Indiana University-Purdue University Indianapolis (IUPUI) / Although the in vivo tibial loading model has been used to study the bone forma- tion response of mice to exercise, little emphasis has been placed on the translation of architectural and compositional modifications to changes in mechanical behaviour. The goals of the studies discussed below were to investigate the mechanical response in both healthy and osteolathrytic mice to this loading model and to determine the dose-depended effects of strain level on these properties. In two separately designed studies, strain levels ranging from 1700 to 2400 were applied to the right tibiae of 8 week old female C57BL/6 mice, while the left tibiae were used as non-loaded control. The first study consisted of loading both PBS- and BAPN-injected mice to 1750 microstrain which resulted in little bone formation but some tissue-level changes in mechanical analyses and an improvement in fatigue-resistance in terms of microdamage accumulation. The second study loaded healthy mice to three strain levels (1700, 2050, and 2400). Results indicated that the low end of the strain range did not engender a robust formation response, while the high end of the strain range resulted in a woven bone response in half of the animals in that group. Future studies will focus on the mid-strain level of 2050 which induced both significant architectural and mechanical improvements.

Excess aluminum has been linked to such diseases as dialysis encephalopathy syndrome and osteodystrophy, in renal dialysis patients. Though the causality relation has not yet been confirmed, aluminum has also been associated with Alzheimer's disease. Aluminum is thought to be stored in bones, and a measure of its deposition should correlate with its total bioaccumulation. To date, only bone biopsies and desferrioxamine tests are available for such measurements. Neutron activation analysis (NAA) has been studied as a non-invasive technique for measuring bone aluminum. Two neutron beam ports from the McMaster nuclear reactor and the KN accelerator have been compared as possible thermal neutron sources. Resin-based phantoms, physiologically resembling a hand, were irradiated using the reactor based neutron source. From the results, a minimum detection limit (MDL) of aluminum in bone was obtained. An Andersson-Braun remmeter measured the dose delivered to the phantoms, due to the radiation exposure. The hand dose equivalent combined with the MDL, in this study, are compared to the results of previous NAA studies. The goal of this study is to measure low aluminum stores In Vivo, while delivering a low dose with respect to natural background levels. / Thesis / Master of Science (MS)

Organic polymers S2218600, S2429901 and S2219200 (referred to as Polymer 1, Polymer 2 and Polymer 3, respectively) of varying toxic potential, designed for use in cosmetic aerosols, were used as model substances to predict inflammatory potential. In vivo inflammogenic potential was evaluated by assessment of inflammatory cell profile (alveolar macrophage (AM), polymorphonuclear neutrophil (PMN)) of broncho-alveolar lavage fluid (BAL) 24hrs after a single instillation of either 0.5 mg or 2 mg polymer in Sprague Dawley rats. Pro-inflammatory Minusil particles and non-inflammatory titanium dioxide (TiO2) particles were used as controls. For comparison, cultured rat NR8383 AM-like cells, human THP-1 monocyte cells or human monocyte derived macrophages were treated with polymer for 24 h and supernatants analysed for indicators of cytotoxicity and inflammatory mediator release. In addition, after 6 h treatment, gene changes in the rat lung tissue and also in the rat NR8383 alveolar macrophage cell line were assessed using microarray to analyse the entire rat genome. The in vivo studies showed that Polymer 1, Polymer 3 and Minusil caused significant PMN influx into BAL. Polymer 3 and Minusil caused a significant increase in AM number in BAL. Polymer 2 and TiO2 had no effect on BAL cell profile. BAL tumour necrosis factor-α (TNFα) and macrophage inflammatory protein-2 (MIP-2) levels were significantly increased following instillation of Polymer 3 and Minusil. Thus the polymers and particles were ranked for potential to cause pulmonary inflammation: Polymer 3 > Minusil > Polymer 1 > Polymer 2 > TiO2. In vitro studies using cultured rat NR8383 AM-like cells showed that the polymers and particles could be ranked similarly for cytotoxic potential and their ability to stimulate the release of both TNFα and MIP-2. Cultured human monocyte derived macrophages detected the pro-inflammatory abilities of Polymer 3, as measured by cytotoxic potential and ability to stimulate TNFα, interleukin-8 (IL-8) and macrophage inflammatory protein-1α (MIP-1α), however, did not detect the pro-inflammatory abilities of Polymer 1. Cultured human THP-1 cells predicted the pro-inflammatory effects of Polymer 3 in rat lungs using the cytotoxicity assay and by changes in IL-1β, MIP-1α and IL-10 levels. The human THP-1 cell line did not predict the pro-inflammatory effects of Polymer 1 that were observed the rat lungs. Electron spin resonance (ESR) detected free radicals produced by the pro-inflammatory polymers and particles which had the ability to break bonds in super-coiled DNA and deplete intracellular glutathione (GSH). Microarray analysis of the canonical pathways activated by the pro-inflammatory polymers, Polymer 1 and Polymer 3, showed that 3 similar pathways were significantly activated in the instilled rats and the rat NR8383 AM-like cells following treatment. ‘Xenobiotic metabolism’, ‘IL-10 signalling’ and ‘leukocyte extravasation signalling’ pathways were significantly changed by the pro-inflammatory polymers. Use of these cell model alternatives in an industrial setting will refine and reduce in vivo testing and as these models are further developed and used alongside future new alternatives they will provide a substantial contribution towards the replacement of animal testing.

