1

ABSTRACT
Glaucoma is a group of eye diseases which in most cases produce increased pressure within the eye. This elevated pressure is caused by a backup of fluid in the eye. Over time, it causes damage to the optic nerve. When the eye pressure is increased and/or other inciting factors exist, the optic nerve becomes damaged and the retinal ganglion cells undergo a slow process of cell death termed "apoptosis." The death of the retinal cells and degeneration of the nerve fibers results in permanent vision loss. First of all a database on Genes and Proteins associated with Glaucoma is developed. This database contains the gene and the nucleotide sequences of the major genes involved in the disease development and progression, as well as the gene function, location, etc. When the database is developed the user can retrieve the information of any gene and its associated protein. Secondly, the target proteins(CYP1B1) structure is derived by homology modeling and the inhibitor(Pinostillbene) is identified for the target protein. The analogues for the inhibitor are made and they are then docked onto the targete. The analogue having the minimum Etotal can act as an effective drug for the disease. Thirdly, a gene with an unknown function (ANGPTL7) is selected and genome analysis is done for the same to predict its structure and function by the help of computational biology tools.

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CHAPTER 1: INTRODUCTION

INTRODUCTION
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3 1.1 WHAT IS GALUCOMA? Glaucoma is optic nerve damage (often, but not always, associated with increased eye pressure) that leads to progressive, irreversible loss of vision.

Damage to the optic nerve can occur when pressure within the eye increases. The vision loss occurs so slowly that it may not be noticed for a long time. People at risk should have a complete eye examination, including measurement of eye

pressures and testing of side (peripheral) vision.

Eye pressure needs to be controlled throughout life, usually with eye drops but sometimes

with eye surgery. Almost 3 million people in the United States and 14 million people worldwide have glaucoma. Glaucoma is the third leading cause of blindness worldwide and the second leading cause of blindness in the United States, where it is the leading cause of blindness among blacks and Hispanics. In the United States, about one third of glaucoma occurs with eye pressures within the average range, a condition called low-tension glaucoma. People at highest risk are those with any of the following:

Age older than 40 African-American race Family members who have (or had) the disease Farsightedness or nearsightedness Diabetes Long-term use of corticosteroid drugs Previous eye injury

Glaucoma occurs when an imbalance in production and drainage of fluid in the eye (aqueous humor) increases eye pressure to unhealthy levels. Normally the aqueous fluid, which nourishes the eye, is produced by the ciliary body behind the iris (in the posterior chamber) and flows through 19/06/2010 Glaucoma

4 the pupil to the front of the eye (anterior chamber), where it exits into drainage canals between the iris and cornea (the angle). When functioning properly, the system works like a faucet (ciliary body) and sink (drainage canals). Balance between fluid production and drainagebetween an open faucet and a properly draining sinkkeeps the fluid flowing freely and prevents pressure in the eye from building up.

Normal Fluid Drainage

Fig 1: Fluid is produced in the ciliary body behind the iris (in the posterior chamber), passes into the front of the eye (anterior chamber), and then exits through the drainage canals.

In glaucoma, the drainage canals become clogged, blocked, or covered. Fluid cannot leave the eye even though new fluid is being produced in the posterior chamber. In other words, the sink backs up while the faucet is still running. Because there is nowhere in the eye for the fluid to go, pressure in the eye increases. When the pressure becomes higher than the optic nerve can tolerate, damage to the optic nerve occurs. This damage is called glaucoma. Sometimes eye pressure 19/06/2010 Glaucoma

5 increases within the range of normal but is nonetheless too high for the optic nerve to tolerate (called low tension glaucoma). 1.2 FORMS OF GLAUCOMA There are many forms of adult and childhood glaucomas. Most glaucomas fall into two categories: open-angle or closed-angle glaucomas. Open-angle glaucoma is more common. In open-angle glaucoma, the drainage canals in the eyes become clogged gradually over months or years. Pressure in the eye rises slowly because fluid is produced at a normal rate but drains sluggishly. Closed-angle glaucoma is less common than open-angle glaucoma. In closed-angle glaucoma, the drainage canals in the eyes become blocked or covered because the angle between the iris and cornea is too narrow. The blockage can occur suddenly or slowly. If the blockage occurs suddenly, pressure in the eye rises rapidly. If the blockage occurs slowly, the pressure in the eye rises slowly like in open-angle glaucoma. In most people, the cause of glaucoma is not known, although both open-angle and closed-angle glaucomas tend to run in families. In others, damage to the eye caused by infection, inflammation, tumor, large cataracts or surgery for cataracts, or other conditions keeps the fluid from draining freely and leads to increased eye pressure and optic nerve damage (secondary glaucoma). 1.3 SYPMTOMS Open-Angle Glaucoma: Open-angle glaucoma is painless and causes no early symptoms. The most important symptom of open-angle glaucoma is the development of blind spots, or patches of vision loss, over months to years. The blind spots slowly grow larger and coalesce. Peripheral vision is usually lost first. Vision loss occurs so gradually that it is often not noticed until much of it is lost. Because central vision is generally lost last, many people develop tunnel vision: they see straight ahead perfectly but become blind in all other directions. If glaucoma is left untreated, eventually even tunnel vision is lost, and a person becomes totally blind.

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6 Closed-Angle Glaucoma: If eye pressure rises rapidly in closed-angle glaucoma (acute closedangle glaucoma), people typically notice an abrupt onset of severe eye pain and headache, redness, blurred vision, rainbow-colored halos around lights, and sudden loss of vision. They may also have nausea and vomiting as a response to the increase in eye pressure. Acute closed-angle glaucoma is considered a medical emergency, because people can lose their vision as quickly as 2 to 3 hours after the appearance of symptoms if the condition is not treated. People who have had open-angle or closed-angle glaucoma in one eye are likely to develop it in the other. 1.4 SCREENING AND DIAGNOSIS Because the most common types of glaucoma can cause slow and silent loss of vision over years, early detection of the disease is extremely important. All people at high risk of glaucoma should have a comprehensive eye examination every 1 to 2 years. There are four parts to a comprehensive eye examination for glaucoma. First, pressure in the eye is measured. This measurement is taken painlessly with an instrument called a tonometer. In general, eye pressure readings of greater than 20 to 22 millimeters of mercury (mm Hg) are considered higher than normal. But measuring eye pressure is not enough, because a third or more of people with glaucoma have eye pressure in the average range. So doctors also use an ophthalmoscope and a slit lamp to look for changes in the optic nerve that indicate damage caused by glaucoma. In addition, visual field (peripheral vision) testing allows a doctor to detect blind spots. Most often, visual field testing is done with a machine that determines the person's ability to see small dots of light in all areas of the visual field. Finally, doctors may also use a special lens to examine the drainage channels in the eye, a procedure known as gonioscopy. The gonioscope allows the doctor to determine whether the glaucoma is of the open-angle or closed-angle type. 19/06/2010

