Metastatic bone cancers cause severe symptoms including pain that compromises patient functional status, quality of life, and survival. Current treatment strategies have limited efficacy and dose-limiting side effects. Cancer-induced bone pain (CIBP) is a unique pain state that shares features with but is distinct from the pathology of neuropathic and inflammatory pain. This dissertation investigates how CIBP is generated and maintained by the direct effects of cancer cells on their metastatic microenvironment and the peripheral nervous system, including unique signaling properties and gene expression changes. In particular, we found that genetic knockdown of the functional subunit xCT of the system xC- cystine/glutamate antiporter can reduce CIBP, further elucidating this as a therapeutic of interest. We found that the neuroprotective voltage-gated calcium channel inhibitors progesterone and pregabalin markedly reduce mechanical hypersensitivity and excitability in sensory neurons of the dorsal root ganglion (DRG) in male rat models of neuropathic pain, but that these effects and less pronounced in females. In cancer pain, these sex differences are reversed, with females but not males demonstrating a delay in time-to-onset of mechanical hypersensitivity. We also analyzed gene expression at the DRG by RNA-Sequencing of rat models of CIBP. Our findings uncovered differential gene expression between CIBP and sham controls and between ipsilateral and contralateral DRGs in CIBP model rats. These studies have identified several promising avenues for therapeutic research for CIBP. / Dissertation / Doctor of Philosophy (PhD) / The tools we have right now to manage severe and chronic pain are insufficient. Patients with advanced cancers including bone cancer can suffer from very severe pain. This pain is generated in a number of ways including by the tumour itself releasing chemicals that activate pain-sensing nerves, by the destruction of the bone in and around the tumour, and by the sensitization of the nervous system, which can make pain worse and longer lasting. We have taken three approaches to researching cancer pain and to investigating new treatments. We have found that by reducing the amount of glutamate that cancer cells can release into their environment, we can reduce cancer pain in mice. We also found that treating rats with pregabalin and progesterone can change nerve signaling and reduce neuropathic pain, but that this effect is most pronounced in male rats with neuopathic pain and smaller in female rats with neuropathic pain, and even smaller in rats with cancer pain. We also analyzed expression of all the protein-coding genes in dorsal root ganglia from rats with cancer pain and found that there are many differences from rats without pain. Some of these differences may be promising new research targets. Going forward this research has provided important evidence necessary for next steps to develop new therapies and research strategies for cancer pain.

Los transportadores heteroméricos de aminoácidos (HAT) están formados por una subunidad ligera (LSHAT) y una subunidad pesada (HSHAT) unidas entre ellas mediante un puente disulfuro.Se han descrito dos HSHATs: rBAT, y 4F2hc. Son glicoproteínas de membrana de tipo II, con un extremo N-terminal intracelular, un único dominio trans-membrana y un extremo C-terminal extracelular homólogo a las alfa-amilasas.Hasta el momento se han identificado 9 LSHATS: 6 se unen a 4F2hc para dar lugar al transportador funcional (LAT-1, LAT-2, y+LAT-1, y+LAT-2, asc-1, xCT), una se une a rBAT (bo,+ AT) y existen dos miembros "huérfanos" (asc-2, AGT-1), que no interaccionan con las HSHATs descritas y que quizás se unen a HSHATs todavía por identificar. Las diferentes LSHAT presentan las siguientes características estructurales y funcionales comunes: 1. Son proteínas altamente hidrofóbicas no glicosiladas. 2. Presentan una predicción de estructura de 12 segmentos trans-membrana, con extremos N y C-terminal intracelulares. Esta topología se ha demostrado para la subunidad ligera xCT, que presenta además un reentrant-loop entre los segmentos trans-membrana 2 y 3, con evidencias funcionales de su proximidad a la vía de translocación de sustrato.3. El residuo de cisteína conservado que interviene en la formación del puente disulfuro se encuentra localizado en el dominio extracelular putativo II.4. Necesitan la coexpresión de la HSHAT para alcanzar la membrana plasmática en un sistema de expresión heterólogo. 