Despite being the most prevalent presentation of Age-Related Macular Degeneration (AMD), dry AMD (dAMD) lacks a therapeutic treatment. Retinal pigment epithelium (RPE) dysfunction preceding the onset of dAMD has inspired interest in regenerative medicine approaches seeking to replenish the RPE and preserve visual acuity. Cell delivery to the subretinal space however has been met with challenges surrounding ease of access and invasive surgical implantation. Two-dimensional scaffolds have made use of natural and polymeric materials to act as carriers for RPE cells and various progenitor lines. These substrates mitigate issues surrounding the handling of delicate cell sheets harvested for transplant. As well, they are often successful in preserving RPE phenotype, supporting growth, and can be fine tuned to possess morphologies comparable to native extracellular matrix (ECM). Despite aiming to act as replacement Bruch’s membrane on which RPE resides, two-dimensional substrates are often notably bulky and require traumatic surgery for implantation. As a result, the use of injectable methods of cell delivery has gained appeal. Bolus injections, despite improved methods of administration, are correlated with issues of inadequate cell localization. In response, three-dimensional hydrogel carriers for retinal applications aim to encapsulate cells, allowing for better cell distribution as these materials spread throughout the subretinal space. Increased viscosity of hydrogels as compared to saline injections, is hypothesized to improve cell loss and reduce aggregation. Of particular interest are in situ gelling systems, which undergo physical changes upon injection. Gelation upon delivery works to further assist in maintaining the cells within their target site. Purity and reproducibility concerns associated with the use of natural materials in the development of hydrogel cell carriers, have inspired the use of synthetic thermoresponsive poly-N-isopropylacrylamide (pNIPAAm). pNIPAAm undergoes a liquid to gel transition at a lower critical solution temperature (LCST) of 32°C. Copolymerization with various hydrophobic and hydrophilic groups can be used to adjust gel properties such as increasing or decreasing LCST, allowing for degradation, and improving water retention. In the work described herein, two NIPAAm-based thermoresponsive hydrogels intended for use as subretinal cell carriers are proposed. / Thesis / Master of Applied Science (MASc)

Huntington's disease is an autosomal dominant neurodegenerative disorder caused by an extended (≥36) CAG repeat in the huntingtin gene. Its hallmark is brain athrophy, but huntingtin is widely deposited in all tissues of the body, most notably in the brain and testes. Its pathogenic effect is conditioned by the formation of cytotoxic forms of aggregates and fragments, which occur in both brain and peripheral tissues. Testicular atrophy has been demonstrated in postmortem samples from human patients with Huntington's disease and in transgenic mouse models. We investigated reproductive decline in a large animal model of Huntington's disease. A transgenic (tgHD) minipig model was created by inserting a lentiviral vector into the genome of a pig. Vector contained a truncated form of the N terminal part of huntingtin gene. Boars of this transgenic line showed a reduced ability to produce offspring from 13 months of age. We confirmed apoptosis of seminiferous epithelial cells and Sertoli cells, and a production of morphologically damaged spermatozoa, which were unable to efficiently fertilize the oocyte under in vitro conditions. We found a reduction of mitochondrial metabolism parameters in the sperm of tgHD boars. These changes were not dependent on the age of the boars., It is directly related to the...

Purpose. In the genetically defective Royal College of Surgeons (RCS) rat model for retinal degeneration, a breakdown occurs in the retinal pigment epithelial (RPE) cell tight junctions just as the photoreceptors begin to degenerate. These experiments sought to determine the impact of the RPE genetic defect on this alteration in the RPE cell tight junctions. Methods. Retinal pigment epithelial cell cultures prepared from RCS and control rats were treated with hormonally defined medium (HDM), base medium conditioned by RCS or control retinas, or unconditioned base medium. The tight junctions formed by these cultures were assayed functionally by measuring transepithelial electrical resistance and permeability. Junction structure was evaluated by immunolocalization of the tight junction protein zonula occludens I and of the junction-associated actin microfilaments. Results. Retinal pigment epithelial cultures from dystrophic rats formed structurally and functionally normal tight junctions when maintained in hormonally defined medium. The junctions remained stable when the medium bathing the apical surface was switched to base medium preconditioned by normal retinas. In contrast, cultures treated with medium preconditioned by degenerating dystrophic retinas or with unconditioned medium exhibited a breakdown in their tight junctions. Conclusions. Retinal pigment epithelial cells isolated from dystrophic RCS rats can form tight junctions normally in vitro. Normal, but not dystrophic, retinas release factors that support RPE tight junctions. Therefore, the junctional abnormality seen in dystrophic rat RPE cells in vivo is probably caused by the loss of trophic factors normally provided by the healthy neural retina rather than by a direct effect of the genetic defect on the tight junctions.

