The hallmark of Gram-negative bacteria is the presence of an outer membrane (OM) surrounding the cytoplasmic membrane (here called the inner membrane [IM]) and the cell wall. The OM is a unique asymmetric bilayer with an inner leaflet consisting of phospholipid and an outer leaflet consisting of lipopolysaccharide (LPS). LPS is a large anionic molecule that typically contains six fatty acyl chains and up to several hundred sugar residues. This chemical structure explains why the OM is relatively impermeable to large hydrophobic molecules, such as detergents, bile salts, and high molecular weight antibiotics, which readily cross a normal phospholipid bilayer. LPS and the OM are essential to the viability of most Gram-negative organisms, including major human pathogens. LPS molecules are biosynthesized at the IM and subsequently exported out of the IM, across the intermembrane space (the periplasm) and through the OM to their final position at the cell surface. In Escherichia coli, the essential LPS transport proteins, LptA-G, are required for this process. This Lpt pathway includes an IM adenosine triphosphate binding cassette (ABC) transporter, LptBFG, which is associated with an additional IM protein, LptC; a periplasmic protein, LptA; and an OM complex consisting of the lipoprotein LptE and the transmembrane \(\beta\)-barrel protein LptD. All seven Lpt proteins associate as a single complex that spans the cell envelope. However, little is known about how these proteins work together to transport LPS. Here, we use in vivo and in vitro biochemical studies to probe the organization, function, and assembly of the Lpt machine. In Chapter 2, we show that LptE forms a plug within the LptD \(\beta\)-barrel and present a model for how this unusual structure can move LPS from the periplasm directly into the outer leaflet of the OM. In Chapter 3, we demonstrate that the Lpt transenvelope bridge consists of a series of structurally homologous domains – LptC, LptA, and the N-terminal domain of LptD – stacked in a head-to-tail orientation, providing a route for LPS from the IM to the OM. Finally, in Chapter 4, we connect these two sets of results by showing how the assembly of the Lpt transenvelope bridge is regulated by that of the LptD/E complex in the OM. Together, these findings explain how the functions of the Lpt proteins are coordinated to ensure delivery of LPS to the correct cellular compartment. A fundamental understanding of LPS biogenesis will contribute to the development of new therapies against Gram-negative infections.

Since the development of knockout and transgenic mouse models, mice have become the most widely used mammalian animal model to study bone. Despite the advances in knowledge of bone biology and function that have occurred from use of mouse models, many studies use primarily qualitative techniques, which may result in overlooking important subtle pathophysiologic changes. The hypothesis of this dissertation is that quantitative techniques to measure bone structure and function could identify the physiologic role of carbonic anhydrase II and calbindin-D28k in mouse bone, despite earlier qualitative studies indicating mice without these proteins have normal bone structure and function. Furthermore, a method to quantify bone function in vivo will be tested in a mouse model.Although carbonic anhydrase II deficient mice are less severely affected than patients, the mice demonstrate features of osteopetrosis including metaphyseal widening and a 50% increase in trabecular bone volume. The mice partially compensate for inhibited osteoclast function by increasing osteoclast number.Calbindin-D28k knockout mice demonstrated an increase in bone volume that results from additional bone at the endosteal surfaces. The higher bone volume results in increased stiffness and failure loads, highlighting the potential use of drugs that inhibit calbindin-D28k to treat diseases such as osteoporosis.Finally, calcium phosphate ceramic and hydroxyapatite particles used as strain gauge coatings demonstrated bone bonding to mouse femora after two months in vivo. The use of hydroxyapatite particles to coat strain gauges is the first time this method has been used with all commercially available materials, and will allow other research groups to use this technique. The major limitation to in vivo bone strain measurement in mice is the relatively large size of the sensors, which resulted in increased second moments of inertia in the implanted bones.Overall, this dissertation demonstrates that the use of quantitative techniques, including histology, histomorphometry, µCT imaging, and mechanical testing can measure subtle changes in bone properties that have been previously overlooked. Development of additional quantitative methods to study bone biomechanics in mouse models may encourage other research groups to quantify bone properties if no changes are noted using primarily qualitative methods.

