We previously reported that the CNGB1 locus encoding the rod photoreceptor cGMP-gated channel β-subunit is complex, comprising non- overlapping transcription units that give rise to at least six transcripts (Ardell, M.D., Aragon, I., Oliveira, L., Porche, G.E., Burke, E., Pittler, S.J., 1996. The beta subunit of human rod photoreceptor cGMP-gated cation channel is generated from a complex transcription unit. FEBS Lett. 389, 213- 218). To further understand the transcriptional regulation of this extraordinarily complex locus, and to develop a screen for defects in the gene in patients with hereditary disease, we determined its genomic organization and DNA sequence. The CNGB1 locus consists of 33 exons, which span approximately 100 kb of genomic DNA on chromosome 16. The β-subunit comprises two domains, an N-terminal glutamic acid-rich segment (GARP), and a C-terminal channel-like portion. Two additional exons encoding a short GARP transcript and a truncated channel-like transcript have been identified. A major transcription start point was identified 79 bp upstream of the initiator ATG. To begin analysis of the basis for the generation of multiple transcripts, and to identify promoters driving expression in retina, approximately 2.5 kb of the upstream region were sequenced. Putative cis- elements, which can bind the retina-specific transcription factors Crx and Erx, were found immediately upstream of the transcription start point, and may be important for gene expression in this tissue. From our analysis, a model is reported to account for at least four of the retinal transcripts.

Postoperative corneal complications from pars plana vitrectomy surgery on the retina have been studied extensively in the literature. Researchers are aware of possible issues with oil tamponades, laser techniques, and other methods used. There could be clear markers to focus on a pattern of retina diagnoses of the patients that seem more prone to these problems in the front of the eye. Studies have noted refractive issues and increased cataract or posterior capsule opacification (PCO) progression but it is inconclusive in many studies on a corneal safety standpoint. Furthermore, a 23 or 27gauge pars plana vitrectomy has varying protocols depending on the diagnosis of the retina patient including endolaser, tamponade exchange, or even endoscopic cyclophotocoagulation. This study was conducted to research data looking at varying combinations of surgical type, diagnosis, and other patient characteristics to gain statistical evidence or relative frequency to better understand what type of retinal demographics cause corneal complications. The list of corneal complications in this study include: visual distortion involving anisometropia or photophobia, increased intraocular pressure including Uveitis-Glaucoma Hyphema syndrome, allergic conjunctivitis, a lens subluxation, herpes virus (zoster or simplex), a corneal scar or lesion, neurotrophic cornea, bullous keratopathy and corneal neovascularization. This was a retrospective case study evaulating 57 patients and 58 eyes that underwent a retinal surgery, with corneal complications at Beth Israel Deaconess Medical Center between October 2013 to December 2016. Number of patients, systemic demographics including frequency of hypertension and diabetes, and frequency of retina surgery per eye was analyzed. Moreover, we looked at different retina diagnoses to view which groups have a higher occurrence of complications after surgery. We used silicone oil as a way to divide the corneal complication patients to determine if the use of oil had an effect on a higher rate of issues after surgery. Eyes were divided into treatment with silicone oil group (n=23) and a non-silicone oil group (n=34), and we found that the silicone group had a significantly higher frequency of retinal surgeries (p<0.001). Moreover, there was no significant evidence between certain systemic factors (p<0.05), that allowed us to include the silicone oil and non-silicone oil patients as a unified group. When looking at our retina diagnoses we saw some groups had a higher percentage of complications when we took total number of problematic postoperative outcomes and divided that by total number of surgeries. Over one quarter of the surgeries per category leading to corneal complications occurred in the categories of subluxed lens, endophthalmitis, trauma, and uveitis-glaucoma hyphema Syndrome or neovascular glaucoma. Vitreomacular traction similarly had a high percentage of patients with corneal complications. Retinal detachment and epiretinal membrane were largest quantities of a specific retinal problem with low percentages of fewer than 15% with complications postoperatively. The study found that in our patient demographic silicone oil did not seem to be a factor in causing more corneal complications but it did cause more retinal surgeries. Moreover, certain retina diagnoses seem more prone to cause challenging outcomes, which leaves room for further studies distinguishing certain factors that could cause such specific issues.

