Lysosomal Storage Diseases …an overview for the non-scientist

Lysosomal Storage Diseases …an overview for the non-scientist Kelley Moremen

Overview of Lysosomal Storage Diseases …defects in degradation of carbohydrates • Result from a genetic alterations… • cause the creation of defective proteins (enzymes) that can not do their job in chewing up carbohydrates… • in a specific compartment in the cell (lysosomes). • Foundation for understanding lysosomal storage diseases: • GENETICS: how are defects inherited • BIOCHEMISTRY: how are normal cellular processes altered in disease. • CELL STRUCTURE: where do things happen

Liver cells blood cells onion root cells • Many different cell types in an organism • Different cell types of cells have different purposes. • They look different and they have different functions. • Different cell types can work together for a common purpose to form a tissue. • Collections of tissues can work together form organs. • A collection of organs are integrated for a common purpose: TO FORM A LIVING ORGANISM.

Cells have a variety of internal structures • Separation of function in different compartments • Nucleus:contains the genetic information for the organism • Mitochondria:generates cellular energy from breakdown of carbon compounds • Cytoskeleton:fibers that give cells shape and allow them to move • Secretory pathway (endoplasmic reticulum, Golgi, secretory vesicles):compartments that that allow the transport of products out of the cell • Endosomal/Lysosomal system:compartments that import materials into the cell, break them down, transport building blocks to the cytosol • Cytosol:space inside of the cell but not inside of any other organelle, where many chemical reactions in the cell occur. http://www.DennisKunkel.com/ http://www.biosci.uga.edu/almanac/bio_103/notes/may_15.html

Proteins and human genetic defects • Proteins are the molecular “machines” inside cells, required for cell growth and survival. They do most of the “work” inside cells. • Proteins create things and break things down. • Proteins are made from building blocks based on instructions from a “blueprint”. • Genetic diseases result from a defective blueprint -> produce defective proteins. • Logical progression: • Proteins do the “work” in the cell • Cells require that all of their proteins to do their “work” correctly for cells to function correctly • If cells don’t work correctly -> tissues and organs fail • Organs fail -> severe disease and possibly death to the organism

What are the “blueprints”, how can they be defective? • Blueprints (genes) stored in the form of DNA. • DNA is a linear molecule, built from linear collection of linked building blocks • Read in segments, just like reading sentences in a book. • Sentences (genes) have clear punctuation that define the beginning and end of the sentence. • If there are defects in sentences: • if the letters get out of order or • if a character is inserted or deleted • then the sentence may make no sense.

