ROLE OF SOX9 IN UTERINE GLAND DEVELOPMENT AND DISEASE INITIATION

The female reproductive tract (FRT) develops midway through embryogenesis, and consists of oviducts, uterine horns, cervix and upper part of the vagina. The uterine horns are composed of an epithelial layer, luminal (LE) and glandular epithelium (GE), surrounded by a mesenchymal layer, the stroma and myometrium. Interestingly, in most mammals the GE forms after birth and it only becomes fully differentiated as the female reaches sexual maturity. Uterine glands (UG) are made up of GE and are present in all mammals. They secrete nutrients, cytokines and several other proteins, termed histotroph, that are necessary for embryo implantation and development. Experiments in ewes and mice have revealed that females who lack UGs are infertile mainly due to impaired implantation and early pregnancy loss, suggesting that UGs are essential for fertility. Fortunately for us, UGs develop after birth allowing us to peer into the genetic mechanism of tubulogenesis and branching morphogenesis; two processes that are disrupted in various adenocarcinomas (cancer derived from glands). We created 3D replicas of the epithelium lining the FRT using optical projection tomography and characterized UG development in mice using lineagetracing experiments. Our findings indicate that mouse UGs develop as simple tubular structures and later grow multiple secretory units that stem from the main duct. The main aim of this project was to study the role of SOX9 in the UGs. Preliminary studies revealed that Sox9 is mostly found in the nucleus of the GE. vii This observation led to the hypothesis that Sox9 plays a role in the formation and/or differentiation of the GE. To study the role of Sox9 in UGs differentiation, we conditionally knocked out and overexpressed Sox9 in both the LE and GE using the progesterone receptor (Pgr) promoter. Overexpressing Sox9 in the uterine epithelium, parts of the stroma, and myometrium led to formation of multiple cystic structures inside the endometrium. Histological analysis revealed that these structures appeared morphologically similar to structures present in histological tissue sections obtained from patients with endometrial polyps. We have accounted for the presence of simple and complex hyperplasia with atypia, metaplasia, thick-walled blood vessels, and stromal fibrosis; all “hallmarks” that indicate overexpressing Sox9 leads to development of a polyp-like morphology. Therefore, we can propose the use of Sox9-cOE mice to study development of endometrial cystic lesions and disease progression into hyperplastic lesions.

Evaluation of Polymer Gel Dosimeters for Measurements of Dose and LET in Proton Beams

This project assessed the effectiveness of polymer gel dosimeters as tools for measuring the dose deposited by and LET of a proton beam. A total of three BANG® dosimeter formulations were evaluated: BANG®-3-Pro-2 BANGkits™ for dose measurement and two BANG®-3 variants, the LET-Baseline and LET-Meter dosimeters, for LET measurement. All dosimeters were read out using an OCT scanner. The basic characteristics of the BANGkits™ were assessed in a series of photon and electron irradiations. The dose-response relationship was found to be sigmoidal with a threshold for response of approximately 15 cGy. The active region of the dosimeter, the volume in which dosimeter response is not inhibited by oxygen, was found to make up roughly one fourth of the total dosimeter volume. Delivering a dose across multiple fractions was found to yield a greater response than delivering the same dose in a single irradiation. The dosimeter was found to accurately measure a dose distribution produced by overlapping photon fields, yielding gamma pass rates of 95.4% and 93.1% from two planar gamma analyses. Proton irradiations were performed for measurements of proton dose and LET. Initial irradiations performed through the side of a dosimeter led to OCT artifacts. Gamma pass rates of 85.7% and 89.9% were observed in two planar gamma analyses. In irradiations performed through the base of a dosimeter, gel response was found to increase with height in the dosimeter, even in areas of constant dose. After a correction was applied, gamma pass rates of 94.6% and 99.3% were observed in two planar gamma analyses. Absolute dose measurements were substantially higher (33%-100%) than the delivered doses for proton irradiations. Issues encountered while calibrating the LET-Meter gel restricted analysis of the LET measurement data to the SOBP of a proton beam. LET-Meter overresponse was found to increase linearly with track-average LET across the LET range that could be investigated (1.5 keV/micron – 3.5 keV/micron).

Multimodal Imaging of Cotton Wool Spots in Branch Retinal Vein Occlusion.

