Over 500 million people worldwide suffer from disease associated with intestinal inflammation, including gastric cancer, inflammatory bowel disease, h. pylori infections, and numerous viral and bacterial infections. Although potentially effective therapeutics exist for many of these pathologies, delivery challenges thwart their clinical viability. The objective of this work was to develop drug delivery platforms that could target toxic immunomodulatory therapeutics to diseased intestinal tissues. To meet this objective, we developed an oral delivery vehicle for siRNA and an NF-κB inhibiting nanoparticle that reduces drug-resistance. Small interfering RNA (siRNA) represents a promising treatment strategy for numerous gastrointestinal (GI) diseases; however, the oral delivery of siRNA to inflamed intestinal tissues remains a major challenge. In this presentation, we describe a delivery vehicle for siRNA, termed thioketal nanoparticles (TKNs), that can orally deliver siRNA to sites of intestinal inflammation, and thus inhibit gene expression in diseased intestinal tissue. Using a murine model of ulcerative colitis, we demonstrate that orally administered TKNs loaded with TNFα-siRNA (TNFα-TKNs) diminish TNFα messenger RNA (mRNA) levels in the colon and protect mice from intestinal inflammation. Activation of nuclear factor-κB (NF-κB) results in the expression of numerous prosurvival genes that block apoptosis, thus mitigating the efficacy of chemotherapeutics. Paradoxically, all conventional therapeutics for cancer activate NF-κB, and in doing so initiate drug resistance. Although adjuvant strategies that block NF-κB activation could potentiate the activity of chemotherapeutics in drug resistant tumors, clinical evidence suggests that current adjuvant strategies also increase apoptosis in non-malignant cells. In this presentation, we present a nanoparticle, formulated from a polymeric NF-κB-inhibiting prodrug, that target the chemotherapeutic irinotecan (CPT-11) to solid tumors, and thus abrogates CPT-11-mediated drug resistance and inhibits tumor growth. In order to maximize the amount of NF-κB inhibitor delivered to tumors, we synthesized a novel polymeric prodrug, termed PCAPE, that releases the NF-κB inhibitor caffeic acid phenethyl ester (CAPE) as its major degradation product. Using a murine model of colitis-associated cancer, we demonstrate that when administered systemically, CPT-11-loaded PCAPE-nanoparticles (CCNPs) are three time more effective than a cocktail of the free drugs at reducing both tumor multiplicity and tumor size.

An online, semi-continuous instrument to measure both total and gas phase atmospheric reactive oxygen species (ROS) and determine the concentration of ROS in the particle phase (ROS(p)) was developed. This instrument was based on a fluorescent probe for quantifying ambient ROS, specifically 2'7'-dichlorodihydrofluorescin, or DCFH probe. This probe was analyzed for sensitivity to a variety of offline and online parameters for efficient use in a field instrument. The ROS(p) instrument measures the peak light intensity at 530 nm to determine ambient ROS concentrations. ROS particles and gases are collected in a mist chamber in a nebulized mist. The instrument alternates measurements of ROS(p+g), or ROS(tot) by means of an inline filter. Fine (PM₂.₅) (ROS(p) is determined by subtraction of the ROS(g) concentration from the ROS(tot), as the ROS(g) signal could not be excluded. This instrument was tested during the summer (May-July) of 2012 at urban and rural sites in the metropolitan Atlanta and surrounding region. Concentrations of ROS(p) determined from this instrument were often below limit of detection. Average concentrations of ROS(p) were found to be 0.25 nmol/m³ in urban Atlanta (Jefferson St. and Georgia Tech), and 0.15 nmol/m³ in Yorkville, a rural site. A side by side comparison of this method with a filter collection method was made in July. The average ROS(p) offline concentrations were 0.15 nmol/m³. These concentrations were comparable to the online average concentrations of 0.21 nmol/m³ for the same period of time. This average and the majority of the measurements comprising it is dominated by the high limit of detection. The ROS instrument as constructed and operated is an efficient way to conduct ROS(p) measurements at the level of a filter study while reducing the labor intensive filter collection and extraction. In order for this instrument to be successful at measuring ambient ROS in the particle phase, the removal of the gas phase from the current sampling scheme is critical as the ROS(g) concentrations are over 90% of the measured ROS. The system as currently operable is best suited for source measurements, including biomass burning plumes or fresh exhaust to capture immediate formation.

