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Durch Ausübung physiologischer Grundfunktionen spielen Ionenkanäle eine entscheidende Rolle für die reguläre Funktion von Zellen. Zum besseren Verständnis ihrer Struktur und Funktion sind Untersuchungen natürlicher und künstlicher Ionenkanäle wichtige Werkzeuge. Großes analytisches und therapeutisches Potential ist in der Untersuchung künstlicher Kanäle in lebenden Zellen vorhanden, was bisher wenig Beachtung fand. In der vorliegenden Arbeit wurde die Wirkung der künstlichen Ionenkanäle THF-gram, THF-gram-TBDPS sowie linked-gram-TBDPS auf elektrophysiologische Eigenschaften boviner Trabekelwerkszellen des Auges anhand von Patch-Clamp-Untersuchungen im Whole-Cell-Modus analysiert. Die Untersuchung brachte folgende Erkenntnisse: 1. Die Inkorporation aller drei Verbindungen war erfolgreich, was sich durch Anstieg der Stromdichte und Verschiebung des Umkehrpotentials zeigte. 2. Einbau von THF-gram und THF-gram-TBDPS war mit dem Überleben der Zellen vereinbar, während linked-gram-TBDPS aufgrund einer sehr potenten Antwort bereits bei sehr geringen Konzentrationen zum raschen Zelltod führte. 3. Eine Asymmetrie der Stromantwort zugunsten stärkerer Auswärtsströme wurde bei THF-gram und in schwächerer Ausprägung bei THF-gram-TBDPS festgestellt. Linked-gram-TBDPS zeigte keine derartige Asymmetrie. 4. Unter Verwendung von Cs+ als Ladungsträger war der beobachtete Anstieg der Stromdichte bei allen drei Verbindungen eindeutig stärker als unter physiologischen Bedingungen (Na+/K+). 5. Die dargestellten Erkenntnisse sind ein erster Schritt zur therapeutischen Anwendung von künstlichen Ionenkanälen. Eine Weiterentwicklung in Richtung höherer Selektivität und besserer Kontrolle ist jedoch genauso erforderlich wie die Klärung der klinischen Umsetzbarkeit. / Ion channels play a pivotal role for regular cell function. To better understand their structure and function, investigation of both natural and artificial ion channels is being performed to date. Investigation of artificial channels in living cells hides a big potential, however, little attention has been paid to this field so far. In this work, the effect of the artificial ion channels THF-gram, THF-gram-TBDPS and linked-gram-TBDPS on electrophysiological properties of bovine trabecular meshwork cells was investigated with the patch-clamp-technique. Following results were obtained: 1. Incorporation of all three compounds was successful, which was proven by increase of current density and cell depolarisation. 2. The cells survived after incorporation of THF-gram and THF-gram-TBDPS but not after linked-gram-TBDPS, which resulted in cell death at very low concentrations. 3. Larger outward currents were observed with THF-gram and, at a lower extent, with THF-gram-TBDPS. Linked-gram-TBDPS did not show such an asymmetry. 4. With Cs+ as charge carrier all three compunds showed a stronger increase of current density than under physiological conditions (Na+/K+). 5. The decribed results are a first step towards therapeutic application of artificial ion channels, however, further development towards higher selectivity and better control is as necessary as clarification of clinical feasibility.

