Advances in Ophthalmology

This book focuses on the different aspects of ophthalmology - the medical science of diagnosis and treatment of eye disorders. Ophthalmology is divided into various clinical subspecialties, such as cornea, cataract, glaucoma, uveitis, retina, neuro-ophthalmology, pediatric ophthalmology, oncology, pathology, and oculoplastics. This book incorporates new developments as well as future perspectives in ophthalmology and is a balanced product between covering a wide range of diseases and expedited publication. It is intended to be the appetizer for other books to follow. Ophthalmologists, researchers, specialists, trainees, and general practitioners with an interest in ophthalmology will find this book interesting and useful

Plasmonically enhanced photonic inactivation of pathogens

Infectious pathogens are a prominent threat to human health in the world. There is a ubiquitous need for safe and reliable pathogen inactivation in the entire health care sector and pharmaceutical industry. Unfortunately, existing chemical treatment methods for virus inactivation have shortcomings as they introduce toxic chemicals or alter the structure of the products, which often pose significant side effects. Furthermore, considering the alarming growth of antibiotic resistances and hospital associated microbial infections, there is an urgent need for alternative pathogen inactivation strategies. Femtosecond (fs) pulsed laser irradiation technique is a promising solution free of added toxic chemicals and does not require the invention of new antibiotics for inactivation of virus contaminations in biological samples. Conventional pulsed laser techniques require relatively long irradiation times to achieve a significant viral inactivation. This thesis is focused on developing a novel photonic inactivation approach that is selective to pathogens, doesn’t compromise the protein-based pharmaceuticals, and is obtained without specific targeting to the pathogens. In our study, we report comparative studies using femtosecond laser pulses generated using Chirped Pulse Amplification (CPA) centered at either 800 nm or frequency-doubled 400 nm wavelengths, on the model bacteriophage φX174. We show that photonic inactivation is wavelength dependent and a Log Reduction Value (LRV) of > 6 in a 2 ml bacteriophage sample volume is achieved with less than 1 min of 400 nm laser exposure. Traditional methods for assaying viral inactivation require cell culture studies that can take up to 48–72 hours. We describe a solid-state nanopore technique that can monitor the effect of this optical viral therapy in under 10 minutes. By developing a statistical model based on the probability distribution function obtained from nanopore data, we monitor the survival fraction of viruses with low sample volume, high precision and fast assay time. Lastly, the purely photonic virus inactivation requires UV fs laser irradiation, which can risk photodamage to biologics. In our research, we introduce a novel inactivation approach that takes advantage of the strong light-matter interactions provided by noble metal nanoparticle (NP) structures that sustain plasmons. We report a plasmonically enhanced virus inactivation of Murine Leukemia Virus (MLV) via 10 s laser exposure with 800 nm fs pulses through gold nanorods, with LRV>3.7. We demonstrate that this NP-enhanced, physical inactivation approach is effective against a diverse group of pathogens, including both enveloped and non-enveloped viruses, and a variety of bacteria and mycoplasma. Importantly, the fs-pulse induced inactivation was selective to the pathogens and did not induce any measurable damage to co-incubated antibodies, or to large mammalian cells. Based on the observations, a model of selective pathogen inactivation based on plasmon enhanced cavitation is proposed

Enhancement of Carotenoid Biosynthesis and Antioxidant Responses in Diatoms by Light Modulation

The ecological success of diatoms is mostly attributed to their ability to adjust biological performances to the variable environmental conditions they experience in the water column. While the efficient photoacclimative and photoprotective mechanisms in diatoms are already reported, the involvement of the antioxidant network in lowering or repairing light stress or damage is poorly understood. My thesis consists of the concomitant investigation of the photoprotective and antioxidant network functioning in the coastal diatom Skeletonema marinoi, and during an experiment at sea in the Gulf of Naples. The aims of my work are to explore the antioxidant network in diatoms to better understand its activation in coping with light variation, and the functional link between antioxidant molecules synthesis/activity and photoprotection. Results showed the spectral light dependent activity of antioxidant enzymes such as ascorbate peroxidase, catalase, and superoxide dismutase. They act in complementarity of the antioxidant molecules synthesis. A high concentration of ascorbic acid, phenolics compounds and among them flavonoids were found in diatoms. These molecules respond to light variations in terms of spectral composition, photon flux density, daily light dose, light shape distribution and photoperiod duration. Also, in a natural microalgal community, these molecules were found at high concentration following a dynamics that relied on light, nutrient stress and photosynthetic regulation. The photoprotective xanthophyll cycle, involving the pigments diatoxanthin and diadinoxanthin, is activated by light and modulated in concordance with antioxidant molecules synthesis. Indeed, under different light climates, a link between these two defense processes was found. I also show that the xanthophyll cycle pigments have a high antioxidant activity. This feature explains why they increase together with antioxidant molecules during cell senescence. More generally, the physiological state of the cells modulates the antioxidant and photoprotective network in diatoms