<p>Methylglyoxal (MG) is a reactive glucose metabolite and a known causative factor for hypertension and diabetes. Hydrogen sulfide (H₂S), on the other hand, is a gasotransmitter with multifaceted physiological functions, including anti-oxidant and vasodilatory properties. The present study demonstrates that MG and H₂S can interact with and modulate each other's functions. Upon <i>in vitro</i> incubations, we found that MG and H₂S can directly interact to form three possible MG-H₂S adducts. Furthermore, the endogenous production level of MG or H₂S was significantly reduced in a concentration-dependent manner in rat vascular smooth muscle cells (A-10 cells) treated with NaHS, a H₂S donor, or MG, respectively. Indeed, MG-treated A-10 cells exhibited a concentration-dependent down-regulation of the protein and activity level of cystathionine &gamma;-lyase (CSE), the main H₂S-generating enzyme in the vasculature. Moreover, H₂S can induce the inhibition of MG-generated ROS production in a concentration-dependent manner in A-10 cells. In 6-22 week-old CSE knockout male mice (CSE^-/-), mice with lower levels of vascular H₂S, we observed a significant elevation in MG levels in both plasma and renal extracts. Renal triosephosphates were also significantly increased in the 6-22 week-old CSE^-/- mice. To identify the source of the elevated renal MG levels, we found that the activity of fructose-1,6-bisphosphatase (FBPase), the rate-limiting enzyme in gluconeogenesis, was significantly down-regulated, along with lower levels of its product (fructose-6-phosphate) and higher levels of its substrate (fructose-1,6-bisphosphate) in the kidney of 6-22 week-old CSE^-/- mice. We have also observed lower levels of the gluconeogenic regulator, peroxisome proliferator-activated receptor-&gamma; coactivator (PGC)-1&alpha;, and its down-stream targets, FBPase-1 and -2, phosphoenolpyruvate carboxykinase (PEPCK), and estrogen-related receptor (ERR)&alpha; mRNA expression levels in renal extracts from 6-22 week-old CSE^-/- mice. Likewise, FBPase-1 and -2 mRNA levels were also significantly down-regulated in aorta tissues from 14-16 week-old CSE^-/- mice. Administration of 30 and 50 &#x00B5;M NaHS induced a significant increase in FBPase-1 and PGC-1&alpha; in rat A-10 cells. We have also observed a significant up-regulation of PEPCK and ERR&alpha; mRNA expression levels in 50 &#x00B5;M NaHS-treated A-10 cells, further confirming the involvement of H₂S in regulating the rate of gluconeogenesis and MG formation. Overall, this unique study demonstrates the existence of a negative correlation between MG and H₂S in the vasculature. Further elucidation of this cross-talk phenomenon between MG and H₂S could lead to more elaborate and effective therapeutic regimens to combat metabolic syndrome and its related health complications.</p>

Despite extensive preventative efforts, the problem of controlling infections associated with biomedical materials persists. Bacteria tend to colonize on biocompatible materials and form biofilms; thus, novel biomaterials with antibacterial properties are of great interest. In this thesis, titanium dioxide (TiO2)-associated photocatalysis under ultraviolet (UV) irradiation was investigated as a strategy for developing bioactivity and antibacterial properties on biomaterials. Although much of the work was specifically directed towards dental materials, the results presented are applicable to a wide range of biomaterial applications. Most of the experimental work in the thesis was based on a resin-TiO2 nanocomposite that was prepared by adding 20 wt% TiO2 nanoparticles to a resin-based polymer material. Tests showed that the addition of the nanoparticles endowed the adhesive material with photocatalytic activity without affecting the functional bonding strength. Subsequent studies indicated a number of additional beneficial properties associated with the nanocomposite that appear promising for biomaterial applications. For example, irradiation with UV light induced bioactivity on the otherwise non-bioactive nanocomposite; this was indicated by hydroxyapatite formation on the surface following soaking in Dulbecco’s phosphate-buffered saline. Under UV irradiation, the resin-TiO2 nanocomposite provided effective antibacterial action against both planktonic and biofilm bacteria. UV irradiation of the nanocomposite also provided a prolonged antibacterial effect that continued after removal of the UV light source. UV treatment also reduced bacterial adhesion to the resin-TiO2 surface. The mechanisms involved in the antibacterial effects of TiO2 photocatalysis were studied by investigating the specific contributions of the photocatalytic reaction products (the reactive oxygen species) and their disinfection kinetics. Methods of improving the viability analysis of bacteria subjected to photocatalysis were also developed.