5. Confieren la especificidad de transporte al complejo heteromérico, representando una gran diversidad de sustratos y acoplamiento a iones: aminoácidos neutros de tamaño grande (LAT-1, LAT-2), pequeño (asc-1, LAT-2), cargados negativamente (xCT) y aminoácidos básicos y neutros (y+LAT-1, y+LAT-2 y bo,+AT).6. Se comportan como intercambiadores obligatorios con una estequiometría 1:1 y con una afinidad intracelular aparente por el sustrato mucho menor que la extracelular, excepto para el caso asc-1/4F2hc, y quizás asc-2, que se comporta como un intercambiador no obligatorio.7. La LSHAT es capaz de mediar el transporte en ausencia de la subunidad pesada cuando se consigue expresarla en superficie, como ocurre en un sistema reconstituido.rBAT (codificada por el gen SLC3A1) y bo,+AT (codificada por el gen SLC7A9) forman el sistema bo,+, que transporta aminoácidos neutros y básicos. Defectos en este sistema de transporte causan cistinuria. La cistinuria (OMIM 220100) es una enfermedad hereditaria, autosómica recesiva, causada por el defecto en la absorción intestinal y la reabsorción renal de aminoácidos básicos (lisina, arginina, ornitina) y cistina. Debido a su baja solubilidad, la cistina precipita formando cálculos a lo largo del sistema urinario. Los cálculos causan obstrucción, infecciones y, en último término, insuficiencia renal. Mutaciones en SLC3A1 causan cistinuria tipo I (recesiva completa: los heterozigotos no presentan hiperexcreción de aminoácidos) y mutaciones en SLC7A9 causan cistinuria tipo no I (recesiva incompleta: los heterozigotos hiperexcretan los cuatro aminoácidos en menor medida que los homozigotos cistinúricos y raramente llegan a desarrollar cálculos).En esta tesis se ha realizado un análisis mutacional de rBAT (SLC3A1) en familias cistinúricas, y estudios de relación estructura-función con las LSHATS bo,+AT y xCT. Debido al solapamiento de fenotipos encontrado en los portadores con mutaciones en SLC3A1 o SLC7A9, se ha establecido una nueva clasificación de la cistinuria, basada en datos genéticos. Los estudios de relación estructura-función han dado lugar a la identificación de un residuo en xCT (C327) próximo al lugar de unión y/o translocación de sustrato, y a la determinación de la unidad funcional mínima en xCT y bo,+AT, formada por una única subunidad ligera. Con estos resultados, y con los conocimientos que tenemos de esta familia de transportadores, proponemos que en una subunidad ligera coexisten dos vías de translocación asimétricas, una para el influjo, y otra para el eflujo. / Heteromeric amino acid transporters (HATs) are composed by disulfide-linked heavy (HSHAT) and light (LSHAT) subunits. HSHATs are type II membrane glycoproteins with a large extracellular domain and are involved in trafficking of the heterodimer to the plasma membrane. The LSHAT is a polytopic membrane protein that confers transport function and specificity. Two HSHATs are known, rBAT and 4F2hc. rBAT forms system b0,+ with the light subunit b0,+AT. 4F2hc forms two different system L isoforms with LAT1 and LAT2, two different system y+L isoforms with y+LAT1 and y+LAT2, system asc with asc1, and system xC- with xCT.Mutations in rBAT (SLC3A1 gene) or b0,+AT (SLC7A9 gene) cause cistinuria, an autosomal inherited metabolic disorder characterised by impaired transport of cystine and dibasic amino acids in the renal tubule and the gastrointestinal tract. High cystine concentration in the urinary tract often causes the formation of cystine stones. SLC3A1 mutations cause type I cystinuria (recessive form) and SLC7A9 mutations cause non-type I cistinuria (dominant form with incomplete penetrance). Mixed cistinuria has also been described. Mutational analysis in SLC3A1 realized in this thesis, together with mutational analysis in SLC7A9 in 164 families of the International Cystinuria Consortium database, established a new genetic classification: type A, with two mutations in SLC3A1; type B, with two mutations in SLC7A9; and type AB, with one mutation on each gene. Digenic inheritance in two mixed families caused partial phenotype.Little is known about the structure-function relationships of the HATs. Structure-function studies realized in this thesis demonstrate that Cys327 in transmembrane domain 8 of xCT is the target for transport inactivation by sulfhydril reagents. Protection and kinetic experiments suggest that Cys327 is close to the substrate permeation pathway. On the other hand, co-injection of xCT or b0,+AT sensitive (wild type) and insensitive (xCT C327S and b0,+AT C321S) to the inactivation by sulfhydryl reagents, and/or the effect of these reagents on concatamers indicate that the heterodimer is the functional unit of systems b0,+ and xc-. Together, with earlier studies on system b0,+, the results suggest that two asymmetric translocation pathways (export and import) co-exist simultaneously on a single LSHAT subunit.