Chapter 7 - Water Relations

Explore the contents of Bryophyte Ecology, Volume 1, Chapter 7 - Water Relations by clicking the links above. For the Contents section of this ebook, as well as Volumes 1 through 5, please visit the Bryophyte Ecology Main Page. Use CTRL+F to easily search within PDF files. Ebook sponsored by Michigan Technological University and the International Association of Bryologists.https://digitalcommons.mtu.edu/bryophyte-ecology1/1006/thumbnail.jp

Construction and molecular characterisation of an improved chloroplast transformation vector system as a versatile delivery and expression platform for in-vitro propagated Nicotiana benthamiana

The objective of this study is to develop a versatile vector system for the delivery and expression of transgenes in the chloroplast genome of N. benthamiana. The successful advent of such a system would vastly streamline the construction process of chloroplast transformation vectors for the expression of recombinant proteins, such as vaccine candidates, in the chloroplasts of N. benthamiana. Transgenes targeted to the chloroplasts of higher plants are expected to be expressed at considerably higher levels as compared to nuclear expression, resulting in more significant accumulation of recombinant proteins. In this study, a 2-part chloroplast transformation vector system was developed and two new GFP vector prototypes, pEXPR-G and pEXPR-UG were generated for preliminary evaluation of functionality. The aadA and GFP expression cassettes of pEXPR-G and pEXPR-UG were evaluated in E. coli prior to actual delivery into N. benthamiana via particle bombardment. Particle bombardment parameters were optimised with particular emphasis on minimising excessive damage to the target tissue in order to facilitate the recovery of antibiotic resistant shoots and calli following transformation. To further evaluate the versatility of the developed system for the expression of vaccine antigens, recombinant vectors, pEXPR-HA and pEXPR-NA were constructed for the delivery of hemagglutinin (HA) and neuraminidase (NA) genes of avian influenza strain H5N1 into the chloroplast genome of N. benthamiana. Experimental results indicated that pEXPR-G and pEXPR-UG were fundamentally functional in E. coli and both the aadA and GFP expression cassettes were active, allowing the bacteria to withstand 500mg/l spectinomycin and express the transgene of interest at the protein level. Similar results were also observed in transplastomic N. benthamiana transformed with pEXPR-UG and pEXPR-NA. In essence, the developed 2-part chloroplast transformation vector system was found to be highly versatile and could be conveniently applied for the construction of transformation vectors for the delivery and expression of HA and NA in the chloroplast of N. benthamiana

Effects of postharvest techniques on nutritional quality of cherry tomatoes

Cherry tomatoes play an important part in the human diet as they have a high content of nutritional component, including ascorbic acid, carotenoids and total phenolics. This study aimed to investigate how postharvest techniques, temperature, light irradiation and Modified atmosphere packaging (MAP) affected nutritional quality of cherry tomatoes, and to identify the strategies to preserve their quality. Three temperatures (5, 15, 20°C) were examined to evaluate the effect of temperature on cherry tomatoes. A storage temperature of 15°C was found to inhibit weight loss and the decrease of lycopene, s-carotene and lutein content when compared with 20°C, whereas a low temperature (5°C) caused chilling injuries, such as pour colour development, and inhibited the increase of lycopene and s-carotene content compared with 15 and 20°C. The effect of postharvest red/far-red (ratio 0.89) and blue light irradiation on fruit quality was also investigated. The results showed that red/far-red light inhibited weight loss, inducing colour changing from green to red, and increased the content of lycopene, b-carotene, and total phenolics compared to darkness. In contrast, blue light induced weight loss, and had little effect on colour change and the content of ascorbic acid, lycopene and total phenolics compared to darkness. The influence of the combination of red/far-red light and modified atmosphere packaging on fruit quality was also examined. The results showed that the combined treatment effectively extended shelf-life of cherry tomatoes by delaying ripening as indicated by the delayed increase of respiration and colour change from green to red, reduced weight loss, and increased the content of lycopene and s-carotene. The role and mechanism of red/far-red light in the regulation of carotenoid biosynthesis were explored. Results showed that gene Phytoene synthase (PSY), Zeta carotene (ZDS) and Chloroplast lycopene beta cyclase (LCY-b) and were overexpressed in fruits treated with red/far-red light during storage compared to the darkness. Moreover, red/far-red light induced expression of 1-aminocyclopropane-1- carboxylate synthase 2 (ACS2) during the first 25 days of storage, Ripening inhibitor (RIN) during the first 28 days and Elongated hypocotyl 5 (HY5) during the whole period of storage. This provided a hypothetical model of red/far-red light in the regulation of carotenoid biosynthesis