Avaliação da \"qualidade\" da microarquitetura óssea contribui na prevenção e risco de fraturas associado a osteoporose. Amostras de ossos trabeculares de corpos vertebrais da região T12, L1 e L4 de 30 de indivíduos (cadáveres humanos), classificados pela técnica de ultrassonometria de calcâneo como normais, osteopênicos e osteoporóticos foram utilizadas. Os parâmetros histomorfométricos, físicos e mecânicos da microarquitetura analisados pela técnica de microtomografia de raios-X, ensaios mecânicos de compressão axial e nanoindentação. Baseado nestes parâmetros, constantes elásticas efetivas globais foram obtidas pela técnica homogeneização assintótica para estrutura tipo-placa. Teste ANOVA apresentou diferenças, muito e extremamente significativas para densidade linear estrutural (p = 0,017), grau de anisotropia (p = 0,042), auto valor (p = 0,045), número de Euler (p = 0,016), idade (p = 0,009), separação trabecular (p = 0,009), densidade de conectividade (p = 0,006), módulo de elasticidade (p = 0,001), tensão máxima no limite de resistência a fratura (0,001), índice de qualidade óssea do calcâneo (p = 0,0001), fração de volume ósseo (p = 0,0001), espessura trabecular (p = 0,0002), dimensão fractal (p = 0,0002), momento de inércia polar médio (p = 0,00005), excentricidade (p = 0,0002), fração de porosidade (p = 0,0001) e densidade mineral óssea aparente (p = 0,00005) entre os grupos. O teste de Spearman identificou correlação moderada, alta e muito alta dos valores da tensão máxima no limite de resistência a fratura com os parâmetros de idade (r = -0,684), fração de volume ósseo (r = 0,762), fator de forma trabecular (r = -0,754), espessura trabecular (r = 0,675), densidade linear trabecular (r = 0,622), autos valores (r = -0,615), dimensão fractal (r = 0,855), momento de inércia polar médio (r = 0,816), excentricidade (r = -0,569) e fração de porosidade (r = -0,762). Estes parâmetros são indicadores da qualidade da microarquitetura óssea trabecular e risco de fratura associado a osteoporose. / Evaluation of the \"quality\" of bone microarchitecture can contribute to the prevention and risk of fractures associated with osteoporosis. Samples of trabecular bone from the vertebral regions T12, L1 and L4 from the bodies of 30 individuals (human cadavers), classified by ultrasonometry technique calcaneus as normal, osteopenic and osteoporotic were used. The histomorphometric, physical and mechanical parameters of the microarchitecture were analyzed by X-ray microtomography technique, mechanical tests of axial compression and nanoindentation. Based on these parameters, globally effective elastic constants were obtained by asymptotic homogenization technique for type-plate structure. ANOVA test showed differences that were very and extremely significant for the following: structural linear density (p = 0.017) anisotropy (p = 0.042), auto value (p = 0.045), Euler number (p = 0.016), age (p = 0.009), trabecular separation (p = 0.009), connectivity density (p = 0.006), elastic modulus (p = 0.001), maximum stress fracture strength limit (0.001), bone quality score of the calcaneus (p = 0.0001), bone volume fraction (p = 0.0001), trabecular thickness (p = 0.0002), fractal dimension (p = 0.0002), mean polar moment of inertia (p = 0.00005), eccentricity (p = 0.0002), porosity fraction (p = 0.0001) and apparent bone mineral density (p = 0.00005) between groups. The Spearman test identified moderate correlation, high and very high values of maximum stress fracture resistance limit for the following parameters: age (r = -0.684), bone volume fraction (r = 0.762), trabecular bone pattern factor (r = -0.754), trabecular thickness (r = 0.675), trabecular linear density (r = 0.622), auto value (r = -0.615), fractal dimension (r = 0.855), mean polar moment of inertia (r = 0.816), eccentricity (r = -0.569) and porosity fraction (r = -0.762). These parameters are quality indicators of the microarchitecture of trabecular bone and fracture risk associated with osteoporosis.

Cette thèse présente l’apport d’une analyse fractale de la texture de l’os trabéculaire sur des clichés de radiographie du genou pour la caractérisation de la gonarthrose. Dans un premier temps cette analyse de texture a été confrontée aux méthodes actuelles de suivi de la pathologie afin de valider leur intérêt dans le suivi clinique des patients. Un second temps a été consacré à la prédiction de l’initiation de la pathologie où aucun marqueur ne fait référence. L’analyse de texture a été menée en utilisant des approches fractales permettant de s’affranchir des problèmes de variation d’acquisition entre différents centres cliniques tout en comparant différentes méthodes de calcul. La texture osseuse trabéculaire a été analysée à différentes plages d’échelle afin de caractériser les mécanismes sous-jacents de modification de la microarchitecture osseuse induits par la gonarthrose. Nous avons pu mettre en évidence l’intérêt prédictif de cette analyse peu coûteuse et largement accessible dans le cadre aussi bien de l’initiation que de la progression de l’arthrose du genou. / This thesis presents the contribution of the trabecluar bone texture fractal analysis on knee radiographs for the knee osteoarthritis characterization. Firstly, this texture analysis was confronted to the actual methods of the pathology clinical monitoring in order to validate their interest in the patients monitoring. Secondly, the prediction of the knee osteoarthritis initiation was investigated, domain in which there are still no markers defined. The texture analysis was performed using fractal approaches avoiding the problems of the variations in the acquisition among different clinical centers and comparing different computing methods. Trabecular bone texture was analyzed at different ranges of scale in order to characterize the underlying mechanisms of the microarchitecture modification induced by the knee osteoarthritis. Thus, we highligthed the predictive interest of this cheap and largely available analysis for both the initiation and the progression of the knee osteoarthritis.

