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Malaria parasite (Plasmodium spp.) infection in the mosquito Anopheles stephensi induces significant expression of A. stephensi nitric oxide synthase (AsNOS) in the midgut epithelium as early as 6 h post-infection and intermittently thereafter. This induction results in the synthesis of inflammatory levels of nitric oxide (NO) in the blood-filled midgut that limit parasite development. However, the Plasmodium-derived factors that can induce AsNOS expression and the signaling pathways responsible for transduction in A. stephensi have not been identified until completion of the work described herein. In my studies, I have determined that P. falciparum glycosylphosphatidylinositol (PfGPIs) can induce AsNOS expression in A. stephensi cells in vitro and in the midgut epithelium in vivo. Based on related work in mammals, I hypothesized that parasite-derived AsNOS-inducing factors signal through the insulin signaling pathway and the NF-kappaB-dependent Toll and Immune deficiency (Imd) signaling pathways. In support of this hypothesis, I have determined that signaling by P. falciparum merozoites and PfGPIs is mediated through A. stephensi protein kinase B (Akt/PKB) and DSOR1 (mitogen activated protein kinase kinase, MEK)/Extracellular signal-regulated protein kinase (ERK), kinases which are associated with the insulin signaling pathway. However, signaling by P. falciparum and PfGPIs is distinctively different from signaling by insulin and these parasite signals are not insulin-mimetic to A. stephensi cells. In other studies, treatment with pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-kappaB, reduced AsNOS expression by P. falciparum merozoites in A. stephensi cells. This result suggested the involvement of Toll and Imd pathways in parasite signaling of mosquito cells. Knockout of Pelle, a proximal signaling protein in the Toll pathway, increased AsNOS expression following parasite stimulation, suggesting that the Toll pathway may negatively regulate signaling by Plasmodium-derived AsNOS-inducing factors. In contrast, knockout of TGF-beta-activated kinase 1 (Tak1), a proximal signaling protein in the Imd pathway, reduced AsNOS expression by 20% relative to the control, suggesting that the Imd pathway is required for signaling by Plasmodium-derived AsNOS-inducing factors. Despite the NO-rich environment of the midgut, Plasmodium development is not completely inhibited. This observation suggests that Plasmodium may have efficient detoxification systems during sexual development in A. stephensi. To identify Plasmodium defense genes that may defend parasites against nitrosative stress caused by AsNOS induction, expression of several antioxidant defense genes known to function in nitrosative stress defense in a variety of organisms were examined during sporogonic development. Notably, increased expression levels of P. falciparum peroxiredoxins containing 1 or 2 cysteines (1-cys or 2-cys PfPrx) were associated with periods of parasite development just prior to and during parasite penetration of midgut epithelium, an event associated with significant AsNOS induction in the midgut. The provision of N omega-L-arginine (L-NAME), a known inhibitor of NOS enzyme activity, to A. stephensi with Plasmodium culture by artificial bloodmeal significantly reduced expression of 1-cys and 2-cys PfPrx indicating that these gene products may function to protect parasites against nitrosative stress induced by AsNOS. / Ph. D.

Both vertebrates and anopheline mosquitoes inhibit <i>Plasmodium</i> spp. (malaria parasite) development via induction of nitric oxide (·NO) synthase. Expression of <i>Anopheles stephensi</i> ·NO synthase (<i>AsNOS</i>) is induced in the midgut epithelium beginning at 6 h following a <i>Plasmodium berghei</i>-infected blood meal. ·NO reacts readily with other biocompounds forming a variety of reactive nitrogen intermediates (RNIs) that may impose a nitrosative stress. These RNIs are proposed to be responsible for the AsNOS-dependent inhibition of <i>Plasmodium</i> development. In my studies, I identified several RNIs that are induced in the blood-filled midgut in response