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants that can be modified to oxygenated PAH (oxyPAHs) derivatives. It is well known that oxyPAHs tend to be much more reactive than their parent compounds. Toxicity can be attributed to direct interaction with target molecules or generation of reactive oxygen species (ROS). Metals are another class of contaminant found ubiquitously throughout the environment. Some metals are toxic at levels below the 1:1 ratio predicted by the biotic ligand model and are thought to manifest toxicity through ROS generation. Often metals and PAHs occur as co-contaminants in industrialized environments, yet little is known about their potential co-toxicity or mechanisms of action in mammalian reproductive function. Previously, we described that a PAH, 9, 10-phenanthrenequinone (PHEQ), inhibited LH-stimulated progesterone secretion in dispersed rat corpus luteal (CL) cells (Nykamp et al., 2001). Viability was decreased in CL cells exposed to PHEQ and 1,2-dihydroxy-anthraquinone (1,2-dhATQ), but not their parent compounds phenanthrene (PHE) or anthracene (ANT). Similarly, LH-stimulated progesterone production in CL cells was inhibited by PHEQ and 1,2-dhATQ, but not PHE. Further investigation revealed that PHEQ, but not PHE, ANT nor 1,2-dhATQ generated ROS in CL cells. Viability experiments were repeated using the choriocarcinoma cell line JEG-3 with similar results. Various metals were assessed for their toxicity to both CL and JEG-3 cells. The endpoints used to measure viability were metabolic activity and membrane integrity. In general, metabolic activity was a more sensitive indicator of toxicity than membrane integrity. The order of toxicity for metals in CL cells was Hg2+ > Cd2+ > Zn2+ > Ni2+ > Cu2+ for metabolic activity and Hg2+ ≈ Zn2+ > Cd2+ > Cu2+ > Ni2+ for membrane integrity. Only Hg2+ and Cu2+ were tested in JEG-3 cells. While Cu2+ was non-toxic, EC50s for Hg2+ metabolic activity and membrane integrity were 20 mM and 23 mM, respectively. Experiments were designed to study the mixtures of metals and PAHs on viability, ROS production, and LH-stimulated progesterone production in CL cells. Mixtures of each metal with either PHEQ or 1,2-dhATQ were incubated with CL cells and their effect on metabolic activity and membrane integrity assessed. Generally, most metal/oxyPAH mixtures displayed only additive toxicity. However, mixtures of Cu2+ and PHEQ showed synergistic toxicity to both metabolic activity and membrane integrity. Mixture studies in JEG-3 cells used only combinations of Cu2+ or Hg2+ with PHEQ or 1,2-dhATQ. Similar results to metabolic activity and membrane integrity in CL cells were observed. Mixtures of Cu2+ and PHEQ or 1,2-dhATQ were tested in CL cells for their effect on LH-stimulated progesterone secretion and ROS production. Additive effects were observed in both LH-stimulated progesterone secretion and ROS production for Cu2+/1,2-dhATQ mixtures while synergistic effects for both parameters were seen with Cu2+/PHEQ. Efforts to determine the site of action for mixtures of Cu2+/PHEQ involved adding the cholesterol analogue, 22-OH cholesterol (22-OHC) to CL cells in the absence of LH. Cytochrome P450 side-chain cleavage (CYP450scc) enzyme operates constitutively and the addition of 22-OHC to CL cells resulting in a 5-fold increase in progesterone production without added LH. Kinetic assays with 22-OHC show that while progesterone secretion was inhibited with PHEQ addition alone, a further significant reduction with both Cu2+ and PHEQ was not observed. The use of forskolin, an activator of adenylate cyclase, did not show any significant enhancement of progesterone secretion with the addition of Cu2+/PHEQ compared to