active oxygen) and vitamin C-containing beverages were all found to produce false negative results at varying degrees with each of the tests mentioned. Increased knowledge of potential negative interfering agents by forensic investigators can help ensure that probative evidence is properly collected and thoroughly analyzed from a crime scene.

Fine particulate matter (PM2.5) is the leading public health risk factor of global disease burden, which has caused 4.2 million deaths in 2015. This thesis aims to improve the scientific understanding on the sources and health impacts of PM2.5 in Hong Kong. Various chemical and biological analytical techniques were applied to characterize the chemical and toxicological properties of PM2.5 samples collected in Hong Kong during 2011-2012. Positive matrix factorization (PMF), together with the quantified chemical markers and water-soluble PM2.5-induced reactive oxygen species (ROS) activity as the input matrix, was performed to apportion the source-specific contributions to ambient organic carbon (OC) and the oxidative potential of water-soluble PM2.5. Zebrafish was applied as in-vivo model to evaluate the PM2.5-induced differential expression genes (DEGs). An L2-normaliztion integrated PMF was developed and applied to quantitatively assess the ability of PM2.5 to induced DEGs in relation to various sources and chemical compositions of PM2.5. The main findings are summarized below: (1) Thirty nine primary organic aerosol (POA) and secondary organic aerosol (SOA) markers of various anthropogenic (i.e. biomass burning (BB)) and biogenic sources (i.e. isoprene, monoterpenes and β-caryophyllene) were identified and quantified. High levels of OC and SOA markers were observed on regional pollution days than long regional transport (LRT) pollution and local emissions days. A kinetic model (Kintecus) was applied to explore the major formation channels of isoprene SOA, and it was found that isoprene SOA was mainly formed through the ring-opening reaction of isoprene epoxydiols (IEPOX) in Hong Kong. (2) PMF analysis, together with the chemical markers measured in Chapter 1 &2, was performed to evaluate the sources of OA in Hong Kong. Sea salt, marine vessels, vehicle emissions, BB/SOA, SOA, and secondary sulfate (SS) were apportioned as the major sources of ambient OC in Hong Kong. Secondary formation, including SOA, BB aging and SS sources, was found to be the major contributor to OC (~51%) throughout the whole year. BB was the major anthropogenic contributor to OC on regional days (28.8%); while marine vessel was the dominated primary source of OC on local days (33.2%). SOC concentrations were estimated using a tracer-based method (SOCTBM) and PMF (SOCPMF). Both SOCTBM and SOCPMF showed highest concentrations on regional days (SOCTBM: 0.74 µg m-3; SOCPMF: 3.27 µg m-3). Among all SOA precursors, monoterpenes had the most abundant contribution (40.9%) to SOCTMB during the whole year. Moreover, sulfate has significant impacts on SS-related SOC and SOA from monoterpenes and naphthalene. Particle acidity (HP+) showed correlation with SOC from BB aging. These results provide us a quantitative understanding on the SOA origins in the region, which lays a foundation for the source apportionment of PM2.5-induced toxicity in the following chapters. (3)Cell-free dithiothreitol (DTT) and ·OH generation assays were applied to measure the ROS activity induced by water-soluble PM2.5 collected in Hong Kong during 2011-2012. Different levels of ROS activity were observed for different chemical fractions of PM2.5 and PM2.5 from various sources. Six factors, i.e. SS, BB, SOA, vehicle emissions, marine vessels and metal factors were apportioned by PMF as the major sources of water-soluble PM2.5 induced ROS potential. Metal factors was found to be the major contributor to both DTT activity (39.1%) and ·OH generation ability (84.5%) throughout the year, especially on LRT (DTT: 54.8%; ·OH generation: 91.1%) and regional days (DTT: 53.9%; ·OH generation: 87.7%). On local days, contribution of marine vessels to DTT oxidation become more significant (48.7%), however its contribution to ·OH generation is negligible. Metal factors is by far the most significant contributor to ·OH generation, even on local days (73.1%). It is interesting to note that all six PMF-resolved sources are associated with DTT oxidation, however only three sources (i.e. metal factor, vehicle emissions and SOA) showed contributions to ·OH generation. Moreover, among these six sources, marine vessels exhibited the highest intrinsic DTT ability; while metal factor was the most effective source in ·OH generation. (4) Zebrafish embryo (AB strain) was applied as the in-vivo model to assess PM2.5 toxicity in Hong Kong through genome-wide gene transcriptional analysis. The results showed that embryonic exposure to PM2.5 could induce remarkable changes in gene expression patterns in zebrafish. DEGs between PM2.5 extract treated and untreated zebrafish embryo samples were identified, and they were found mainly associated with responses to xenobiotic stimulus, and muscle and heart development and functions. The correlation analysis between co-expressed gene modules and chemical species of PM2.5 implied the different chemical compositions and sources of PM2.5 have significant influences on the PM2.5-induced biological responses. (5) An L2-normalizaiton integrated PMF was developed to analyze the high throughput biological and chemical data simultaneously, which quantitatively evaluated the ability of PM2.5 to induce DEGs in relation to sources and compositions. In this chapter, nine sources associated with PM2.5-induced DEGs were well apportioned, i.e. fresh sea salt, aged sea salt, SS, SOA, BB, coal combustion, vehicle emissions, marine vessels and metal factors. Among these sources, metal factors (annual mean: 26.5%, range: 17.6-39.3%) and vehicle emissions (annual mean: 16.3%, range: 0.0-25.3%) are the two leading contributors to PM2.5-induced DEGs levels. PM2.5 from combustion related sources (e.g. vehicle emissions, metal factors, BB) and sea salt exhibited stronger ability to induce DEGs than those from secondary sources. Although secondary formation (including SOA and SS) has a significant contribution to ambient PM2.5 (12 μg m-3, 40%), its capacity of DEGs induction is quite low. Moreover, several biological functions and pathways influenced by PM2.5 from various sources have also been well evaluated. In this study, large scales of biological and chemical data were analyzed for the first time by a L2-normalizaiton integrated PMF to apportion the PM2.5-induced DEGs, and this thesis work firstly reported the major sources of water-soluble PM2.5-induced ROS in Hong Kong. Results from this study provide a scientific basis for the prediction of PM2.5-associated adverse health outcomes and can help the policy makers to formulate cost-effective and targeted PM2.5 mitigation strategies to protect public health.;Fine particulate matter (PM2.5) is the leading public health risk factor of global disease burden, which has caused 4.2 million deaths in 2015. This thesis aims to improve the scientific understanding on the sources and health impacts of PM2.5 in Hong Kong. Various chemical and biological analytical techniques were applied to characterize the chemical and toxicological properties of PM2.5 samples collected in Hong Kong during 2011-2012. Positive matrix factorization (PMF), together with the quantified chemical markers and water-soluble PM2.5-induced reactive oxygen species (ROS) activity as the input matrix, was performed to apportion the source-specific contributions to ambient organic carbon (OC) and the oxidative potential of water-soluble PM2.5. Zebrafish was applied as in-vivo model to evaluate the PM2.5-induced differential expression genes (DEGs). An L2-normaliztion integrated PMF was developed and applied to quantitatively assess the ability of PM2.5 to induced DEGs in relation to various sources and chemical compositions of PM2.5. The main findings are summarized below: (1) Thirty nine primary organic aerosol (POA) and secondary organic aerosol (SOA) markers of various anthropogenic (i.e. biomass burning (BB)) and biogenic sources (i.e. isoprene, monoterpenes and β-caryophyllene) were identified and quantified. High levels of OC and SOA markers were observed on regional pollution days than long regional transport (LRT) pollution and local emissions days. A kinetic model (Kintecus) was applied to explore the major formation channels of isoprene SOA, and it was found that isoprene SOA was mainly formed through the ring-opening reaction of isoprene epoxydiols (IEPOX) in Hong Kong.

