Background: Primary cilia are cellular organelles project from the cell surface of mammalian cell and play important roles in vertebrate development, organogenesis, health, and others genetic diseases. Primary cilium functions as a mechano-sensor and chemo-sensor. Defect in primary cilia causes the progression of polycystic kidney disease (PKD) which further leads to the inflammatory responses. We, therefore, investigated the role of Toll-like receptors 4 and 9 (TLR) in primary cilia towards PKD. Purpose: The main purpose of the proposed study is to identify and target the immune reactive proteins i.e. TLRs in the primary cilia. By targeting those primary cilia immune reactive proteins using suitable agonist and antagonists to study the control of cystic formation and their progression mechanisms. Methods: To target the ciliary immune TLR proteins (TLR4 and TLR9), we did immunostaining to evaluate their localization on primary cilia. Cilia lengths were measured and compared using differential interference contrast (DIC) and fluorescent imaging techniques. The in vitro3D cyst progression was monitored by adding agonists lipopolysaccharide (LPS) and oligodeoxynucleotides (ODN) and antagonist 4-hydroxy chloroquine (HCQ). Results: From our results we found that the TLR antagonist HCQ increases ciliary length in treated scrambled control, Pkd2knockout (KO) and TLR4KOcells as an immune response, whereas opposite results were observed with TLR9KO. However, the selected agonists for TLRs (LPS/ODN) increases cilia length in TLR9KO cells and decreases scrambled control, Pkd2KO and TLR4KO. In our 3D cyst cultures, we used agonists and antagonist for both the TLRs and observed that the cyst formations and progressions were inversely related to the cilia lengths. From these observations, we speculated that the new ciliary TLR proteins have a role in cystic progression. In conclusion, we found that the TLRs agonists/antagonist can modulate cilia length and TLRs role in inflammatory actions. The primary cilium already has central roles throughout cell biology, but here we propose, for the first time, that the cilium and the regulation of its structural importance in inflammation of PKD.

<p>Heavy water moderated thermal nuclear fission reactors have a greater inherent neutron economy than light water or graphite moderated reactor designs. Consequently, such units, operating on a variety of fuel cycles, may play an ever-increasing role in meeting future global energy demands. This thesis explores, analytically, the operational advantages and challenges associated with the use of a low enriched uranium (LEU) fuel cycle in advanced reactors based on the CANDU heavy water moderated, pressure tube design concept. The flexibility afforded through the use of LEU fuel is applied to enhance the operational and safety characteristics of reactors utilizing this fuel cycle. An investigation of factors influencing coolant void reactivity is conducted. Design modifications are introduced to reduce the coolant void reactivity, while maintaining the continued capability of high power operation. An enrichment and element radius graded fuel bundle design is developed with a central graphite core, an inner ring of 14 fuel elements, and an outer ring of 21 fuel elements. Fuel and lattice design perturbations are investigated to examine the effect of lattice pitch variations, capability of radioisotope production, the use of burnable poisons, and light water coolant. Xenon override requirements with LEU fuel are addressed. The efficacy of using modified two group (M-2) neutron diffusion theory for LEU fuel management studies is investigated. A modelling strategy is developed for the simulation of reactivity devices and fuel lattice properties using the M-2 methodology with a fixed energy cut-off. Detailed fuel management studies are conducted to examine the operational intricacies of LEU fuelling. Improved checkerboard type fuelling strategies are developed. Finally, the CANDU - Spectral Shift Advanced Thermal Reactor (CANDU-SSATR) is introduced and characterized. This multi-spectrum high burnup advanced reactor design utilizing simplified fuel management strategies holds great promise for the future.</p> / Doctor of Philosophy (PhD)