The retina is one of the most metabolically active tissues in the body and the high energetic demand is met by a well-organized vascular network. Aberrant vasculature is a prominent feature of many vision-threatening diseases, and although angiogenic pathways have been extensively studied the limited efficacy of therapies currently available for the treatment of these diseases suggests that there is more to be elucidated. The caspase family of proteases is best known for their roles in programmed cell death and inflammation, however members of this family have been found to have essential functions independent of cell death. Caspase-8, in particular, has been previously shown to be essential for embryonic vascular development, however, a requirement for caspase-8 in postnatal vascular development has not been established and it is unclear how caspase-8 exerts its function. In this study, we investigate the cell specific roles of caspase-8 in the development of the retinal vasculature using the postnatal mouse retina as our model and identified endothelial caspase-8 as a mediator of canonical Wnt signaling. Inducible endothelial cell-specific caspase-8 knockout (Casp8 iECKO) resulted in a delay in early angiogenesis and barrier establishment, and an increase in inflammation and premature vascular remodeling compared to littermate controls. Assessment of Lef1, a downstream effector of the Wnt pathway, confirmed that this phenotype was a result of inhibited Wnt signaling. We additionally show that caspase-8 mediates this pathway through degradation of its substrate HDAC7. HDAC7 has been shown previously to bind to β-catenin blocking its nuclear translocation. Caspase-8 mediated HDAC7 degradation restores β-catenin translocation and downstream Wnt signaling. We also explore the function of caspase-8 in myeloid cells – microglia and macrophages – during angiogenesis. We used an inducible myeloid-specific caspase-8 knockout (Casp8 imGKO) mouse and found that loss of caspase-8 in these cells did not affect angiogenesis. However, Casp8 imGKO resulted in a reduction in microglia number and a change in their morphology specifying a role for caspase-8 in mediating cell survival and activation in microglia. Altogether we show that caspase-8 exerts cell specific functions during retinal angiogenesis that are independent of cell death. We elucidate a novel role of caspase-8 in mediating Wnt/β-catenin signaling, and implicate caspase-8 as a potential therapeutic target in pathological angiogenesis.

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The increasing incidence of disc degeneration and its correlation with lower back pain is an alarming trend in modern society. The research of intervertebral disc degeneration and low back pain would greatly benefit from additional methods to study its etiology and possible treatment methods. A large animal model that maintains the biological and mechanical environment that is most similar to the human lumbar spine could provide substantial improvements in understanding and resolving the problem of intervertebral disc related low back pain.This dissertation presents my doctoral work of investigating the potential for the camelid cervical spine to serve as a suitable animal model for advancing biomechanical research of low back pain and intervertebral disc degeneration in the human lumbar spine. Specifically, this work identifies the cellular, morphological and biomechanical characteristics of the camelid cervical spine and intervertebral disc as compared to the human lumbar spine. My results demonstrate that there are remarkable similarities in all aspects. Many of the similarities with respect to the cellular environment of the intervertebral disc are a consequence of the camelid status as a large mammal. Additional testing of the cellular makeup of the camelid intervertebral disc cells revealed that many human qRT-PCR primers associated with disc degeneration are suitable for use in alpacas without modification. From a biomechanics standpoint, the camelid cervical spine also has a vertically oriented spinal posture and is unsupported near the end in an open kinetic chain, providing a mechanical parallel with the human lumbar spine. The camelid cervical intervertebral disc size is closer to the human lumbar intervertebral disc than all other currently used animal models available for comparison in the literature. Average flexibility (range of motion) of a camelid spinal motion segment showed similarities in all modes of loading. Based on magnetic resonance imaging and radiologic grading of the intervertebral disc, almost 90% of elderly camelids exhibited advanced degeneration (Pfirrmann grade 3 or higher) in their cervical spine, and about half of aged camelids have developed severe degeneration (Pfirrmann grade 4 or higher) in at least one or more of their cervical segments, most commonly within the two lowest cervical segments (e.g. c6c7 and/or c7t1). Thus, while there remain differences, the remarkable similarities between the camelid and human spine strengthen the case for using camelids as a model for human disc degeneration, normal and pathological biomechanics and fluid transport, and potentially as a pre-clinical model for investigating the efficacy of novel spinal devices.

