Doctor of Philosophy

dissertationFovea centralis is a depression located in the back of the primate retina. This region is crucial for sharp central vision. Age Related Macular Degeneration (AMD) is a debilitating disease that affects several million people in the world. In this disease, the retina and RPE surrounding the fovea undergoes degeneration, thereby compromising visual acuity. The fovea contains significant amounts of three carotenoids - lutein, zeaxanthin, and meso-zeaxanthin. Carotenoids are plant-derived pigment molecules. Vertebrates are unable to synthesize these compounds de novo, and have to obtain these through the diet. Carotenoids protect the foveal region from oxidative stress, light damage, and improves vision. Carotenoid supplementation is shown to alter the course of AMD. Hundreds of carotenoids are present in nature, and the regular human diet consumes about 50 of these. Among these, only 15 are absorbed by the gut and present in the serum. However, only lutein, zeaxanthin, and meso-zeaxanthin are present in the retina. Retinal concentrations of these carotenoids are 5000 times greater than observed levels in the serum, suggesting a very specific transport mechanism into the retina. There are gaps in our knowledge of the mechanisms involved in carotenoid transport into the eye. meso-Zeaxanthin is a retina-specific carotenoid that is rarely encountered in nature. No dietary sources of meso-zeaxanthin are identified. At the foveal pit, there are equal amounts of lutein, zeaxanthin, and meso-zeaxanthin. However, meso-zeaxanthin is absent in the peripheral retina. The mechanism by which meso-zeaxanthin is produced in the eye and the physiological significance of its presence is not understood

AN INTRODUCTION TO THE ART OF MASSAGE

Massage, or systematic rubbing and manipulation of the tissues of the body, is probably one of the oldest of all means used for the relief of bodily infirmities. There are various evidences for the practice of massage in different forms in medieval times also. Later on, Dr. Mezger and Kellogg developed the two types of massage viz, Swedish and Kellogg massage. The basic movements and procedures are the same in both. The four major procedures in Swedish massage are effleurage (stroking), frictions, petrissage (kneading) and tapotement (percussion). Swedish massage is not much popular in India but it is widely practiced in other countries. The practice of massage produces mechanical, reflex and metabolic effects in the body. Massage influences the circulation, respiratory activities, nervous coordination and digestive activities which are detailed in the paper. Massage helps in lymph and blood circulation and absorption of waste and effused products. Massage can be indicated in arthritic pain, paralysis etc. and contra indicated in skin diseases, fever, after surgery etc. Care should be taken to make all the movements from wrist otherwise it generates pain. Only few studies have been conducted on massage and its effects. Moreover not much authentic books are available on massage except Kellog’s ‘The Art of Massage’. More and more studies should be done in order to make the practice of massage popular

COMPARISION OF PHYTOCHEMICALS IN THE FLOWER BUDS, PEDICELS AND LEAVES OF SYZYGIUM AROMATICUM (L.) MERRIL AND PERRY

Objective: To analyse and compare the major chemical components in the flower buds, pedicels and leaves of Syzygium aromaticum by Gas-Chromatography Mass spectrometry technique. Methods: Healthy and mature flower buds, pedicels and leaves were shade dried and pulverized using a mechanical grinder. The powder was successively extracted with ethanol (40-60o C). The extracts were concentrated under reduced pressure in a rotary evaporator. The ethanolic extracts of the plant parts such as leaves, pedicels, and buds were used for GC-MS analysis.Results: The major constituent is eugenol. Pedicels contain 79.75% eugenol, buds contain 74.12% eugenol and leaves contain 51.03% eugenol. In addition to eugenol, other important components are Acetyl eugenol, Caryophyllene, Humulene and Caryophyllene oxide.Conclusion: Eugenol has a wide range of medicinal properties such as antiseptic, anaesthetic, analgesic anti-inflammatory. Commercially pedicel is not used for eugenol extraction. Present study has revealed that it could be used as a promising one in pharmaceutical industry in addition to flower buds

T Cell Receptor Gamma and Delta Gene Rearrangements in T-cell Acute Lymphoblastic Leukemia in South India and Quantitation of Minimal Residual Disease

Background: T cell-Acute Lymphoblastic Leukemia (T-ALL) arises by clonal proliferation of lymphoid precursors arrested at particular stage of differentiation. The incidence of T-ALL in India is 37-43% of ALL. In this study, TCR gamma (TCRG) and TCR delta (TCRD) gene rearrangements were detected in diagnostic samples of ALL. Those clonal rearrangements detected at diagnosis are used as clonal markers for the quantitation of minimal residual disease (MRD) in follow up samples. 