PURPOSE: To describe and follow cotton wool spots (CWS) in branch retinal vein occlusion (BRVO) using multimodal imaging. METHODS: In this prospective cohort study including 24 patients with new-onset BRVO, CWS were described and analyzed in color fundus photography (CF), spectral domain optical coherence tomography (SD-OCT), infrared (IR) and fluorescein angiography (FA) every 3 months for 3 years. The CWS area on SD-OCT and CF was evaluated using OCT-Tool-Kit software: CWS were marked in each single OCT B-scan and the software calculated the area by interpolation. RESULTS: 29 central CWS lesions were found. 100% of these CWS were visible on SD-OCT, 100% on FA and 86.2% on IR imaging, but only 65.5% on CF imaging. CWS were visible for 12.4 ± 7.5 months on SD-OCT, for 4.4 ± 3 months and 4.3 ± 3.4 months on CF and on IR, respectively, and for 17.5 ± 7.1 months on FA. The evaluated CWS area on SD-OCT was larger than on CF (0.26 ± 0.17 mm(2) vs. 0.13 ± 0.1 mm(2), p < 0.0001). The CWS area on SD-OCT and surrounding pathology such as intraretinal cysts, avascular zones and intraretinal hemorrhage were predictive for how long CWS remained visible (r(2) = 0.497, p < 0.002). CONCLUSIONS: The lifetime and presentation of CWS in BRVO seem comparable to other diseases. SD-OCT shows a higher sensitivity for detecting CWS compared to CF. The duration of visibility of CWS varies among different image modalities and depends on the surrounding pathology and the CWS size.

Acute, posterior multifocal placoid pigment epitheliopathy: a case of 11 recurrences over 15 years

PURPOSE: To report the case of a patient with recurrent, acute posterior multifocal placoid pigment epitheliopathy. To the best of our knowledge, this is the longest documented course with the greatest number of recurrences reported. METHODS: Observational case report of one patient. A 27-year-old otherwise healthy male patient presented with recurrence of new scotomata over 15 years. Fundus photography, fluorescein angiography, indocyanine green angiography, fundus autofluorescence, and optical coherence tomography documented his clinical course. RESULTS: Over the course of 15 years, the patient developed 11 symptomatic (5 imaging-documented) recurrences of acute, posterior multifocal placoid pigment epitheliopathy affecting both eyes. Each episode manifested with new subjective scotomata and new lesions noted on imaging. Symptoms mostly resolved after each episode, and visual outcome remained excellent (20/20 in the right eye and 20/25 left eye at the last follow-up). CONCLUSION: Although typically monophasic, acute posterior multifocal placoid pigment epitheliopathy can rarely present with a recurrent course over a prolonged period of time and should be considered as a diagnosis in patients presenting with recurrent visual symptoms and new placoid lesions on imaging. In recurrent cases, visual recovery may still remain excellent.

Idiopathic multifocal choroiditis/punctate inner choroidopathy with acute photoreceptor loss or dysfunction out of proportion to clinically visible lesions.

PURPOSE To report acute/subacute vision loss and paracentral scotomata in patients with idiopathic multifocal choroiditis/punctate inner choroidopathy due to large zones of acute photoreceptor attenuation surrounding the chorioretinal lesions. METHODS Multimodal imaging case series. RESULTS Six women and 2 men were included (mean age, 31.5 ± 5.8 years). Vision ranged from 20/20-1 to hand motion (mean, 20/364). Spectral domain optical coherence tomography demonstrated extensive attenuation of the external limiting membrane, ellipsoid and interdigitation zones, adjacent to the visible multifocal choroiditis/punctate inner choroidopathy lesions. The corresponding areas were hyperautofluorescent on fundus autofluorescence and were associated with corresponding visual field defects. Full-field electroretinogram (available in three cases) showed markedly decreased cone/rod response, and multifocal electroretinogram revealed reduced amplitudes and increased implicit times in two cases. Three patients received no treatment, the remaining were treated with oral corticosteroids (n = 4), oral acyclovir/valacyclovir (n = 2), intravitreal/posterior subtenon triamcinolone acetate (n = 3), and anti-vascular endothelial growth factor (n = 2). Visual recovery occurred in only three cases of whom two were treated. Varying morphological recovery was found in six cases, associated with decrease in hyperautofluorescence on fundus autofluorescence. CONCLUSION Multifocal choroiditis/punctate inner choroidopathy can present with transient or permanent central photoreceptor attenuation/loss. This presentation is likely a variant of multifocal choroiditis/punctate inner choroidopathy with chorioretinal atrophy. Associated changes are best evaluated using multimodal imaging.