Carisbamate (CRS) and lamotrigine (LTG) are anticonvulsants which act mainly on neuronal voltage-gated sodium channels, that have been shown to have antidepressant-like effects in animal models of depression. In vivo electrophysiological recordings were carried out following 2 and 14 days of CRS or LTG administration. Overall firing activity in the dorsal raphe, locus coeruleus and ventral tegmental area were decreased with CRS. Similarly, a decrease in the dorsal raphe was also observed with LTG. Despite these presynaptic decreases in firing activity, both anticonvulsants exhibited significant enhancement of serotonergic transmission in the hippocampus as demonstrated by increased tonic activation of postsynaptic 5-HT1A receptors. This may be attributed to the observed desensitization of the terminal 5-HT1B autoreceptors. This study suggests that the enhanced serotonergic effect may be associated with an antiglutamatergic effect, and may contribute to the antidepressant-like effect of CRS in the forced swim test and the antidepressant properties of LTG.

Aus pharmakokinetischer Sicht sind neben Parametern wie der oralen Bioverfügbarkeit und der systemischen Clearance, für die Effektivität und Sicherheit eines inhalativ angewendeten Wirkstoffes unter anderem das Ausmaß der pulmonalen Deposition und seine pulmonale Umverteilungskinetik entscheidend. Wird eine topische Wirkung des Arzneistoffes angestrebt, so trägt eine lange Verweilzeit des Arzneistoffes im Zielgewebe, verbunden mit einer langsamen Umverteilung in den systemischen Kreislauf zu einer Wirkungsoptimierung mit gleichzeitiger Minimierung systemischer Nebenwirkungen bei. In-vitro- und ex-vivo-Modelle eignen sich hervorragend zur isolierten Untersuchung solcher pharmakokinetischer Vorgänge ohne den Einfluss verschiedener in-vivo-Faktoren, wie der Verteilung in andere Gewebe, Metabolisierungs- oder Eliminationsprozessen. Das Ziel der vorliegenden Arbeit war es daher, Modelle der humanen Lunge zu etablieren bzw. weiterzuentwickeln, die möglichst realitätsnah die Untersuchung der Pharmakokinetik pulmonal applizierter Wirkstoffe ermöglichen. / From a pharmacokinetic point of view, the extent of pulmonary deposition and the pulmonary redistribution are crucial for an inhaled drug’s effectiveness and safety besides parameters such as oral bioavailability and systemic clearance. Aiming at a local effect, a long residence time in the target tissue combined with a slow redistribution into systemic circulation contribute to a drug’s optimal potency while simultaneously minimizing systemic adverse effects. In-vitro and ex-vivo models are particularly suitable for examining single pharmacokinetic aspects without the influences occurring in-vivo such as distribution into other tissues and processes of metabolism or elimination. Therefore, the aim of the present thesis was to establish, respectively enhance models of the human lung, which were able to describe the pharmacokinetics of pulmonary applied drugs close to reality.