DNA double strand breaks (DSBs ) are extremely toxic to cells because they can lead to genomic rearrangements and even cell death. Two main pathways can repair DSBs: the homologous recombination repair (HRR) pathway and the non-homologous end-joining (NHEJ) pathway. NHEJ is the primary repair pathway in mammalian cells. HRR repairs single strand breaks (SSBs) or DSBs, mostly during late S phase and G2 phase of the cell cycle, by using an undamaged copy of the DNA sequence, and is therefore largely error-free, while the NHEJ pathway repairs DSBs without the requirement for sequence homology, may be error-free or error-prone, and is most active during G1 phase. Thymine glycol (Tg), the most common oxidation product of thymine. It produced endogenously as a consequence of aerobic metabolism or via exogenous factors such as ionizing radiation (IR), it is one of the predominant types of base modifications produced by ionizing radiation. Due to clustering of radiation – induced ionizations, Many DSBs induced by ionizing radiation bear damaged bases, including (Tg) moieties at or near the DSB ends that may interfere with subsequent gap filling and ligation. Artemis is a nuclease that is involved in the processing of termini during repair of DSBs and in modifying termini of complex DSBs. It has 5′–3′ exonuclease activity specific for single- stranded DNA, but, in the presence of DNA-PK, Artemis demonstrates endonuclease activity that is utilized in the removal of 3′ phosphoglycolate termini and 5′ overhangs, in the shortening of 3′ overhangs at DSBs, and in the opening of hairpin ends. To assess the ability of NHEJ to rejoin DSBs accompanied by Tg lesions and to elucidate the aspects of the possible role of Artemis in DSB repair, linearized plasmids with Tg either at the 3’ terminus of a blunt end (designated Tg1) or three or two bases from the end (Tg3 and Tg2, respectively), were subjected to a repair assay using XRCC4-like factor (XLF) deficient cell extracts, with or without the addition of XLF and/or Artemis, EndoIII and ddTTP. The data indicated that, the cell extract could ligate the plasmids with Tg1 and Tg2 with extremely low efficiency but could repair plasmid with Tg3 as efficiently as unmodified plasmid. In addition, Plasmids with Tg1and Tg2 were treated with Endonuclease III and ddTTP to test whether the end joining occurred before or after Tg removing, neither one had any effects on plasmids with Tg1. However, plasmids with Tg2 showed reduced in the intensity upon treatment with Endonucleases III and ddTTP, which suggested some ligation occur while Tg still present. In Artemis reaction, substrate with Tg2 and Tg3 could stimulate Artemis mediated trimming but not Tg1. Addition of EndoIII or ddTTP to plasmid with Tg3 resulted in a significant decrease in the intensities of the bandsrepresenting ligated products compared to XLF alone, suggesting that in some of the ligated products Tg are still present, while in others Tg had been removed and replaced by polymerization with normal nucleotides. Taken together, our results indicated that cell extract could ligate the plasmid with Tg located at the third base to DSB with high efficiency compared to plasmids with Tg1 and Tg2 which apparently this ability was severely inhibited when it located at or in the second position to DSB ends. Moreover, Artemis is also capable of trimming of thymine glycol at the second or third position from DSB ends with limited capability but inhibited by the presence of thymine glycol at the break site.

According to the World Health Organization, mental disorders represent the leading cause of disability in the US generating ~58 billion dollars in medical costs annually. Additionally, among the US population, ~40 million adults suffer from an anxiety disorder and ~14 million suffer from a major depressive disorder. The association between the persistence of these neurobehavioral conditions and central nervous system (CNS) levels of biogenic amines and metabolites has been studied for half a century. Further, a number of drugs interfering with neurotransmission/metabolism are used clinically for treatment of these disorders. Recently, some members of the solute carrier (SLC) superfamily, the SLC22 transporter family, which includes organic anion transporters (Oat1, Oat3), were found to be expressed and functional on the apical membrane of the choroid plexus, a component of the blood-cerebrospinal fluid barrier. The cells of this epithelia form tight junctions, which slows penetration of solutes into the brain and limits passive efflux of endogenous solutes from the brain. Therefore, Oat1 and Oat3 are poised to play an active role in the removal of NTs and metabolites from the CSF. Thus, a better understanding of the underlying roles of OATs in regulating CNS neurotransmitters and connecting their activity to complex behaviors may result in improved understanding of the processes governing CNS homeostasis. Basal locomotor, anxiety-like and depressive-like behaviors in mice of three genotypes (WT, Oat1-/-, and Oat3-/-) across ages (3-18 mo.) were evaluated using behavioral paradigms (e.g. open field activity (OFA), light-dark (LD), marble burying (MB), and tail suspension test (TST)). Secondly, a simple high performance liquid chromatography-ultraviolet/electrochemical detection (HPLC-UV/ECD) method was developed for quantitation of monoamines and metabolites in mouse whole brain. Following completion of behavioral assessments, whole brain concentrations of monoamines and metabolites were determined using the developed method. Lastly, a novel gas chromatography tandem mass spectrometry (GC-MS/MS) method was developed for quantitation of amino acid neurotransmitters, L-glutamic acid (GA) and γ-aminobutyric acid (GABA), in mouse whole brain. The developed method was used for measurement of whole brain concentrations of GA and GABA in a small subset of WT, Oat1-/-, and Oat3-/- mice at 3 and 18 mo.