Blood is considered one of the most widely tested biological matrices. The first step in blood identification involves visual examination followed by presumptive testing. Once a positive presumptive result is observed, confirmatory tests are performed to determine that a stain is human blood, thus reducing the time and resources spent on forensically irrelevant samples. When interfering agents are present, this general workflow is hindered as presumptive tests can render false-negative results. General awareness of these interfering agents can help analysts to recognize forensically relevant evidence that may have otherwise been deemed immaterial. The main objective of this study was to understand various interfering agents and their effects on presumptive blood tests such as Kastle Meyer (KM) and Orthotolidine (O-tol) reagents and confirmatory tests such as HemaTrace® and Rapid Stain Identification (RSID™) Blood. Additional experiments explored the effects on downstream DNA analysis. In the first part of the study, bloodstains in varying concentrations were exposed to ten chemical interferents over a period of time to understand how blood dilution, age of the stain, and the chemical nature of the interferent affect presumptive blood test results. Antioxidants, active oxygen, and tannins are known to interrupt the mechanism of presumptive tests. Thus, ten interfering agents (ascorbic acid, chlorogenic acid, catechin, sodium percarbonate, hydrogen peroxide, oxalic acid, proanthocyanidins, quebracho extract, chestnut extract, and theaflavin) were selected based on these characteristics. Six blood dilutions (neat, 1:10, 1:50, 1:100, 1:500, and 1:1000) were exposed to the interferents, and presumptive tests for blood were conducted on six days (day 1, 8, 22, 43, 71, and 106). The second part of the study examined bloodstains deposited on real-world samples (wines, citrus fruit juices, teas, coffee, cleaning agents, and leather products) containing chemical interferents. In addition, confirmatory testing for human blood was conducted with HemaTrace® and RSID™-Blood on day 106 using the 1:500 dilution. Finally, DNA analysis of 1:10 dilution stains was performed on day 150 to study whether downstream DNA analysis was compromised due to the presence of the interferents. The results showed that as blood concentration reduced, more false-negative results were observed when chemical interferents were present. Further, chemical interferents produced frequent atypical color changes in tests with KM and O-tol reagents, while only some atypical color changes were observed with the household products tested. Immunochromatographic assay results indicated both HemaTrace® and RSID™-Blood could detect the presence of blood when interfering agents are present, although the positive result bands with RSID™-Blood were faint and sometimes difficult to visualize. Poor DNA results from the untreated blood sample limited any interpretation of the DNA results obtained from bloodstains deposited on household products. Overall, the data indicates that valuable blood evidence may be overlooked due to faint or false-negative results when these interferents are present. Future studies should focus on how these interferents may affect downstream DNA analysis.

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