Effect of fibers and whole grain content on quality attributes

Incorporation of fiber in cereals may lead to quality issues, thus decreasing consumer acceptance. This is partially due to deterioration of the microstructure, one of the primary quality attributes of cereals. The objective of this study was to better understand the mechanisms by which dietary fibers affect the quality of cereal products during extrusioncooking. The study quantified the effect of amount and type of fiber and whole grain on (i) texture, (ii) structure, and (iii) rehydration properties of extruded cereals. New innovative methods were applied and combined with traditional techniques to characterize both the structure and the rehydration properties. Extruded cereals were produced using a starch-based recipe (whole and wheat flours) and two sources of fibers (oat bran concentrate and wheat bran). The oat and wheat bran levels used in this study were 0, 10, and 20%. The different mixtures were extruded in a pilot twinscrew extruder BC21 (Clextral) and then sugar coated after drying. Mechanical properties of extruded cereals were investigated by compression test. The cellular structure was observed by X-ray tomography. The quality of coating (thickness, homogeneity) was analyzed by optical coherence tomography. The rehydration properties of such cereals in milk were evaluated by magnetic resonance imaging and optical coherence tomography. This work revealed that structure assessment of extruded cereals may lead to a better understanding of the effect of fiber addition on texture and rehydration properties. The application of innovative methods, such as optical coherence tomography and magnetic resonance imaging, was found to be useful to quantify the structural properties.