Platelet activation and adhesion resulting in thrombus growth is essential for normal hemostasis, but can lead to irreversible, life-threatening vessel occlusion. In the current study, the contribution of platelet integrins, activation receptors and the contact system of blood coagulation in such pathological conditions was investigated in mice. / Plättchenaktivierung, -adhäsion und nachfolgende Thrombusbildung ist ein für die Hämostase essentieller Prozess, der jedoch zu irreversiblem lebensbedrohlichen Gefäßverschluss führen kann. In der vorliegenden Arbeit wurde die Rolle von Thrombozyten-Integrinen, aktivierenden Rezeptoren, sowie dem Kontaktsystem der Koagulation unter pathologischen Bedingungen im Maussystem untersucht.

Understanding how memory, learning and reward work in unison to form adaptive and sometime maladaptive behaviour is at the forefront of modern neuroscience. The largest unmet need in treating maladaptive reward learning behaviours such as addiction is maintaining long-term abstinence and preventing relapse after re-exposure to drug-associated cues. Nicotinic acetylcholine receptors (nAChR) have been implicated in responses to drugs of abuse other than nicotine (Rahman et al., 2015) and the aim of this work was to characterise the role of α7 nAChRs in morphine reward learning using conditioned place preference (CPP). The α7 nAChR antagonist methyllycaconitine (MLA) was used to determine if these receptors contribute to specific stages of drug-paired learning, namely acquisition, expression, reconsolidation or reinstatement of morphine-CPP. In 7-8week old C57BL/6J mice MLA (4mg/kg, s.c), given 20 minutes prior to a conditioning dose of morphine (10mg/kg, i.p) or post-test trial, had no effect on the acquisition, reconsolidation or expression of morphine-CPP. However, when given 20 minutes prior to a priming dose of morphine (5mg/kg, i.p), MLA (4mg/kg, s.c) significantly inhibited drug-induced reinstatement. The mechanisms of this effect were investigated using glutamate receptor autoradiography. Changes in 2-Amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid (AMPA) and N-methyl-D-aspartate (NMDA) binding were examined in mice treated with either saline or MLA at morphine reinstatement. There were no significant changes in NMDA receptor binding (using [3H]MK-801) but morphine reinstatement significantly increased [3H]AMPA binding in the CA1/2 of the ventral but not dorsal hippocampus, or in any other brain regions examined (including mPFC, nucleus accumbens, amygdala and VTA). The selective increase in the hippocampus was partially antagonised by MLA, linking α7 nAChR activation to glutamatergic synaptic plasticity in the hippocampus. Intracranial infusions of MLA into the ventral but not the dorsal hippocampus or medial prefrontal cortex blocked reinstatement to morphine-CPP in male Wistar rats.