PHEQ alone. The potential targets of Cu2+/PHEQ mixture include any step in the steroidogenic cascade from activation of protein kinase A onward with the proteins of the mitochondria, cytochrome P450 side chain cleavage enzyme and steroidogenic acute regulatory protein, being the most likely. Differential display polymerase chain reaction (ddPCR) was a molecular approach taken to determine the effect of PHEQ on JEG-3 gene expression. The genes whose expression appeared to be up-regulated with PHEQ exposure were serine protease inhibitor, Alu repeat sequence, heterogeneous ribonuclear ribonucleoprotein C (hnRNP C), eukaryotic translation initiation factor 3 (eIF3), nucleoporin-like protein, eukaryotic translation elongation factor 1a1 (eEF1 a 1), autophagy-linked FYVE domain (Alfy), spectrin, and proteasome. Apparent down-regulated genes in JEG-3 cells after PHEQ exposure included poly(ADP-ribose) polymerase 10 (PARP10), polyglutamine binding protein-1 (PQBP-1), heterogeneous ribonuclear ribonucleoprotein C (hnRNP C), eukaryotic translation initiation factor 5A (eIF5A), and keratin. In both cell types, oxyPAHs were more toxic than their parent compounds. Metals showed greater toxicity to metabolic activity than to membrane integrity. Of the combinations tested, only PHEQ and Cu2+ exhibited synergistic toxicity. ROS generation was the likely mechanism behind PHEQ/Cu2+ toxicity. Both cell types used represent critical roles in human reproductive health. The proper production of progesterone, a critical hormone for the maintenance of pregnancy in mammals, represents a unique endpoint for the assessment of toxicity. These results illustrate the need to study modified oxyPAHs, metals and metal/oxyPAH mixtures for their potential impact on human reproductive health.

<p>Methylglyoxal (MG) is a reactive glucose metabolite and a known causative factor for hypertension and diabetes. Hydrogen sulfide (H₂S), on the other hand, is a gasotransmitter with multifaceted physiological functions, including anti-oxidant and vasodilatory properties. The present study demonstrates that MG and H₂S can interact with and modulate each other's functions. Upon <i>in vitro</i> incubations, we found that MG and H₂S can directly interact to form three possible MG-H₂S adducts. Furthermore, the endogenous production level of MG or H₂S was significantly reduced in a concentration-dependent manner in rat vascular smooth muscle cells (A-10 cells) treated with NaHS, a H₂S donor, or MG, respectively. Indeed, MG-treated A-10 cells exhibited a concentration-dependent down-regulation of the protein and activity level of cystathionine &gamma;-lyase (CSE), the main H₂S-generating enzyme in the vasculature. Moreover, H₂S can induce the inhibition of MG-generated ROS production in a concentration-dependent manner in A-10 cells. In 6-22 week-old CSE knockout male mice (CSE^-/-), mice with lower levels of vascular H₂S, we observed a significant elevation in MG levels in both plasma and renal extracts. Renal triosephosphates were also significantly increased in the 6-22 week-old CSE^-/- mice. To identify the source of the elevated renal MG levels, we found that the activity of fructose-1,6-bisphosphatase (FBPase), the rate-limiting enzyme in gluconeogenesis, was significantly down-regulated, along with lower levels of its product (fructose-6-phosphate) and higher levels of its substrate (fructose-1,6-bisphosphate) in the kidney of 6-22 week-old CSE^-/- mice. We have also observed lower levels of the gluconeogenic regulator, peroxisome proliferator-activated receptor-&gamma; coactivator (PGC)-1&alpha;, and its down-stream targets, FBPase-1 and -2, phosphoenolpyruvate carboxykinase (PEPCK), and estrogen-related receptor (ERR)&alpha; mRNA expression levels in renal extracts from 6-22 week-old CSE^-/- mice. Likewise, FBPase-1 and -2 mRNA levels were also significantly down-regulated in aorta tissues from 14-16 week-old CSE^-/- mice. Administration of 30 and 50 &#x00B5;M NaHS induced a significant increase in FBPase-1 and PGC-1&alpha; in rat A-10 cells. We have also observed a significant up-regulation of PEPCK and ERR&alpha; mRNA expression levels in 50 &#x00B5;M NaHS-treated A-10 cells, further confirming the involvement of H₂S in regulating the rate of gluconeogenesis and MG formation. Overall, this unique study demonstrates the existence of a negative correlation between MG and H₂S in the vasculature. Further elucidation of this cross-talk phenomenon between MG and H₂S could lead to more elaborate and effective therapeutic regimens to combat metabolic syndrome and its related health complications.</p>