Ovarian cancer is the fifth leading cause of cancer related deaths among women. Early detection improves the chances of survival following diagnosis, and new imaging modalities have the potential to reduce deaths due to this disease. The confocal microendoscope (CME) is a non-destructive in-vivo imaging device for visualization of the ovaries that operates in real-time. Two components of the CME system are evaluated in this paper, and initial results from an ongoing clinical trial are presented.Fiber-optic imaging bundles are used in the CME imaging catheter to relay images over distances of up to 20 feet. When detecting fluorescent signals from investigated tissue, any fluorescence in the system can potentially reduce contrast in images. The emission and transmission properties of three commercially available fiber optic imaging bundles were evaluated. Emission maps of fluorescence from bundles were generated at multiple excitation wavelengths to determine the profile and amount of fluorescence present in bundles manufactured by Sumitomo, Fujikura, and Schott. Results are also presented that show the variation of transmittance as a function of illumination angle in these bundles. Users of high-resolution fiber-optic imaging bundles should be aware of these properties and take them into account during system design.Contrast is improved in images obtained with the CME through the application of topical dyes. Acridine orange (AO) and SYTO 16 are two fluorescent stains that are used to show the size, shape, and distribution of cell nuclei. Unfortunately, little is known about the effects of these dyes on living tissues. This study was undertaken to evaluate the effects of dye treatment on peritoneal tissues in mice. Seventy-five Balb/c mice were split into five groups of fifteen and given peritoneal injections of dye or saline. The proportions of negative outcomes for the control and test groups were compared using confidence intervals and the Fisher's exact test. No significant difference was determined between the groups. These data provide preliminary results on determining the effect of these dyes on living tissues.Preliminary results of a clinical trial are presented showing in-vivo use of the CME for imaging of the ovaries. This is the first portion of a two part study to demonstrate the clinical diagnosis potential of the CME system. A mobile version of the bench-top CME was modified to be used in the clinic. Fluorescein sodium is used as an initial contrast agent in these studies to demonstrate fluorescence imaging. Twenty patients were successfully imaged, and results of this study have allowed progression to a clinical validation study showing the diagnostic capabilities of the CME.

Olfaction is vitally important to animals in all environments and is used to identify food, habitat, conspecifics and predators. Some odors, like pheromones or the pungent smell of spoiled foods, can trigger pre-existing behavioral responses that appear to require no learning. Most odors, however, are only attended to as a result of prior experience. It is believed that different types of odors are processed in different olfactory pathways in the forebrain. This thesis examines the relationship between innate and learned olfactory behaviors and the anatomy of the neural pathways that underlie them, using the zebrafish olfactory system as a model. I first characterized an appetitive olfactory behavior, which is displayed promptly by zebrafish when they encounter amino acid odors. A similar appetitive behavior can also be learned by the fish for another, initially neutral odorant, if it is repeatedly paired with food rewards. Zebrafish can therefore respond to, and learn to respond to certain odors. I then conducted an in-depth anatomical analysis of the structure and distribution of glomeruli in the zebrafish olfactory system. Glomeruli are spheroidal synaptic aggregates that organize and shape olfactory information that arrives in the brain. Throughout the development of zebrafish, I identified two distinct populations of glomeruli. One population consisted of 25 individually identifiable, anatomically stereotypic glomeruli that closely resembled specialized glomeruli in mammals and insects. These glomeruli were already formed during embryonic development and persisted in remarkably stable configurations throughout later developmental stages. I hypothesize that the 25 individually identifiable glomeruli constitute stable olfactory pathways (i.e., for innate olfactory behaviors). Most glomeruli, however, were anatomically variable and displayed different distributions within coarsely circumscribed regions in the zebrafish olfactory bulbs. The development of these glomeruli could be modified by sensory experience, suggesting that they may comprise plastic olfactory pathways that subserve the establishment of learned olfactory behaviors. Collectively my results show that innate and learned olfactory behaviors may indeed be represented in different olfactory pathways, and that these types of pathways may be located in both main and accessory olfactory systems.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Glaucoma is a leading cause of world blindness, often associated with elevated eye pressure. Current glaucoma treatments aim to lower eye pressure by improving aqueous humor outflow from the eye. Ocular lymphatics have been demonstrated to contribute to aqueous humor outflow in human and sheep. It is not known whether any glaucoma drugs target this lymphatic drainage. The mouse is a valuable model with similar aqueous humor dynamics and pharmacology as human. Using in vivo hyperspectral fluorescence imaging combined with intracameral quantum dot injection, we identified an ocular lymphatic drainage in mouse. Immunofluorescence and confocal microscopy revealed lymphatic channels in the ciliary body, sclera, and orbit that may be responsible for this lymphatic drainage. We showed that latanoprost, a prostaglandin F2α analog widely used to treat glaucoma, increases this ocular lymphatic drainage. Our findings provide the framework for future development of novel glaucoma drugs that stimulate the ocular lymphatic drainage.