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7 1.5 TREATMENT Once a person loses vision because of glaucoma, the loss is permanent. But if glaucoma is detected, proper treatment can prevent further vision loss. So the goal of glaucoma treatment is to prevent the onset of vision loss or stop its progression. Treatment of glaucoma is lifelong. It involves decreasing eye pressure by increasing fluid drainage out of the eyeball or by reducing the amount of fluid produced inside the eyeball. Some people with high eye pressure who do not have signs of optic nerve damage (known as glaucoma suspects) can be monitored closely without treatment. Eye drops and surgery are the main treatments for open-angle and closed-angle glaucomas. Eye drops containing beta-blockers, prostaglandin-like compounds, alpha-adrenergic agonists, carbonic anhydrase inhibitors, or cholinergic drugs are commonly used to treat glaucoma. Most people with open-angle glaucoma respond well to these drugs. These drugs are also used for people with closed-angle glaucoma, although surgery, not eye drops, is the main treatment. Glaucoma eye drops are generally safe, but they may cause a variety of side effects. People need to use them for the rest of their lives, and regular check-ups are necessary to monitor eye pressure, optic nerves, and visual fields. Sometimes a kind of diuretic (osmotic diuretic) given by mouth or by vein is also used briefly to help decrease eye pressure rapidly in acute closed-angle glaucoma. Surgery may be needed if eye drops cannot effectively control eye pressure, if a person cannot take eye drops, or if people develop intolerable side effects from the eye drops. Laser surgery can be used to increase drainage in people with open-angle glaucoma (laser trabeculoplasty) or to make an opening in the iris (laser peripheral iridectomy or iridotomy) in people with acute closed-angle glaucoma. Laser surgery is done in the doctor's office or in a hospital or clinic. Anesthetic eye drops are used to prevent pain. People are usually able to go home the same day of any of these surgical procedures. Glaucoma filtration surgery is the other form of surgery doctors use to treat glaucoma. With traditional glaucoma filtration surgery, doctors manually create a new drainage system 19/06/2010 Glaucoma

8 (trabeculectomy or tube shunt) to allow fluid to bypass the clogged or blocked canals and filter out of the eye. Glaucoma filtration surgery is generally performed in a hospital. Newer filtration procedures (viscocanalostomy and Trabectome) remove only part of the drain to enhance the outflow of fluid. People are usually able to return home the day of the procedure. The most common complication of glaucoma laser surgery is a temporary increase in eye pressure, which is treated with glaucoma eye drops. Rarely, the laser used in laser surgery may burn the cornea, but these burns usually heal quickly. With laser and glaucoma filtration surgery, inflammation and bleeding within the eye may occur but are usually short-lived. Glaucoma filtration surgery may occasionally lead to double vision, cataracts, or infection. Because severe closed-angle glaucoma is a medical emergency, doctors may use very strong and fast-acting drugs that affect the eye pressure more rapidly than the standard eye drops or surgery. Doctors may use glycerin or acetazolamide pills or drugs given by vein (such as mannitol) if they think the eye is vulnerable to high pressure. Eye drops are also given as soon as possible. Emergency surgery is performed if necessary. The treatment of glaucoma caused by other disorders depends on the cause. For infection or inflammation, antibiotic, antiviral, or corticosteroid eye drops may provide a cure. A tumor obstructing fluid drainage should be treated, as should a cataract that is so large it causes eye pressure to rise. High eye pressure that results from cataract surgery is treated with glaucoma eye drops that reduce eye pressure. If eye drops do not work, glaucoma filtration surgery can be performed.

Medications: Nearly all glaucoma medications are prescribed for reducing eye pressure. Beta-blockers (Timolol and Others) 19/06/2010

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9 Topical beta adrenoceptor blockers (commonly called beta-blockers) are the drugs most often prescribed to treat glaucoma. They lower the pressure inside the eye by inhibiting the production of aqueous humor. Brands. These drugs are categorized as either nonselective or selective beta-blockers: Nonselective adrenoceptor beta-blockers. Timolol (Timoptic, Betimol) has been the standard beta-blocker for years. Newer nonselective drugs include levobunolol (Betagan), carteolol (Ocupress), and metipranolol (OptiPranolol). A few studies suggest some are more beneficial than timolol with similar side effects. Selective beta1-adrenoceptor blockers. Betaxolol (Betoptic) and levobetaxolol (Betaxon) are selective beta-blockers. These drugs appear to have fewer adverse effects on the heart than the nonselective beta-blockers, although they still have widespread effects. Studies also suggest that they slow progression more than timolol, although timolol is more effective at lowering IOP. Selective beta-blockers may also have nerve-protecting properties. All beta-blockers work well and generally well tolerated. Because they cause less eye irritation than many other glaucoma medications, they are often prescribed for patients who also have cataracts. Side Effects and Complications. After the beta-blocker is administered, only a tiny amount of the drug is absorbed by the cornea. Most of it enters in the bloodstream. These drugs, therefore, can cause side effects in parts of the body other than the eyes ("systemic" side effects): Systemic side effects may include reduced sexual drive, fatigue, depression, anxiety, and breathing difficulties. Beta-blockers affect the heart. They lower heart rate and reduce blood pressure. Beta-blockers can worsen severe asthma or other lung diseases. A patient switching to a beta-blocker from other glaucoma medication may feel a sudden rise in eye pressure. It is important that the pressure be checked shortly after the other drug has been withdrawn. 19/06/2010 Glaucoma

10 When beta-blockers are used to treat one eye, the other (contralateral) eye also experiences a lesser, but still significant reduction in IOP. Interactions with Other Medications- The effects of the eye medication can interact with other oral medications, such as oral beta-blockers, calcium-channel blockers, or the antiarrhythmic drug quinidine. People with diabetes who take insulin or hypoglycemic medications should realize that beta-blocker side effects may mask the symptoms of hypoglycemia (low blood sugar). Prostaglandins Prostaglandins are hormone-like substances that help open blood vessels. Drugs that resemble prostaglandins increase outflow of aqueous humor (the watery substance in the eye). Drainage of aqueous humor helps reduce intraocular pressure. Brands. Latanoprost (Xalatan) and unoprostone (Rescula) are the standard brands. Latanoprost was the first prostaglandin to be approved as first-line treatment for elevated eye pressure. Two newer prostaglandins, travoprost (Travatan) and bimatoprost (Lumigan), may help some patients who do not respond to latanoprost. These drugs may also benefit patients with normal-tension glaucoma. Side Effects. These drugs do not slow down the heart rate and also appear to be safe for people with asthma. Side effects include itching, redness, and burning during administration. Muscle and joint pain may also occur. All of these drugs may permanently change eye color from blue or green to brown.

Carbonic Anhydrase Inhibitors Carbonic anhydrase inhibitors (CAIs) decrease eye pressure by reducing the fluid in the chambers of the eye (aqueous humor). These drugs are used for glaucoma when other drugs do not work. They may be combined with other medications.

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11 CAIs may also improve blood flow in the retina and optic nerve (beta-blockers do not). Improving blood flow can keep the disease from getting worse. Brands and Side Effects. CAIs are available in the following forms: Eye-drop CAIs include dorzolamide (Trusopt) and brinzolamide (Azopt). About 10% of patients report fatigue, stinging in the eye, and loss of appetite using dorzolamide. Taste changes can occur. Brinzolamide is a newer medication that may cause less stinging than dorzolamide. Forms taken by mouth (oral) include acetazolamide (Diamox), methazolamide (Neptazane), and dichlorphenamide (Daranide). Although they are more effective than eye drops, they have significantly more side effects and are rarely used for long-term treatment. The oral forms have very unpleasant side effects, including frequent urination, depression, stomach problems, fatigue, weight loss, sexual dysfunction, and, in infants, failure to thrive. Long-term use of the oral forms, in rare cases, can cause serious anemia and kidney problems, including the risk for stones. They can also produce a toxic reaction when taken with large doses of aspirin. Adrenergic Agonists Adrenergic agonists activate muscles in the eye that dilate pupils and, therefore, increase outflow of aqueous fluid. Newer variations called alpha 2-adrenergic agonists reduce production of aqueous humor and also increase outflow through the uveoscleral pathway (the alternative channel to the trabecular meshwork). Apraclonidine (Iopidine) and brimonidine (Alphagan) are alpha 2-adrenergic agonists. These are generally been used before glaucoma surgery, but may be useful as primary therapy when used in combination with beta-blockers or other standard drugs. Brimonidine is proving to be particularly effective for long-term therapy. (Apraclonidine is used for the short term.) It also may have nerve-protecting properties and may be safer than other drugs during pregnancy and for patients with asthma.