This thesis discusses pyridinium bisretinoid compounds (PBRs), which were prepared for two purposes: 1) to use them as standards for detection of novel fluorophores in human RPE cells, which may be involved in age-related macular degeneration (AMD), and 2) to use them in the development of a targeted and triggered drug delivery system for cancer therapy. We prepared a selection of PBRs using a one-pot biomimetic method; synthesis, mechanisms for formation, and characterization of these compounds is described. We also explored the photoreactivity of three novel PBR compounds and found that these PBRs form oxidation products under blue-light irradiation. The photoinduced cytotoxity of A2P and A2EE was examined in HL-60 cells. Results from this work suggest that the PBRs presented have the potential to be involved in AMD and to be developed into a targeted and triggered drug delivery system for cancer therapy.

There is a need for spinal implants that have nonlinear stiffness to provide stabilization if the spine loses stiffness through injury, degeneration, or surgery. There is also a need for spinal implants to be customizable for individual needs, and to be small enough to be unobtrusive once implanted. Past and ongoing work that defines the effects of degeneration on the torque rotation curve of a functional spinal unit (FSU) were used to produce a spinal implant which could meet these requirements. This thesis proposes contact-aided inserts to be used with the FlexSuRe™ spinal implant to create a nonlinear stiffness. Moreover, different inserts can be used to create customized behaviors. An analytical model is introduced for insert design, and the model is verified using a finite element model and tests of physical prototypes both on a tensile tester and cadaveric testing on an in-house spine tester. Testing showed the inserts are capable of creating a non-linear force-deflection curve and it was observed that the device provided increased stiffness to a spinal segment in flexion-extension and lateral-bending. This thesis further proposes that the FlexSuRe™ spinal implant can be reduced in size by joining LET joint geometries in series in a serpentine nature. An optimization procedure was performed on the new geometry and feasible designs were identified. Moreover, due to maintaining LET joint geometry, the contact-aided insert could be implemented in conjunction with this new device geometry.

The primary objective of this research is to associate lumbar segmental mechanical response with intervertebral disc degeneration under physiologic testing conditions. Because no mathematical model exists for lumbar spine segmental rotations, a portion of this thesis evaluates potential methods for curve fitting the torque-rotation curves. The Dual Inflection Point (DIP) Boltzmann equation was developed during the course of this research and is presented here as a method for fitting spinal motion data wherein a physical meaning can be assigned to each of the model coefficients. This model can tell us more about the effects of degeneration, testing conditions, and other factors that are expressed in the change in spinal motion. Previous studies have investigated the relationship between the degeneration grade and flexibility of the intervertebral disc, but were completed without the presence of a compressive follower load. This study builds on past work by performing the testing under a compressive follower load. Segmental stiffness, range of motion (ROM), hysteresis area, and normalized hysteresis (hysteresis area/ROM) were evaluated and the effect of degeneration, segment level, temperature, and follower load were analyzed. Twenty-one functional spinal units (FSUs) were tested in the three primary modes of loading at both body temperature and room temperature in a near 100% humidity environment. A compressive follower load of 440 N was applied to simulate physiologic conditions. Fifteen of the twenty-one segments were also tested without the follower load to determine the effects of the load on segmental biomechanics. The grade of degeneration for each segment was determined using the Thompson scale and the torque-rotation curves were fit with the DIP-Boltzmann sigmoid curve.The effect of degeneration was statistically significant (α = 0.05) for stiffness, ROM, and hysteresis area in axial rotation (AR) and lateral bending (LB); it was also statistically significant for ROM and normalized hystersis in flexion-extension (FE). The lumbosacral joint (L5-S1) was significantly stiffer in AR and LB; the decrease in ROM and hysteresis area in AR and LB were also statistically significant for the lumbosacral joint compared to L1-L2 and L3-L4. Temperature had a significant effect on stiffness and hysteresis area in AR and on hysteresis area in LB. The follower load increased stiffness in all three modes of loading, but was significant only in AR and LB; it also reduced ROM and increased normalized hysteresis in all three modes of loading.