Extraction and Biochemical Characterization of Alligator mississippiensis glycosaminoglycans and an Ex-vivo Murine Pilot Study to Test their Potential Effect on a Selected Panel of Genes Associated with Cystic Fibrosis

Cystic fibrosis (CF) is a genetic disorder with a median survival age of 40.7 years. Chronic airway inflammation and dehydration are critical features of CF. The size and structure-dependent hydration and anti-inflammatory properties of glycosaminoglycans (GAGs) such as hyaluronic acid (HA) may help ameliorate these symptoms. The GAGs contained in farmed Alligator mississippiensis by-products offer a potential to improve waste management practices and increase revenue in the alligator industry, through their development for use in biomedical applications. This study aimed to efficiently extract and characterize GAGs from alligator carcasses (ACS), backstraps (ABS), feet (AFT) and eyeballs (AEB), to evaluate their effects on CF-like airways ex-vivo. Samples were collected from local alligator processing facilities and extractions were conducted according to a randomized complete block design. The contents of sulfated and non-sulfated GAGs were determined using dimethylmethylene blue and HA ELISA assays, respectively. Total GAG content was confirmed by the carbazole reaction assay. Protein content, mineral content and molecular weight (MW) were determined by the bicinchoninic acid assay, inductively coupled plasma and 0.75% agarose electrophoresis, respectively. FTIR spectroscopy was conducted to fingerprint the structures of the extracted GAGs. Multi-step processes for the extraction of GAGs from alligator by-products were developed and ABS had the highest (p≤0.05) total GAG content (15.53±0.27 mg/g), followed by AFT (4.72±0.05 mg/g), AEB (0.79±0.01 mg/g) and ACS (0.60±0.00 mg/g). These results are equivalent to ~2.13 g GAGs per harvest-size farmed alligator or an estimated GAG production of ~0.73 tons/year in Louisiana. The GAGs in all samples were predominantly (\u3e97%) HA with a poly-disperse MW of up to 1600 kDa. FTIR revealed spectra showing characteristic GAG features such as -OH, -NH, -CH and amide signals from residual protein, comparable to previous studies on animal-sourced GAGs. Ex-vivo gene expression analysis of Scnn1b-Tg mice tracheal epithelial cells indicated that AEB GAGs (0.5 mg/12 mm insert) have the potential to regulate the expression of genes which may aid in restoring the protease/anti-protease balance, reducing inflammation, and regulating ASL osmotic homeostasis. Alligator GAGs may also aid in the treatment of other inflammatory conditions such as wound healing and arthritis in humans and animals

A history of physiological optics from 1650 to 1800

Imperial Users onl

Magnetic Resonance Imaging of the Rat Retina

The retina is a thin layer of tissue lining the back of the eye and is primarily responsible for sight in vertebrates. The neural retina has a distinct layered structure with three dense nuclear layers, separated by plexiform layers comprising of axons and dendrites, and a layer of photoreceptor segments. The retinal and choroidal vasculatures nourish the retina from either side, with an avascular layer comprised largely of photoreceptor cells. Diseases that directly affect the neural retina like retinal degeneration as well as those of vascular origin like diabetic retinopathy can lead to partial or total blindness. Early detection of these diseases can potentially pave the way for a timely intervention and improve patient prognosis. Current techniques of retinal imaging rely mainly on optical techniques, which have limited depth resolution and depend mainly on the clarity of visual pathway. Magnetic resonance imaging is a versatile tool that has long been used for anatomical and functional imaging in humans and animals, and can potentially be used for retinal imaging without the limitations of optical methods. The work reported in this thesis involves the development of high resolution magnetic resonance imaging techniques for anatomical and functional imaging of the retina in rats. The rats were anesthetized using isoflurane, mechanically ventilated and paralyzed using pancuronium bromide to reduce eye motion during retinal MRI. The retina was imaged using a small, single-turn surface coil placed directly over the eye. The several physiological parameters, like rectal temperature, fraction of inspired oxygen, end-tidal CO2, were continuously monitored in all rats. MRI parameters like T1, T2, and the apparent diffusion coefficient of water molecules were determined from the rat retina at high spatial resolution and found to be similar to those obtained from the brain at the same field strength. High-resolution MRI of the retina detected the three layers in wild-type rats, which were identified as the retinal vasculature, the avascular layer and the choroidal vasculature. Anatomical MRI performed 24 hours post intravitreal injection of MnCl2, an MRI contrast agent, revealed seven distinct layers within the retina. These layers were identified as the various nuclear and plexiform layers, the photoreceptor segment layer and the choroidal vasculature using Mn54Cl2 emulsion autoradiography. Blood-oxygenlevel dependent (BOLD) functional MRI (fMRI) revealed layer-specific vascular responses to hyperoxic and hypercapnic challenges. Relative blood volume of the retina calculated by using microcrystalline iron oxide nano-colloid, an intravascular contrast agent, revealed high blood-volume in the choroidal vasculature. Fractional changes to blood volume during systemic challenges revealed a higher degree of autoregulation in the retinal vasculature compared to the choroidal vasculature, corroborating the BOLD fMRI data. Finally, the retinal MRI techniques developed were applied to detect structural and vascular changes in a rat model of retinal dystrophy. We conclude that retinal MRI is a powerful investigative tool to resolve layer-specific structure and function in the retina and to probe for changes in retinal diseases. We expect the anatomical and functional retinal MRI techniques developed herein to contribute towards the early detection of diseases and longitudinal evaluation of treatment options without interference from overlying tissue or opacity of the visual pathway