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12 The most common side effects of brimonidine and apraclonidine are dry mouth and altered taste. They also commonly trigger an allergic reaction that causes red and itching eyes and lids,. Brimonidine causes less of an allergic response than apraclonidine. Unlike apraclonidine, however, it can cause lethargy and mild low blood pressure. Miotics (Pilocarpine and Others) Miotics, also called cholinergic agonists, narrow the iris muscles and constrict the pupil. This action pulls the iris away from the trabecular meshwork and allows the aqueous humor to flow out through the drainage channels, reducing the pressure inside the front of the eye. Brands. Pilocarpine (Pilocar, Adsorbocarpine, Almocarpine, Isoptocarpine, Ocusert) was the most widely used anti-glaucoma drug before timolol was introduced. It is the preferred miotic. Because pilocarpine is used up by the body fairly quickly, however, patients must take it several times a day; many people, therefore, fail to take their medication regularly. A combination of timolol or latanoprost with pilocarpine is more effective than either drug used alone. Carbachol is another miotic. Epinephrine and its derivatives are the older anticholinergics. Epinephrine is now rarely prescribed because of side effects. Dipivefrin (Dipivefrin), a newer form of epinephrine, remains inactive until it reacts with enzymes in the cornea. It is effective in low doses and causes few systemic side effects.

Side Effects. Side effects include:

Teary eyes, brow-aches, eye pain, and allergic reactions. A miotic narrows the pupil and so can cause nearsightedness. Vision can also become dim and it may difficult to see in darkened rooms or at night, when driving could be hazardous. Anticholinesterase miotics increase the risk of cataract development and are therefore used mostly in patients in whom cataracts have already been removed. Retinal detachment is an 19/06/2010 Glaucoma

13 uncommon but dangerous side effect in susceptible individuals. Excessive use of these miotics may cause toxic reactions, including convulsions, muscular paralysis, and even death from respiratory failure.

Epinephrine can produce burning in the eyes, enlarged pupils, and allergic reactions. Occasionally it can cause anxiety and headaches. Rare side effects include high blood pressure and disturbances in heart rhythm. It is rarely prescribed now. Although dipivefrin, the newer form of epinephrine, has fewer systemic side effects, it still causes problems in the eyes similar to those of epinephrine.

Managing Drug Regimens Studies indicate that many patients skip doses of their glaucoma medications, sometimes because of side effects and sometimes because of confusing or time-consuming regimens. Skipping even a few doses can greatly increase the risk of visual loss. It is essential that patients tell their doctor if they are not regularly taking their medication. Otherwise, the doctor may increase the dosage, thereby causing unwelcome side effects. Patients who do not regularly take their glaucoma medication are at high risk for blindness. If you have problems taking your medications or sticking to the dosing regimen, talk with your doctor.

Hints for Managing a Regimen.

Pharmaceutical manufacturers use colored tops, yellow for timolol, for example, and green for pilocarpine, to help prevent mix-ups. Creating a chart scheduling each drug by color can be helpful.

Small electronic timers are available that will signal times for taking the medications. The timing of these combinations is important. Some patients may be candidates for single medications that combine two drugs, such as Cosopt, which contains both dorzolamide and timolol. This medication requires only one 19/06/2010

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14 drop twice per day. Patients who need additional glaucoma drugs, however, will need to take these two drugs separately.

When using any drug for a long period of time, side effects are a potential problem. If they become intolerable, patients should discuss with the doctor reducing the dosage or trying other drugs.

1.6 DATABASE A Database is an integrated collection of logically related records or files consolidated into a common pool that provides data for one or more multiple uses. One way of classifying databases involves the type of content, for example: bibliographic, full-text, numeric, and image. Other classification methods start from examining database models or database architectures. Software organizes the data in a database according to a database model. As of 2009 the relational model occurs most commonly. Other models such as the hierarchical model and the network model use a more explicit representation of relationships. The field of biology is vast and fast growing informations regarding the specific fields are day by day increasing in a fractional manner. Thus retrieving and understanding those data becomes tedious and time consuming factor. Each and every day new web sources with surplus information are rising. Human pathogens and their complications is broad field which has enormous sources which provide vast information. The experimental research results are day by day increasing. It needs a single platform for retrieving all these information. Thus bioinformatics paves the way for it using information technology many databases are created and the experimental results are deposited frequently. NCBI (National Center for Biotechnology Information) is a genomic database where the information regarding different nucleotide sequence and their properties are deposited frequently. genome analysis and different diseases like OMIM. Need for the database: To club the information scattered in the web source and to provide complete information about glaucoma starting from genes involved, associated proteins to their functions and sequences. A database management system (DBMS) consists of software that organizes the storage of data. A DBMS controls the creation, maintenance, and use of the database storage structures of social 19/06/2010 Glaucoma Along with this information SNP, ORF s can also be presented in the databases. It also has link to many tools for

15 organizations and of their users. It allows organizations to place control of organization wide database development in the hands of Database Administrators (DBAs) and other specialists. In large systems, a DBMS allows users and other software to store and retrieve data in a structured way. Database management systems are usually categorized according to the database model that they support, such as the network, relational or object model. The model tends to determine the query languages that are available to access the database. One commonly used query language for the relational database is SQL, although SQL syntax and function can vary from one DBMS to another. A common query language for the object database is OQL, although not all vendors of object databases implement this, majority of them do implement this method. A great deal of the internal engineering of a DBMS is independent of the data model, and is concerned with managing factors such as performance, concurrency, integrity, and recovery from hardware failures. In these areas there are large differences between the products. A relational database management system (RDBMS) implements features of the relational model. In this context, Date's "Information Principle" states: "the entire information content of the database is represented in one and only one way. 1.7 SQL SERVER

SQL (Structured Query Language) is a database computer language designed for managing data in relational database management systems (RDBMS), and originally based upon Relational Algebra. Its scope includes data query and update, schema creation and modification, and data access control. SQL was one of the first languages for Edgar F. Codd's relational model in his influential 1970 paper, "A Relational Model of Data for Large Shared Data Banks" and became the most widely used language for relational databases.

1.8 HTML

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16 Hyper Text Markup Language is a programming language for creating the web page. Through HTML one designed web page can be linked to another. HTML is a basic language and its syntaxes are easy to understand. Unlike C++ and Java software development is not possible through HTML. The data created using SQL server can be extrapolated as a web page through HTML. It acts as a platform for the display of data deposited in the database. HTML (HyperText Markup Language) and XHTML (Extensible HTML) are composed of tags, i.e. commands in angle-brackets (< >). HTML tags are case-insensitive, that is, it doesn't matter whether you type them in upper or lower case. However, in XHTML tags need to be lower case, so it is a good idea to get into the habit of always using lower case. Tags typically occur in begin-end pairs. These pairs are in the form <tag> ... </tag> where the <tag> indicates the beginning of a tag-pair, and the </tag> indicates the end. (The three dots indicate an arbitrary amount of content between the tags). These pairs define containers. Any content within a container has the rules of that container applied to it. For example, the text within a "boldface container" would be boldfaced. Similarly, paragraphs are defined using a "paragraph container."