Le taxon du Pléistocène inférieur Paranthropus robustus, dont l'holotype est le spécimen TM 1517, a été défini en 1938 par le paléontologue R. Broom suite à la découverte d'éléments crâniens et postcrâniens sur le site de Kromdraai, Gauteng, en Afrique du Sud. Depuis, d'autres sites sud-africains ont contribué à l'extension de son hypodigme et fourni la preuve de sa contemporanéité à l'échelle macro-régionale avec des représentants des taxons Australopithecus et Homo. L'identification des hominines étant principalement basée sur l'analyse de la variation morphologique des éléments cranio-dentaires, un enjeu majeur dans l'étude des assemblages fossiles des sites sud-africains concerne donc l'identification et l'attribution taxinomique de restes isolés et/ou fragmentaires du squelette axial et appendiculaire non associés à des éléments cranio-dentaires. Il en résulte que plusieurs aspects fonctionnels et paléobiologiques du squelette postcrânien de P. robustus restent à préciser. Au moyen de la microtomographie à rayons X, de l'imagerie virtuelle et d'analyses quantitatives en deux-trois dimensions, nous avons entrepris un projet de recherche visant à explorer, extraire et les patrons d'organisation endostructurale de spécimens fossiles communément, ou préliminairement, attribués à P. robustus. Sur une base comparative, nous visons à (i) identifier quelques caractéristiques osseuses endostructurales propres à ce taxon, qui pourraient fournir un cadre de référence pour l'attribution de spécimens fossiles isolés; (ii) déconstruire l'environnement biomécanique ayant façonné l'arrangement de l'os cortical et trabéculaire au niveau des articulations du coude, de la hanche et du genou; (iii) évaluer le degré de variation et, dans la mesure du possible, les différences liées au sexe et à l'âge. L'échantillon étudié comprend quatre huméri distaux (TM 1517g, SK 24600, SKX 10924, SKX 34805), cinq fémurs proximaux (SK 82, SK 97, SK 3121, SKW 19, SWT1/LB-2), une patella (SKX 1084), des éléments additionnels échantillonnant l'assemblage TM 1517 (l'ulna proximale TM 1517e, la phalange distale d'hallux TM 1517k) et deux ilia (TM 1605, SK 50) provenant des sites de Kromdraai et Swartkrans. [...] / The Early Pleistocene taxon Paranthropus robustus, represented by the holotype TM 1517, was established in 1938 by the paleontologist R. Broom following the discovery of craniodental and postcranial remains at the cave site of Kromdraai, in Gauteng, South Africa. Since, other Southern African sites have contributed to the extension of its hypodigm, providing evidence for its chronological overlap in the macro-region with representatives of the taxa Australopithecus and Homo. As species identification in the hominin fossil record is commonly based on the comparative assessment of craniodental anatomy and morphological variation, the rarity in the hominin-bearing South African cave assemblages of unambiguously associated craniodental and postcranial remains usually complicates the task of identifying isolated and fragmentary elements from the axial and the appendicular skeleton. Consequently, different functionally- and paleobiologically-related aspects of the P. robustus postcranial skeleton remain poorly known. By means of techniques of high resolution X-ray micro-tomography and virtual imaging coupled with two-three-dimensional quantitative analyses, in this research project we explored, extracted and assessed the patterns of endostructural organisation in some fossil specimens commonly, or tentatively, attributed to P. robustus. On comparative ground, we aim at (i) identifying some endostructural bony features characteristic of this taxon, if any, thus tentatively providing a reference framework for the attribution of isolated fossil specimens; (ii) deconstructing the biomechanical (loading) environment having shaped the cortical and cancellous bone arrangement at the elbow, the hip, and the knee joints; (iii) assessing variation and, whenever possible, sex- and age-related differences. The investigated sample consists of four distal humeri (TM 1517g, SK 24600, SKX 10924, SKX 34805), five proximal femora (SK 82, SK 97, SK 3121, SKW 19, SWT1/LB-2), a patella (SKX 1084), some additional elements from the assemblage labelled TM 1517 (the proximal ulna TM 1517e, the distal hallucial phalanx TM 1517k), and two ilia (TM 1605, SK 50) from the sites of Kromdraai and Swartkrans. [...]