to <i>Plasmodium</i> infection. Stable end products of ·NO (NO₃⁻ and NO₂⁻), measured using a modified Griess assay, are elevated in infected midguts at 24 h post-blood meal (pBM). Further studies using chemical reduction-chemiluminescence with Hg displacement showed that infected midguts contained elevated levels of potentially toxic higher oxides of nitrogen (NO<SUB>x</SUB>), but <i>S</i>-nitrosothiol (SNO) and nitrite levels did not differ between infected and uninfected midguts at 12.5 and 24 h pBM. Thus, nitrates contributed to elevated NO<SUB>x</SUB> levels. SNO-biotin switch westerns indicated that <i>S</i>-nitrosated midgut proteins change over the course of blood meal digestion, but not in response to infection. Photolysis-chemiluminescence was used to release and detect bound ·NO from compounds in blood-filled midguts dissected from 0-33 h pBM. Results showed increased ·NO levels in <i>Plasmodium</i>-infected midgut lysates beginning at 8 h, with significant increases at 12.5-13.5 h and 24-25.5 h pBM and peak levels at 20-24 h. Photolyzed ·NO is derived from SNOs and metal nitrosyls. Since SNO concentrations did not change in response to infection, I proposed that metal nitrosyls, specifically Fe nitrosyl hemoglobin (nitrosylHb) based on the concentration of hemoglobin, were elevated in the infected midgut. At 12-24 h pBM, levels of midgut RNIs in infected mosquitoes were typical of levels measured during mammalian septic inflammation. The inverse relationship between AsNOS activity and parasite abundance indicates that nitrosative stress has a detrimental effect on parasite development. However, nitrosative stress may impact mosquito tissues as well in a manner analogous to mammalian tissue damage during inflammation. Elevated levels of nitrotyrosine (NTYR), a marker for nitrosative stress in many mammalian disease states, were detected in tissues of parasite-infected <i>A. stephensi</i> at 24 h pBM. Greater nitration of tyrosine residues was detected in the blood bolus, midgut epithelium, eggs and fat body. In the midgut, Hb remained in an oxygenated state for the duration of blood digestion. The reaction between ·NO and oxyhemoglobin (oxyHb) can result in the formation of nitrate and methemoglobin (metHb). Although nitrate levels were elevated in response to parasite infection, there was little to no metHb present in the mosquito midgut. The simultaneous presence of nitrates, nitrosylHb, oxyHb, and NTYR, together with a lack of elevated nitrites and metHb, suggested that alternative reaction mechanisms involving â ¢NO had occurred in the reducing environment of the midgut. In addition, I proposed that nitroxyl and peroxynitrite participated in reactions that yielded observed midgut RNIs. To cope with the parasite-induced nitrosative stress, cellular defenses in the mosquito may be induced to minimize self damage. I proposed that peroxiredoxins (Prx), enzymes that can detoxify peroxides and peroxynitrite, may protect <i>A. stephensi</i> from nitrosative stress. Six Prx genes were identified in the <i>A. gambiae</i> genome based on homology with known <i>D. melanogaster</i> Prxs. I identified one <i>A. stephensi</i> Prx, AsPrx, that shared 78% amino acid identity with a <i>D. melanogaster</i> 2-Cys Prx known to protect fly cells against various oxidative stresses. <i>AsPrx</i> was expressed in the midgut epithelium and is encoded by a single-copy, intronless gene. Quantitative RT-PCR analyses confirmed that induction of <i>AsPrx</i> expression in the midgut was correlated with malaria parasite infection and nitrosative stress. To determine whether AsPrx could protect against RNI- and ROS-mediated cell death, transient transfection protocols were established for AsPrx overexpression in <i>D. melanogaster</i> (S2) and <i>A. stephensi</i> (MSQ43) cells and for <i>AsPrx</i> gene silencing using RNA interference in MSQ43 cells. Viability assays in MSQ43 cells showed that AsPrx conferred protection against hydrogen peroxide, ·NO, nitroxyl and peroxynitrite. These data suggested that the ·NO-mediated defense response is toxic to both host and parasite. However, AsPrx may shift the balance in favor of the mosquito. / Ph. D.

Experiments were conducted to evaluate nitric oxide (NO) involvement in the leptin effect on food intake in both broiler and Leghorn chickens. The first experiment studied the effect of leptin combined with L-arginine on the food intake in broilers. Intracerebroventricular (ICV) administration of human recombinant leptin injection decreased (P=.01) food intake from 15 to 150 minutes compared to the control group treated with artificial cerebrospinal fluid ( aCSF) while food intake was increased by L-arginine. Food intake between the group receiving leptin and L-arginine was similar to the control group. Therefore, broilers were sensitive to the anoregenic effects of leptin, while L-arginine, a NO precursor appeared to attenuate the leptin effect on food intake. The effect of leptin and L-NNA on food intake in broilers was measured in the second experiment. Lepin, L-NNA and leptin plus L-NNA decreased food intake. The NO inhibitor L-NNA tended to enhance the suppression of leptin on food intake. In the third experiment, using Leghorns instead of broilers, the ICV injection of leptin decreased food intake from 15 to 60 minutes postinjection (P=.05). However, food intake was not affected by injection of L-arginine plus leptin. Therefore, L-arginine appeared to antagonize the leptin inhibitory effect on food intake. A small increase food intake induced by L-arginine was also observed (P=.09). The change of food intake in Leghorns administered leptin and L-NNA were measured in Experiment 4. Food intake was decreased by L-NNA and leptin with the effects lasting 60 minutes, similar to that observed in broilers (P<0.0l). For group B (leptin treatment), there was decreased food intake within 45 minutes (P=.04) and the effect disappeared 60 minutes, post injection. Also, the results along with Experiment 2 demonstrated that NO mediated the effect of leptin in Leghorns. The fifth experiment investigated the change in concentration of metabolites of nitric oxide after injection of leptin within 30 minutes. The group treated with the leptin had a lower level of metabolites of nitric oxide in the hypothalamus than the control group (P=.004). This effect further demonstrates that leptin modulated feeding activity through its inhibition on nNOS activity in the hypothalamus. These results showed that both leptin and NO participated in the regulation of food intake in broiler and Leghorn chickens, and the effect of hypothalamic neuropeptidergic circuitry leptin on food intake was mediated by NO. / Master of Science

Conventional measurements of NO and NO₂ produced by a diffusion flame around a cotton ball wetted by heptane have been performed and prove that NO is oxidized to NO₂ on a mole for mole basis when the air of the flame is doped with hydrogen and that the NO to NO₂ mechanism does not require carbon atoms in the dopant. In-situ spectroscopic measurements of NO in a laminar H₂-air diffusion flame were performed and compared to data obtained with probe sampling procedures. Ultraviolet absorption of the (1,0) gamma bands of nitric oxide near 214.8 nm were used for the spectroscopy. Spectroscopic measurements were possible only when the air stream was seeded with ca. 100 ppm NO. A conventional sampling system was operated at a probe pressure of 0.3 atmosphere and was used to sample from both the high temperature combustion zone and relatively cool regions on both sides of the flame. Spectroscopic and probe measurements of NO agree to within 30%, with probe concentrations being greater. The air of the flame was doped to give 1200 ppm methane and the NO concentrations were measured again, using probe and spectroscopic techniques. Both techniques confirm that even small unburned hydrocarbon concentrations cause the disappearance of NO on the air side of the visible reaction zone. / Master of Science

Nitric oxide (NO) is a minor species in the Earth’s atmosphere whose densities have been measured to closely reflect solar energy deposition above 100 km. It is an efficient emitter in the infrared where the thermosphere is optically thin, and serves as an important source of radiative cooling between 100 - 200 km. The primary mechanism of this cooling involves the conversion of kinetic energy from the background atmosphere into vibrational energy in NO, followed by the radiative de-excitation of the NO molecule. This results in the production of a 5.3 µm photon which escapes the thermosphere and results in a net cooling of the region. While this process causes the excitation of ground state NO to its first vibrational level, nascent vibrational excitation to the (v≥ 1) levels may also occur from the reactions that produce NO in the thermosphere. The NO(v≥ 1) molecules produced from this secondary process can undergo a radiative cascade and emit multiple photons, thus forming a significant fraction of the 5.3 µm emission from NO in the thermosphere. Existing thermospheric models consider the collisional excitation of NO to be the only source of the 5.3 µm emission and assume the contribution from nascent excitation to be negligible. These models also tend to use a rate coefficient for the collisional excitation that is significantly larger than the values suggested in literature in order to obtain a temperature profile that is in agreement with empirical data. We address these discrepancies by presenting an updated calculation of the chemically produced emission by accounting for the v ≤ 10 level populations. By incorporating this process into a three dimensional global upper atmospheric model, it is shown that the additional emission contributes between 5 − 40% of the daytime emission from nitric oxide under quiet solar conditions, and is a significant source of energy loss during periods of enhanced solar energy deposition. Accounting for this process however does not resolve the model-data discrepancy seen with regards to the recovery times of thermospheric densities following geomagnetic storms, suggesting that an improved treatment of nitric oxide chemistry is required to resolve this issue. In order to improve our understanding of the thermospheric energy budget, we also develop the Atmospheric Chemistry and Energetics (ACE) 1D