Magnetic Resonance Imaging of the Rat Retina: a Dissertation

The retina is a thin layer of tissue lining the back of the eye and is primarily responsible for sight in vertebrates. The neural retina has a distinct layered structure with three dense nuclear layers, separated by plexiform layers comprising of axons and dendrites, and a layer of photoreceptor segments. The retinal and choroidal vasculatures nourish the retina from either side, with an avascular layer comprised largely of photoreceptor cells. Diseases that directly affect the neural retina like retinal degeneration as well as those of vascular origin like diabetic retinopathy can lead to partial or total blindness. Early detection of these diseases can potentially pave the way for a timely intervention and improve patient prognosis. Current techniques of retinal imaging rely mainly on optical techniques, which have limited depth resolution and depend mainly on the clarity of visual pathway. Magnetic resonance imaging is a versatile tool that has long been used for anatomical and functional imaging in humans and animals, and can potentially be used for retinal imaging without the limitations of optical methods. The work reported in this thesis involves the development of high resolution magnetic resonance imaging techniques for anatomical and functional imaging of the retina in rats. The rats were anesthetized using isoflurane, mechanically ventilated and paralyzed using pancuronium bromide to reduce eye motion during retinal MRI. The retina was imaged using a small, single-turn surface coil placed directly over the eye. The several physiological parameters, like rectal temperature, fraction of inspired oxygen, end-tidal CO2, were continuously monitored in all rats. MRI parameters like T1, T2, and the apparent diffusion coefficient of water molecules were determined from the rat retina at high spatial resolution and found to be similar to those obtained from the brain at the same field strength. High-resolution MRI of the retina detected the three layers in wild-type rats, which were identified as the retinal vasculature, the avascular layer and the choroidal vasculature. Anatomical MRI performed 24 hours post intravitreal injection of MnCl2, an MRI contrast agent, revealed seven distinct layers within the retina. These layers were identified as the various nuclear and plexiform layers, the photoreceptor segment layer and the choroidal vasculature using Mn54Cl2emulsion autoradiography. Blood-oxygenlevel dependent (BOLD) functional MRI (fMRI) revealed layer-specific vascular responses to hyperoxic and hypercapnic challenges. Relative blood volume of the retina calculated by using microcrystalline iron oxide nano-colloid, an intravascular contrast agent, revealed a superfluous choroidal vasculature. Fractional changes to blood volume during systemic challenges revealed a higher degree of autoregulation in the retinal vasculature compared to the choroidal vasculature, corroborating the BOLD fMRI data. Finally, the retinal MRI techniques developed were applied to detect structural and vascular changes in a rat model of retinal dystrophy. We conclude that retinal MRI is a powerful investigative tool to resolve layerspecific structure and function in the retina and to probe for changes in retinal diseases. We expect the anatomical and functional retinal MRI techniques developed herein to contribute towards the early detection of diseases and longitudinal evaluation of treatment options without interference from overlying tissue or opacity of the visual pathway

Penyediaan modul pembelajaran secara komputer (Computer aided fixed learning module-CAFLM)

FLM (Fixed Learning Module) telah dijadikan bahan untuk dimasukkan ke dalam program CAFLM (Computer aided fixed learning module) di mana isi kandungannya dipindahkan dari poster ke dalam komputer. Segala isi kandungannya masih lagi dikekalkan. Modul-modul yang berkaitan dengan fisiologi telah dipilih dan dibahagikan mengikut tajuk tertentu seperti sistem pernafasan, sistem percernaan dan sistem penglihatan. Proses menyiapkan CAFLM dilakukan dengan menaip semula teks, melukis serta mengedit gambar yang terkandung dalam poster FLM