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants that can be modified to oxygenated PAH (oxyPAHs) derivatives. It is well known that oxyPAHs tend to be much more reactive than their parent compounds. Toxicity can be attributed to direct interaction with target molecules or generation of reactive oxygen species (ROS). Metals are another class of contaminant found ubiquitously throughout the environment. Some metals are toxic at levels below the 1:1 ratio predicted by the biotic ligand model and are thought to manifest toxicity through ROS generation. Often metals and PAHs occur as co-contaminants in industrialized environments, yet little is known about their potential co-toxicity or mechanisms of action in mammalian reproductive function. Previously, we described that a PAH, 9, 10-phenanthrenequinone (PHEQ), inhibited LH-stimulated progesterone secretion in dispersed rat corpus luteal (CL) cells (Nykamp et al., 2001). Viability was decreased in CL cells exposed to PHEQ and 1,2-dihydroxy-anthraquinone (1,2-dhATQ), but not their parent compounds phenanthrene (PHE) or anthracene (ANT). Similarly, LH-stimulated progesterone production in CL cells was inhibited by PHEQ and 1,2-dhATQ, but not PHE. Further investigation revealed that PHEQ, but not PHE, ANT nor 1,2-dhATQ generated ROS in CL cells. Viability experiments were repeated using the choriocarcinoma cell line JEG-3 with similar results. Various metals were assessed for their toxicity to both CL and JEG-3 cells. The endpoints used to measure viability were metabolic activity and membrane integrity. In general, metabolic activity was a more sensitive indicator of toxicity than membrane integrity. The order of toxicity for metals in CL cells was Hg2+ > Cd2+ > Zn2+ > Ni2+ > Cu2+ for metabolic activity and Hg2+ ≈ Zn2+ > Cd2+ > Cu2+ > Ni2+ for membrane integrity. Only Hg2+ and Cu2+ were tested in JEG-3 cells. While Cu2+ was non-toxic, EC50s for Hg2+ metabolic activity and membrane integrity were 20 mM and 23 mM, respectively. Experiments were designed to study the mixtures of metals and PAHs on viability, ROS production, and LH-stimulated progesterone production in CL cells. Mixtures of each metal with either PHEQ or 1,2-dhATQ were incubated with CL cells and their effect on metabolic activity and membrane integrity assessed. Generally, most metal/oxyPAH mixtures displayed only additive toxicity. However, mixtures of Cu2+ and PHEQ showed synergistic toxicity to both metabolic activity and membrane integrity. Mixture studies in JEG-3 cells used only combinations of Cu2+ or Hg2+ with PHEQ or 1,2-dhATQ. Similar results to metabolic activity and membrane integrity in CL cells were observed. Mixtures of Cu2+ and PHEQ or 1,2-dhATQ were tested in CL cells for their effect on LH-stimulated progesterone secretion and ROS production. Additive effects were observed in both LH-stimulated progesterone secretion and ROS production for Cu2+/1,2-dhATQ mixtures while synergistic effects for both parameters were seen with Cu2+/PHEQ. Efforts to determine the site of action for mixtures of Cu2+/PHEQ involved adding the cholesterol analogue, 22-OH cholesterol (22-OHC) to CL cells in the absence of LH. Cytochrome P450 side-chain cleavage (CYP450scc) enzyme operates constitutively and the addition of 22-OHC to CL cells resulting in a 5-fold increase in progesterone production without added LH. Kinetic assays with 22-OHC show that while progesterone secretion was inhibited with PHEQ addition alone, a further significant reduction with both Cu2+ and PHEQ was not observed. The use of forskolin, an activator of adenylate cyclase, did not show any significant enhancement of progesterone secretion with the addition of Cu2+/PHEQ compared to PHEQ alone. The potential targets of Cu2+/PHEQ mixture include any step in the steroidogenic cascade from activation of protein kinase A onward with the proteins of the mitochondria, cytochrome P450 side chain cleavage enzyme and steroidogenic acute regulatory