The ability to resolve multiple fluorescent emissions from different biological targets in video rate applications, such as endoscopy and intraoperative imaging, has traditionally been limited by the use of filter-based imaging systems. Hyper and multispectral imaging facilitate the detection of both spatial and spectral information in a single data acquisition, however, instrumentation for spatiospectral data acquisition is typically complex, bulky and expensive. This thesis seeks to overcome these limitations by using recently commercialised compact and robust hyper/multispectral cameras based on spectrally resolved detector arrays. Following sensor calibrations, which devoted particular attention to the angular sensitivity of the sensors, we integrated spectrally resolved detector arrays into a wide-field and an endoscopic imaging platform. This allowed multiplexed reflectance and fluorescence imaging with spectrally resolved detector array technology in vitro, in tissue mimicking phantoms, in an ex vivo oesophageal model and in vivo in a mouse model. A hyperspectral linescan sensor was first integrated in a wide-field near-infrared reflectance based imaging set-up to assess the suitability of spectrally resolved detector arrays for in vivo imaging of exogenous fluorescent contrast agents. Using this fluorescence hyperspectral imaging system, we could accurately resolve the presence and concentration of seven fluorescent dyes in solution. We also demonstrated high spectral unmixing precision, signal linearity with dye concentration, at depth in tissue mimicking phantoms, and delineation of four fluorescent dyes in vivo. After the successful demonstration of multiplexed fluorescence imaging in a wide-field set-up, we proceeded to combine near-infrared multiplexed fluorescence imaging with visible light spectral reflectance imaging in an endoscopic set-up. A multispectral endoscopic imaging system, capable of simultaneous reflectance and fluorescence imaging, was developed around two snapshot spectrally resolved detector arrays. In the process of system integration and characterisation, methods to characterise and predict the imaging performance of spectral endoscopes were developed. With the endoscope we demonstrated simultaneous imaging and spectral unmixing of chemically oxy/deoxygenated blood and three fluorescent dyes in a tissue mimicking phantom, and of two fluorescent dyes in an ex vivo oesophageal porcine model. With further developments, this technology has the potential to become applicable in medical imaging for detection of diseases such as gastrointestinal cancers.

O carrapato Rhipicephalus microplus é um ectoparasito hematófago de grande importância para a pecuária por ser responsável por perdas massivas na produção animal, de forma que o seu controle é economicamente relevante. Este carrapato, além dos danos que causa por si só, é também um importante vetor para a transmissão de microorganismos patogênicos, entre eles o hemoprotozoário intraeritrocítico Babesia bovis. O presente trabalho descreve o desenvolvimento de uma linhagem de B. bovis capaz de expressar um antígeno protetor, uma glutationa S-transferase do carrapato Haemaphysalis longicornis (HlGST), e o teste desta linhagem como uma vacina de vetor vivo para o controle do carrapato R. microplus. B. bovis, em cultivo, da linhagem S74-T3B foram eletroporados em presença de plasmídeo contendo o promotor bidirecional de B. bovis Ef-1 aresponsável pela expressão independente de dois genes: o repórter fusionado ao agente para seleção (GFP-BSD) e HlGST fusionada à sequência codificadora do peptídeo sinal de MSA-1 (merozoite surface antigen-1). Após a eletroporação, foi feita a seleção com blasticidina para obtenção da linhagem nomeada HlGST. A linhagem HlGST é composta por parasitos contendo diferentes padrões de inserção dos genes exógenos, tanto dentro quanto fora do locus Ef-1. Uma linhagem clonal denominada HlGST-Cln expressando HlGST e GFP-BSD foi obtida a partir da linhagem HlGST. Dois ensaios, independentes, de imunização de bovinos com os parasitos clonais foram realizados, sendo usado como controle uma linhagem clonal previamente caracterizada denominada GFP-Cln. Todos os animais inoculados desenvolveram uma forma branda de babesiose, indicando que ambas as linhagens clonais são atenuadas, mas apenas os animais imunizados com a linhagem HlGST-Cln foram capazes de produzir anticorpos anti-HlGST. O segundo procedimento de imunização foi seguido por um desafio com larvas de R. microplus. O desenvolvimento dessas larvas no hospedeiro levou a fêmeas adultas de menor peso e fertilidade. Coletivamente, esses dados mostram a possibilidade de uso de linhagens transfectadas de B. bovis como vacinas de vetor vivo. / The tick Rhipicephalus microplus is a notorious blood-feeding ectoparasite of cattle, responsible for massive losses in animal production. It is the main vector of pathogenic microorganisms, including Babesia bovis, an intraerythrocytic apicomplexan protozoan parasite responsible for bovine babesiosis. This study describes the development and testing of a live B. bovis vaccine expressing the protective tick antigen glutathione S-transferase from Haemaphysalis longicornis (HlGST). The B. bovis S74- T3B parasites were electroporated with a plasmid containing the bidirectional Ef-1 promoter of B. bovis controlling expression of two independent genes, the selectable marker GFP-BSD, and HlGST fused to the MSA-1 (merozoite surface antigen-1) signal peptide from B. bovis. Electroporation followed by blasticidin selection resulted in the emergence of a mixed B. bovis transfected line (termed HlGST) in in vitro cultures, containing parasites with distinct patterns of insertion of both exogenous genes, either in or outside the Ef-1 a locus. A B. bovis clonal line termed HlGST-Cln expressing HlGST and GFP-BSD was then derived from the mixed parasite line HlGST. Two independent calf immunization trials were performed via intravenous inoculation of the HlGST-Cln and a control consisting of an irrelevant transfected clonal line of B. bovis designated GFP-Cln. The control GFP-Cln line contains a copy of the GFP-BSD gene inserted into the Ef-1 locus of B. bovis in an identical fashion as the HIGST-Cln parasites. All animals inoculated with the HlGST-Cln and GFP-Cln transfected parasites developed mild babesiosis indicating that both transfected cloned parasite lines are attenuated. All animals immunized with HlGST-Cln produced detectable anti-glutathione-S-transferase antibodies. After immunization with HlGST-Cln, calves were challenged with R. microplus larva. Development of these larva produce fully engorged female tick with reduced weight and fertility. Collectively, these data show that transfected B. bovis parasites can be used as vectors in live vectored vaccines.