model using up-to-date aeronomic results. The model self-consistently solves the 1D momentum and energy equations to produce a global average profile of the coupled thermosphere and ionosphere system in terms of its constituent densities and temperatures. The model calculations of neutral densities and exospheric temperatures are found to be in good agreement with empirical data for a wide range of solar activity. It is concluded from the present work that while the magnitude of the chemically produced emission from nitric oxide has previously been underestimated, its effect on the thermospheric energy budget is relatively small. Including the secondary emission in thermospheric models results in an average reduction of 3% in the exospheric temperatures, which does not completely offset the change introduced by using a smaller rate coefficient for the collisional excitation of NO. However, thermospheric temperatures can still be accurately modeled by including these changes as part of broader improvements to calculations of the thermospheric energy budget. / Ph. D. / Nitric oxide (NO) is a molecule that is produced in the Earth’s thermosphere (the region of the atmosphere above 100 kilometers) as a consequence of solar energy deposition. As an important source of radiative cooling, its presence significantly influences the temperature structure of the region. An accurate understanding of the associated energetics is thus vital towards the development of numerical models used to describe the thermosphere. Energy loss from the thermosphere due to nitric oxide begins with the vibrational excitation of the molecule either due to collisions or chemical processes, followed by the emission of one or more infrared photons which returns the molecule to the ground state. The photons produced escape the thermosphere resulting in a net energy loss from this region of the atmosphere. Existing thermospheric models generally account for the vibrational excitation of nitric oxide only via collisions, and have assumed chemical processes to be a negligible source of thermospheric energy loss. These models also assume a rate of collisional excitation that is significantly larger than the values suggested in literature in order to obtain a temperature profile that is in agreement with empirical data. The present work demonstrates that the chemical excitation in fact contributes to between 5 − 40% of the total energy loss due to nitric oxide under quiet solar conditions on the dayside of the Earth, and is also an important energy loss mechanism during periods of enhanced solar activity. However, including this mechanism into existing models does not resolve outstanding model-data discrepancies regarding the rate at which the thermosphere returns to equilibrium following sudden enhancements in solar energy deposition. This suggests the need for an improved treatment of the nitric oxide chemistry in current thermospheric models. This work also presents the Atmospheric Chemistry and Energetics (ACE) 1D model, a new one dimensional upper atmospheric model developed in order to obtain a better understanding of the thermospheric energy budget. The model includes the effects of the chemically produced emissions from nitric oxide, and also uses a collisional cooling rate that is in line with the value suggested in literature. The model calculations of thermospheric densities and temperatures are shown to be in good agreement with empirical data over a wide range of solar activity.

The design of new, improved NO-donor drugs is an important pharmacological objective due to the biological importance of nitric oxide. N-Hydroxyguanidines represent a useful class of NO donors where the mechanism of action is based on the biosynthetic pathway for NO. Thirty new N-arylalkyl-N’-hydroxyguanidines were synthesized and their vasodilatation activity examined by myography in rat aortic rings. The observed relaxations were reversed by ODQ, which is an inhibitor of the guanylate cyclase, implying that this was an NO dependent vasodilatation. The most active compounds were also tested in the isolated perfused kidney (IPK) giving the vasodilatation properties. Preliminary results indicated that N-phenyl-N’- hydroxyguanidine showed the best pharmacological profile with EC₅₀= 19.9 μM and ca. 100% reversibility with ODQ. A series of N-phenylalkyl-N’-hydroxyguanidines were synthesised. NO donor activity was found to be fairly constant up to three methylene groups, and then decreased. Substitutions in the benzene ring of N-phenylethyl-N’-hydroxyguanidine demonstrated that various electron-withdrawing and electron-donating groups in the para position did not significantly affect the NO donor activity of this series of analogues. The nitro and trifluoromethyl substituted compounds gave the best biological profiles. Additionally, a novel heterocyclic, N–furfuryl-N’–hydroxyguanidine possessed very promising vasodilatation properties. In general, almost all the N-arylalkyl-N’-hydroxyguanidines behaved as potent NO donors in the rat aorta assay. In order to establish the influence of the free NH₂ group in the hydroxyguanidine functionality on the vasodilatation properties, N,N-dimethyl and N-methyl-N’- hydroxyguanidines were successfully synthesised. Unfortunately, they have not been tested yet in the biological assay. However, their NMR spectra showed some unusual features and their detailed analysis and X-ray data are presented herein. In addition a series of hydroxamic acids was synthesised and the NO donor activity investigated using the same biological methodology. It was found that the 3-phenylpropionohydroxamic acid was the most potent compound with EC₅₀ = 6 μM and ODQ = 96%. However, behavior in the IPK indicated that hydroxamic acids did not undergo the same biological pathway as in the rat aorta. Two different types of enzyme-activated pro-drugs were designed using N-hydroxyguanidines as the NO donating molecule. Synthetic studies towards these targets were carried out using various synthetic approaches. The desired molecules have not yet been synthesised but the chemistry explored so far has indicated potentially more successful approaches that could be attempted.