Patients and Methods: BM/PB from 54 T-ALL patients (34 pediatrics and 20 adults) at diagnosis, treated by MCP 841 protocol were studied. Median age of the patients was 13. The frequency of clonal TCRG and TCRD gene rearrangements were studied by Polymerase Chain Reaction (PCR) coupled with Heteroduplex analysis (HD). Allele Specific Oligos (ASO) was designed by sequencing the junctional region sequence of clonal TCRG and TCRD gene rearrangements. MRD was studied in 4 patients with follow up samples. Diagnostic DNA with almost 100% tumor involvement as standard was serially diluted (50ng to 5ng) in 500ng control DNA to give a final concentration of one leukemic cell in 101 (1 in 101) cells to one leuekemic cell in 105 (1 in 105) cells. The serially diluted diagnostic standard in triplicates was amplified with TaqMan probe for respective TCRG/TCRD gene rearrangements in Real-time PCR along with 500ng of follow up DNA samples at different time points (Unknown) in triplicates. Analysis was done to calculate the amount of leukemic cells in follow up samples. 

Results: Using PCR-HD analysis, TCRG gene rearrangements were detected in 37 of 54 cases (68.5%) and TCRD gene rearrangements in 16 of 54 cases (29.6%). VgI-Jg1.3/2.3 was more commonly rearranged in 29 cases (53.7%) of T-ALL; VgII-Jg1.3/2.3 in 14 cases (26%). Both VgIII-Jg1.3/2.3 and VgIV-Jg1.3/2.3 rearrangements were detected in 4 cases respectively and VgI-Jg1.1/2.1 was detected in 3 cases. Vd1-Jd1 rearrangement was detected in 9 cases (16.6%); Vd2-Dd3 in 5 cases and Dd2-Dd3 in 4 cases of T-ALL. Both TCRG and TCRD gene rearrangements were detected in 8 cases (14.8%). The junctional region in TCRG rearrangements ranged from 1nucleotide to 11nucleotide (average 7.6 nt) and in TCRD rearrangements ranged from 14 nucleotide to 42 nucleotide (average 27 nt). In patient 1, the initial leukeimia load of 1 (before treatment) was reduced to 3 leukemic cell in 104 cells at the lost follow up. In patient 2, the initial leukemia load was reduced to 3.7 in 104 cells. In Patient 3, the initial leukemia load 1 was reduced to 6 in 102 cells at end of M2 and disease relapsed at M5. In patient 4, the initial leukemia load was reduced to 1.6 leukemic cell in 104 cells. 

Conclusion: Real-time PCR experiments reached a reproducible sensitivity of detecting one leukemic cell in 104 normal cells. Real-time PCR analysis showed that Patient 3 was not all responded to treatment and it was detectable by Real-time PCR at RI1 stage itself though the disease was clinically evident only at M5 stage of treatment. Thus, monitoring the MRD using Real-time PCR will help to quantitate the accurate amount of residual leukemic load, predate relapse and assess the response to treatment. 
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Impairment of Protein Trafficking upon Overexpression and Mutation of Optineurin