1.8 IN-SILICO APPROACHES FOR DRUG DISCOVERY Target and lead discovery constitute the main components of today's early pharmaceutical research. The aim of target discovery is the identification and validation of suitable drug targets for therapeutic intervention, whereas lead discovery identifies novel chemical molecules that act on those targets. With the near completion of the human genome sequencing, bioinformatics has established itself as an essential tool in target discovery and the in silico analysis of gene expression and gene function are now an integral part of it, facilitating the selection of the most relevant targets for a disease under study. In lead discovery, advances in chemoinformatics have led to the design of compound libraries in silico that can be screened virtually. Moreover, computational methods are being developed to predict the drug-likeness of compounds.The use of computers and computational methods permeates all aspects of drug discovery today and forms the core of structure-based drug design. High-performance computing, data management software and internet are facilitating the access of huge amount of data generated and transforming the massive 19/06/2010 Glaucoma

17 complex biological data into workable knowledge in modern day drug discovery process. The use of complementary experimental and informatics techniques increases the chance of success in many stages of the discovery process, from the identification of novel targets and elucidation of their functions to the discovery and development of lead compounds with desired properties. Computational tools offer the advantage of delivering new drug candidates more quickly and at a lower cost. Major roles of computation in drug discovery are; (1) Virtual screening & de novo design, () in silico ADME/T prediction and (3) Advanced methods for determining protein-ligand binding. Thus, drug discovery is already on the road towards electronic R&D. 1.9 GENOME ANALYSIS Genome is defined as all the genetic content contained within an organism. An organisms genome is made up of molecules of Deoxyribonucleic Acid (DNA) that form long strands that are tightly wound into chromosomes, which are found in the nucleus of eukaryotic organisms and in the cytoplasm of prokaryotic organisms. Chromosomes that are unique to certain organelles within a cell, such as mitochondria or chloroplast are also considered a part of an organisms genome. A genome includes all the coding (regions that are translated into molecules of protein) of DNA that form discrete genes, as well as the non-coding stretches of DNA that are often found on the areas between genes. The sequence, structure, and chemical modifications of DNA not only provide the instructions needed to express the information held within the genome but also provide the genome with the capability to replicate, repair, package, and otherwise maintain itself. The human genome contains approximately 25,000 genes within its 3,000,000,000 base pairs of DNA, which form the 46 chromosomes found in human cell. In contrast, Nanoarchaenum equitans, a parasitic prokaryote in the domain Archea, has one of the smallest known genomes, consisting of 552 genes and 490,885 base pairs of DNA. The study of the structure, function, and inheritance of genomes is called genomics. Genomics is useful for identifying genes, determining gene function, and understanding the evolution of organisms.

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CHAPTER 2: LITERATURE REVIEW

LITERATURE REVIEW
2.1 Update on Glaucoma Genetics While great progress has been made in identifying the underlying genetic components of glaucoma, there is still a lot of ground to cover before we have a complete picture of the many different genes that cause glaucoma, contribute to its severity, or affect treatment outcomes.As research groups around the world are carrying out the search for glaucoma 19/06/2010 Glaucoma

20 genes, each new discovery draws additional research groups into the study of the genes that are found. With more than a dozen genetic locations known for genes that can cause a broad array of different kinds of glaucoma, and more than a half dozen of the genes themselves identified, it is clear that most glaucoma genes remain to be found. For primary open-angle glaucoma, of special importance because it affects so many people, there are now eight mapped gene locations, and two of those genes (MYOC and OPTN) have been shown to account for a small fraction of open-angle glaucoma cases. Many more regions around the genome have been identified that appear to be predictive of risk for primary open-angle glaucoma, called genetic risk factors, but the actual genes involved have not been found. In addition, among the several congenital glaucoma genes that have been found, the CYP1B1 gene has been found to be responsible for more than half of cases of congenital glaucoma in some populations studied. The identification of the first two open-angle glaucoma genes, MYOC and OPTN, and the first congenital glaucoma gene, CYP1B1, has provided important means to develop diagnostic tools and explore the mechanisms by which these genes participate in the initiation and progression of the glaucoma disease process. Biochemical and cell biology studies are examining the role of these three genes in cellular and developmental processes. Genetic studies are evaluating how large a role each of these genes plays in different populations. Future studies are expected to examine the roles of such glaucoma genes in populations that have participated in some of the largest glaucoma clinical trials studies, in an effort to determine whether genetic testing might help predict not only whether someone will develop glaucoma, but also perhaps predict things about the clinical course of the disease or even the optimal treatment for that particular patient. As a preliminary part of such clinical trials studies, the Glaucoma Research Foundation is currently supporting a study of family history of glaucoma in the population participating in the Collaborative Initial Glaucoma Treatment Study. Since family history is already one of the pieces of information that physicians obtain during an eye examination, it will be important to determine whether such information can predict more than risk of glaucoma and perhaps also give the ophthalmologist important information that will assist in discussing prognosis and in suggesting the optimum treatment. 19/06/2010

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21 2.2 Complex genetics of glaucoma susceptibility. Glaucoma describes a group of diseases that kill retinal ganglion cells. There are different types of glaucoma, and each appears to be genetically heterogeneous. Different glaucoma genes have been identified, but these genes account for only a small proportion of glaucoma. Most glaucoma cases appear to be multifactorial, and are likely affected by multiple interacting loci. A number of genetic susceptibility factors have been suggested to contribute to glaucoma. These factors fit into two broad groups, those affecting intraocular pressure and those important in modulating retinal ganglion cell viability. Defining the complex genetics of glaucoma will require significant further study of the human disease and animal models. Genetic approaches are essential and will be enhanced by recently developed genomic and proteomic technologies. These technologies will provide valuable clues about pathogenesis for subsequent testing. In this review, we focus on endogenous genetic susceptibility factors and on how experimental studies will be valuable for dissecting the multifactorial complexity of their interactions. 2.3 The Primary open-angle glaucoma gene WDR36 functions in ribosomal RNA processing and interacts with the p53 stressresponse pathway Primary open-angle glaucoma (POAG) is a genetically complex neuropathy that affects retinal ganglion cells and is a leading cause of blindness worldwide. WDR36, a gene of unknown function, was recently identified as causative for POAG at locus GLC1G. Subsequent studies found disease-associated variants in control populations, leaving the role of WDR36 in this disease unclear. To address this issue, we determined the function of WDR36. We studied Wdr36 in zebrafish and found it is the functional homolog of yeast Utp21. Utp21 is cell essential and functions in the nucleolar processing of 18S rRNA, which is required for ribosome biogenesis. Evidence for functional homology comes from sequence alignment, ubiquitous expression, sub-cellular localization to the nucleolus and loss-of-function phenotypes that include defects in 18S rRNA processing and abnormal nucleolar morphology. Additionally, we show that loss of Wdr36 function leads to an activation of the p53 stressresponse pathway, suggesting that co-inheritance of defects in p53 pathway genes may influence the impact of WDR36 variants on POAG. Although

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22 these results overall do not provide evidence for or against a role of WDR36 in POAG, they do provide important baseline information for future studies. 2.4 Role of CYP1B1 in glaucoma. Glaucoma is a leading cause of blindness, estimated to affect 60 million people by 2010, and represents a heterogeneous group of neurodegenerative disease. The two major types of glaucoma include primary open-angle glaucoma (POAG) and primary congenital glaucoma (PCG). A genetically heterogeneous group of developmental disorders known as anterior segment dysgenesis (ASD) have been reported to be associated with increased intraocular pressure (IOP) and glaucoma. These include Peters' anomaly, Rieger's anomaly, aniridia, iris hypoplasia, and iridogoniodysgenesis. Genetic linkage analysis and mutation studies have identified CYP1B1 as a causative gene in PCG, as a modifier gene in POAG, and, on rare occasions, as causative gene in POAG as well as in several ASD disorders. CYP1B1deficient mice exhibit abnormalities in their ocular drainage structure and trabecular meshwork that are similar to those reported in human PCG patients. Accordingly, it is speculated that diminished or absent metabolism of key endogenous CYP1B1 substrates adversely affects the development of the trabecular meshwork. CYP1B1 protein is involved in the metabolism of steroids, retinol and retinal, arachidonate, and melatonin. The conserved expression of CYP1B1 in both murine and human eyes, its higher expression in fetal than adult eyes, and its biochemical properties are consistent with this hypothesis. The exact role of CYP1B1 in the pathogenesis of glaucoma and other ASD disorders remains to be elucidated. 2.5 Discovery of cytochrome P450 1B1 inhibitors as new promising anti-cancer agents Human cytochrome P450 (CYP)1B1 is a major enzyme for carcinogenic estrogen metabolism and involved in the metabolic activation of procarcinogens of the polycyclic aromatic hydrocarbons (PAHs). CYP1B1 is known to be expressed at a high frequency in various human cancers, but not in normal tissues. It also plays an important role in the metabolism of various anti-cancer drugs. These findings suggest inhibition of CYP1B1 as a new oncological therapeutic strategy. Several natural and synthetic compounds have been studied in an effort to find the isoform-specific inhibitors of the CYP1 subfamily. A survey