Investigation into the potential of methylene blue and its derivatives to be used in the photodynamic therapy of non-pigmented and pigmented cells

Photodynamic therapy (PDT) is a novel treatment for malignant disease. The first step is intravenous injection of a light-absorbing, cytotoxic drug (the photosensitizer) that is then allowed time to accumulate in malignant tissue. The second step involves local activation of the photosensitizer with long (red) wavelength light, delivered usually from a laser. Subsequent to irradiation, highly reactive singlet molecular oxygen (Type II mechanism) is likely to be the most damaging cytotoxic species in vivo. The porphyrin molecule, Photofrin®, is the only photosensitizer currently registered for clinical use but is associated with several problems. Most disappointing is the fact that Photofrin® accumulates not only in malignant tissue but also in other organs, such as the liver, kidney and spleen. Its long persistence in the skin commonly causes severe photosensitization reactions in patients for up to three months post-treatment. Photofrin® also has poor light absorption properties within the therapeutic window (600 to 800 nm) for PDT. Furthermore, PDT with Photofrin® has proved of no use in the treatment of malignant melanoma due, possibly, to competition between the photosensitizer and melanin for light absorption. Second-generation photosensitizers have tended to be porphyrin-based molecules, many of which have reached various stages of clinical trial. Of non porphyrin-based compounds, the cationic dye, methylene blue (MB), used traditionally as a nuclear stain in histology, has proved also to be an efficient photosensitizer, with maximum light absorption properties within the therapeutic window for PDT. Its use as a selective stain for tumour tissue in the bladder led first to its investigation in humans for the PDT of bladder cancer and inoperable tumours of the oesophagus. Radiolabelled MB has also recently been used as a tracer for metastatic melanoma in humans. The disadvantages of MB are an inherent (dark) toxicity and its rapid reduction in vivo to the inactive form, leuco-methylene blue (LMIB). This study found the cytotoxicity of MB to be enhanced by illumination and that successive methylation of the molecule corresponds to both increased light and dark toxicities in the EMT-6 (murine mammary), the SK-23 (murine melanoma) and SKMEL-28 (human melanoma) cell lines. The increased toxicities may be due to increased resistance to reduction (MBcNMB.cMMB<DMMB), improved singlet oxygen quantum yield (MB.cMMBcDMMBcNMB), increased hydrophobicity (MB.cMMBCDMMBcNMB), improved cellular uptake (MBctvHv1BcDMMBNTvffi) and/or changes in intracellular targeting and localisation. MB is known to target the nucleus but it was proposed that the increased hydrophobicities could lead to the mitochondrial targeting of the derivatives. The intracellular localisation of the photosensitizers following incubation was studied using both fluorescence and confocal microscopy. Here, confocal microscopy showed that none of the four photosensitizers, including MB itself, target the nucleus prior to illumination. DMMB and NIMB in particular appear to be confined to small subcellular bodies within the cytoplasm. However, the precise locations of the photosensitizers, prior to illumination, were not established during the course of this study. Nevertheless, confocal microscopy showedthat, upon illumination, all four photosensitizers rapidly relocalise within the nucleus. Since photosensitizers that localise in mitochondria are found to be more efficient inducers of apoptosis than photosensitizers that target other subcellular sites, the ability of the photosensitizers, MB, MMB, DMMB and NMB, to induce apoptosis in EMT-6, SK-23 and SK-MEL-28 cells in culture was investigated in this study. The methods used were visual examination of cell morphology, by use of the cyanine dye, JC-1, and the use of the FluorAce® Apopain Assay Kit from Biorad, in cells that had been exposed to the photosensitizers. From these, it was concluded that an apoptotic cell killing mechanism might play an important role in the photocytotoxicity of the photosensitizers, moreso for DMMB and NMB. The overall purpose of this project was to assess the potential of the derivatives of MB to be used in the PDT of cancer, with a special emphasis on malignant melanoma, since this is a field of cancer treatment where PDT has had no success. Although methyl substitution of MB did not abolish the inherent toxicity of the molecule, it is possible to assess the potential usefulness of the photosensitizers by examination of the light: dark differential. In fact, the light:dark differential was improved for all the derivatives of MB in all three cell lines. Nevertheless, NMB consistently performed the best, achieving maximum photocytotoxicity at concentrations that caused very little toxicity in the dark. The presence of melanin made no difference to the photosensitizing capabilities of the photosensitizers in melanoma cells. In terms of a clinical application of the current work, PDT employing phenothiazinium photosensitizers is not suggested procedurally for the removal of primary melanoma, since this is routinely performed by excision. However, due to the demonstrated efficacy of MB in tracing microsatellites and its use in sentinel lymph node tracing, it may be of use in the photodynamic treatment of local metastatic lymph infiltration immediately post-surgery, as an alternative to lymphadenectomy. At present, MB is used routinely in various tracing or demarcation procedures, either visible or scintillographic, without reported toxicity. The derivatives used in the present in vitro study were all more effective in terms of the photodynamic effect and it is thus possible that future clinical developments in this direction may be feasible