3D Wavelet-based Fusion Techniques for Biomedical Imaging

Hoy en día las técnicas de adquisición de imágenes tridimensionales son comunes en diversas áreas, pero cabe destacar la relevancia que han adquirido en el ámbito de la imagen biomédica, dentro del cual encontramos una amplia gama de técnicas como la microscopía confocal, microscopía de dos fotones, microscopía de fluorescencia mediante lámina de luz, resonancia magnética nuclear, tomografía por emisión de positrones, tomografía de coherencia óptica, ecografía 3D y un largo etcétera. Un denominador común de todas esas aplicaciones es la constante necesidad por aumentar la resolución y la calidad de las imágenes adquiridas. En algunas de dichas técnicas de imagen tridimensional se da una interesante situación: aunque que cada volumen adquirido no contiene información suficiente para representar el objeto bajo estudio dentro de los parámetros de calidad requeridos por algunas aplicaciones finales, el esquema de adquisición permite la obtención de varios volúmenes que representan diferentes vistas de dicho objeto, de tal forma que cada una de las vistas proporciona información complementaria acerca del mismo. En este tipo de situación es posible, mediante la combinación de varias de esas vistas, obtener una mejor comprensión del objeto que a partir de cada una de ellas por separado. En el contexto de esta Tesis Doctoral se ha propuesto, desarrollado y validado una nueva metodología de proceso de imágenes basada en la transformada wavelet disc¬reta para la combinación, o fusión, de varias vistas con información complementaria de un mismo objeto. El método de fusión propuesto aprovecha la capacidad de descom¬posición en escalas y orientaciones de la transformada wavelet discreta para integrar en un solo volumen toda la información distribuida entre el conjunto de vistas adquiridas. El trabajo se centra en dos modalidades diferentes de imagen biomédica que per¬miten obtener tales adquisiciones multi-vista. La primera es una variante de la micro¬scopía de fluorescencia, la microscopía de fluorescencia mediante lámina de luz, que se utiliza para el estudio del desarrollo temprano de embriones vivos en diferentes modelos animales, como el pez cebra o el erizo de mar. La segunda modalidad es la resonancia magnética nuclear con realce tardío, que constituye una valiosa herramienta para evaluar la viabilidad del tejido miocárdico en pacientes con diversas miocardiopatías. Como parte de este trabajo, el método propuesto ha sido aplicado y validado en am¬bas modalidades de imagen. En el caso de la aplicación a microscopía de fluorescencia, los resultados de la fusión muestran un mejor contraste y nivel de detalle en comparación con cualquiera de las vistas individuales y el método no requiere de conocimiento previo acerca la función de dispersión puntual del sistema de imagen. Además, los resultados se han comparado con otros métodos existentes. Con respecto a la aplicación a imagen de resonancia magnética con realce tardío, los volúmenes fusionados resultantes pre-sentan una mejora cuantitativa en la nitidez de las estructuras relevantes y permiten una interpretación más sencilla y completa de la compleja estructura tridimensional del tejido miocárdico en pacientes con cardiopatía isquémica. Para ambas aplicaciones los resultados de esta tesis se encuentran actualmente en uso en los centros clínicos y de investigación con los que el autor ha colaborado durante este trabajo. Además se ha puesto a libre disposición de la comunidad científica la implementación del método de fusión propuesto. Por último, se ha tramitado también una solicitud de patente internacional que cubre el método de visualización desarrollado para la aplicación de Resonancia Magnética Nuclear. Abstract Nowadays three dimensional imaging techniques are common in several fields, but es-pecially in biomedical imaging, where we can find a wide range of techniques including: Laser Scanning Confocal Microscopy, Laser Scanning Two Photon Microscopy, Light Sheet Fluorescence Microscopy, Magnetic Resonance Imaging, Positron Emission To-mography, Optical Coherence Tomography, 3D Ultrasound Imaging, etc. A common denominator of all those applications being the constant need for further increasing resolution and quality of the acquired images. Interestingly, in some of the mentioned three-dimensional imaging techniques a remarkable situation arises: while a single volume does not contain enough information to represent the object being imaged within the quality parameters required by the final application, the acquisition scheme allows recording several volumes which represent different views of a given object, with each of the views providing complementary information. In this kind of situation one can get a better understanding of the object by combining several views instead of looking at each of them separately. Within such context, in this PhD Thesis we propose, develop and test new image processing methodologies based on the discrete wavelet transform for the combination, or fusion, of several views containing complementary information of a given object. The proposed fusion method exploits the scale and orientation decomposition capabil¬ities of the discrete wavelet transform to integrate in a single volume all the available information distributed among the set of acquired views. The work focuses in two different biomedical imaging modalities which provide such multi-view datasets. The first one is a particular fluorescence microscopy technique, Light-Sheet Fluorescence Microscopy, used for imaging and gaining understanding of the early development of live embryos from different animal models (like zebrafish or sea urchin). The second is Delayed Enhancement Magnetic Resonance Imaging, which is a valuable tool for assessing the viability of myocardial tissue on patients suffering from different cardiomyopathies. As part of this work, the proposed method was implemented and then validated on both imaging modalities. For the fluorescence microscopy application, the fusion results show improved contrast and detail discrimination when compared to any of the individual views and the method does not rely on prior knowledge of the system’s point spread function (PSF). Moreover, the results have shown improved performance with respect to previous PSF independent methods. With respect to its application to Delayed Enhancement Magnetic Resonance Imaging, the resulting fused volumes show a quantitative sharpness improvement and enable an easier and more complete interpretation of complex three-dimensional scar and heterogeneous tissue information in ischemic cardiomyopathy patients. In both applications, the results of this thesis are currently in use in the clinical and research centers with which the author collaborated during his work. An imple¬mentation of the fusion method has also been made freely available to the scientific community. Finally, an international patent application has been filed covering the visualization method developed for the Magnetic Resonance Imaging application.

Cystoid Macular Edema: Causes, Diagnosis and Treatment

The purpose of this paper is to conduct a review of studies on cystoid macular edema published in the last seven years. Cystoid macular edema is a major cause of loss of visual acuity. It is the final common pathway of many diseases and can be caused by numerous processes including inflammatory, vascular, adverse drug reactions, retinal dystrophy or intraocular tumors. These processes disrupt the blood-retinal barrier, with fluid extravasation to the macular parenchyma. Imaging tests are essential for both detection and monitoring of this pathology. Fluorescein angiography and autofluorescence show the leakage of liquid from perifoveal vessels into the tissue where it forms cystic spaces. Optical coherence tomography is currently the gold standard technique for diagnosis and monitoring. This allows objective measurement of retinal thickness, which correlates with visual acuity and provides more complete morphological information. Based on the underlying etiology, the therapeutic approach can be either surgical or medical with anti-inflammatory drugs. We found that disruption of the blood-retinal barrier for various reasons is the key point in the pathogenesis of cystoid macular edema, therefore we believe that studies on its treatment should proceed on this path.