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23 of the inhibitors of CYP1B1 and other related inhibitors of the CYP1 subfamily is provided in this review. 2.6 Thiomethylstilbenes as inhibitors of CYP1A1, CYP1A2 and CYP1B1 activities Resveratrol (3,5,4 -trihydroxy-trans-stilbene) is a natural stilbene derivative occurring in grapes, peanuts and red wine. Its chemopreventive action has been established in studies on animal models. Recently, numerous classes of compounds with stilbene backbone have been investigated for their biological activity concerning cancer prevention; e. g. resveratrol methyl ethers appeared to be specific and potent inhibitors of cytochromes P450 (CYP) family 1 involved in the activation of procarcinogens. Since the replacement of the 4 hydroxyl with a thiomethyl group is supposed to reduce toxicity of stilbene derivatives, the purpose of this study was the synthesis and evaluation of a series of 4-thiomethyl-transstilbene derivatives differing in a number and position of additional methoxy groups. Their inhibitory potency toward human recombinant CYPs: CYP1A1, CYP1A2 and CYP1B1 have been studied and compared with the effect of resveratrol and its analogues. Among compounds tested, 2-methoxy-4 -thiomethyl-trans-stilbene and 3-methoxy-4 -thiomethyltrans-stilbene demonstrated the most potent and selective inhibitory effect on CYP1A1 and CYP1B1 activities. The results of our study indicate that modification of stilbene derivatives with thiomethyl group may influence the selectivity and inhibitory potency of these compounds toward P450 isozymes. Thus, it should be considered in developing new chemopreventive agents based on their mechanism of action.

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24

CHAPTER 3: AIMS AND OBJECTIVES

AIMS AND OBJECTIVES

The aim of this project was as follows:1. To develop a database of genes and proteins associated with Glaucoma which could give all the valuable information regarding these genes including its function, gene ID, 19/06/2010

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25 Accession number, sequences etc, and to form a platform of HTML pages to make this data represent able. 2. The next target was to design a lead molecule which can act as an effective drug for glaucoma gene CYP1B1 using in-silico drug desigining tools and softwares. 3. Lastly, a gene associated with Glaucoma with an unknown function was chosen and its genome was studied extensively using tools of bioinformatics, so as to predict its possible function in the human body.

CHAPTER 4: MATERIALS AND METHODOLOGY 1 9 / 0 6 / 2 0 1 0

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CHAPTER 4: MATERIALS AND METHODOLOGY

MATERIALS AND METHODOLOGY

4.1 MATERIAL A) DATABASE DEVELOPMENT 19/06/2010 Glaucoma

27

WEB SOURCES USED:There are various web resources where the information about glaucoma and their clinical pathology can be collected. The information about glaucoma is surplus in the following web sources: NCBI(National Center For Biotech Information) This is used to retrieve the nucleotide and the protein sequences. OMIM(Online Medical Inheritence in Man)

Fig:2 Print Screen Of OMIM OMIM is a comprehensive, authoritative, and timely compendium of human genes and genetic phenotypes. The full-text, referenced overviews in OMIM contain the information on all known Mendelian disorders and over 12,000 genes. OMIM focuses on the relationship between phenotype and genotype. It is updated daily, and the entries contain copious links to other genetics resources. Available resource:http://www.ncbi.nlm.nih.gov/omim 19/06/2010 Glaucoma

28 WIKIPEDIA Wikipedia is an open source which gives the complete information on the query.

Fig 3: Print Screen Of Wikipidea Available source: http://www.wikipedia.org/ THE MEDLINE DICTIONARY PAGE It is a website that gives complete information about many kinds of disorders. It contains all the information about glaucoma. Available source: http://www.themedicalbiochemistrypage.org/inborn.html

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29

Fig 4: Print Screen Of Medlineplus B) DRUG DESIGNING WIKIPEDIA GENO3D This web server is used to derive the protein structure in case it is not available on Protein Data Bank (PDB). The technique is known as Homology Modeling. The amino acid sequence is submitted in raw form on the server which then generates templates matching the submitted sequence. The template having minimum energy is selected as a model for the target protein structure. The outline of the server is shown as a print screen on the next page.

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30

Fig 5: Print Screen Of Geno3D PubChem Compound This is a tool of NCBI in which the chemical compound its structure and their chemical properties and interactions are generated. The structure of the

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31 inhibitor in this case (Pinostillbene) is retrieved by the help of PubChem Compound:

Fig 6: Print Screen Of Pubchem Compound

C) GENOME ANALYSIS NCBI (National Center For Biotech Information) This is used to retrieve the nucleotide and the protein sequences.

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32

Fig 7: Print Screen Of NCBI

BLAST (Basic local Alignment search Tool) This is a computational biology tool used to search for the similarity amongst the sequences of the query with the other sequences

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33 already present in the database. In this project both the protein and the nucleotude BLAST is carried out.

Fig 8 : Print Screen Of BLAST ORF FINDER(Open Reading Frame Finder) This genearates all the open reading frames present in the submitted nucleotide sequence.

Fig 9 : Print Screen Of ORF Finder 4.2 TOOLS USED

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34 A) DATABSE CREATION Collecting information about a specigic topic (glaucoma) from various sources and clubbing that informationb in a unique database is a tedious task. Various application softwares and rogramming languages are used to create the databases. MICROSOFT SQL SERVER 2005: Microsoft SQL SERVER 2005 is a relational model database server produced by Microsoft. It runs on the server providing the multi-user environment. It uses C and C++ as a basic and its primary query languages are T-SQL and ANSI SQL. SQL Server Screen shot is given on the next page:-

FIG 10 : Print Screen Of MSSQL Server 2005 HTML: Hyper Text Markup language is the web programming language. Database created using SQL server can be made as a web page using HTML coding. Using various types of tags the webpage for the database can be designed. For the successful construction of the web pages the HTML coding should be used well. By the use of few more tags the HTML pages can be used in an interaction way where more than 2 web pages are linked by the help of the following coding: 19/06/2010 Glaucoma

35 <HTML> <a href------------------> </a href> </HTML> This is the HTML coding used for building the home page. <html> <head> <title>Glaucoma</title> </head> <body background="http://www.layoutstar.com/images/allbackgrounds/bgs/dark/dark_backgroun d_a2.gif"> <center><img src="http://macfarlaneoptometrist.com.au/blogs/media/blogs/Optometry/glaucoma.jpg"></ center></img> <marquee width="100%" hieght="0%"><font color="#EEE8AA" size="4" style="verdana"><center><u>A group of disease that can steal sight without warning or symptoms </u></center></head></marquee> <p><font color="white" size="4"><center><b>Glaucoma is a disease of the major nerve of vision, called the optic nerve. The optic nerve receives light from the retina and transmits impulses to the brain that we perceive as vision. Glaucoma is characterized by a particular pattern of progressive damage to the optic nerve that generally begins with a subtle loss of side vision (peripheral vision). If glaucoma is not diagnosed and treated, it can progress to loss of central vision and blindness. </center></font><b></p></body> </html>

B) DRUG DESIGNING

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36 ACD CHEMSKETCH This is a software used to generate the chemical compounds manually. The structure is drawn on the screen, cleaned and 3D optimized and saved as a .MOL file which can be easily converted into PDB format using MN Convert server. The layout of the software is shown on the next page.