Submitted by Luciana Sebin (lusebin@ufscar.br) on 2016-10-10T13:15:34Z No. of bitstreams: 1 DissGMS.pdf: 1736443 bytes, checksum: 9e6aa510fda128c4e4722add3ed4b7ed (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-10-13T20:11:02Z (GMT) No. of bitstreams: 1 DissGMS.pdf: 1736443 bytes, checksum: 9e6aa510fda128c4e4722add3ed4b7ed (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-10-13T20:11:13Z (GMT) No. of bitstreams: 1 DissGMS.pdf: 1736443 bytes, checksum: 9e6aa510fda128c4e4722add3ed4b7ed (MD5) / Made available in DSpace on 2016-10-13T20:11:25Z (GMT). No. of bitstreams: 1 DissGMS.pdf: 1736443 bytes, checksum: 9e6aa510fda128c4e4722add3ed4b7ed (MD5) Previous issue date: 2016-02-26 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Ethanol addiction is a serious public health problem that still needs more effective pharmacological treatment. A key factor in the development and maintenance of this disease is the development of neural plasticity that occurs in the mesocorticolimbic brain pathway upon chronic ethanol abuse. These plasticity events are, in general, maladaptive and affect numerous neurotransmitter systems and intracellular molecules. One of these molecules is ΔFosB, a transcriptional factor that is altered after chronic drug use. Behavioral sensitization is a phenomenon resulting from repeated administration of abuse drugs useful for the study of the neural alterations related to addiction. Recent works have shown a role for the imbalance of glutamatergic neurotransmission in the symptoms found in addicted people. In this line, the treatment with N-acetylcysteine, a L-cysteine prodrug that acts restoring extrasynaptic concentrations of glutamate through the activation of cystine-glutamate antiporter, has shown promising results in the treatment of psychostimulant addiction. Thus, we evaluated the effects of N-acetylcysteine treatment in behavioral and molecular alterations induced by chronic ethanol administration. Swiss mice were subject to thirteen days of daily ethanol administration to induce the development of behavioral sensitization. Two hours before each ethanol administration and locomotor activity assessment, animals received N-acetylcysteine injections i.p.. Right after the last test session, their brains were removed for ΔFosB and cystineglutamate antiporter quantification. We found that N-acetylcysteine treatment blocked ethanol-induced behavioral sensitization, the increase of ΔFosB content in the medial prefrontal cortex and its reduction in the nucleus accumbens. Our results suggest a possible use of N-acetylcysteine in the ethanol-related disorders. / A dependência ao etanol é um grave problema de saúde pública que ainda necessita de tratamentos farmacológicos mais efetivos. Um fator chave no desenvolvimento e manutenção dessa doença são as plasticidades neurais que ocorrem na via mesocorticolímbica mediante o abuso crônico de etanol. Estas plasticidades são, em geral, maladaptativas e afetam inúmeros sistemas de neurotransmissores e moléculas intracelulares. Uma dessas moléculas é a ΔFosB, um fator de transcrição que é alterado após o uso crônico de drogas de abuso. A sensibilização comportamental é um fenômeno decorrente da administração repetida de drogas muito útil no estudo das alterações neurais relacionadas à dependência. Trabalhos recentes tem demonstrado um papel do desequilíbrio da neurotransmissão glutamatérgica nos sintomas encontrados em indivíduos dependentes. Neste sentido, o tratamento com a N-acetilcisteína, um pró-fármaco da L-cisteína que atua restaurando as concentrações extrasinápticas do glutamato através da ativação do trocador cistina-glutamato, tem mostrado resultados promissores no tratamento da dependência de psicostimulantes. Assim, avaliamos os efeitos do tratamento com a N-acetilcisteína nas alterações comportamentais e moleculares induzidas pela administração crônica de etanol. Camundongos suíços machos foram submetidos a administrações diárias de etanol por 13 dias a fim de induzir o desenvolvimento da sensibilização comportamental. Duas horas antes de cada administração, os animais receberam uma administração intraperitoneal de Nacetilcisteína. Imediatamente após a última sessão de teste, os cérebros dos animais foram removidos para quantificação de ΔFosB e do trocador cistinaglutamato. Nós encontramos que o tratamento com a N-acetilcisteína bloqueou o desenvolvimento da sensibilização comportamental ao etanol, o aumento de ΔFosB no córtex pré-frontal medial e a sua redução no núcleo acumbens. Nossos resultados sugerem um possível uso da N-acetilcisteína nas desordens relacionadas ao uso de etanol. / FAPESP: 2015/01026-9

No description available.