Choroidal Thickness And Volume In Healthy Young Whites And Their Relationship With Axial Length, Ammetropy And Sex

Purpose: To evaluate choroidal thickness in young subjects using Enhanced Depth Imaging Spectral Domain Optical Coherence Tomography (EDI SD-OCT) describing volume differences between all the defined areas of the Early Treatment Diabetic Retinopathy Study (ETDRS). Design: Prospective, clinical study. Methods: Seventy-nine eyes of 95 healthy, young (23.8±3.2years), adult volunteers were prospectively enrolled. Manual choroidal segmentation on a 25-raster horizontal scan protocol was performed. The measurements of the nine subfields defined by the ETDRS were evaluated. Results: Mean subfoveal choroidal thickness was 345.67±81.80μm and mean total choroidal volume was 8.99±1.88mm3. Choroidal thickness and volume were higher at the superior and temporal areas compared to inferior and nasal sectors of the same diameter respectively. Strong correlations between subfoveal choroidal thickness and axial length (AL) and myopic refractive error were obtained, r = -0.649, p<0.001 and r = 0.473, p<0.001 respectively. Emmetropic eyes tended to have thicker subfoveal choroidal thickness (381.94±79.88μm versus 307.04±64.91μm) and higher total choroidal volume than myopic eyes (9.80± 1.87mm3 versus 8.14±1.48mm3). The estimation of the variation of the subfoveal choroidal thickness with the AL was-43.84μm/mm. In the myopic group, the variation of the subfoveal choroidal thickness with the myopic refractive error was -10.45μm/D. Conclusions: This study establishes for the first time a normal database for choroidal thickness and volume in young adults. Axial length, and myopic ammetropy are highly associated with choroidal parameters in healthy subjects. EDI SD-OCT exhibited a high degree of intraobserver and interobserver repeatability.

Technologies for Anatomical and Geometric Characterization of the Corneal Structure and Anterior Segment: A Review

Corneal and anterior segment imaging techniques have become a crucial tool in the clinical practice of ophthalmology, with a great variety of applications, such as corneal curvature and pachymetric analysis, detection of ectatic corneal conditions, anatomical study of the anterior segment prior to phakic intraocular lens implantation, or densitometric analysis of the crystalline lens. From the Placido-based systems that allow only a characterization of the geometry of the anterior corneal surface to the Scheimpflug photography-based systems that provide a characterization of the cornea, anterior chamber, and crystalline lens, there is a great variety of devices with the capability of analyzing different anatomical parameters with very high precision. To date, Scheimpflug photography-based systems are the devices providing the more complete analysis of the anterior segment in a non-invasive way. More developments are required in anterior segment imaging technologies in order to improve the analysis of the crystalline lens structure as well as the ocular structures behind the iris in a non-invasive way when the pupil is not dilated.

Repeatability and reproducibility of corneal thickness using SOCT Copernicus HR

Purpose: The aim of this study is to determine the reliability of corneal thickness measurements derived from SOCT Copernicus HR (Fourier domain OCT). Methods: Thirty healthy eyes of 30 subjects were evaluated. One eye of each patient was chosen randomly. Images were obtained of the central (up to 2.0 mm from the corneal apex) and paracentral (2.0 to 4.0 mm) cornea. We assessed corneal thickness (central and paracentral) and epithelium thickness. The intra-observer repeatability data were analysed using the intra-class correlation coefficient (ICC) for a range of 95 per cent within-subject standard deviation (SW) and the within-subject coefficient of variation (CW). The level of agreement by Bland–Altman analysis was also represented for the study of the reproducibility between observers and agreement between methods of measurement (automatic versus manual). Results: The mean value of the central corneal thickness (CCT) was 542.4 ± 30.1 μm (SD). There was a high intra-observer agreement, finding the best result in the central sector with an intra-class correlation coefficient of 0.99, 95 per cent CI (0.989 to 0.997) and the worst, in the minimum corneal thickness, with an intra-class correlation coefficient of 0.672, 95 per cent CI (0.417 to 0.829). Reproducibility between observers was very high. The best result was found in the central sector thickness obtained both manually and automatically with an intra-class correlation coefficient of 0.990 in both cases and the worst result in the maximum corneal thickness with an intra-class correlation coefficient of 0.827. The agreement between measurement methods was also very high with intra-class correlation coefficient greater than 0.91. On the other hand the repeatability and reproducibility for epithelial measurements was poor. Conclusion: Pachymetric mapping with SOCT Copernicus HR was found to be highly repeatable and reproducible. We found that the device lacks an appropriate ergonomic design as proper focusing of the laser beam onto the cornea for anterior segment scanning required that patients were positioned slightly farther away from the machine head-rest than in the setup for retinal imaging.