Pharmacokinetic evaluation of 5-chloro-2-methoxy-N-[2-(4- sulfamoylphenyl)ethyl]benzamide in mouse plasma demanded for a suitable bioanalytical method. No reported bioanalytical method exists to-date that can quantify concentration of this compound in any biological matrix. The purpose of this study was 1) to develop and validate a new bioanalytical method using a micro-extraction and LC-MS/MS to quantify the target analyte in mouse plasma and 2) to partially validate the method in whole blood. A bioanalytical method was developed and validated in both matrices for a linear concentration range of 2-1000 ng/ml. For both matrices, the reverse predicted concentration of calibration standards (-8.95% to 12.16% and -9.54% to 12.90% respectively) and precision and accuracy (QCs) were within ±15% (%RSD and %BIAS). Four-hour bench top stability and post preparative stability results for plasma and whole blood matrices were within ±15% and ±20% respectively. Blood –plasma concentration correlation co-efficient was 0.9956 with a slope value of 1.018.

Studies were carried out on the feline adrenal gland to ascertain the role of prostaglandins in the mechanism of action of ACTH. Using tritiated arachidonic acid as a prostaglandin (PG) precursor , it was demonstrated by column and thin layer chromatography techniques that isolated trypsinized adrenocortical cells possess an active PG synthetase capable of synthesizing radiolabeled PGE, PGF, and PGA/B-like substances. Concentrations of ACTH (125 - 250 μU) which stimulate steroidogenesis enhanced the conversion of radiolabeled arachidonic acid to PGE, PGF and the PGA/B products extracted from cortical cells and incubation media. PG biosynthesis by isolated cortical cells was studied by radio immunoassay (RIA) using antisera generated against conjugates of PGE2 , PGF1α and PGF2α. PGF2α and PGE2 were identified as the primary PGs released by feline cortical cells, and steroidogenic concentrations of ACTH (50-250 μU) enhanced their release in a dose related manner. Indomethacin (10-5 M) inhibited PG and steroid release, whereas low indomethacin concentrations (10-9 M) potentiated ACTH-evoked PG and steroid release. The steroidogenic response to exogenous PGE2 was not markedly altered by indomethacin. 5 , 8, 11, 14- Eicosatetraynoic acid (ETA) inhibited PGE and PGF release , and elicited a concentration-dependent inhibition of ACTH-induced steroid release. Therefore, there appears to be a functional relationship between PG and steroid release. Such a relationship was further supported by studies on the perfused adrenal gland, which demonstrated that maximal PGF2α release in response to ACTH preceded the maximal steroidogenic response. Moreover, pregnenolone (3 μM) elicited a 30-fold increase in steroid release from isolated cortical cells but failed to augment PGF2α and PGE2 release; this study further supports the concept that PG synthesis occurs prior to the steroidogenic response to ACTH. Cycloheximide did not block the steroidogenic response to pregnenolone, but completely blocked the steroidogenic effects of ACTH. Cycloheximide also depressed basal PGF2α and PGE2 release , while ACTH-facilitated PG release was not significantly impaired. Thus, the enzymes responsible for increasing PG synthesis are activated rather than formed de novo in response to ACTH. Three steroidogenic agents, ACTH, an ACTH analogue NPS-ACTH, and monobutyryl cyclic AMP (BCAMP), increased PGF2α and PGE2 release from isolated adrenocortical cells. Calcium deprivation blocked PG and steroid release evoked by ACTH and NPS-ACTH, but only inhibited PG release elicited by BCAMP without affecting steroid release. These studies suggest a functional role for PGs in the mechanism of action of ACTH. Although the nature of this role remains to be elucidated, it appears to involve some complex interaction with calcium and cyclic nucleotides.