Concrete is the most widely used construction material in the world. Given its relative availability, strength, economy, and versatility to fit various applications, the material has been incorporated in roadways, bridges, buildings, and a host of other infrastructure projects. Oftentimes, concrete will be exposed to several environmental conditions that ultimately affect its durability and lifespan. These conditions include repeated freezing and thawing, chloride intrusion, sulfate attack, alkali-silica reaction, and many others. Given the age and condition of American infrastructure, concrete structures throughout the country need repair or rehabilitation. Often this repair includes the removal of degraded or damaged concrete and the application of an overlay material. There are several factors affecting the bond performance of the newly formed substrate-repair concrete, such as surface roughness, overlay material, and substrate moisture condition. The work presented in this thesis is dedicated to understanding the effect of substrate moisture condition on the overlay transition zone (OTZ) of the substrate-repair concrete. The substrate moisture condition can significantly impact the microstructure characterization of the OTZ. If the substrate is too dry, then it may absorb water from the repair material, reducing the local water-to-cement (w/c) ratio in the OTZ. Conversely, if the substrate is too wet, then the w/c ratio of the OTZ will be locally increased. In both scenarios, the interfacial bond strength is expected to be modified due to the change in the local w/c ratio. To understand this effect, various test methods and degradation mechanisms were explored. Initially, substrate-repair concrete specimens were prepared utilizing three separate substrate moisture conditions: saturated surfaced dry (SSD), sub-saturated surface dry (Sub-SSD), and oven dry (OD). After allowing these samples to cure, the strength and ion penetration risk were evaluated. The bond strength of the samples was evaluated through flexural strength testing and fracture energy determined through the RILEM draft tests. The OTZ ion penetration risk was evaluated by conducting rapid chloride penetration test (RCPT) on samples prepared with the three substrate moisture conditions. Furthermore, to determine the effect of repeated freezing and thawing on the OTZ and flexural strength, additional samples were created with the three moisture conditions. After allowing these samples to cure, they were subjected to ASTM C666 and were tested to observe their flexural strength. Another important performance indicator of concrete elements is its resistance to chloride ion penetration and corrosion. Since many structural elements are designed with steel reinforcement, chloride ion penetration represents a critical parameter in projecting material performance, since chloride ions will accelerate the rate of steel corrosion. Oftentimes, a key element in projecting this performance is identifying the rate at which ions diffuse through the material. There remain many established techniques to identify this rate of diffusion and derive a chloride diffusion coefficient; however, many of them are either destructive or qualitative in nature. In recent years, transmission X-ray microscopy (TXM) has been employed to non-destructively track diffusion and develop diffusion coefficients. The work presented in this thesis surrounds the efforts of incorporating TXM experiments at Virginia Tech. This work initially utilized a SkyScan 1174 μCT, and additional work in this thesis presents the design and construction of a dental X-ray system based on the checking ion penetration (CHIP) design. This system can conduct TXM experiments utilizing a dental X-ray as the source. The research, design, and construction of the CHIP system is discussed in this thesis. Ultimately, the research in this thesis has not observed any significant relationship between substrate moisture condition and overlay bond strength. There does appear to be an increase in chloride ion resistance for drier substrates, suggesting that pre-wetting the surface increases penetrability of the interface. / Master of Science / Concrete is the most widely used construction material in the world. Given its relative availability, strength, economy, and versatility to fit various applications, the material has been incorporated in roadways, bridges, buildings, and a host of other infrastructure projects. Oftentimes, concrete will be exposed to several environmental conditions that ultimately affect its durability and lifespan. These conditions include repeated freezing and thawing, chloride intrusion, sulfate attack, alkali-silica reaction, and many others. These environmental conditions ultimately degrade the material by inducing cracks, exposing steel reinforcement, and spalling. When the concrete has experienced significant deterioration, repair and rehabilitation of the