Fig 11 : Print Screen Of ACD Chemsketch MN Convert This is used to convert .mol files generated by ACD chemsketch of the analogues into PDB formats so as to be easily available for docking and for viewing purposes in Swiss PDB Viewer.

Fig 12 : Print Screen Of MN Convert MolInspiraton This server is used to know about the molecules chemical properties like molecular weight, logP vales, No of H donors, No of H Acceptors. It is basically used to 19/06/2010 Glaucoma

37 determine the drug likeness of a molecule. A molecule in order to act as a potnt drug molecule must obey the Rule of 5 given by Lipinsky. The rule is that a particular molecule can act as a drug if it satisfies the following criteria:1. Its ClogP value should be <5 2. Its molecular weight should be <500 daltons 3. It should not have more than 10 H bond acceptors 4. It should not have more than 5 H bond donors The chemical properties of the inhibitor molecule (Pinostillbene) is as shown as generated by MolInspiration:-

Fig 13 : Parent Structure Properties Generated By MolInspiration

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38 SWISS PDB VIEWER This software helps in modifying and viewing the PDB files. The view is as shown below:-

Fig 14 : Print Screen Of Swiss PDB Viewer RAMACHANDRAN PLOT It is generated by Swiss PDB Viewer software itself. It is used to optimize the structure of the protein derived by homology modeling energetically by formation of loops. The loop formation changes the orientation of the amino acid residues in such a way so as to bring them inside the favorable regions of the phi and psi angle orientations. The view is :-

Fig 15 : Print Screen Of Ramchandran Plot

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39 LIGSITE This server is used to identify the pockets present in the protein. Pockets are those sites where a ligand can attach itself to the protein molecule and give rise to a product. The outline is :-

Fig 16 : Print Screen Of Ligsite Server C) GENOME ANALYSIS SOPMA This is used for secondary structure prediction in a protein. The server view is as follows:-

Fig 17 : Print Screen Of SOPMA Server 19/06/2010 Glaucoma

40 PROSITE This is used to determine the domain of the protein. Domain is the functional region/part of the protein. The server view is as shown:-

Fig 18 : Print Screen Of Prosite Server CLUSTALW2 This server is used to check the alignment and similarity between multiple sequences of genes and proteins. The view is as shwon below :-

Fig 19: Print Screen Of CLUSTALW Server

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41 BC Preds This is known as B-Cell epitope finder. It helps in locating all the epitope regions in the submitted protein sequence. Its view is generated below:-

Fig 20 : Print Screen Of BC Preds Server 4.3 METHODOLOGY A) DATABASE DEVELOPMENT STEP 1: DATA COLLECTION The very first step of the project was data collection. In this step first of all the information about the genes and proteins associated with glaucoma are collected from various web resources and literatures. The gene function, proteins encoded, accession number, gene IDs, mRNA sequence, protein sequence etc becomes part of this collection. STEP 2: DATA SEPARATION 1. In this step collected information is separated based upon their types. The separation procedures are: (a) Gene Name (b) Gene Function 19/06/2010

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42 (c) Gene Location (d) Number of Base Pairs in mRNA (e) Gene Id (f) Accession Number (g) mRNA Sequence (h) Number of Base Pairs in Nucleotide (i) Nucleotide Accession Number (j) Protein Name (k) Mass (l) Alternative Names (m)Number Of Amino Acid in protein sequence (n) Protein gene ID (o) Protein Accession Number (p) Kegg ID (q) Uniprot ID (r) Amino Acid sequence STEP 3: DATABASE CREATION This is the main step of our project. After the data is separated according to the required columns the next step is to create the database in SQL server. Create database<Database name> is the command used for creating database in SQL server. Here the name of the database is Galucoma. In this database a table named glaucoma_info is created with the above mentioned column names. Required datatypes are defined for different columns. After that SQL coding is done to populate the table with their respective values. The syntax for creating a table in SQL server is :- create table [table name] ([column definitions]) The syntax for insering values in the table is:INSERT INTO table(column1, [column2,.]) values (value1, value2)

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43 STEP 4: WEB PAGE CREATION After the SQL coding part is finished the next and the most important step in database creation is the web page designing using HTML. HTML acts as a platform for the display of data in the database. For creating the web page HTML coding is done. STEP 5: DATABASE CONNECTIVITY Once the database and the webpages are created, the next step is the linking of those web pages to the database. For this ASP coding is done. Using this ASP code we can connect our database and HTML page and retrieve the data from the database. The connection code is as shown below:-

Fig 21 : Print Screen Of Constring For Databse Connectivity STEP 6 : DATA RETRIEVAL Once our database is connected to the HTML web page, next and final step is to retrieve the data. Here if we select a gene from drop down menu the information related to that gene are retrieved from the database on the web page.

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44 B) DRUG DESIGNING STEP 1:HOMOLOGY MODELING The target proteins structure (CYP1B1) was searched in for on Protein Data bank, but it was available in complexed form along with a fatty acid molecule. Therefore, the structure had to be derived by comparative homology modeling technique. For this purpose, the sequence of the amino acid in protein structure was retrieved from NCBI and fed into the Geno3D server. The model having minimum energy optimization was chosen as the target protein structure. STEP 2:POCKET IDENTIFICATION The next task was to determine the pockets in the target. This was done by Ligsite server where PDB file of the target was fed in. it generated the pockets in the structure and gave result in PDB format. The target protein along with its pockets as viewed by the Swiss PDB Viewer is as shoen on the next page:-

Fig 22 : Print Screen Of Swiss PDB Viewer Showing Pocket In The Target Protein STEP 3:RAMACHANDRAN PLOT In this step the protein structure derived by homology modeling is manipulated by loop formation so as to bring all the residues under the favorable phi and psi angle orientations. The resultant protein structure is used finally as a target for docking purpose. The resultant Ramachandran plot after energy optimization is shown on the next page. 19/06/2010 Glaucoma

45

Fig 23 : Print Screen Of Ramachandran Plot for Target protein after Energy Optimization STEP 4 : PREARATION OF THE ANALOGUES In this step, the inhibitor molecules structure is drawn on ACD Chemsketch softwar. It was cleaned, 3D optimized and saved as .MOL file. The file was converted into PDB form via MN Convert. Its chemical and molecular properties was also calculated using MolInspiration server so as to check whether it satisfies the Lipinskys rule of five or not. Two more analogues of inhibitor were generated by following the same procedure but changing the side chains and the functional groups. These PDB files are used finally for the purpose of docking as ligands. The structure of inhibitor in DB format is :-

Fig 24 : Print Screen Of Analogue I As Seen In Swiss PDB Viewer

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46 STEP 5 : DOCKING Docking is a process wherein, the receptor (target) molecule is hit with the ligand (analogue) molecule so as to bind at a specific residues. Out of the three pockets generated by Ligsite server , only one was present at the surface and hence that can only be used for docking purpose. Three residues nearest to the pocket were selected and docking of the ligand was done onto them and checked for binding. In case binding was achieved the ETotal would prove to be the most effective drug molecule for the target.

