This thesis represents an attempt to further our knowledge of the ophthalmic complications of spina bifida and hydrocephalus by means of literature review and a long term clinical study, and to determine whether regular ophthalmic supervision can assist in the general management of affected children. The ophthalmic complications of spina bifida have often been reported in the literature and thought to merit regular supervision of affected children, yet few centres currently offer this service. In this study 322 children attending one regional centre were examined repeatedly over a six year period by one ophthalmologist. Ophthalmic complications were found to be very common. They frequently provided evidence of raised intracranial pressure due to shunt dysfunction even when other objective evidence was lacking. Every spina bifida and hydrocephalus clinic should have an ophthalmalogist in its medical team. Preservation of visual function and early diagnosis of raised intracranial pressure in these children should result from this arrangement.

Limbal Epithelial Crypts (LECs) are stem cell niches located in the limbus, the area between the cornea and the conjunctiva, and are responsible for cornea epithelium regeneration. Their anatomical structures were identified and described by Dua et al (2005). Stem cells migrate from this area toward the cornea where they differentiate into corneal epithelial cells. The lack of the LECs determines a pathology called Limbal Stem Cell Deficiency (LSCD) that can cause conjunctivalization, neovascularisation, corneal opacification, eventually resulting in vision loss. Currently, the main carriers to support limbal stem cell proliferation and differentiation used in clinics do not include enclosed structures able to act as cell reservoirs. In addition, the driving forces that determine the migration and the differentiation of limbal stem cells are not clarified yet. The hypotheses of this thesis were that the stiffness and the geometry of the scaffold influence the limbal stem cell differentiation process. Understanding these key factors could help to inform the design of scaffolds for future cornea regeneration strategies. The effect of the stiffness on cell differentiation is a well-known mechanism, but is less investigated in the limbus-corneal tissue. In this work, it was studied by producing macro-scaffolds (d=8 mm) obtained by UV-crosslinking. The stiffness was varied by increasing the concentration of Gelatin Methacrylate (GelMA), a biocompatible, photocrosslinkable material. Compression tests, rheology, AFM, and swelling analysis were performed on the developed hydrogels and compared to the ex vivo human cornea values. The gels with a concentration between 5% and 15% (w/v) exhibited mechanical properties in the same order of magnitude obtained for the cornea. The results indicated that the increased stiffness did not have a significant impact either in the expression of cytokeratin (CK3/CK14, stem cell marker, and CK14, differentiation marker), or in the gene expression (KRT3 and KRT19). However, the secondary key factors explored, differentiation media and oxygen concentration, supported cell differentiation. Inkjet printing was investigated as an additive manufacturing technique to produce 3D architectures. Although with this technique it was possible to develop a 3D structure with a stiffness gradient, its resolution was not sufficiently high to obtain a structure with dimensions comparable to the in vivo limbal stem cell niche. The second hypothesis was verified by developing a biocompatible scaffold mimicking the structure of the limbal stem cell niche and by evaluating the impact of its architecture on stem cell differentiation. The crypt geometry was modelled as U-shaped scaffolds with a diameter narrowing from 200 μm to 20 μm and was micro-fabricated from GelMA/PEGDA-based hydrogels using a Two-Photon Polymerization system (2PP). However, it was proven that the use of riboflavin as photoinitiator was inefficient at 780 nm, the wavelength used in the 2PP system. For this reason, P2CK was used as photoinitator to obtain stable hydrogels. The 2PP system allowed the precise recreation of the exact dimensions of the native crypts. Swelling, susceptibility to enzymatic degradation and stiffness were all evaluated. The biocompatibility of the printed scaffolds was assessed using immortalized human corneal epithelial cell proliferation up to 14 days. The ability of limbal stem cells to repopulate the crypts was demonstrated via two strategies. In the first strategy, human limbal stem cells were seeded directly inside the niche whilst in the second strategy, primary human limbal explants were placed adjacent to the printed structures and cells migrated towards the scaffold. Cell distribution and differentiation along the z-axis were evaluated using confocal microscopy. Cytokeratin 14 (CK14) with p63 and Cytokeratin CK3/12 (CK3/12) were used as limbal stem cell and differentiated corneal epithelial cell markers, respectively. Limbal epithelial stem cells were cultured in two conditions: xeno-free media, and with primary cells in serum containing media on a feeder layer. Both conditions showed the zonation of markers along the z-axis, which was not observed on flat scaffolds, demonstrating that the geometry alone influences cell phenotype. This suggests that the enclosed geometry results in the generation of a microenvironment inside the niche that influences cell phenotype. The presence of soluble factors, generated by cellular secretions, a specific oxygen concentration, and a more ‘stressful’ biomechanical milieu for the cells are some hypotheses that need further investigation and will be the basis of future work. In conclusion, the hypotheses of this thesis were partially confirmed. The variation in gel stiffness did not allow for the control of the hLESC differentiation process. However, the results demonstrated the influence of the geometry on stem cell differentiation without the use of signaling molecules. Further studies are necessary to have a description of the detailed spatial variability of the scaffold’s characteristics. Overall, the 2PP approach is flexible and could be applied to the generation of stem cell niches of other tissues, and could represent a significant advance in regenerative medicine.