damaged section must be performed. Most often, this repair consists of the removal of damaged concrete and the application of an overlay material to prevent further deterioration. The topics discussed in this thesis evaluate the optimum substrate conditions prior to an overlay application and the implementation of techniques to evaluate deterioration mechanisms. There are several substrate conditions that will affect bonding with the overlay material, including surface roughness, moisture conditions, and overlay type. This paper focused on the moisture condition and what effect this had on bond strength and resistance to chloride intrusion. This effect was studied in laboratory conditions and under environmental conditions such as rapid freezing and thawing. Several different deterioration mechanisms may contribute to concrete degradation. The research presented in this thesis also aimed to evaluate chloride ion diffusion. To evaluate this mechanism, two systems were explored with the intent of conducting transmission X-ray microscopy (TXM). With TXM, chloride ion diffusion can be tracked to determine the rate at which ions diffuse through the concrete. The two systems explored were an X-ray computed tomography scanner and a dental X-ray system. Both systems can conduct TXM, and this paper presents the efforts dedicated to developing them for this technique at Virginia Tech. Ultimately, the research in this thesis has not observed any significant relationship between substrate moisture condition and overlay bond strength. There does appear to be an increase in chloride ion resistance for drier substrates, suggesting that pre-wetting the surface increases penetrability of the interface.

No description available.

Im Rahmen der vorliegenden Dissertationsschrift „Semi-quantitative röntgentomographische Untersuchungen zur Biodistribution von magnetischen Nanopartikeln in biologischem Gewebe“ wurden tomographische Untersuchungen an biologischen Objekten durchgeführt. Bei diesen Objekten handelt es sich um Gewebeproben nach minimal-invasiven Krebstherapien wie zum Beispiel magnetischem Drug Targeting und magnetischer Wärmebehandlung. Der Erfolg dieser Therapien ist sowohl abhängig von der korrekten Verteilung der magnetischen Nanopartikel als auch von der Tatsache, dass diese in der Zielregion in einer ausreichenden Menge vorhanden sind. Das Vorliegen dieser beiden Voraussetzungen ist in der vorliegenden Arbeit untersucht worden. Dabei lag der Schwerpunkt der Arbeit auf der Quantifizierung von magnetischem Material in unterschiedlichen biologischen Gewebeproben mittels Röntgenmikrocomputertomographie (XµCT). Für diesen Zweck wurde ein Kalibrationssystem mit speziellen Phantomen entwickelt, mit dessen Hilfe eine Nanopartikelkonzentration einem Grauwert voxelweise zugewiesen werden kann. Mit Hilfe der Kalibration kann der Nanopartikelgehalt sowohl in monochromatischen als auch in polychromatischen tomographischen Daten im Vergleich zu magnetorelaxometrischen Ergebnissen mit wenigen Prozent Abweichung ermittelt werden. Trotz Polychromasie und damit einhergehenden Artefakten können 3-dimensionale röntgentomographische Datensätze mit einer geringfügigen Konzentrationsabweichung im Vergleich zur quantitativen Messmethode Magnetorelaxometrie semi-quantitativ ausgewertet werden. / The success of the minimal invasive cancer therapies, called magnetic drug targeting and magnetic heating treatment, depends strongly on the correct distribution of the magnetic nanoparticles on one side. On the other side it depends on the fact that a sufficient amount of magnetic nanoparticles carrying drugs is accumulated in the target region. To study whether these two requirements are fulfilled motivates this PhD thesis „Semi-quantitative X-ray-tomography examinations of biodistribution of magnetic nanoparticles in biological tissues“. The analysis of the distribution of the magnetic nanoparticles in tumours and other tissue examples is realized by means of X-ray-micro computer tomography (XμCT). The work focuses on the quantification of the magnetic nanoparticles in different biological tissue samples by means of XµCT. A calibration of the tomographic devices with adequate phantoms, developed in the frame of this work, opens now the possibility to analyze tomographic data in a semi-quantitative manner. Thus, the nanoparticle concentration can be allocated voxel-wise to the grey values of the three-dimensional tomographic data. With the help of calibration of the tomography equipments used, polychromatic as well as monochromatic three-dimensional representations of objects can be analyzed with regard to the biodistribution of magnetic nanoparticles as well as with regard to their quantity. The semi-quantitative results have been compared with results obtained with a quantitative measurement method magnetorelaxometry (MRX). Thereby a good agreement of the semi-quantitative and quantitative data has been figured out.