L’imagerie médicale et la 3D, en pleine expansion dans le champ de l'anthropologie biologique, permettent d’explorer les structures internes tout en les préservant. L’étude de la micro-architecture osseuse trabéculaire permet d’appréhender la variabilité de l'os humain à une échelle jusqu'à présent peu explorée. Dans le cadre de cette recherche, cette variabilité est analysée et caractérisée en termes de croissance et de maturation, en fonction des critères individuels d’âge et de sexe, ainsi que dans des contextes pathologiques variés. Les images microtomodensitométriques des métaphyses humérales proximales de 43 sujets immatures (provenant de 3 collections ostéologiques de référence et couvrant l’ensemble des âges du développement) et celles de 8 cas paléopathologiques (représentant 5 étiologies différentes) ont été analysées pour quantifier la micro-architecture osseuse trabéculaire. Nos résultats montrent que cette micro-architecture varie pendant et entre les différentes phases de la croissance. Des corrélations avec l’âge sont mises en évidence, si elles n’expliquent pas suffisamment la variabilité observée pour en faire des estimateurs d'âge précis, il apparaît néanmoins que les variations relevées entre les différents volumes d’intérêt pourraient caractériser différentes périodes de la croissance. Les variables mesurées présentent des différences sexuelles significatives pendant l’adolescence, mais ne peuvent pas en l'état être utilisées pour la diagnose sexuelle. L'étude de la microarchitecture trabéculaire osseuse des sujets pathologiques atteste d’un développement anormal de l’os et donc du statut pathologique de l’individu observé. / Medical imaging and 3D reconstructions are used increasingly by anthropologists; they allow both investigating and preserving internal structures. Study of trabecular bone microarchitecture allows understanding variability of human skeleton at a smaller scale. This variability is observed and characterized in terms of normal growth and maturation according to age and sex, and for several pathological conditions. μCT scans of proximal metaphysis of humerus from 43 immature individuals (coming from 3 identified skeletons collections and representing all periods of age development) and 8 paleopathological cases (corresponding to 5 different etiologies) have been analyzed to quantify bone microarchitecture. Our results show that this microarchitecture varies during and between different phases of growth. Correlations with age are highlighted, even if they do not sufficiently explain the observed variability in order to represent specific age estimators; it nevertheless appears that the variations observed between the different volumes of interest could characterize different periods of growth. The measured variables showed significant sex differences only during the adolescence period, but they cannot be used, in the present state, for sex determination. The study of the trabecular bone microarchitecture of pathological individuals attests of the abnormal development of bone and therefore of their pathological status.

Bone is a composite material consisting of hydroxyapatite crystals deposited in an oriented manner on a collagen backbone. The arrangement of the mineral and organic phases provides bone tissue with the appropriate strength, stiffness, and fracture resistance properties required to protect vital internal organs and maintain the shape of the body. A remarkable feature of bone is the ability to alter its properties and geometry in response to changes in the mechanical environment. However, in cases of metabolic bone diseases or aging, bone can no longer successfully adapt to its environment, increasing its fragility. To elucidate the mechanisms of bone microdamage, this research project developed a specimen-specific approach that integrated 3D imaging, histological damage labeling, image registration, and image-based finite element analysis to correlate microdamage events with microstructural stresses and strains under compressive loading conditions. By applying this novel method to different ages of bovine and human bone, we have shown that the local mechanical environment at microdamage initiation is altered with age. We have also shown that formation of microdamage is time-dependent and may have implications in age-related microdamage progression with cyclic and/or sustained static loading. The work presented in this dissertation is significant because it improved our understanding of trabecular bone microdamage initiation and unlocked exciting future research directions that may contribute to the development of therapies for fragility diseases such as osteoporosis.