Glaucoma is a major blinding disease characterized by progressive loss of retinal ganglion cells (RGCs) and axons. Optineurin is one of the candidate genes identified so far. A mutation of Glu(50) to Lys (E50K) has been reported to be associated with a more progressive and severe disease. Optineurin, known to interact with Rab8, myosin VI and transferrin receptor (TfR), was speculated to have a role in protein trafficking. Here we determined whether, and how optineurin overexpression and E50K mutation affect the internalization of transferrin (Tf), widely used as a marker for receptor-mediated endocytosis.Human retinal pigment epithelial (RPE) and rat RGC5 cells transfected to overexpress wild type optineurin were incubated with Texas Red-Tf to evaluate Tf uptake. Granular structures or dots referred to as foci formed in perinuclear regions after transfection. An impairment of the Tf uptake was in addition observed in transfected cells. Compared to overexpression of the wild type, E50K mutation yielded an increased foci formation and a more pronounced defect in Tf uptake. Co-transfection with TfR, but not Rab8 or myosin VI, construct rescued the optineurin inhibitory effect, suggesting that TfR was the factor involved in the trafficking phenotype. Forced expression of both wild type and E50K optineurin rendered TfR to colocalize with the foci. Surface biotinylation experiments showed that the surface level of TfR was also reduced, leading presumably to an impeded Tf uptake. A non-consequential Leu(157) to Ala (L157A) mutation that displayed much reduced foci formation and TfR binding had normal TfR distribution, normal surface TfR level and normal Tf internalization.The present study demonstrates that overexpression of wild type optineurin results in impairment of the Tf uptake in RPE and RGC5 cells. The phenotype is related to the optineurin interaction with TfR. Our results further indicate that E50K induces more dramatic effects than the wild type optineurin, and is thus a gain-of-function mutation. The defective protein trafficking may be one of the underlying bases why glaucoma pathology develops in patients with E50K mutation

Biochemistry and Molecular Biology Developmentally Regulated Production of meso- Zeaxanthin in Chicken Retinal Pigment Epithelium/ Choroid and Retina

PURPOSE. meso-Zeaxanthin is a carotenoid that is rarely encountered in nature outside of the vertebrate eye. It is not a constituent of a normal human diet, yet this carotenoid comprises onethird of the primate macular pigment. In the current study, we undertook a systematic approach to biochemically characterize the production of meso-zeaxanthin in the vertebrate eye. METHODS. Fertilized White Leghorn chicken eggs were analyzed for the presence of carotenoids during development. Yolk, liver, brain, serum, retina, and RPE/choroid were isolated, and carotenoids were extracted. The samples were analyzed on C-30 or chiral HPLC columns to determine the carotenoid composition. RESULTS. Lutein and zeaxanthin were found in all studied nonocular tissues, but no mesozeaxanthin was ever detected. Among the ocular tissues, the presence of meso-zeaxanthin was consistently observed starting at embryonic day 17 (E17) in the RPE/choroid, several days before its consistent detection in the retina. If RPE/choroid of an embryo was devoid of mesozeaxanthin, the corresponding retina was always negative as well. CONCLUSIONS. This is the first report of developmentally regulated synthesis of mesozeaxanthin in a vertebrate system. Our observations suggest that the RPE/choroid is the primary site of meso-zeaxanthin synthesis. Identification of meso-zeaxanthin isomerase enzyme in the developing chicken embryo will facilitate our ability to determine the biochemical mechanisms responsible for production of this unique carotenoid in other higher vertebrates, such as humans

Autophagy in the eye:from physiology to pathophysology

Autophagy is a catabolic self-degradative pathway that promotes the degradation and recycling of intracellular material through the lysosomal compartment. Although first believed to function in conditions of nutritional stress, autophagy is emerging as a critical cellular pathway, involved in a variety of physiological and pathophysiological processes. Autophagy dysregulation is associated with an increasing number of diseases, including ocular diseases. On one hand, mutations in autophagy-related genes have been linked to cataracts, glaucoma, and corneal dystrophy; on the other hand, alterations in autophagy and lysosomal pathways are a common finding in essentially all diseases of the eye. Moreover, LC3-associated phagocytosis, a form of non-canonical autophagy, is critical in promoting visual cycle function. This review collects the latest understanding of autophagy in the context of the eye. We will review and discuss the respective roles of autophagy in the physiology and/or pathophysiology of each of the ocular tissues, its diurnal/circadian variation, as well as its involvement in diseases of the eye