In this thesis, the propagation characteristics of the biological optical waveguides, considering the materials as lossy in the optical frequencies, have been analysed. It has been found that the losses present in the biological materials in optical frequencies are not negligible, and the loss values have significant effects on the propagation characteristics of these waveguides. In biological optical waveguides, each waveguide is surrounded by parallel waveguides so that the propagation characteristics would be different from that of single waveguide present in a homogeneous material. In this thesis, the impacts of the presence of the neighbouring waveguides on the propagation characteristics of a waveguide are studied in details. Dispersion characteristics of the waveguides have been investigated, and the effects of the material loss, presence of the neighbouring waveguides and the presence of multi-layer W-fibre like structure on the dispersion characteristics have also been studied. The modal characteristics, the time-domain evolution of the signal and the diffraction characteristics have been integrated to explain some of the still unanswered questions in the visual systems. An attempt has been made to explain the Stiles-Crawford effect of human retina in light of the findings of this thesis. A full-vectorial H-field based finite element method (FEM) is used for the modal solutions, Finite Difference Time Domain (FDTD) is used to study the time evolution of the signals through the waveguides, and the Diffraction profiles have been obtained by Rayleigh-Sommerfeld(RS) diffraction integral.

The fundamental corneal properties of mechanical rigidity, maintenance of curvature and optical transparency result from the specific organisation of collagen fibrils in the corneal stroma. The exact arrangement of stromal collagen is currently unknown but several structural models have been proposed. The purpose of the present study is to investigate inconsistencies between current x‐ray derived structural models of the cornea and optically derived birefringence data. Firstly, the thesis reviews the current understanding of corneal structure, particularly in relation to corneal birefringence. It also reviews and develops the different analytical approaches used to model optical biaxial behaviour, particularly as applied to predict corneal optical phase retardation. The second part develops a novel technique of elliptic polarization biomicroscopy (EPB), enabling study of corneal birefringence in vivo. Using EPB, the pattern of corneal retardation is recorded for a range of human subjects. This dataset is then used to investigate both central and peripheral corneal birefringence as well as the corneal microstructure. A key finding is that the central parts of the cornea exhibit a retardation pattern compatible with a negative biaxial crystal, whereas the peripheral corneal regions do not. Furthermore, within the central regions of the cornea, orthogonal confocal conic fibrillar structures are identified which resemble the analytically derived contours of equal refractive index of an ideal negative biaxial crystal. The third part of this work presents a synthesis of previous published experimental, anatomical and theoretical findings and the experimental results presented in this thesis. Based on these findings, a novel corneal structural model is proposed that comprises overlapping spherical elliptic structural units. Finally, ensuing biomechanical and clinical consequences of the spherical elliptic structural model and of the EPB technique are discussed including their potential diagnostic and surgical applications.

How do we know where objects are in the environment and how do we use this information to guide our actions? Recovering the three-dimensional (3D) structure of our surroundings from the two-dimensional retinal input received from the eyes is a computationally challenging task and depends on the brain processing and combining ambiguous sources of sensory information (cues) to depth. This thesis combines psychophysical and computational techniques to gain further insight into (i) which cues the brain uses for perceptual judgments of depth and motion-in-depth; and (ii) the processes underlying the combination of the information from these cues into a single percept of depth. The first chapter deals with the question which sources of information the visual system uses to estimate the time remaining until an approaching object will hit us; a problem that is complicated by the fact that the variable of interest (time) is highly correlated to other perceptual variables that may be used (e.g. distance). Despite these high correlations we show that the visual system recovers a temporal estimate, rather than using one or more of its covariates. In the second chapter I ask how extra-retinal signals (changes in the convergence angles of the eyes) contribute to estimates of 3D speed. Traditionally, extra-retinal signals are reputed to be a poor indicator of 3D motion. Using techniques to isolate extra-retinal signals to changes in vergence, we show that judgments of 3D speed are best explained on the basis that the visual system computes a weighted average of retinal and extra-retinal signals. The third and fourth chapters investigate how the visual system combines binocular and monocular cues to depth in judgments of relative depth and the speed of 3D motion. In chapter three I show that differences in retinal size systematically affect the perceived disparityde defined depth between two unfamiliar targets, so that a target with a larger retinal size is seen as closer than a target with a smaller retinal size at the same disparity-defined distance. This perceptual bias increases as the retinal size ratio between the targets is increased but remains constant as the absolute sizes of the targets change concurrently while keeping the retinal size ratio constant. In addition, bias increases as the absolute distance to both targets increases. I propose that these findings can be explained on the basis that the visual system attempts to optimally combine disparity with retinal size cues (or in the case of 3D motion: changing disparity information with looming cues), but assumes that both objects are of equal size while they are not. In chapter 4 these findings are extended to 3D motion: physically larger unfamiliar targets are reported to approach faster than a smaller target moving at the same speed at the same distance. These findings cannot be explained on the basis of observers' use of a biased perceived distance, caused by differences in the retinal size (as found in chapter 3). I conclude that, in line with contemporary theories of visual perception, the brain solves the puzzle of 3D perception by combining all available sources of visual information in an optimal manner, even though this may lead to inaccuracies in the final estimate of depth.

Ten experiments examined the way that automatic processing of the visual perspectives and eye gaze of others affects adults‘ perception and encoding of the social world. I investigated the amount of flexibility that automatic visual perspective computation accommodates. Experiments 1, 2, and 3 demonstrate that automatic visual perspective-computation shows some flexibility for enumerating and representing perspective contents. Experiments 4 and 5 further indicate that automatic visual perspective-taking allows selection of relevant perspective information. I also examined whether observing others‘ eye gaze affects adults‘ visual working memory encoding. Experiments 6, 7, and 8 indicate that agents‘ object-oriented gaze does not lead to more efficient encoding of agent and object information. Experiments 9 and 10 demonstrate that observing others‘ participant-oriented gaze disrupts visual working memory encoding. I argue that although adults have minimal conscious control over the activation of visual perspective-computation and processing of participantoriented gaze, the efficient mindreading system shows some flexibility.