Controlled hydrogel dissolution allows for: 1) atraumatic material removal after it served its function, 2) site-specific delivery of encapsulated therapeutics (e.g., proteins, small molecules), and 3) a tailored administration of an agent with high efficiency. Dissolution of covalently crosslinked hydrogels has been accomplished by incorporating cleavable moieties that undergo ester hydrolysis or enzymatic degradation. Recently, thiol-disulfide exchange, retro Michal-type reactions, retro Diels-Alder reactions, and thiol-thioester exchange chemistries have gained attention, as they provide a responsive synthetic handle for engineering hydrogel dissolution rates. We synthesized, characterized and tested in vivo two on-demand dissolvable dendritic thioester hydrogel dressings for second-degree burn care and hemorrhage control. The hydrogels are composed of lysine-based dendrons and PEG-based crosslinkers, which were prepared in high yields. In context of hemorrhage, there is an unmet clinical need for an on-demand dissolvable sealant for non-compressible hemorrhage or areas of body not amenable to treatment with a torniquet. In a model of in vivo hemorrhage control of intra-abdominal wounds, our hydrogel reduced blood loss by 33% in severe hepatic hemorrhage and by 22% in aortic injury, as compared to untreated controls. There is an unmet clinical need for a second-degree burn dressing that can be removed atraumatically and serve as a barrier to bacterial infection. When our hydrogel was used as a dressing, local and systemic bacterial proliferation after wound contamination was significantly lower than in the untreated group. The total bacterial burden of the burn wound in the positive controls was significantly higher than in the hydrogel group and the negative controls (1.39x10E8 ± 8.30x10E7 CFU/g v. 4.04x10E3 ± 3.99x10E3 CFU/g v. 6.88x10E2 ± 6.38x10E2 respectively; P = 0.009). Also, the total systemic bacterial burden in the positive controls was significantly higher than the hydrogel group and the negative controls (9x10E2 ± 7.76x10E7 CFU/g v. 5x10E1 ± 0 CFU/g v. 5x10E1 ± 0 CFU/g, respectively; P = 0.031). A unique feature of both hydrogel systems is their capability to be dissolved on-demand via thiol-thioester exchange reaction with a biocompatible solution following its initial application – thus the wound area can be re-exposed to allow for definitive surgical care.