The goal of this project was to investigate the effect of input-rate (oral and intravenous) and degree of exposure on the pharmacokinetics of ethanol and on subjective and objective measures of impairment, as well as on the development of acute tolerance to the effects of ethanol in young, healthy volunteers. The primary objective of this research was to test the following hypotheses: 1) the rate and degree of ethanol exposure (oral and intravenous) in normal healthy males and females affect the pharrnacokinetics (PK) and pharmacodynarnics (PD) of ethanol in a non-linear fashion; 2) the EEG changes after ethanol administration correlate with changes in psychometric performance and subject-rated impairment, as well as serum ethanol concentrations; and 3) acute tolerance develops to the subjective effects of ethanol which is not reflected in changes in electroencephalographic (EEG) activity or psychometric performance. This study was conducted in two parts. Part I was a five-way crossover pilot study in six healthy male volunteers to evaluate the effect of dose and dose-rate on the PK and PD of ethanol. This study evaluated changes in EEG activity, psychometric performance and subjective impairment to evaluate the relationship between these subjective and objective measures, and the relationship between these measures and serum ethanol concentrations. Part II was a 4-way crossover study in 16 healthy male and female subjects to study the PK-PD relationship for intravenous (IV) ethanol and acute tolerance development to the effects of ethanol. In this study, subjects were administered individualized intravenous ethanol infusions, to achieve a target concentration of 1000 mg/L after different durations of exposure. This study was designed to i:nvestigate the PK of ethanol, as well as to assess changes in EEG activity, psychometric performance and subjective impairment. This study evaluated the relationship between these measures, and the relationship between these measures and serum ethanol concentrations, as well as the development of acute tolerance to the effects of ethanol. Results from both studies showed that: 1) Ethanol, after oral and intravenous administration, follows capacity-limited pharmacokinetics. Intrinsic PK parameters, V max• Km and Vd were independent of dose and input-rate, but were associated with fairly high inter-individual variability. 2) Ethanol, after oral and intravenous administration, induced a transient slowing of the EEG and impairment in psychometric performance. The magnitude of the changes in these measures appeared to be dose-related as well as inputrate- related (observed in the IV study), however there was a fairly large degree of variability in response between individuals. 3) Ethanol, after oral and intravenous administration, induced transient subjective impairment, which was dose-related and input-rate-related, and correlated with serum ethanol concentrations across treatments. 4) A subset of subjects (2/6 males in the oral ethanol study, and 2/8 males and 4/8 female subjects in the IV study) were classified as "non-responders" based on their lack of subjective response to ethanol, despite serum ethanol concentrations, psychometric impairment and EEG changes that were consistent with the other subjects. 5) There was significant exposure-related acute tolerance development to the subjective effects of ethanol observed in both studies. This acute tolerance development could be characterized by a PK-PD model incorporating tolerance as a compensatory feedback mechanism to counter-regulate the direct subjective impairment effect of the drug. Acute tolerance was not observed for the psychometric impairment or changes in EEG activity, indicating that there was a temporal disparity be~een objective and subjective impairment following ethanol administration. 6) The EEG changes were not correlated with the psychometric or subjective impairment. 7) There was a significant gender difference observed in the Cmax and Vdss for ethanol, probably due to gender differences in body weight and body water content. There was also a significant gender difference observed in the magnitude of ethanol-induced subjective impairment, with females showing a lower degree of subjective impairment, despite achieving similar concentrations and demonstrating similar psychometric impairment and EEG changes. This gender difference may be partly confounded by the larger proportion of female "non-responders" compared to the male "non-responders" in the study.

The effects of ethanol administration on the antinociceptive activity, lethal properties and brain concentration of methadone, were investigated. The effect of ethanol on the antinociceptive activity of methadone was assessed by the hot-plate and tail-flick tests. Concentrations of methadone in the brain were determined by the use of 3H-methadone as well as by gas liquid chromatographic analysis. The study showed that moderate doses of ethanol did not alter tail-flick or hot-plate response by themselves. However, when combined with methadone, ethanol produced a significant increase in the antinociceptive effectiveness of methadone as measured by both a decrease in the ED50 of methadone and by an increased intensity and prolonged duration of methadone antinociception. Ethanol increased the antinociceptive activity of methadone in both naive and methadone-tolerant mice. This increased activity was not due to simple addition of subthreshold effects of ethanol nor was it due to an ethanol-mediated increase in whole brain·concentrations of methadone. It is hypothesized that the increased antinociceptive activity was the result of an ethanol-mediated increase in central nervous system sensitivity to the antinociceptive activity of methadone. Ethanol pretreatment produced significantly lower brain concentrations of methadone compared to controls when methadone was administered subcutaneously. When both drugs were administered orally, ethanol administration resulted in brain concentrations of methadone initially less than control and at later times greater than control. In both ethanol and water-pretreated mice there was an excellent correlation between the whole brain concentration of methadone and antinociceptive effect, but the antinociceptive effect at any brain concentration of methadone was greater in ethanol-pretreated mice. Although ethanol produced significant alterations in the brain concentration of methadone, the brain concentration of ethanol was generally not altered by methadone administration. Investigations of the excretion of methadone and its metabolites and the half-life of methadone in the brain failed to reveal any significant ethanol-induced alterations. A dose of ethanol which increased the antinociceptive activity of methadone did not alter the oral or subcutaneous LD50 of methadone, although mice that died as a result of ethanol and methadone administration died at lower whole brain concentrations of methadone than those that died as a result of methadone alone. The LD50 of ethanol was significantly decreased in mice maintained on a methadone dose of 100 mg/kg/day.