Vitreomacular traction (VMT) is a condition characterized by an incomplete posterior vitreous detachment resulting in traction on the macula and decreased visual acuity. The process of vitreomacular traction can lead to a number of maculopathies including cystoid macular edema, epiretinal membranes, and macular holes. VMT is more common in patients over 40 years of age; however no population-based studies documenting the prevalence of VMT in this age group has been published. Purpose: The current study was undertaken to evaluate the prevalence of vitreomacular traction syndrome in the population aged above 40. Clinical examination does not adequately illustrate the extent of vitreomacular adhesion; therefore, Optical Coherence Tomography (OCT) was used to determine the presence of VMT. High-definition OCT has provided new insight into vitreomacular traction syndrome by allowing for better visualization of the tractional forces at the vitreoretinal interface. This investigation will analyze the vitreomacular interface with regard to VMT in correlation to age through use of the Spectral Domain OCT. Methods: One hundred and thirty-one eyes of 67 patients (36 females/31 males) were examined by optical coherence tomography (OCT). OCT was performed to obtain high-resolution cross-sectional images of the vitreoretinal interface in the posterior fundus. Results: The status of the posterior vitreoretinal interface was evaluated as follows; (1) Intact Vitreous- No posterior vitreous abnormality is depicted on OCT. (2) Vitreomacular adhesion/incomplete PVD- the posterior vitreous is partially attached and can be seen in contact with the macula on OCT. (3) Vitreomacular traction- the posterior vitreous is partially adhered to the inner retina resulting in distortion of the retinal architecture. (4) Complete posterior vitreous detachment, indicating no contact between the vitreous and retina. Conclusion: OCT results found no vitreous abnormality in 39.7% of all eyes; PVD in 6.87% of eyes, VMA in 51.1% of eyes, and VMT in 2.29% of eyes. Age was a significant risk factor to the development of VMT, while gender was not. As age increases, the probability of having VMT increases by 1.2%. Due to the association between vitreomacular traction and a variety of maculopathies, recognition and diagnosis of this entity is crucial. High definition OCT has allowed for outstanding visualization of the vitreomacular interface, which has allowed for a better understanding of this entity.

Indiana University-Purdue University Indianapolis (IUPUI) / Human pluripotent stem cells (hPSCs) have the ability to self renew indefinitely while maintaining their pluripotency, allowing for the study of virtually any human cell type in a dish. The focus of the current study was the differentiation of hPSCs to retinal ganglion cells (RGCs), the primary cell type affected in optic neuropathies. hPSCs were induced to become retinal cells using a stepwise differentiation protocol that allowed for formation of optic vesicle (OV)-like structures. Enrichment of OV like structures allowed for the definitive identification of RGCs. RGCs displayed the proper temporal, spatial, and phenotypic characteristics of RGCs developing in vivo. To test the ability of hPSC-RGCs to serve as a disease model, lines were generated from a patient with an E50K mutation in the Optineurin gene, causative for normal tension primary open angle glaucoma. E50K RGCs displayed significantly higher levels of apoptosis compared to a control lines. Apoptosis was reduced with exposure to neuroprotective factors. Lastly, hPSC-derived RGCs were studied for their ability to develop functional features possessed by mature in vivo RGCs. hPSC-derived RGCs displayed a few immature functional features and as such, strategies in which to expedite synaptogenesis using hPSC-derived astrocytes were explored. Astrocyte and RGG co-cultures displayed expedited synaptic and functional maturation, more closely resembling mature in vivo RGCs. Taken together, the results of this study have important implications for the study of RGC development and by extension, the advancement of translational therapies for optic neuropathies.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第12800号 / 論医博第2072号 / 新制||医||1001(附属図書館) / 80844 / (主査)教授 髙橋 淳, 教授 吉村 長久, 教授 江藤 浩之 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Sight is the most powerful of all human senses. For the vast majority of people on Earth, the loss of that sense would be unimaginable. Without assistive technology, it would separate them from their ability to work, their ability to travel, and their ability to interact with their loved ones. And yet, this extraordinary process, carefully refined by billions of years of evolution, is threatened for millions of people all over the world from a wide array of diseases of the retina. Many of these diseases arise from malnutrition and infection and are being rapidly eradicated. However, many dozens more result from convoluted permutations of genetics, age, and diet that threaten blindness for millions more with little hope of treatment, even with the best of modern medicine. As our life expectancies extend and our population ages, these diseases will only become more prevalent. In humanity's ever-present pursuit of medicine and knowledge to improve our quality of life, cutting-edge treatments offer promise that one day soon, even these diseases may be eradicated. One key technology capable of treating these devastating illnesses, on the precipice of being translated to real-world clinical treatments, is pluripotent stem cell-derived therapies. Patient-specific pluripotent stem cells, meaning pluripotent stem cells sourced directly from the patient, have a wealth of applications ranging from drug identification to disease modeling to implantation and regeneration. This research has been developed and advanced remarkably in the approximately two decades since the early isolation of pluripotent stem cells. Naturally, this advancement has predominantly been focused on cell and molecular biology. However, this focus has left significant research questions to be answered from engineering perspectives across a wide latitude of sub-disciplines. This dissertation explores three independent avenues of engineering principles as they relate to improving 2D and 3D retinal tissues derived from pluripotent stem cells in materials, devices, and computation. The first aim explores how plant protein-based nanofibrous scaffolds can marry the advantages and minimize the disadvantages of synthetic and animal-derived scaffolds for the culture of 2D retinal pigment epithelium (RPE) constructs. The second aim describes the development and testing of a novel, perfusing rotating wall vessel (RWV) bioreactor to support culture of 3D retinal organoids. Finally, the third aim performs a meta-analysis of published RNA-Seq datasets to determine the precise mechanisms by which bioreactors support organoid growth and extrapolate how these conclusions can support future experiments. / Bioengineering