Decreased Perifoveal Sensitivity Detected by Microperimetry in Patients Using Hydroxychloroquine and without Visual Field and Fundoscopic Anomalies

Purpose. To evaluate the usefulness of microperimetry in the early detection of the ocular anomalies associated with the use of hydroxychloroquine. Methods. Prospective comparative case series study comprising 14 healthy eyes of 7 patients (group A) and 14 eyes of 7 patients under treatment with hydroxychloroquine for the treatment of rheumatologic diseases and without fundoscopic or perimetric anomalies (group B). A comprehensive ophthalmological examination including microperimetry (MP) and spectraldomain optical coherence tomography was performed in both groups. Results. No significant differences were found in mean MP foveal sensitivity between groups (P = 0.18). However, mean MP overall sensitivity was significantly higher in group A (29.05 ± 0.57 dB versus group B, 26.05 ± 2.75 dB; P < 0.001). Significantly higher sensitivity values were obtained in group A in comparison to group B for the three eccentric loci evaluated (P < 0.001). Conclusion. Microperimetry seems to be a useful tool for the early detection of retinal damage in patients treated with hydroxychloroquine.

Phosphorylated α-synuclein-immunoreactive retinal neuronal elements in Parkinson's disease subjects

Visual symptoms are relatively common in Parkinson's disease (PD) and optical coherence tomography has indicated possible retinal thinning. Accumulation of aggregated α-synuclein is thought to be a central pathogenic event in the PD brain but there have not as yet been reports of retinal synucleinopathy. Retinal wholemounts were prepared from subjects with a primary clinicopathological diagnosis of PD (N = 9), dementia with Lewy bodies (DLB; N = 3), Alzheimer's disease (N = 3), progressive supranuclear palsy (N = 2) as well as elderly normal control subjects (N = 4). These were immunohistochemically stained with an antibody against α-synuclein phosphorylated at serine 129, which is a specific molecular marker of synucleinopathy. Phosphorylated α-synuclein-immunoreactive (p-syn IR) nerve fibers were present in 7/9 PD subjects and in 1/3 DLB subjects; these were sparsely distributed and superficially located near or at the inner retinal surface. The fibers were either long and straight or branching, often with multiple en-passant varicosities along their length. The straight fibers most often had an orientation that was radial with respect to the optic disk. Together, these features are suggestive of either retinopetal/centrifugal fibers or of ganglion cell axons. In one PD subject there were sparse p-syn IR neuronal cell bodies with dendritic morphology suggestive of G19 retinal ganglion cells or intrinsically photosensitive ganglion cells. There were no stained nerve fibers or other specific staining in any of the non-PD or non-DLB subjects. It is possible that at least some of the observed visual function impairments in PD subjects might be due to α-synucleinopathy.

Optic neuritis in pediatric population: A review in current tendencies of diagnosis and management

Optic neuritis is an inflammation of the optic nerve and may be related to different systemic conditions. The clinical presentation of this pathology usually includes sudden loss of visual acuity (VA) which may be unilateral or bilateral, visual field restriction, pain with eye movements, dyschromatopsia, a relative afferent pupillary defect and optic disk swelling. Optic neuritis in children has specific clinical features and a better prognosis than in adulthood. Although usually appears an underlying viral disease, the main concern for practitioners is the relationship of optic neuritis with multiple sclerosis. In addition to the classical techniques as magnetic resonance imaging (MRI), current tendencies of diagnosis for eye practitioners include new imaging devices as optical coherence tomography (OCT), useful to show a thinning of the retinal fibers layer (RFL) after the inflammatory episode. Regarding the management of these patients, short-term intravenous steroid dosages seem to be the best option to treat acute attacks characterized by a very poor bilateral VA.