A review of the emerging role of silk for the treatment of the eye

Silk is a remarkable biopolymer with a long history of medical use. Silk fabrications have a robust track record for load-bearing applications, including surgical threads and meshes, which are clinically approved for use in humans. The progression of top-down and bottom-up engineering approaches using silk as the basis of a drug delivery or cell-loaded matrix helped to re-ignite interest in this ancient material. This review comprehensively summarises the current applications of silk for tissue engineering and drug delivery, with specific reference to the eye. Additionally, the review also covers emerging trends for the use of silk as a biologically active biopolymer for the treatment of eye disorders. The review concludes with future capabilities of silk to contribute to advanced, electronically-enhanced ocular drug delivery concepts

Cloning and Sequencing of Bovine Na+/K= Atpase α-1 Sub-Unit

The major focus of this project was cloning and sequencing the α-1 sub-unit of Na+/K+-ATase gene in bovine corneal endothelium. The Na+/K+-ATase, also called the Na+ pump, is a crucial transmembrane protein. By transporting water and ions from and through the cornea into the aqueous humor, it is responsible for maintenance of structural integrity, corneal hydration and thereby transparency of the cornea. The Na+ pump is characterized by a complex molecular heterogeneity that results from differential association of multiple isoforms of both a (the catalytic) and [3 (glycoprotein) sub-units. In the corneal endothelium, α-1 α-2, β-l and β-2 sub-units are expressed. Pathological and mechanical causes can disrupt the endothelial morphology and deregulate the pump function leading to corneal swelling and opacity. Recent works have focused on the study of pump expression, and the influence of different factors on the upregulation of its expression. For example, hypersaline and hyperosmotic treatment significantly increases pump expression

Molecular characterization of the interaction between peripherin-2 and opsins in rod and cone photoreceptors

The tetraspanin peripherin-2 is a glyco-membrane protein exclusively expressed in the outer segments of rod and cone photoreceptors. Mutations in peripherin-2 are associated with retinal disorders characterized by Degeneration of rod or cone cells. Previous unpublished work identified peripherin-2 as a potential novel part of the protein complex comprising the B-subunit of the cyclic nucleotide-gated channel (CNGB1a and the light detector rhodopsin. In the first part of this study, using a combination of protein biochemical and FRET approaches in transfected HEK293 cells and in virally transduced murine rod outer segments, it could be demonstrated that peripherin-2 simultaneously binds to both, CNGB1a and rhodopsin. The interaction between peripherin-2 and rhodopsin was not described in previous studies. The binding domain mediating the peripherin-2/rhodopsin interaction could be narrowed down to the fourth transmembrane domain (TM4) of peripherin-2. Finally, the data revealed that the G266D point mutation in TM4 of peripherin-2 that is linked to a rod degenerative disease selectively disrupts the peripherin-2/rhodopsin interaction. To analyze if peripherin-2 also binds to cone opsins in the second part of this study, a similar experimental approach was conducted as used for the investigation of the peripherin-2/rhodopsin interaction. In this context, it was unveiled that peripherin-2 binds to both, short wavelength-and medium wavelength-sensitive cone opsin (S-opsin and M-opsin, respectively) in transfected HEK293 cells and in outer segments of transduced murine cones. Co-immunoprecipitation and quantitative FRET analysis revealed that binding of peripherin-2 to M-opsin was stronger than the peripherin-2/S-opsin interaction. This result was supported by transmission electron microscopy studies using gold particles coupled to opsin- and peripherin-2-specific antibodies. Finally, quantitative FRET analysis in transfected HEK293 cells and in transduced cone outer segments demonstrated that the V268I Point mutation in TM4 of peripherin-2 associated with a degenerative cone disease significantly attenuates the peripherin-2/M-opsin interaction. Taken together, this study provides a proof-of-principle for FRET-based analysis of protein-protein interactions in the outer segments of rod and cone photoreceptors. This approach led to the identification of hitherto unknown Protein complexes between peripherin-2 and opsins suggesting a novel physiological role of peripherin-2 in rods and cones. Finally, Analysis of disease-linked point mutations unveiled the molecular determinants of the peripherin-2/opsin interaction. These results might contribute to understanding the differential penetrance of certain point mutations in rods and cones