protein, being the most likely. Differential display polymerase chain reaction (ddPCR) was a molecular approach taken to determine the effect of PHEQ on JEG-3 gene expression. The genes whose expression appeared to be up-regulated with PHEQ exposure were serine protease inhibitor, Alu repeat sequence, heterogeneous ribonuclear ribonucleoprotein C (hnRNP C), eukaryotic translation initiation factor 3 (eIF3), nucleoporin-like protein, eukaryotic translation elongation factor 1a1 (eEF1 a 1), autophagy-linked FYVE domain (Alfy), spectrin, and proteasome. Apparent down-regulated genes in JEG-3 cells after PHEQ exposure included poly(ADP-ribose) polymerase 10 (PARP10), polyglutamine binding protein-1 (PQBP-1), heterogeneous ribonuclear ribonucleoprotein C (hnRNP C), eukaryotic translation initiation factor 5A (eIF5A), and keratin. In both cell types, oxyPAHs were more toxic than their parent compounds. Metals showed greater toxicity to metabolic activity than to membrane integrity. Of the combinations tested, only PHEQ and Cu2+ exhibited synergistic toxicity. ROS generation was the likely mechanism behind PHEQ/Cu2+ toxicity. Both cell types used represent critical roles in human reproductive health. The proper production of progesterone, a critical hormone for the maintenance of pregnancy in mammals, represents a unique endpoint for the assessment of toxicity. These results illustrate the need to study modified oxyPAHs, metals and metal/oxyPAH mixtures for their potential impact on human reproductive health.

Plant programmed cell death (PCD) is an essential process in plant-pathogen interactions. Importantly, PCD can have contrasting effects on the outcome depending on context. For example, plant PCD in plant-biotroph interactions is clearly beneficial to plants, whereas it could be detrimental to plants in plant-necrotroph interactions. Sclerotinia sclerotiorum is an agriculturally and economically important necrotrophic pathogen. Previous studies have shown that S. sclerotiorum secretes oxalic acid (OA) to enhance Sclerotinia virulence by various mechanisms including induction of PCD in plants. A recent study has also shown that reactive oxygen species (ROS) generation correlates with induction of PCD during disease development. These studies focus on links between ROS, oxalate, and PCD, and how they impact S. sclerotiorum disease development. I examined the involvement of ROS in pathogenic development of S. sclerotiorum. I identified and functionally characterized two predicted S. sclerotiorum NADPH oxidases (Nox1 and Nox2) by RNAi. Both nox genes appear to have roles in sclerotial development, while only Nox1-silenced mutants showed reduced virulence. Interestingly, the reduced virulence of the Nox1-silenced mutant correlated with decreased production of OA in the mutant. This observation suggests that regulation of ROS by S. sclerotiorum Nox1 may be linked to OA. The next study details the phenotype of plants inoculated with an S. sclerotiorum oxalate deficient mutant (A2), which showed restricted growth at the infected site. This response resembles the hypersensitive response (HR), and is associated with plant resistance responses including cell wall strengthening, plant oxidative burst, and induction of defensin genes. Conversely, leaves infected with wild type showed unrestricted spreading of cell death and were not associated with these resistant responses. Furthermore, previous work had shown that a Caenorhabditis elegans anti-apoptotic gene (ced-9) conferred resistance to wild type S. sclerotiorum, while this gene had negligible effects on the phenotype of plant leaves inoculated with A2 mutants. These findings suggest that HR-like cell death by A2 and PCD by wild type S. sclerotiorum may be regulated by different pathways. As a whole, these results reveal the importance of ROS, oxalate, and PCD in Sclerotinia disease development as well as the significance of interplay between them. These studies contribute to the understanding of the underlying mechanisms of Sclerotinia disease.

Thesis (Ph. D.)--West Virginia University, 2002. / Title from document title page. Document formatted into pages; contains xii, 216 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.

Lic.-avh. (sammanfattning) Stockholm : Karolinska institutet, 2010.