Fig 25 : Print Screen Of Hex4.5 Software Showing Binding Between His22 of Target and Ligand The binding of His22 to parent molecule is as shown below:-

Fig 26 : Print Screen Of Binding Between His 22 and Analogue I As Seen In Swiss PDB Viewer 19/06/2010

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47 GENOME ANALYSIS STEP 1: SEQUENCE RETRIEVAL The gene whose function is unknown (ANGPTL7 in this case) was found and its nucleotide and protein sequence was retrieved from NCBI. STEP 2: BLAST BLAST(Basic Local Alignment Search Tool) was performed for both of them. STEP 3: ORF FIND The Open Reading Frames was found out in the gene by submitting the nucleotide sequence on RF finder server. STEP 4: SOPMA SECONDARY STRUCTURE PREDICTION The protein sequence was submitted on SOPMA server which generated all the secondary structure information of the protein. STEP 5 : PROSITE DOMAIN FINDER The protein sequence in its raw format was submitted on ExPASy Prosite server which generated the region of protein which formed the domain(functional region) of the protein. STEP 6 : CLUSTALW Clustal W was performed for the alignment of sequences (4 other species and one of the chosen AGPTNL7 Homo Sapien gene) generated by protein Blast having best similarity with the query sequence. STEP 7 : FUNCTION PREDICTION The function of the domain of the chosen gene could be predicted by looking out the function of the similar regions corresponding to the domain in other organisms by submitting the gene ID of those organisms on NCBI. STEP 8 : EPITOPE FINDER The epitopes present on the protein could be found out by submitting the protein sequence on B-Cell Epitope finder (BcPreds) server and these eptopes were then marked with a particular color on the protein generated by Geno3D(Hmology Modelling)

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48

CHAPTER 5: RESULT AND DISCUSSION

RESULT AND DISCUSSIONS

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4.1 DATABASE DEVELOPMENT RESULTS RESULT OF SQL SERVER MANAGEMENT

Fig 27 : Print Screen Of Database Generated Using SQL Server 5.0 Using SQL programming codes the data collected from various sources are segregated and clubbed together in to a database. The information collected was arranged in order. Their synonyms, gene symbol, functions, gene sequences, protein sequences, gene ID, accession number, uniprot ID, Kegg ID, Mass etc form the realm of the database. After the successful deposition of the data in SQL server it was successfully connected to database engine. RESULT FOR HTML WEB DESIGN HTML coding was used to create a web page. According to the groups various web pages were designed.

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50

Fig 28 : Print Screen Of Home Page Generated Using HTML Above is the resultant home page created for home page of our project. This page contains all the important links to the information of the project. Similarly various web pages were designed using HTML coding. Below is the HTML coding used to create home web page: <html> <head> <title>Glaucoma</title> </head> <body background="http://www.layoutstar.com/images/allbackgrounds/bgs/dark/dark_backgroun d_a2.gif"> <center><img src="http://macfarlaneoptometrist.com.au/blogs/media/blogs/Optometry/glaucoma.jpg"></ center></img>

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51 <marquee width="100%" hieght="0%"><font color="#EEE8AA" size="4"

style="verdana"><center><u>A group of disease that can steal sight without warning or symptoms </u></center></head></marquee> <p><font color="white" size="4"><center><b>Glaucoma is a disease of the major nerve of vision, called the optic nerve. The optic nerve receives light from the retina and transmits impulses to the brain that we perceive as vision. Glaucoma is characterized by a particular pattern of progressive damage to the optic nerve that generally begins with a subtle loss of side vision (peripheral vision). If glaucoma is not diagnosed and treated, it can progress to loss of central vision and blindness. </center></font><b></p></body> </html>

Fig 29 : Print Screen Of Introdcution Page generated Via HTML

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52

Fig 30 : Print Screen Of Types Page generated Via HTML

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53

Fig 31 : Print Screen Of Glaucom Genetics Page Generated Via HTML The nucleotide sequence for specific genes present in the database link is also provided on this page because they could not be put into the database due to non0-avaialability of any data type for entries containing more than 8000 characters.

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54

Fig 32 : Print Screen Of Diagnosis Generated Via HTML

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55

Fig 33 : Print Screen Of Treatment Page Generated Via HTML

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56

Fig 34 : Print Screen Of Drugs Information Page Generated Via HTML

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57

Fig 35 : Print Screen Of Drug Design Page Generated Via HTML Above result shows the drug design result lnks for glaucoma. It also contains links to web pages showing binding of various analogues to various residues as well as ETotal of the bindings.

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58

Fig 36 : Print Screen Of Genome Analysis As Generated Via HTML RESULT OF ASP CONNECTION Various web pages were created based on the specific information and they are connected to the database through ASP connection. The coding used to make connection link between browser and the database is shown on the next page.

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59

Fig 37 : Print Screen Of ASP Coding Done To Connect The Database To HTML Pages The above coding helps to select all the information contained in a database in SQL to be available on the web pages if All is selected. Particular gene information can also be obtained by selecting that particular gene from the drop down box.

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60

Fig 38 : Print Screen Of Local Host Showing The Glaucoma Data from The Database Thus Proving That The Connection Was Achieved Successfully The above web page is the result of ASP connection. Information about the genes responsible for glaucoma is retrieved from the database to the web site. All the unique information are also retrieved from the database.

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61

5.2 INSILCO DRUG DESIGNING RESULTS After comparative homology modeling and loop formation for optimizing the structure of CYP1B1 molecule, the resultant target which acted as a receptor for docking is as shown below:RESULT OF ACD CHEMSKETCH, MOLINSPIRATION AND MN CONVERT The following are the three analogues generated which act as ligand for the docking process.

Fig 39 : Print Screen Of Analogues As Viewed Under Swiss PDB Viewer RESULT OF DOCKING The following table shows the ETotal of all the dockings carried out. The analogue I bounded to Val378 proves out to be the best molecule which is having a potential to act as a drug for the treatment of glaucoma where CYP1B1 protein is not carrying out its normal monooxygenase activity.

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62 Pocket Residue ID Distance Analogue From I Pocket(Angst (ETotal) rom)

2.06 4.47 4.98 -137.81 -137.84 -153.96

Analogue II (ETotal)

Analogue III (ETotal)

144 144 144

His-22 Ser-377 Val-378

-131.85 -146.36 -152.37

-127.76 -140.07 -145.34

Table 1 : ETotal Of All The Dockings Performed The binding of analogue I to valine is also being produced below:-

Fig 40 : Print Screen Of The Binding between Analogue I to Val 378

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63 5.3 GENOME ANALYSIS RESULTS RESLT OF BLAST PERFORMED FOR NUCLEOTIDE AND PROTEIN :- BLAST was performed to find similarity of the unknown proteinss structure with those already present in the database. This similarity was further used o predict the function of the unknown gene. The BLAST results are produced below:PROTEIN

Fig 41 : Print Screen Of Performed Protein BLAST NUCLEOTIDE

Fig 42 : Print Screen Of Performed Nucleotide BLAST 19/06/2010 Glaucoma

64 RESULT OF ORF FINDER Open reading frames ere found out in the given gene by the help of ORF Finder- an application tool used in NCBI. The result is as shown below:A)

B)

Fig 43: Print Screen Of ORF Finder Results

RESULT OF SOPMA SECONDARY SRUCTURE FINDER SOPMA was used to find out the secondary structure present in a given protein. The number of alpha helices, 19/06/2010

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65 bete sheets etc could be easily found in the protein using this tool and the result is as shown:A)

B)

Fig 44 : Print Screen Of SOPMA Results The secondary structures present are as follows:Sequence length : 346 19/06/2010

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66 SOPMA : Alpha helix 310 helix Pi helix Beta bridge Beta turn Bend region Random coil Other states (Hh) : 130 is 37.57% (Gg) : (Ii) : (Bb) : 0 is 0.00% 0 is 0.00% 0 is 0.00%

Extended strand (Ee) : 77 is 22.25% (Tt) : 23 is 6.65% (Ss) : 0 is 0.00% 0 is 0.00% (Cc) : 116 is 33.53% : 0 is 0.00%

Ambigous states (?) :

RESULT OF ExPASy PROSITE This tool was used to search for the functional domain in the protein. The domain is involved in the functioning of protein. Our protein has just one domain and that lis between the amino acid residue number 122-343.