Matrix metalloproteinases (MMPs) are known to be the mediators of extracellular matrix remodeling. Increased levels of matrix metalloproteinases, particularly MMP-9, have been found in the aqueous humor of patients with glaucoma. However the exact role of MMP-9 in glaucomatous changes is not understood. Previous results from the West-Mays’ lab indicated that MMP-9 deficient (knockout - KO) mice exhibit elevated IOP, in the absence of distinct morphological changes in the anterior chamber. In the current thesis, I investigated whether the elevated IOP in MMP-9KO mice leads to RGC death. Wild type and KO littermates at different age groups: 2-3 months, 3-4 months, 6-8 and 9-12 months were studied. IOP was measured using TonoLab rebound tonometer. My results demonstrated that IOP was significantly increased in MMP-9KO mice compared to control littermates at all ages examined. To investigate if the elevated IOP was due to a difference in central corneal thickness (CCT), CCT measurements were made between WT and KO mice using ultrasound pachymeter. There was no difference in CCT demonstrating that the elevated IOP observed in MMP-9KO mice was not related to changes in corneal thickness. To determine whether the elevated IOP led to RGC death, the animals were sacrificed, eyes were enucleated and retinas (n=4) from both WT and KO animals were dissected and stained with Brn-3a antibody. Additional eyes were harvested from both WT and KO mice for histological and immunofluorescence studies. I found no observable difference in Brn3a+ RGC count between MMP9-WT and KO mice. Furthermore, no difference in retinal morphology, glial reactivity and laminin expression between WT and KO mice was observed. In the future it will be important to investigate whether elevated IOP in the MMP-9KO mice leads to optic nerve axonal loss and further investigate the possibility that the MMP-9KO retina is neuroprotected. / Thesis / Master of Science (MSc)

Previous studies from our lab have shown that the Activating Protein- 2 (AP-2) transcription factors, AP-2α and AP-2β, are important in retinal development. It was discovered that these are co-expressed in developing horizontal cells and postmitotic amacrine cells. To understand their role in retinogenesis, and the impact of their deletion on the adult retina, a double mutant mouse model was created, AP-2αKI/flox/AP-2β-/flox. The neural retina of the AP-2αKI/flox/AP-2β-/flox mice was examined in the current study using histological, immunofluorescent and electron microscopy (EM) techniques at embryonic, post-natal and adult stages. These double mutants displayed a variety of abnormalities in the inner retina. Loss of AP-2α and AP-2β at E10.5 led to a complete absence of developing and mature horizontal cells. This loss was associated with changes in the outer plexiform layer, which diminished from two to four months of age. There were also defects with photoreceptor ribbons in which triad synapses failed to form, and instead led to rudimentary, spherical-shaped ribbons. There was also significant retraction of photoreceptor axons. Furthermore, this study was able to infer a role of AP-2α and AP-2β as acting upstream of the Onecut-1 protein, which targets Lim1 and Prox1 to direct horizontal cell genesis. Examining amacrine cells of the double mutants shows evidence that AP-2α and AP-2β are involved in the mosaic arrangement pattern of amacrine cell bodies and axons. Previous work on embryonic double mutants displayed clustering of amacrine cells. This study observed abnormalities in the dendrites of the inner plexiform layer, which consists of amacrine cell processes. Taken together, the work presented in this thesis implicates the redundant requirement of both AP-2α and AP-2β in development of horizontal cells and patterning of amacrine cells in the neural retina. / Thesis / Master of Health Sciences (MSc)

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24792号 / 医博第4984号 / 新制||医||1066(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 椛島 健治, 教授 辻川 明孝, 教授 山中 伸弥 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Indiana University-Purdue University Indianapolis (IUPUI) / Photoreceptors are a class of sensory neuronal cells that are deleteriously affected in many disorders and injuries of the visual system. Significant injury or loss of these cells often results in a partial or complete loss of vision. While previous studies have determined many necessary components of the gene regulatory network governing the establishment, development, and maintenance of these cells, the necessary and sufficient profile and timecourse of gene expression and/or silencing has yet to be elucidated. Arduous protocols do exist to derive photoreceptors in vitro utilizing pluripotent stem cells, but only recently have been able to yield cells that are disease- and/or patient-specific. The discovery that mammalian somatic cells can be directly reprogrammed to another terminally-differentiated cell phenotype has inspired an explosion of research demonstrating the successful genetic reprogramming of one cell type to another, a process which is typically both more timely and efficient than those used to derive the same cells from pluripotent stem cell sources. Therefore, the emphasis of this study was to establish a novel system to be used to determine a minimal transcriptional network capable of directly reprogramming mouse embryonic fibroblasts (MEFs) to rod photoreceptors. The tools, assays, and experimental design chosen and established herein were designed and characterized to facilitate this determination, and preliminary data demonstrated the utility of this approach for accomplishing this aim.