Glaucoma is a leading cause of world blindness, often associated with elevated eye pressure. Current glaucoma treatments aim to lower eye pressure by improving aqueous humor outflow from the eye. Ocular lymphatics have been demonstrated to contribute to aqueous humor outflow in human and sheep. It is not known whether any glaucoma drugs target this lymphatic drainage. The mouse is a valuable model with similar aqueous humor dynamics and pharmacology as human. Using in vivo hyperspectral fluorescence imaging combined with intracameral quantum dot injection, we identified an ocular lymphatic drainage in mouse. Immunofluorescence and confocal microscopy revealed lymphatic channels in the ciliary body, sclera, and orbit that may be responsible for this lymphatic drainage. We showed that latanoprost, a prostaglandin F2α analog widely used to treat glaucoma, increases this ocular lymphatic drainage. Our findings provide the framework for future development of novel glaucoma drugs that stimulate the ocular lymphatic drainage.

Cortical bone is a dynamic tissue which undergoes adaptive and pathological changes throughout life. An improved understanding of the spatio-temporal process of remodeling holds great promise for improving our understanding of bone development, maintenance and senescence. The use of micro-computed tomography (µCT) on living animals is relatively new and allows the three dimensional quantification of change in trabecular bone microarchitecture over time. The use of in vivo µCT is limited by the radiation dose created by the x-ray beam, with commercially available in vivo systems generally operating in the 10-20 um resolution range and delivering an absorbed dose between 0.5-1 Gy. Because dose scales to the power of four with resolution, in vivo imaging of the cortical canal network, which requires a higher resolution, has not been achieved. I hypothesized that using synchrotron propagation phase contrast µCT, cortical porosity could be imaged in vivo in rats at a dose on the same level as those used currently for trabecular bone analysis. Using the BMIT-BM beamline, I determined the optimal propagation distance and used ion chamber and lithium fluoride crystal thermoluminescent dosimetry to measure the absorbed dose of my in vivo protocol as well as several ex vivo protocols using synchrotron phase contrast µCT at 5 µm, 10 µm, and 11.8 µm and conventional desktop in vivo protocols using commercial µCT systems. Using synchrotron propagation phase contrast µCT, I scanned the forelimb of two adult Sprague-Dawley rats and measured an absorbed dose of 2.53 Gy. Using two commercial µCT system, I measured doses between 1.2-3.6 Gy for protocols at 18µm that are in common use. This thesis represents the first in vivo imaging of rat cortical porosity and demonstrates that an 11.8 µm resolution is enough to visualize cortical porosity in rats, with a dose within the scope of those used for imaging trabecular bone in vivo.

Introduction - Malaria is a major global health problem, with more than 500 million reported cases and at least 1 million deaths each year. The main problem with malaria control is the emerging drug resistance. Plasmodium falciparum (P. falciparum) developed widespread resistance to antimalarial drugs such as chloroquine (CQ) and mefloquine, but not to the artemisinins. The World Health Organization (WHO) recommended artemisinin combination therapy (ACT) for the treatment of uncomplicated malaria in all chloroquine resistance areas. However, P. falciparum has recently started to display resistance to these ACTs, highlighting the need for new chemotherapeutic approaches for the treatment of P. falciparum infections. Aims - The aims of this study were: (i) to design and synthesise a new series of antimalarial hybrid drugs, consisting of dihydroartemisinin (DHA) and aminoquinoline moieties bound covalently through different, very distinctive linkers; (ii) to determine the in vitro antiplasmodial activity and cytotoxicity of the synthesised series; (iii) to ascertain whether the in vitro antiplasmodial activity of the promising compounds would be carried over in vivo against Plasmodium vinckei (P. vinckei); and, (iv) to obtain an indication of the pharmacokinetic properties of this class of antimalarial drugs by performing snapshot pharmacokinetic analysis. Methods - DHA was coupled via an aminoethylether bond to various aminoquinolines to give hybrids and hybrid-dimers. CQ-susceptible (D10 and 3D7) and CQ-resistant (Dd2) strains of P. falciparum were used to determine the in vitro antiplasmodial activity. In vitro cytotoxicity was assessed using a mammalian cell-line (Chinese Hamster Ovarian, CHO). The antiproliferative activity of the hybrid-dimers was tested against three cell lines; renal adenocarcinoma (TK-10), breast adenocarcinoma (MCF-7) and melanoma (UACC-62). P. vinckei-infected mice were treated with the hybrid drugs for four days at a dosage of 0.8 mg/kg, 2.5 mg/kg, 7.5 mg/kg or 15 mg/kg intraperitoneally (ip) or orally (po), with 2.7 mg/kg, 8.3 mg/kg, 25 mg/kg or 50 mg/kg, in order to determine their antimalarial activity. A snapshot oral and intravenous (IV) pharmacokinetic study was performed. Results - All compounds were obtained as the 10-β-isomers and were isolated as the oxalate salts. Low nanomolar in vitro antiplasmodial activities were displayed by several compounds in this series, with IC50 values ranging from 5.15 to 29.5 nM, in comparison with the values of 2.09–5.11 nM and 21.54–157.90 nM for each of DHA and CQ respectively. All compounds displayed good selectivity towards P. falciparum in vitro (selectivity index (SI) ≥ 20). Two of the hybrids, featuring non-methylated and methylated two-carbon diaminoalkyl linkers, exerted potent in vivo antimalarial activities, with ED50 values of 1.1 and 1.4 mg/kg by ip route and 12 and 16 mg/kg po, respectively. Long-term monitoring of parasitaemia showed a complete cure of mice (without recrudescence) at 15 mg/kg ip and at 50 mg/kg po for these two hybrids, whereas artesunate was able to provide a complete cure only at 30 mg/kg ip and 80 mg/kg po. Conclusions - These compounds may provide a lead into a new class of antimalarial drugs so badly needed for treatment of resistant strains. Despite shorter half-lives and moderate oral bioavailability in comparison with DHA, two of the compounds of this series were able to cure malaria in mice at very low dosages, implicating extremely active metabolites. The optimum linker length for antimalarial activity was found to be a diaminoalkyl linker consisting of two carbon atoms, either unmethylated or bearing a single methyl group. / Thesis (PhD (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2013

Introduction - Malaria is a major global health problem, with more than 500 million reported cases and at least 1 million deaths each year. The main problem with malaria control is the emerging drug resistance. Plasmodium falciparum (P. falciparum) developed widespread resistance to antimalarial drugs such as chloroquine (CQ) and mefloquine, but not to the artemisinins. The World Health Organization (WHO) recommended artemisinin combination therapy (ACT) for the treatment of uncomplicated malaria in all chloroquine resistance areas. However, P. falciparum has recently started to display resistance to these ACTs, highlighting the need for new chemotherapeutic approaches for the treatment of P. falciparum infections. Aims - The aims of this study were: (i) to design and synthesise a new series of antimalarial hybrid drugs, consisting of dihydroartemisinin (DHA) and aminoquinoline moieties bound covalently through different, very distinctive linkers; (ii) to determine the in vitro antiplasmodial activity and cytotoxicity of the synthesised series; (iii) to ascertain whether the in vitro antiplasmodial activity of the promising compounds would be carried over in vivo against Plasmodium vinckei (P. vinckei); and, (iv) to obtain an indication of the pharmacokinetic properties of this class of antimalarial drugs by performing snapshot pharmacokinetic analysis. Methods - DHA was coupled via an aminoethylether bond to various aminoquinolines to give hybrids and hybrid-dimers. CQ-susceptible (D10 and 3D7) and CQ-resistant (Dd2) strains of P. falciparum were used to determine the in vitro antiplasmodial activity. In vitro cytotoxicity was assessed using a mammalian cell-line (Chinese Hamster Ovarian, CHO). The antiproliferative activity of the hybrid-dimers was tested against three cell lines; renal adenocarcinoma (TK-10), breast adenocarcinoma (MCF-7) and melanoma (UACC-62). P. vinckei-infected mice were treated with the hybrid drugs for four days at a dosage of 0.8 mg/kg, 2.5 mg/kg, 7.5 mg/kg or 15 mg/kg intraperitoneally (ip) or orally (po), with 2.7 mg/kg, 8.3 mg/kg, 25 mg/kg or 50 mg/kg, in order to determine their antimalarial activity. A snapshot oral and intravenous (IV) pharmacokinetic study was performed. Results - All compounds were obtained as the 10-β-isomers and were isolated as the oxalate salts. Low nanomolar in vitro antiplasmodial activities were displayed by several compounds in this series, with IC50 values ranging from 5.15 to 29.5 nM, in comparison with the values of 2.09–5.11 nM and 21.54–157.90 nM for each of DHA and CQ respectively. All compounds displayed good selectivity towards P. falciparum in vitro (selectivity index (SI) ≥ 20). Two of the hybrids, featuring non-methylated and methylated two-carbon diaminoalkyl linkers, exerted potent in vivo antimalarial activities, with ED50 values of 1.1 and 1.4 mg/kg by ip route and 12 and 16 mg/kg po, respectively. Long-term monitoring of parasitaemia showed a complete cure of mice (without recrudescence) at 15 mg/kg ip and at 50 mg/kg po for these two hybrids, whereas artesunate was able to provide a complete cure only at 30 mg/kg ip and 80 mg/kg po. Conclusions - These compounds may provide a lead into a new class of antimalarial drugs so badly needed for treatment of resistant strains. Despite shorter half-lives and moderate oral bioavailability in comparison with DHA, two of the compounds of this series were able to cure malaria in mice at very low dosages, implicating extremely active metabolites. The optimum linker length for antimalarial activity was found to be a diaminoalkyl linker consisting of two carbon atoms, either unmethylated or bearing a single methyl group. / Thesis (PhD (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2013