Phenolic Compounds

Phenolics are commonly available compounds in foods, beverages, and spices. They have great importance in all aspects of daily life including industry, health, and research. As such, this book presents a comprehensive overview of phenolic compounds and their potential applications in industry, environment, and public health. Chapters cover such topics as the production of these compounds and their uses in environmental sustainability, climate change, green industry, and treatment of human disease

Construction and molecular characterisation of an improved chloroplast transformation vector system as a versatile delivery and expression platform for in-vitro propagated Nicotiana benthamiana

The objective of this study is to develop a versatile vector system for the delivery and expression of transgenes in the chloroplast genome of N. benthamiana. The successful advent of such a system would vastly streamline the construction process of chloroplast transformation vectors for the expression of recombinant proteins, such as vaccine candidates, in the chloroplasts of N. benthamiana. Transgenes targeted to the chloroplasts of higher plants are expected to be expressed at considerably higher levels as compared to nuclear expression, resulting in more significant accumulation of recombinant proteins. In this study, a 2-part chloroplast transformation vector system was developed and two new GFP vector prototypes, pEXPR-G and pEXPR-UG were generated for preliminary evaluation of functionality. The aadA and GFP expression cassettes of pEXPR-G and pEXPR-UG were evaluated in E. coli prior to actual delivery into N. benthamiana via particle bombardment. Particle bombardment parameters were optimised with particular emphasis on minimising excessive damage to the target tissue in order to facilitate the recovery of antibiotic resistant shoots and calli following transformation. To further evaluate the versatility of the developed system for the expression of vaccine antigens, recombinant vectors, pEXPR-HA and pEXPR-NA were constructed for the delivery of hemagglutinin (HA) and neuraminidase (NA) genes of avian influenza strain H5N1 into the chloroplast genome of N. benthamiana. Experimental results indicated that pEXPR-G and pEXPR-UG were fundamentally functional in E. coli and both the aadA and GFP expression cassettes were active, allowing the bacteria to withstand 500mg/l spectinomycin and express the transgene of interest at the protein level. Similar results were also observed in transplastomic N. benthamiana transformed with pEXPR-UG and pEXPR-NA. In essence, the developed 2-part chloroplast transformation vector system was found to be highly versatile and could be conveniently applied for the construction of transformation vectors for the delivery and expression of HA and NA in the chloroplast of N. benthamiana

Polymer Processing and Surfaces

This book focuses on fundamental and applied research on polymer processing and its effect on the final surface as the optimization of polymer surface properties results in the unique applicability of these over other materials. The development and testing of the next generation of polymeric and composite materials is of particular interest. Special attention is given to polymer surface modification, external stimuli-responsive surfaces, coatings, adhesion, polymer and composites fatigue analysis, evaluation of the surface quality and microhardness, processing parameter optimization, characterization techniques, among others