Skeletal fragility is characterized by low bone mass, negative changes in bone microarchitecture, and compromised tissue matrix properties, including accumulation of microdamage. Microdamage accumulates in vivo from daily physiological loading and is targeted for repair through a normal remodeling process, thus preventing microcrack growth and potential fracture. However, impaired remodeling is associated with aging and osteoporosis, resulting in an increased accumulation of microdamage which contributes to reduced bone mechanical properties. The current clinical method for assessing increased risk of fracture involves measuring bone mineral density (BMD) of the hip and spine, locations of trabecular bone where high rates of remodeling occur. The bisphosphonate alendronate (ALN) reduces clinical risk for fracture by significantly increasing BMD, but studies have shown a concomitant reduction in intrinsic properties that may be the underlying cause for recent reports of spontaneous fractures with long-term alendronate use. Another anti-resorptive agent called raloxifene (RAL) is a selective estrogen receptor modulator (SERM) and has been shown to modestly improve BMD while decreasing fracture risk to a similar degree as alendronate. The combination of RAL and ALN as a treatment for osteoporosis may provide the benefits of each drug without the negative effects of ALN. Therefore, the overall goal of this thesis was to address the effects of aging and anti-resorptive agents on the properties of bone through the formation of microdamage. Assessment of age-related effects on bone was conducted through quantification of microdamage progression. It was found that old bone results in greater incidences of microdamage progression, reflecting a compromised tissue matrix with reduced resistance to crack growth. Effects of combination treatment with RAL and ALN were evaluated through biomechanical testing, micro-CT imaging, and microdamage quantification. Results showed improved trabecular bone volume and ultimate load with positive effects on trabecular architecture. Combination treatment reduced the proportion of microdamage that may lead to catastrophic fracture, indicating an improvement in the local tissue matrix properties.

Postmenopausal osteoporosis is a disease characterized by bone loss and increased risk of fracture, and represents a significant burden on the Canadian health care system. Current treatments lack the ability to simultaneously address the therapeutic needs for promoting bone formation and inhibiting resorption. Our approach employs a novel conjugate drug in which an anabolic agent (EP4 receptor agonist) is reversibly joined with an anti-resorptive agent (alendronate) through a linker. This allows the bone-targeting ability of alendronate to deliver the EP4 agonist to bone sites, thereby mitigating the side effects associated with systemic administration of the EP4 agonist. This study investigated the in vivo efficacy of this drug in a curative experiment to treat postmenopausal osteoporosis using an ovariectomized rat model. Results showed that conjugate treatment dose-dependently stimulated bone formation and restored ovariectomy-induced bone loss, and conjugation between alendronate and the EP4 agonist was crucial to the drug’s anabolic effect.

Postmenopausal osteoporosis is a disease characterized by bone loss and increased risk of fracture, and represents a significant burden on the Canadian health care system. Current treatments lack the ability to simultaneously address the therapeutic needs for promoting bone formation and inhibiting resorption. Our approach employs a novel conjugate drug in which an anabolic agent (EP4 receptor agonist) is reversibly joined with an anti-resorptive agent (alendronate) through a linker. This allows the bone-targeting ability of alendronate to deliver the EP4 agonist to bone sites, thereby mitigating the side effects associated with systemic administration of the EP4 agonist. This study investigated the in vivo efficacy of this drug in a curative experiment to treat postmenopausal osteoporosis using an ovariectomized rat model. Results showed that conjugate treatment dose-dependently stimulated bone formation and restored ovariectomy-induced bone loss, and conjugation between alendronate and the EP4 agonist was crucial to the drug’s anabolic effect.