Fig 45 : Print Screen Of ExPASy Prosite Results RESULT OF CLUSTAL W This was performed to align sequences of best similarity as generated by BLAST and to later on predict the function using NCBI. The function which the protein may have came out to be it may be involved in blood clotting, being activated by thrombin to assemble into fibrin clots which is somewhere similar to fibrinogens. The cladogram generated could tell the evolutionary relationship amongst the organisms and the JalView gave the regions of 19/06/2010 Glaucoma

67 conserved and unconserved sequences. The yellow regions indicate conservation indicating that these regions have not changed with time during evolution. The brownish regions show changes in sequences over time.

Fig 46 : Print Screen Of CLUSTAL W Results Showing Similarity

Fig 47 : Print Screen Of CLUSTALW Showing Cladogram 19/06/2010 Glaucoma

68

Fig 48 : Print Screen Of Jal View Showing Conserved Regions

CHAPTER 6: SUMMARY AND CONCLUSION

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69

SUMMARY AND CONCLUSION

A. DATABASE DEVELOPMENT There are various databases which are very broad in their won respect such as whole about diseases, whole about diagnosis, about genes and genomes, but this created database is specific for glaucoma. All the information such as gene name, function, location, alternative names, Gene IDs, mRNA sequence etc. are clubbed together in to a single database. This database can serve as a remark of information about glaucoma. Complete information about the genes and proteins associated with glaucoma both ongoing and finished projects are uploaded in the database which has promising quantity and quality for the data retriever. This database is the unique database where all different information is retrieved in one single web page. This database definitely has the scope of additional information and updates and it also stands unique among the other databases. B. INSILICO DRUG DESIGN

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70 This was done to find out a molecule which can act as an effective drug for the treatment of glaucoma where the CYP1B1 gene was found to be defective. The first analogue i.e. (5,4dihydroxy-3-methoxystillbene) with the ETotal of -153.96 is the potential molecule which needs to be considered for further lab work to generate a drug which can finally enter the market. C. GENOME ANALYSIS The gene ANGPTL7 (Angiopoietion-related protein 7 precursor) after analysis was predicted to have role in blood clotting, being activated by thrombin to assemble into fibrin clots which is somewhere similar to fibrinogens.

CHAPTER 7: FUTURE PROSPECTS

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71

FUTURE PROSPECTS
The future prospect of my project is that the database thus generated can be constantly updated with any new informations discovered, by the help of the SQL queries. Database creation and management is not a single step process. It is an endless process. Each and everyday the information about the gene and the associated protein are updated. As a future work more number of genes can be added. Currently there are only 10 genes and it can be extended up to 100 or even more. Apart from mass storage this database can be linked to various sequences and structure database where the data retriever can download the structure of the causative proteins and enzymes for analysis from this database. Also this database can be connected to the laboratories from where the raw data can be constantly updated. The database can also be updated with the drugs already discovered and those under review so as to give the complete information about the disease, its causes and the cure.

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72 The ligand molecule having minimum ETotal can be studied further for carrying out the laboratory work on it so as it can be transformed into a stable drug which can easily hit the specified target into the body. ADME characteristics of this molecule need to be optimized so as the drug can be easily absorbed, metabolized and excreted without the formation of toxic products in the body. It should be tested for its side effects and if found to be inappropriate for administration, the side chains can be manipulated so as to reduce the same. Lastly, the unknown gene can be studied extensively on the basis of the generated results to check whether it has the same function as predicted in the project or is it involved in some other function in the body. The epitopes found should be studied as to what shape they have and what kind of antigens they can interact with.

CHAPTER 8: REFERENCES
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REFERENCES
1. Asrani S, Zeimer R, Wilensky J, Gieser D, Vitale S, Lindenmuth K. Large diurnal fluctuations in intraocular pressure are an independent risk factor in patients with glaucoma. J Glaucoma. 2000;9:13442. [PubMed] 2. Beltrame JF, Sasayama S, Maseri A. Racial heterogeneity in coronary artery vasomotor reactivity: differences between Japanese and Caucasian patients. J Am Coll Cardiol. 1999;33:144252. [PubMed] 3. Drance SM. The visual field of low tension glaucoma and shock-induced optic neuropathy. Arch Ophthalmol. 1977;95:135961. [PubMed] 4. Ferreira SM, Lerner SF, Brunzini R, Evelson PA, Llesuy SF. Oxidative stress markers in aqueous humor of glaucoma patients. Am J Ophthalmol. 2004;137:629. [PubMed] 5. Flammer J, Drance SM. Correlation between color vision scores and quantitative perimetry in suspected glaucoma. Arch Ophthalmol. 1984;102:389. [PubMed] 19/06/2010 Glaucoma

74 6. Flammer J, Eppler E, Niesel P. Quantitative perimetry in the glaucoma patient without local visual field defects. Graefes Arch Clin Exp Ophthalmol. 1982;219:924. [PubMed] 7. Flammer J, Prunte C. Ocular vasospasm. 1: Functional circulatory disorders in the visual system, a working hypothesis. Klin Monatsbl Augenheilkd. 1991;198:4112. [PubMed] 8. Flammer J, Orgul S. Optic nerve blood-flow abnormalities in glaucoma. Prog Retin Eye Res. 1998;17:26789. [PubMed] 9. Flammer J, Pache M, Resink T. Vasospasm, its role in the pathogenesis of diseases with particular reference to the eye. Prog Retin Eye Res. 2001;20:31949. [PubMed] 10. Flammer J. Glaucoma. A guide for patients. An introduction for care providers. A quick reference. 3rd revised edition. Bern, Hogrefe and Huber Publishers, 2006. 11. Flammer J. Glaucomatous optic neuropathy: a reperfusion injury. Klin Monatsbl Augenheilkd. 2001;218:2901. [PubMed] 12. Flammer J. Innovative glaucoma therapy. Ophthalmologe. 2001;98:9234. [PubMed] 13. Flammer J, Mozaffarieh M. What is the present pathogenetic concept of glaucomatous optic neuropathy? Surv Ophthalmol. 2007;52:S16273. [PubMed] 14. Flammer J, Orgul S, Costa VP, Orzalesi N, Krieglstein GK, Serra LM, Renard JP, Stefansson E. The impact of ocular blood flow in glaucoma. Prog Retin Eye Res. 2002;21:35993. [PubMed] 15. Freeman BA, Crapo JD. Biology of disease: free radicals and tissue injury. Lab Invest. 1982;47:41226. [PubMed] 16. Gasser P, Meienberg O. Finger microcirculation in classical migraine. A video-microscopic study of nailfold capillaries. Eur Neurol. 1991;31:16871. [PubMed] 17. Gasser P. Raynaud's syndrome: diagnosis and therapy in general practice. Schweiz Rundsch Med Prax. 1989;78:86873. [PubMed] 18. Gasser P, Stumpfig D, Schotzau A, Ackermann-Liebrich U, Flammer J. Body mass index in glaucoma. J Glaucoma. 1999;8:811. [PubMed] 19. Gherghel D, Griffiths HR, Hilton EJ, Cunliffe IA, Hosking SL. Systemic reduction in glutathione levels occurs in patients with primary open-angle glaucoma. Invest Ophthalmol Vis Sci. 2005;46:87783. [PubMed]

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