Recent evidence implicates anandamide as the endogenous ligand for the cannabinoid receptor. One purpose of this study was to determine the structural requirements for anandamide's receptor interaction and the influence of phenylmethylsulfonyl fluoride (PMSF), an enzyme inhibitor, on receptor affinity. A second objective was evaluation of the correlation between affinities of the analogs and in vivo pharmacological activities. The ability of anandamide and analogs to displace [3H]- CP 55,940 was determined by a filtration assay. Displacement curves for anandamide in the presence of PMSF produced a Ki of 89 ± 10 nM; without PMSF the Ki increased to 5400 ± 1600 nM. Anandamide analogs were evaluated for their ability to produce antinociception and hypomotility. The levels of saturation and substituents for the ethanolamide and hydroxyl groups of the anandamide structure were critical to receptor affinity and in vivo potency. Increasing the length of the N-substituent by one or two carbons decreased receptor binding affinity. Methylations at carbons 2 and l' produced compounds stable in the absence of PMSF. Addition of larger alkyl groups at these positions or nitrogen methylation reduced receptor affinity and behavioral potency. These results indicate that methylations at specific carbons of anandamide confer stability in vitro. A final objective was to characterize anandamide's binding to the cannabinoid receptor in the CNS. Anandamide's receptor binding affinities and binding densities, as determined from autoradiographic experiments in rat brain, from selected brain areas were compared to the receptor binding densities and patterns of two other compounds, CP 55,940 and SR 147116A, that bind to the central cannabinoid receptor. The lack of difference between receptor affinity, receptor distribution and parallelism of the displacement curves indicate that anandamide, SR 141716A and CP 55,940 are binding to the same receptor in the same manner.

An important principle of pharmacokinetic modeling is to use the least complicated model possible that adequately describes the process studied. Pharmacokinetic studies in peritoneal dialysis often attempt to describe the time course of drug concentration in the dialysate. Peritoneal dialysis patients are subjected to the inconvenience and risk of infection associated with sampling peritoneal fluid. Alternative pharmacokinetic models were used to study drug distribution during continuous ambulatory peritoneal dialysis (CAPD). Efficient pharmacokinetic models of distribution in the peritoneal cavity were described for three drugs and the classes they represent. Procainamide pnarmacokinetics were determined in six CAPD patients. The time course of procainamide or N-acetylprocainamide (NAPA) in the peritoneal fluid is of less interest than the amount cleared through the peritoneum. Therefore the peritoneal cavity is treated as a terminal compartment. Procainamide and NAPA exhibited mean elimination half-lives of 26 and 42.9 hours respectively. CAPD accounted for 1.1% and 13% of total body clearance of procainamide and NAPA respectively. Phenytoin pharmacokinetics were studied to determine the time course of dialysate phenytoin concentrations with respect to drug unbound to plasma proteins (free phenytoin). The peritoneal cavity was modeled as part of the peripheral compartment. CAPD did not prove to be an effective elimination pathway for phenytoin, accounting for only 2.1% of total body clearance of phenytoin. Closed compartment pharmacokinetic theory was investigated as an alternative modeling method when the drug has no other elimination pathway than the peritoneal cavity. In this case, vancomycin closed compartment pharmacokinetics were compared to open model analysis. Both models were used to predict an end of dwell concentration by fitting the data from a previous dwell. It was found that the closed model approach required less data and provided prediction close to the open model predictions. The model used depends on the underlying pharmacokinetic characteristics of the drug and the aims of the study. A terminal compartment method is sufficient when only clearance due to CAPO is required. When the drug is not administered intraperitoneally, the peritoneal space can be described as part of the peripheral compartment. Closed compartment pharmacokinetics are useful when the drug in question is eliminated only through the peritoneum. Whatever model is used, the investigator must consider the integrity of the peritoneal membranes, ultrafiltration, and volume shifts within the peritoneal cavity.