Im Rahmen der vorliegenden Dissertationsschrift „Semi-quantitative röntgentomographische Untersuchungen zur Biodistribution von magnetischen Nanopartikeln in biologischem Gewebe“ wurden tomographische Untersuchungen an biologischen Objekten durchgeführt. Bei diesen Objekten handelt es sich um Gewebeproben nach minimal-invasiven Krebstherapien wie zum Beispiel magnetischem Drug Targeting und magnetischer Wärmebehandlung. Der Erfolg dieser Therapien ist sowohl abhängig von der korrekten Verteilung der magnetischen Nanopartikel als auch von der Tatsache, dass diese in der Zielregion in einer ausreichenden Menge vorhanden sind. Das Vorliegen dieser beiden Voraussetzungen ist in der vorliegenden Arbeit untersucht worden. Dabei lag der Schwerpunkt der Arbeit auf der Quantifizierung von magnetischem Material in unterschiedlichen biologischen Gewebeproben mittels Röntgenmikrocomputertomographie (XµCT). Für diesen Zweck wurde ein Kalibrationssystem mit speziellen Phantomen entwickelt, mit dessen Hilfe eine Nanopartikelkonzentration einem Grauwert voxelweise zugewiesen werden kann. Mit Hilfe der Kalibration kann der Nanopartikelgehalt sowohl in monochromatischen als auch in polychromatischen tomographischen Daten im Vergleich zu magnetorelaxometrischen Ergebnissen mit wenigen Prozent Abweichung ermittelt werden. Trotz Polychromasie und damit einhergehenden Artefakten können 3-dimensionale röntgentomographische Datensätze mit einer geringfügigen Konzentrationsabweichung im Vergleich zur quantitativen Messmethode Magnetorelaxometrie semi-quantitativ ausgewertet werden. / The success of the minimal invasive cancer therapies, called magnetic drug targeting and magnetic heating treatment, depends strongly on the correct distribution of the magnetic nanoparticles on one side. On the other side it depends on the fact that a sufficient amount of magnetic nanoparticles carrying drugs is accumulated in the target region. To study whether these two requirements are fulfilled motivates this PhD thesis „Semi-quantitative X-ray-tomography examinations of biodistribution of magnetic nanoparticles in biological tissues“. The analysis of the distribution of the magnetic nanoparticles in tumours and other tissue examples is realized by means of X-ray-micro computer tomography (XμCT). The work focuses on the quantification of the magnetic nanoparticles in different biological tissue samples by means of XµCT. A calibration of the tomographic devices with adequate phantoms, developed in the frame of this work, opens now the possibility to analyze tomographic data in a semi-quantitative manner. Thus, the nanoparticle concentration can be allocated voxel-wise to the grey values of the three-dimensional tomographic data. With the help of calibration of the tomography equipments used, polychromatic as well as monochromatic three-dimensional representations of objects can be analyzed with regard to the biodistribution of magnetic nanoparticles as well as with regard to their quantity. The semi-quantitative results have been compared with results obtained with a quantitative measurement method magnetorelaxometry (MRX). Thereby a good agreement of the semi-quantitative and quantitative data has been figured out.