The human genome is HUGE!!! 255 characters/line x 160 lines = 40,800 character/page 40,800 characters/page x 3,200 pages = 130,560,000 characters/book 130,560,000 characters/book x 23 books = 3,002,880,000 characters Like a library in every cell in your body!! Every cell has the same library. AAGCTTGGTGATTCCATCTTGATAGATTGTGTTTCATTTGCCTTGTAAGCACCATGTAATTTATGTCTGGAGTTGAATTCTATCAGAGGAAATCTGAGCTCTGCAGGATTTGCTGTCGGGCTTCTCTTTGTTTAAGTTACCCCGTGAAGGCCTAGGATCTGTCTTCTGGTCGAGTCTGGTGTTTCCTTGGATCAGCTGCACACATGCTATAAATACAATGAATCTCCAAACTGATCTCACATAATTACAAAGAGGCAGTGGGCATTAGAGACTTCTAACAAACTTTCAAAATTTCTGAAATGGCCGTAGACTTCTATTTTCTATGATGATTTTTAAACTAGAAGATCACTGTCTCTAACAGAGCATAGGAAGATGGACAGGAAACCATATTATTCCCAAGGCCTTAGCTGATCTCTGGAGATGTGGAGAAAACTGAGACTGAGTGCCTACTGTGTGCTGCTAATGCTACTTCAAAGTATCACATATGTTATCTTATTTAACAAACTTGTACAGTGTTGGCATTCTTCTTTCCATTTTCCAAGATGGGACACTGAGGTACTAAGAAATTTGGTCATATGACATAATACAATGTATTATGTGTCAGAGACAGAATTTGAACCTGGATTTGTCTAAATCATAAGTCCAGACTCTTTCCACAAAATAACCACGAAATATTATATTAATTTAGTCAGTCTGTTCACATGGTATTTGAGATAATATTGAGCTATTCTGTTCAATAGAAAGTGATCAGAATAATTTACTGACTTTCTTAATATGCACAACCCTAATTGAGGACCAGGCAAGGGGAGAAAGTATACAGGACTGTTCTTGTGCCTGTGATTTCAATATACTCATACACCTTACAATGTAGTACGCTGAATTCAGGCAGCCGCCACAGGAAACTACCTGAAGCAGAGTTACATTGGCATTGGGAAAGTGTGGCCCATTCAGGTGAAGCCCATGTTCATTTTTCATGCTGCACCACACTGAAGTGGGATGGATTTCTTTCAGCCCCACCAGTCCTCACTATCACCTGCCGCTCCTATCAACTGTCCCTGTTTGCTCCCACTAGGACCAATTGCCCAGAGTGTGGGCTGCTTGATCTCATTATCAGAGGAAAGCAAACGGTAGTTGCAAAGGAAGAATGTGTCCTCCTTGTCCCCTTAAAGCCATGACAATCAATGGCAGGCATATTCTTCAATAACAAGCCCAAGGCCACACTGGCAGAACCTGAAGCTCTTATCAGGCCTGAGGCTGCTGCATCCTGCCAGAGAAGAGACTTGAAACAGCTCAGAAGAAAGTGCAAAGGATGGAGTGATTCATTAACTCACTCTTTCTCAGTAGCGACTGTGGCTCTGCTCTGCCTGGAATCTGAGGGCATTCAGTTTAGCATAAAACTGAACATCCTTTTAGGAGCTTCTGGGCACTTTAACATTCCAGCCATCTTTTATTAAAGTATTTATTTATGACATTTACAACGTGCCAGGTACTGTTTTAGGTACTTAATGGATATTCACCAAGGGGAAAATAAAAACAGTGAACAACCACTAAAAGGCATTCATGAAAATAATCAAAGTAATATACATTTGCGTTTGTGCTACAATTGTGACTATATAAGATATGTATGCACCAGGCTCAAGAATGCAAGAGCAGATACGCAACGGAAAATAATTATATTGGTATCCAGAAAGACAGTCGATTTTTTTCAACTTTTCAACAATTTTCCTTGATGTTGATTTTATGTTGATTTTATAATTTAAAGAAAACAAACAAAAATAGTAGTCAAATTAGGCTCTCCAGGTAACTTGTTTGAACTGTATGTTGTGAGGGTCTCTGATTTTTTTATGTAGTGGGGTGTAATTTTTTTTCTAATTTTAATTAGGAACTTGGATTATCAGAGTATTGGAACTTAGTGTCATTTGGAATAACAGCTGGGCATGATTGTAGTAATAAATATATAAAGAAGAGGAAAATCTAGCTTCTAAAAGAAGCCTCTATTGAATTTTGATGTTCATTTGTTATATTGTCAAATTGTAATTTCTAATTATGGGGGGGGATAAGTGGATACTGTAGCCAAGAATTACAAAATAGGGCTATGCATTGAAGTATATTCTCATATACTCTTGTCTAACTGAATTTATTATTTGAGACATTGTTGTAGAAACCATCCCAGGTTGCCCAAAATTTATGTTAATGTATTGTTTCTGAAGAGCCTTTATGCATATGTTAGTTATGCTATTTAACCAGTCAAACATTATGTGTTATTGGCAAATAGATGATGATAAACTACTATATTTGAGTTTTGTGCTTTCTGTCCTTTACCATCATCTTTTCATCTGTGTGAATAGCAGCGGATATAGGCTCATCCTTGAGGCTAAGTGCCATCTCATCTAGGTTGTTGGTTTTTTTGTGTCATTTATTTAACAAGCAAAGATCTTGTACTGTTTTATTTATATATTTTGTATTAATGAAAGAATTAATATATTATCACCACCAACTCATTTGACCAATTTCTCATTATGAATTTGCAACATTTCTGACTATTAAATGCCTTCAAGCATTACATAAACAATTTCCAATTGAGCAGGTCTTCAGGAACGTTTATGTAGTACCAAACATCACTTTTAATGTTTTTTAGTTAAGTGTGTAGAGGTTTTTGTGCTATCCCTGAAAACAATCTGGTGGTTCATTTTACTGCATCTAGAAAGAATTCAGTCTCATACTATGGTTAAAAAAATAAATAAAATGCACAGAATGAGAAGCATTTCCACGAGTAATTTCACACATGCTGCTAAGTACTGCAGAGTATTGCCAAGCCTAGCCTTTAATCTCTATGGGAAGCCATACAGCTGTAAACTCTTATTTGCTGTCACATGTCTTTTGCAAGGTTATTAGAAAAGATAGGAGACCAGAAGTCCTTCCATGTTACCTTACTTTATTTTTTGACCAGGTTTTATTACATTATCCCTTTATATCTGTTGATAGCTCAACATGTGCATATTTCACCAGCCTCAGGCAAAATTATGAAGTAGGATTCATGAGGCTTTGGTGTTCTTCCCACTCTGCCTTCTGGGTCCTCGCCTGCATTCTCTCCCCGGAAGTTTCTCACTTTCTGAACCCAGCCCCTGCTCTCTGTCCAATCTACTTATTGACAATTTTAAGGGTGTATTTTCGGCCAAATGCAGACCAGCGTGGTCCATTCATGGATCACCGTGTTTTGTCTCCCAGGTAAGAATCTCATGTGATAAAACTTGGGCTTGCTGAAGTGGTCTATCTCTTGGAGTGGGGATGCATACTTCTAGATCTTCAGCAGGCTCTAAATTTGCTTCAGCCTGTCTTCTTGCAAGACTCAACAGATGTGCTTTGGAGTATTACTGAATTCCTTTGCCTTGACTTCTGTCACTCTGGGCTAAAACTCAAACTGCCGTGCAGTTTCTCAGCTTTGATGTTCCCCTCCTAGTACTGGAAACCGTTCCTGAAATCCTTTAGAACTTGTAAGAAATAATAATTTCCACTGTTAACTATGTGAGGAGGGTAGGGTGAGTGGGGTAGGGGATGGTTTCAGCAGTGTGCTGAGCCTGGAGAACTGATTGTTAAATTTTCAGGAATTTTGTGAGCCAATTCCCATCACCTTGATAGCTTACAATCAGCCACAGGTGGGAGAATTTACACCACGAACTGGCAAACTAGCTGTCCCCCACCTCTAGCCAGTGTAAGAAGAGCCAAGATTGGCATGAAAAGCAGTGTTACTGCTTCCTCTCCACACCATGCTCTCAGTGTGTCTAGCTGGCCTGCATCCATATTTCTCACTCTTGGACCCCTCCTTGCTTCGCACATTAAGCATTCATTAAGAATTTATTGAATGAATGAATCAGTTTGAGACAGAGGATTTTTTTTACACAGTGTGTGCTAGTTGTTCTGATCTTTGCCAAAATGTCAAAAAGTCAAGGCCTCTTCCCTCCTTGCTCCTATTTGCCTATTTAGAAGTTCCCATGGTTAATTGCCTCCTCGCCTACGACTTCTATCGTTCTTTTATTCCTGGAACTAGAGTTCACTATCCACTGTATTGCCCCCTTGCTCACTCATATCCTATAGGATATATTCTAATAAGGTTAGGAAGAAAGAATAAAGGAATTTTAGCTTTTCAGAAAACCTGGTTTCTAGTCTCAATTCAGCCACATGATATCCTTGTGACCTCAGAGAACACACGGCCTCTCAGATCCACAGTGCCTTCATGTGTAAGTGAAAATGTTGTTACTGCTATGCTCACCTCAGGTGGGCTCCCGAGGATCAAAGGAGTTGGTGCATGTGACAGGGCTTTCTAGACTATAAAGTGCTCCACAAATGTTAGTTCTTGTTAAAAGTAGACAGACGCAATGAAGTATCATTGACTTCAGAAACAACAAGAGTTAGTTGAAATCTACCTCTATGATTGTAAGCAAAACTCACCTTTCCTGAATTTCTGTTTCTTCATCCTCAAAACAGAAATAATACCCACTAACACTCAGGGTTGTCAGAGTTCAGTGACACAATGTCAACAAAGGGCCTGACTAGCACAGTTTCCCAGTAACCTCTTCTCTTCTCTGATTTCCTGGCTCAAATGTCTTTGGGATGATATTATCATCATTGTTATTGGTCCATCCTTAAGTTTCCCCTGTCCTCAAGAAAGTATATTTATATATTATTATTGGTTATGTGCAGGCTCTGGAGTAGGACTATCTGGCTTTAATCCTGGCTCTGCACTTGCTAATGCTGTGTTCTTGAGCAAGTCACTGTGCTTCTCTTGGCCTCTGTTATCTTTTATAGGGAACAGTACCTCCCTGAAGTGACAAACCATCCCAGTTTGTCTGTGACAGGACTTTCAGTGCTGAAATCAGGAAAGTCCTAAGCAAACAGGGATGAGCTGGTGACCCTATTACCTACCTCACAGGTTTTTTAACGAGGATGAAAGGAGTTATAAATGTGAAGTGCTTAGAACAACACCTGCAGTTAGTATTACATCTCACTATTATTTCTCCAGAAACAGTCCTTTGGTCCAAAAATTGAATAGTGGGTTTAAGGGCACAGGGACAAATCACAGCAGAGACAGAGAGGCCTCCTCAGAATCACTCATCCAAGCCCAGGCACAGCCACCTCCCCGGGGCCTGCTGCTGACACTGTTCTGAATCATCCTCTGCTTTCCAGGGTCATACGCAGAGGGGAACCACCGCAACTGCCATCCTTGTCTACAGCTACGACTGTCCTTCTGGTCACTTTGAACCTGCTGCCCGAAGGGAAGAGAGGGGATGAAGAAAAAAAAAAACCTACCTCATTGTTTGATACTTCTTGTGAGGATTGACGAAGCCCTAGAGGGTGAGGGGAGGTGAGGCCAGGGTTGAGCTTCAAATCCCTGACTTCCCTATCCTGCCTCTGAACAGGCTCCAGTGGGGGCCATGAAGTATATTTCTGATGCTGTATATTGCACATGATTGAGGGAACTTGACATATTAATTCTGTGCTTCTCCCTGGGTCTGAAATAGCACTGCCTGGAGTCCAGTCCTGCATCTGCCTCTGCTAGGTAGGTGACCTTGGAATTGTTATTTAACTTCTCAGTTCTTTCATCTGTAACACAGGGAATAGTAAAAACAGGACCAACACCATAAAAGTGTTGTGTTATATGAGATAATTCATGTCAGGAAATTTTCTCAGAGCCTGGCCTAGTATGGCAAACTAGGTGAAACTATAGTAAATTATATATAGGCACTGAGGGGAAATGAATATGTGTTTTGTTGGTATGCCTACATTTCCAAATATTAACATCTTCCTTTTAGATAACTTACATATAAAATTTATTCACTCATTTTTACCATCCCTTTGAAATTAGAATGTTAAAATTATGACAATATTTTTCCAGGCCTAATGTTGAAATGGAAATCCTGTGTGGTTTCTATGCTTTGTTAAGAGTTTGCTCATTGTCAATAACCAATAAGAATTTAAAATCTGAGCTTTCAAAAACTGGTTCCAGAAATTAGGTTTTAGTGTGTCATCAGTTCTACCAGAAAGAATGCCAGGAATAAACATCAGACAATAAAGTTATATCTTGCATGTTTAATTGCTTGCATGTTTACCAAAATCACTCTTTTCATAACAATGAAATTCTTGCTCTTCTCCCTTATGAGTGTTTCCAAAGCAGTTTTAATTGTGACAAGGCAATCAAACAATGTCCTAAGTTCCTCGTTCTATTTTCCCTACCTTAATGCTTTTCTGGGTTTGATTCCTTCATACTCCCTGGTATATTTCCATGCAGATGATCTTATGTTTGGAAGCACAAAACAAGGCAAGGCTTTTGCCTTTAGACATGCTGTTGCTGCCAGAATCCCCTGTGAGCTCCCTTTTCCATTGTTTCCAAGCATAATAACAGGATCACCCAAGGGTCTTTATTAAAGCATTGGGGCCAAGATTGTACCACCAAAAACAGCCCTCTGGACTCACTTTTTTATACAATCTGGCTCCTCTATAACAAGCTTTCAGGTTATGTCGGTGATATGACTCACAAAGGGAAAGAATCTTCTGGCCTCCTGATGCACTGTCTTCCCAGAGTTAACTGATGTGCAGTGTGAATCCAGTGTTTCAGCATACATAACATAAAATGAAAAGGATTATATATAGTGAGCATTGCCAAACTGTACTGTCTGAGTAAGTGCAGGCCAACATACCCTTTAAAGGTAATTTGAACAATTATTTCCATTTCTGCTACATCTCCGAATTTCCCCTATCCTTATAGAATTCAGCCTGCAAATTGAAGGTCAAAGAATGTTCATCTCTGAATTTTATGGCATCTAAGTTTAAAAAACTGCACCCGTTCATCAAACTTTAGACCAAAAATCTATGTGTACTACCCTGTGTAAATTAAACCTCATTTTAGAAATGGGGTGAGGTGTTGATGGGGAGGAATGCCCAAAGATCAAGTAATTCAGAACCTCTTGGTGGTGGGGCCTGGATATGGGTATATTTTCAAAACTCTCCAGGTGACTCTAATACACAATTGGCAGGAATACCCTATGATAAAGGAAAATTGAGAGTGGTTTAGGTTCAGGGTTTGGGCACTCCTGCAATATCTGGACGTATCTATGTCTCCTAGCACTGAACTCCTACCATCAACCTCAATTAGTATGCTACAGAAACTTCAGTAATGCCATTGCCTTTTAAGCTGGTTGCCACCCTTCTGACCCTTTCCATCTAGACCATCTAGAAATTTTATTAGGTATGCTTTTGTGAAGGGTATTTATGAAGTGGCAATGACCCCAGACTCTCTCCTCCTTTCATGGCCAGAATAAACATTGGCCTTACTATAAAAAGAAATGCATGAAAAAACTGAGCTTACTCAGGGAGTAAAATTATTATTTGAATTACAGCAAAACAAGTTTGACTCTTTTTCTTGCCTTGCTTCTCTTTTCGTGTCGCCATGGGAGCACTTCCACATGACACATTTAGACCTAAGAAGGAACTGCCAGTTTCACACAAGAAGGAATGAACATGTTCACCGCAAACCACAGGAAGCATGACTGGCAGCTTCCTGGGCCAACCCAGAAATGTGGCTTGCACCAGGAATGATGGAATAGATTGTATCTCTTCCTTCTGTCATGCTTTTGTACTCTACTGGCAGAATTTTTTTTTGAAAAATGACATTCTTTATCCCATGTCCTTGATCAGAATGTTCCAGTAGAACTGCCGGTTGCAGAAAAAACTGTAATCTTCACAGTTCACCACTAAATGGTCTTACTCCTCGAGCCCAAAGCTACTAGTTCTAGCCTGATTTTCCACTACTTGTCTGCACTATTTCTTGCTGTTCCTCAAGTGAGTTCTTCTCTCTCCCACCCCACTTCTTTCCCTGTGCCTTATTCCTGGAATACCTTCTCTCCAAACCTCACTTCTCTTTCCAGCCTTGGTGTCCCCGACCACATCAGCATAGGCCTTCTCCTTTCCTGAAAGTTTGAGCCTGTATTCCTGCCAGACTGAGCATGAGCTTTGGACTCAGAGAGATCCACTTTAAAATCCTATGACCTTCAGGAAGTTCTTTCATTTTTTATCTTTAAAAAAAGGGGATGATAATAATACTCCTGGGGGTGGATTGCTGTAAGAATTATATGAGATCACATGTGAATGCACTTAGATGATCAATACATATTTGCCTTCCCTTTACCACTCAAATAGCATTGCTTTCTACTGTGGGTTTTTGAAAGCACATCTTACTGTCTCTATCAAAGCCTAGGGTCCTTGAAAGCAAGGGTTTTTTTTTTTTTTAAAAAATAACTTCTTTGAATACTCTCTAACACTAAGAAGAGTGCCTTGCATGTAGAAAGTACTCCACAAATATTTTTGATGGCTGAATAAATAAATAAATGAATTTTCTGGCTCTCTGTTGGATAAAAGATAATGGCATGGAGGGGGATATGATGGAGAGAGCCAGTTTACTTAAAATATAGTGCCTTGACAAAATGTTGTAACAATTAGCATCAATGATAATTATGGAAAACATATTAAATAACTTATCCGGTTATATACTAACCTATTTAGGCTTCCTGGGCAAAAAGAAATGTTGAATGTTCCATACAACAGCAAAGAGGTACTCAGGATCACACAAAAATAAATTCATTGACATTTGATGTTCTTATGCTTACAATGTAAAGAATAAAATGGGTTCTTGTCGTTTCCTTTTTAAAAACTTCCCTTTAATAAACTACCCACGACTGTGTGGTCCAGATGGTTGTTTGGTATATCAGTGTGAAATAATGCTTTACATTCTGTGAGAATAGAGAGGAAGCACACAAAGGGATGCCTTATGGACAATCCAGGCAGAGATAGTGCCTCTCTCGCAATAGGGACACACTTTTTTTTTTTTTTTAAAGATTCTGTATATAGAAAGGTTATTGCTTCATTATGAGGTGAACTGTTTGACAAAGAGATGATGCCAGAGAGTTCATATTCCTTAGGAGGAGACATCTTAGTCCTCTCTCCAGAGAGAAAGAAAAAAGAATTAGATGCGATGTTTTTGAGTATTTACTGCCAATGCTGCTAAAAAAGCACTCATAGTCTTATTAGATGACACAAGAATAGACTCAGTTTTATATTGCAAGGTAGAAATGGGTCATTTATTGATGTTTAAGATCCATCAAGACTTGCAGGTAAATTGAGACTCTTGACAGATGACCTTGAAACAAAACAGTGCAATATGATATTTCTCTACCACCAGAAAAAAAGACATTTTTGACAGCTGAGAAATAGTGGCCAGATCTTGAAGTCATTTTTATCCCTCTTAGAGGAATTTGAAATATAAAACTTAGGACAGAGGTCACATCTTTGCTAGGCTCTTCAAGAGCTTAAAGTAATATCAAGATCCCACAGATAAACAATGACTTCTAATCATTATGTTTTTAAGGAAAAATATGTGATAGGATTTAGTGAGAAGTAAATTTATACCATGTTTTAACAAATGCCTCATCATTTTATATGTTTACTCAGTTGTGCCTACATTTTCATATCTAATACTGATGGAAAAATATAGGTTAATGTAAAAGTAGTTGATGTAAATGCATGATAATAGTACCATGGTTATCTCCTCACCTAGTAGTCTTTTGGGTAATAAGTATGACCAACATTAAACAATGCTACAGTTTATTAGAACCTTCAGAAATTTTAAAATCTGGAGTATTCTTTCAGGTCTCATAAAACTATTTCAAAAGGGAGAAAATAACTTTTATATTAATAGTATATTAGAGCTAATGGACAATCAATTACACATTTTTTTTTTCTTCTAGTGGATTAGATTTGCAGCAGGAAGGAAATTTGAAAGGAAAGAAAAGAATAAAATTCTAGGACCTATCACTCTCACCAAGATACAAAACATTTACCAAGAATCAACTGATGTAATGTTATTGTTACCCTCTTTACCAAATACGGATACTAATGCTAGAGATTTTTTCCCAGTTTCTTTCTAATCTAAGTCTGACTCTAAAACATAAATGAAGAAAAATATATCACAATGTGTTATTTTACTCTCTTACTGGAGGAAGGCTTTTGCTGACACCCTATCTGAAATGGATACCTTTTGCAGCATAAGCTGTGCTTCTGAACTTTTGTCTCATGATGTGCAAGCATAACAGACATTCAAAAGACTGAGGATACAAAAATGGGGATTCATAGTTTAATGTATGCCCTTTTTCATATTTTTCTTCAATCACACACTGTCATTTTGAACCACATTGCTTTCCTAATCAATGATAGTGTAGATGGAAGAATTTTCAAAAAATGTTATACCTAAAACTGATAAAAAGGTATCAGTCCATGTTTCTCCCTAATTATCCTAAATCAACACTGTTTTCTAGAAGTTTTCATTTCTTTATTTTTTTCCTTTTAATCTCCAAGGTACTCTAATTGCTTCCATATTATAAAGATTGTTAATATTGTCATGACAACTAAGGAAACTTTGCCACCAGAAGTTATTCAAAATACAATGAAATACTTACTGTGGGGTTTTCTAGTGTTGACTGAGATTATAAAAATATGCTTTGTGCTTCCCACCTCAATCTAGATCAAAGTGTTTTGGACTAATTTGTCTGCTTACATCAGGAAAAAGTGTTTGCTTTTAATCATTGTGTGGGTTTGGTTGAATATATTTTAGCTGTAGTAATGATACACTGAAGGAAAGAATGTTTAAGGCAATAAAACCTAATTACTTTTCATGGATAGGAAAGGTAGTGAACATTCAATACGATAAACATGCAGTCTGTCTTAAGGGATATGGAGCTTCAGAAACATATACCAAGATTTTAAAAAATGTGCAAGGCTAAACCATCAAATCTAGGTTTGCAAGCTTGGATAATAAAACTATAAAGCAAAGCAAGATGGAATAATTATAAAGATCAGGTGTCTGGCTGCTTTTCTGGGAAGGGAATGGCTGTGGTTGAGACAGTTCGCATGAGAGGCTTTTGAGGTGACCTAGGAGGCGGTTAAAAGGACATTTGTCTTATAATAAATCGTTAAGCTATGCACTCATCTAGCCTGGTCTATCTGCATTTATTTTACAATGAAGTGATTTTTAAAATGTTAAGGCTACGCAGCTTCAGATTCTGAGCATAACTCTGTAGGCTATTTATAATCTGGAGGAGGTTTCTTGGACTTTAAGCAATGCCTCTGCCTTCGTATGTCTCAATATTGTACTGACAGGGTCAGAGATCAGAGAAAGATTAAGAACTGCCATAAAGACAAAGACAAAAGAATTTGAAAGTAAAATTTCAATTTAAAATTGTTTTAAAATTATTATTAAAATATTTGATGTAGGCTGGGCGCAGTGGCTCACGCCTCTAATCCCAGCACTTTGGAAGGCTGAGGTGGGTGGATCATGAGGTCAGAAGATCCAGACCATCCTGGCTAACACGGTGAAACCCCGTCTCTACTAAAAATACAAAAAATTAGCTGGGCGCGGTGGCAGGCGCCTGTGGTCCCAGCTACTCGGGAGGCTGAGGCAGGAGAATGGCGTGAACCTGGGAGGCAGTGAGCCGAGATCACGCCACTGCACTCTAGCCTGGGCGACAGAGCAAAACTCTGTCTCAAAAAAAAAAAAAAAATTTTGATGTAATAATTATACTTTTTCCTAATAGACACATGCTGAAGTATTTATAAATGAAATGAAATAATATAGTATCTGAGCTTTGCTTCAATAATAATTTAGGTATATACACAATGTATGTACACAATGGAATACTGTCCAGCCTTATAAAAGAAGGAAATCCTGTCATTTGCAACAACATGGATGAACCTGAAGGACATCATGTTAAGAGAAACAAGCCAGGCACAGAAAGGCAAGTGCTGCATGACCTTACCTACATGTGAAATCCTAAAAAATTGAACTTACAAGTTGAGAGTAGAATTATGGTTACCAAGGGTGGGCGAGGAGCGCACTGGGGAGAAAGTCAAGAGATACAAAATTAAAAAAAAAAGAATTGAGCTTATAGTTGAAACCATGTTGATAACTGGTGAAGCTGCATTATAGGTTCATGAGAGTTCATTATATGATGCTCTCTACTTTTGTATATGTTTGAACACTTCCATAATAAAATAGTTTTAAAAGTAAAGCCTAAAGTATACAGTAGGAACATACATATTTTAAAAGTTGCCTTAGGATACTATAGTCTACATGGGGTTTGTGGCGTTTCAATCAATTTTATATACTTGAATATGACTATTTGTAAAAATTCTAACTGAACGATCTTAGAGATTAGTCCTTCATTTGGGACTAGTTCAATTACAAATTAGTGGCTTAATATAATTACAATAAAATAATCCTAAAACTGAAATAAAATAAATCTAAGTGGATGAGCACATGGGCGTGTTTGCAGTTCCTGGCATTCAGAGAATCTTTGCATTATTATTAACCCACAATGAGGAAAGGAGACACAAGGAAAGACACAATTAAAATGGAGTAGAGAATTGGATTAATTTCTTAGGTAACTCACGGGAAAATAAAATACAGTGATTGTTTCTAGAGAAGCATTGTAATTTTTGCTTTTTGTCTTCTCAAGATATATCTGTACCATGGAAAGTGAAGTAGTTTGTTTATTCTCACATATCATCATTTATTTAACATAAAATGTTGAAGCTATATATAAATAAATGTGCTGTAATTCACGATCTTATGTCCTTCCTGTTAAAATCTTGTTCATTCTTGAGCTGAGCTAACTCCTTAATGTAAGAAAATATCTGTCATCTATCTCCACATAGTGTGTGTATGTGTCTGCATATACATATTTTTTTCTAAACAATTTGAGATTACACATCATGCTCTTTACCCCCTAAATATTTTAATGTATATCTCATAGTAATAGACACTTTTATATACATAACTGCAATATGATGTTCAACTTCAGTAAATATAACAATCAATTTTATCCTAGTCTACCATCTAGATGTCTGTTTTATCCATTAACACAATAACTAACATCTTTACAGCATTTTTTCTTGCAGTATGTGGGGGTGGAGTTCAGTCTAGGATCAAATATTGCATTGAATTATTAACCTTTCATTAAAATTTTTTCACAGGTTTTCTTTGTCTTCCGTGATATTGATGTTTTGAAGTACACAAAAATATACAATATTCCTTATTTGGAGTTATTTGACATTTCCTCATGATTTAACTCAAATTATAAACTCCTGGCTAGAATATCACATAAGCAGCATTGTGTCTTTTTCAATGTATCACATCTGGAAGCAAATGATTTCCATTTGTTCTTCATTAGCGATATTAATTTTGATGACACAGTGAAGCTGTTACACTATTTGTTCACTGTATAATTATTTTTTTCCTTGTAACGAATAAGAAACCCAAGGATTACCTGAAAAACATCCTGCTCCCCATCAAAATTTCCCCTATGTTTAGCGTTCATTGGTGACTTTTGCCTGAGTCAATCTTTGCAAATGAATAATTATTCAACTCCAGCCTTTCTTCTACATTTGCCACTTGACACAATAGTCTACTGTAAGCAAGAGGCCTCACTTTTTACTTGTGGATTTATCTATTAATATTTATTATTAGTATGAATGCATGGACTTCAATCTTATAATTTGGTTTATAATTCATTACTATACTCACTTATTTTGTCATTAAAAATTTTCCAGATTTGGCCAGTATCAGCTCCTTCAAGCTGGTCTCCATCTTTTTTTTTTTCTAGTACTTTATTTTTGTTGTAACAAAACTTTGTAGGCTCATCTTGTACCTATCCCACTCCAATCTTGTAATCAATCATTTCTCTGAGAAGCCCTAGTTCCTTTTAGGGAGAAATATTAGATACGAAAATAGGCATGATAAGTGTGCTAATTGCTACTGTGGTGGCTTTATTTCTAGGTGCTTTCAGTGGCCAGAGCTAGGAAATATGTGCATGTAGGTAGACACAGTCAGGCACACCTCTACATATACATATACATATACATGGGTACTTATATATGCCTATACACATATATGCATATTTTAGAAGTCATAAATTCACACTGATCCTTTTCATTTTAATTCATTCCCATAGAATTGTTTATTGTCTTTTTCAGTTCTATATTTGTAAGTTCCTTCTTCCACAGTGAGAACTCTGGATCTCAACAACATCAGCACATTTATTCATTTGCTTAATTCTGTAATGCACCTAAAATGATCCCCAAATTGCTCTGCCCATATCACTACAAAATAAATAAATAAACCTACTAAACAGAATTCAGGATTTGTTTTTAGTATTTATTCCATTACATCCCCAACGAAGACTGACAATACATAGTCAAATACCGTGTTTATAAATTACTTGAATTAGTTCTTTTGCCTGCTTCAGTTTAGCTATGGCATACATTTGAATTAAAATTGCAGTTTAATTTTTGTTTCAGTTTTCCCCTTCTCATCTTGGTTGATTTAATTTTATTTTTGATTAGTAAAACATTAACATGCTTCCAAAAGTTAAAACTATGTGAAGAGTATATCTGTATCCCTTCTGTATCACTTCCACCCTATTTTCTGATCCCATTGTAGGTAACCAACTTGATTGCCTTCATAGTCTTGATAATTATTAAACTGTGTGACCTCTCTGAGCTCTTTTTTTAAACAGAGTTATGATAATTTGGAAAATATGTGCAGTTTGGTTGGCACGGTGCTTGACATATACTAGGTTGCCAATAAGTGGTTATAAGAATCATCATTGGTAATAATATTTACCAGTAAACACACAAAATCTTTTATATAAGGAAGAGATCTTTTTCTTATGCCAGTGTGGAACTTGGCAGAAAAGGACATGAGAGAAGATTCAAATGGGACCAGCTCAAGGCTTCCTTTGGTGAAGGATTTGGGGGTTATGTTTAGAACCATACTATATGATATTTGTAGGTCTAATACCTATTTATGATCCTGCATATTCATTCCCCAGTGTGGATTCAGGCATTCTTATTTTCTTCTTTCCATTGTTATAACAAAGTTAGGCATTTTGATAATCCCCTGTGGTTTTCAACATGAATGCTGAAAGGAGTGAAACAGTTGACACATCTGATGATAACAGACACAGTTTGGCCCAGTGCGGGTAAAGCAATGGAAGAGCTTCTCTCACCCTGAAGCCTGTCTATCCTTTTGCACGTGTCACATGTTAAATGACCATTTGACATGCCACAAAAAATGTATATGTTCATTTGAAGTGTTTCTTTACCTTGTAAGAGTATGGCTGCAGCAGCTATTCCAGTGAAAAGAATGTCTACATTTAGATGTCGATATTTTAGGTGGGTCATGAAATTAAATTTCTCTTCAGGAGTGAGCATATGTTGATAGTGCATGGCAGAGCTGTGTGTGCGTGAGTGTGGTTAGGAAGAAGGAAAAGAGGCCTTCTGTCTTTTAGATGCATATTCATAGGGAAACTCATAAAGTTCTTTGAGAAAAGTTCCATTTATATATTTGATTCTTGATAATTAAAGCAAATCGACTGAAAGGGAACCATTCGCTTCATAGTTTTGTTTGTAGTTTAATGCTGCTAACACATGGCAACTTATGTGTTTAATGTATTGTTTAGCATTTATATTCTGGTTGTTTTATTTTAGAGTCATACGACATTTCTTCTTATTGAAAATTATCTCTTTTTTTGGACTAAATAATGATTTCACTTTCTTAAGCCTTCATTTTTTTCTTTGATATATTTGATATTTGCCATTCACTGCATTTACAGATTTTGTATTAATTTTTTTTTTCGGTTTTAAAATAGTTTGTATTATGGTAATTCATGCTCATGGTTAAAAACCTCAAAGACATGAAAGTATATAAGCTGAAAAGCACAAATATACTCCTTACCATGGTCTTACTCCTAGAGATTGTCACTGTTCATAATTTCTTGTGTATCCTTCCAGAAATAATTATACATATATGTATTTTCCTTTTATTAATTTAATTTTTTTTTTAGAGACAGAGTTTTACACTGTTGCTCCAGCTAGAGTATAGTAGCATGATCATAGCTCACTGTAACCTCGAACTTCTGTGCTCAAGGAATCCTCCTGCTTCAGCCTCCCAAGTAGCTAGGACTACAGGTACATGCCAGCACATTCAGCTAATTATTATTATTTTTTTTTTCAGAGCTGGGATTTTGCTTTGTTACCCAGGCTGGTCTCGAACTCCTGGCCTCAAGTCATCCTCCCCCCTTGGCCTCTCAAAGTGCTGGGATTATAGGCCTGAGCCACCATGGTTGGCCTGTATTTTTCTTTTAAAAAAGAGTATCAAACTATGCATATGATACAATAGTATAACTAGAATTTTAACATTTAATACCTATCTTGAATATCTTTCCATATCTGTGAGATGTATATATATATATACTAATTATTCATGTGATTCCCATAACATTTTAAAATACATTTGTGTGTGTCTGTGTTATTTTAACAGCTGATTTGTATTCCATTGCATTGGAATTATAATTTTTAGTGTTCAATTTATGAAGATTTAAGATAGTTTAAGGGGCATTTTTTGCAAGATTACTTTTAATAAAAACAATTGACATTAATTAAATTGTCCCTCCTGTTAGTGAATAATGGACATCAACTGACCAGAAGAAAGACTTAACATTCACCAATACTCAACCTATGCCACCCTCTGACCCAACAATAATTCACATGCTGCTTCTCTAGCAGTGTTAGGTAATCATCTAAGGCAAGTAAAATACATTTACTCTCTCATTTCTGGGTGACAGTTTGCTAAGATCTAAGAAGGTTAATCATTTTGATGATCAATTGTACCATTTCTTTTTGATGAGAATGAATGCTTCTCAGAAAGACATGGATCAGTAAGATCTCAGTGTAGCCTGTGTACATCGTAATTGTTCATGTGATTTCCATAAGAAATGCAATAGCAGCAAGACCATTCAAAGTAAAAAAGAGGATTCGAAAATTATCTTACAAAATGTGCAAAAAGCTTCCCATTTATTTATTTCTAGGGAGTTTTTGTTGTTATTTTGTTTGGGCATTTAACAATTTTTTAAAAATTGTCATTGTATTCTTCCCATTCTGTTAATGGGTAGATTTCCCTTTTTTGGTTGACACTTCCTATTTTGTGATACCTATCAACATTAAAGTAATAGTAAATACAAATGATTTTAACTTTAAGTTTTTGAGCTTCTTTTTCTTTTCTTTCATTTCCTTAAGGCCTCAATTTCATAAATAGTCCTTTTTTCATATTTTTATTGTCTCTTTATTTCTGTTTATTTCTAAGACATCACTATTGCTGATATCCCATGATGCACAATATGTTACATGAACCACTTTTATTCTTCTTCTTTTAATAGTATTATTCTTACCTTTACCTTTTCTGTAACATAAATAATTCCTAATGCCTCTTACAGTGGGCCCCGCTAGTATACATAAATTGGTCAGCCCTTCTTTAGTCACTTCTATATTTTACTACCAGCCCTGAAATAGGCGTTACCTTCTGTTTCTTAACTATTCCTCTAATTCTGTAGCAGTATGTGAAAATCCTTACACTGTGAAAAAAAAAAGACATGCTAGCAAAACAAAAAACAATATTTTATTTAGATTGACTGTAAATTTTTCAAGGTGTCCATTCTCACCAGCTAGAGTGTAGTGGTGTGTTCCTGTTTCCTTAGGATAGGGCCTGATACTTAGTAGGCAGTCAACACTTGCTCATCAGGGGACTATTTGCTTTTTGGATGAATCCAAACTGTTCTAAGAGCAACTGCAAAATTATGGTTGAGTATAGACAATGTTGATAAAGAATATAGAAAAATAGTGGTTGATTTGTTAGGTGGGGGAGTAGTAAGTACTTTACTGCCATTTTTGTCTTTTGGTATTGTTATAGTGCTGATTGAAAAATAAAAAGTTCTGTTAGAGCTTGTCTGTTACAGGTCCATGCCTATGGATGACTGAAGTCAGCATGCTTCCTCCGTTTTACTATTGGCTGATCACATTGTAGCCTTCCTCTGACTTACAAAATTTTTTTGATATAGTGAAATTGAACTAAAGTGAATATCCAGATTAAGATTAAGAACAATGATGAAAATAAAAGCCAATATTGCATATTCACTCATGGGCCAAGTACAATATTTAGCACTTTGCTTGTATTACTTCATTTAATCCTCCCAACATCGTTAGGAGGTAGGATTATTATTGTCTCTATTTAGAGATGAGGAAGGACAGATTCTGAGAGGCTAAATAACTTATTTAGAAAAACACAACATCTTGAGTCTGTCTCCAAAGTTCTTGAAAGTAACTGTAACAGAGCCTGAGATAGTGGGTTAGAACTGAGGGCAGGCAGGCACATCTACTTTGCTCCAACAGGGATTAATGGTTTGACTGACCACTATATATTTTTTTTCAGAGATGGTGGCTGTAGCCTCTGCTTTGTTTACCCAAGACTCAGACAAGTCAGAATATAACTGAAGAACGAATAGGTGTACCCATCAGGAAAATTATTTGCATAAAAGACCAAGGTACTATCTGGGGAGATGAAAAATAAGGAAAACAACAAAAAATAACAAAAATCACATTTCACTCACAGGAATGAGAGAGTGAAGACTACAGATTTGATCTGGCTAATCACTCAGGATCACAGGAAAGGTCTGTTCAGATTCACAACAGTCCCTGAGTCGAAGGTCCTGGTGCTATGAGCAAGAAATGATGGGGCTCACTCATGCCATTTCTCTAAGACTTTGGGAACACTGCTTTTAAGACGCATAGCAACAGACCCCAGGTGGACTAAAGAATAGAGCGTGGGGAGAGGAATTCTTTACACTGCAGCCTCAAGAGTCATGGTCACAGAAAGCCAAAACAGGTGAGGAAAAAAAAAAAAGATTGGTTGAAGTCACAGGGTATCTGGGATGGATCTGCTCATGGGGTTCACGTGAGATAGCCCTAGTGTTCACTATTCAGAGCTACCATGTTTCCTTCCTGGCCTTCTGACTGCTTCCCTGAGTCATGCCGCACAGGATAGGGGGTCAGCCCTACCGGAATTCACTATACCCGAGGTACAGCAACATTTTCTTGTTTTTGTTATTAAATATTGTTGTTCCAAGAAATGGTGAAGTCTTCAAGGGCAAAGACCATATTATTTTTCATATGTCTGGTAGGGCTTAGCACAATGTTGGGTATAGAGCAAGAAGTTGCTAATTCTTCTTTAACCTGATTTGACATTAATTTGGTTCTTATTGTTAGCCATTCAAAACTACAAGGTTGCAAAAAGGTATAAATGAAAAAAAAATCCCTCCTTCGTGGCTATCACCAAGGACAACATTGCTTGGAAAGGACTGAAACATTTTATATCTCTACCAGCCATTTCCCTGGGAAGCAAAGCCAAGCCTGTAATGCTGATTAACAATGTTAATGTTCTCCTGTAGGCGGACAGGCAACTCAGAGGCTTGCTTCTCACCTTCCCCCTGTTCCTTAGATAGAATGACTTAACTCCTCTAATTTTTTAGAGAGAGAATGAGACAGAGAGAGACACACACACAGAAAGAGACTTATTTACAGTTATGCAGTCTCTACTTGGTATAACACATATCCTACAGGAATTTAGAAAGCAGTGAACTTTAAACTGCAACAACACAAATACCTCGTCTACCATATCAGAATAAATTAGGAACCAAGAACTGAGAAGGGAACTGATGCTTTAGGCCATTCAAGGGTCTTCTTGCCAAAGCCAATGAGGTTTCAGTATCAGAAAAAGCTGCAGCCCTTAGATTTCAGTGACTGTGGAAAGAGATAAAGTACCACAACACACCTGGCAGATTATAGTTTTTCAGCTCAGTTGTTTACTTACATTTGACCGTATACTTATTTGACTCAGAGACTCTGTTACTCTCACACATTTTACTGCCAGACCTTACACATTGCCAGGATTTCCCTGATGATGACTGGATATTTGTGAGTTTGTTCATCCTTTGACAGAATTTAAATGGTGGGATGATTTGTGGTTACAACTAAATTTGATAAAGTGTGTTTATCCATGGCATTAAGGAGTGAGCACTGGTCTAGAGGTCAATACATTTTAGCCTAATTCCATTCTGACCTTTGGCAAGAACCTCACCTCTCTGGGCCCCGTTTCCTCACGGGGAAATTAGCAGGTTACACTAGCTTGTTTAGAGGATCTTTGCCAACTCTAATATGCTGTGCTATTGTTTATAAGATGAGGTGTAACAGAGAGATGTCCAAACGAAATCAAGAAGACATGACTTGAAGTCCCTGCATTTGCCATTTACCAACTCATTAAATCTCTCTGAGCCTTGGTTTCTTTCATCTGTTAAGGTTCATTCCAGTTCTAAATTCTACGATTTTATGACATTGCTTATTGAGTCTGGAAATATCACAAACATAAGGTCTCAGAAATTACCTTTAAGATTTTCGTACTGCACAGATTGTACCACATTAACAACCCAATTTGATCGTCAACTGCCAGCATTTTTGTACAAATGGTTTATCTGCACAACTGTGCAGGGGCGAGAAGATGCGTATTTTGCAAATACCATTTAGTAGTGGTGGTTTTACTTTCCCCATCATAACTGAGTTTTAAATCACCATTAATATTTAGCATCAATTACCACCACACGCTTTTGTTTTCATGAGGTTAAACACCATGTCAAGTTGATAAATTCTATATTTTTAACACTGTGGATTGCCTGTTCTCTAGGATGGTGCTATTGGCAAGTAGCCAGACAAACACAGATTGGTGCCTTCATACAATTGGCCCTAAGAAAATTGCATGCCTGTTGATGTGCTTTTGGAGTCTGCATCTTGCAGTAGGTTCCACTTTAGGGTTTAAACAATTCAGATCCTTCCTCCTCCAGAAAATAAATTGTCTCTTTTTTCTTGTATCAGCTTGAGTCTGGCTAGGCTGATGTCTGAGATGTGCACTGAGCAAAGAATGGCTGTTTTTAAAGGAATATTGGTAAAATGCAGAGGGAGCTTACATTGTTTTAGGGTGTGACTTTGTGCTCATTATTTTCCATTCTATCTTACTGGCTTTCCTCTGAAAGCCAGAAACAGAAGCATTGGCTGCCAAAGATGGTTTTAATCCTTGTCTGGCAGCTGCCAAAGTCAAAGAAGGAGGGAGAGATGGAAAGTAACATTTATTGAGCATCTCTTGTATTGCCAGATGGTTTAACTCATTCAGTGCTCATGAGGTTACATCAGACATGACAGAATGCAGGGACCTGGACAATGCACTCAGTACTCTGATTTTCTCCATCTCTCAGTTCTGCTTTTTCCTACTTGATTTCTTCTCAACAGGCTCTCTCCATAGGGTGGCAAGGCCACCACCAGCAAATCTACTCTTACATTCACCAAGTTTAGTAAGCCCAGAGATGGAGAACCTTCCCATGCCTGTATTAGCAACCCCAAGGAAGATTCACATTGGCCTTGCTGCATCACATGTTGATGATGGAATCAATCACTCTGCCAGGTCAGGCAAGCCTGTTCAGGAGTCAGGGTATGAGGCACAGACGGGGGGAGCCCCTTTGTGGAGTGGAGGAGGAGTTTGTTCAAGGAACTAAAGGAAGAGGGTGGCAACCCTTCTTAGACAAAAATTCTTACCATACTCCACGGCAATGGACTACTTAAGAATACTGTTGGTTTGAGTTGTCAGTTTGAATGTCTTCTCTGGTAGAACTGAATCAAGAACCCAAAAATAAAGTTAAGGGTAGTTTACCAAGTCTCATTGGACATATACATTAACATATATTTGTCAGACAAGCTACATAAATCTGTCTTTTTTTTCATCTTTCAATGACTCTTGCTGTGCAGTAGTGGCAGTTAGGTTTCTCTTGGATATGTATGCAGTCATCTAGATTTAAAGGCATATGGTCTGCCTTGGATCACTTACAGATTGCAAGATGGCTCTGAAGCCAAACTTGCTGCTTATTTCATGCCCTGCACATTTAATTACTGCTTGATGTGAATGAAGTGTTTTATCTGAAGATATGCGCCACTGAAATTTTAGCTAGGTAGAATCGTAAAACAGTTTTCAGCAGATTGAGGCAATCTTAGAGGGAACTTTGTAAAGAAAGTTTGCTTGAAGTAGCTCACCGTAGGTCACAAATGTTCTGAGTTAGTTTGATCATTGCTAGATAGGACAACGAGTACAAGTGTTCCTAGTTTCCTAATTTCCTGGAAAACTCTAGGGATAAGACAAAATTCTTCTACTGAGGTTAAACAAATGGTAAATTGTTGATCATTATTCAGGTGACTTCTGGAAGAAAGGTAGGGCATTTTCCAAAGGCACTTGTTTCTTTTGATTACACTTACCTGGTGCAAACAAATTTCTCTGCAATACAAGTTTTTTTAGTTTTTCTCTTTGGCTTACCTGCTATGTCTTCATGTCAGTTAAATCAATTCTCTAGCTGGTCACGACAGCTAGTACTAGCCAGGGCTAGGGCAGTAACAATAACCTAGAAGCCTGTATAGCCTGGTATTTAAGCACATAGACAAAGAGCTCAAAAGTTCAGGTTGCTCTATCTCATGGGTTTCTGTAGCAATTTCCAATTATGCAAATGTGAATGTCGGGAAATAGGTAGGCTTGTGGTGAGGATTGAACTAAATAATGCTAGTAAATTACCCAGCATATGTCCTTTATCCAAGGTGACTGCTGTGTTGTAATCAATCCAAGGCTACTGTCCATGCACGTTCTTCACACTGAAGCATGTGGGATGGTGGGAGGCTATAGTAGTTATTAGCCACATCTGGCTCCAAGTGATCCTTTGTTTTTTCCTTCCCTACTGCCTTCCCTTCTGTGCTAAGTAACTTGGATAAAATGTTAAAACAATGATCAAAACTCTAATCTCAAGAGATGGAATGGATGAAGAAAGTAATTTTACTGACTTCTTTAAATTCTTCCAAAGCATGGCATGCTCTCTCACCTTTCTGAGTCTTCCTACAGACTCTTCCTTAATTCAGAAACACTTTCCCTCTTTTTTGTTAATGAGGCTTCTCACAGTCAAGGCCCTAGTTTACATTTTCCCTCTCCAGAAAGCCTTGCCTCACCTCTGCCCCAAAGTCGGGGTTAGGTGTCCCATAAAAAACTAGTAGGTTTTTTATGCCTCCTACTAGTTGAACCTCTCTTACCAAAGGAGTGTTCTCACTGGAGCCTGGCTTGGCAGGACTATGAGCTCCAAAAGGGGTGGAAATCATGCATACCTCATTGATCCTTATTGATCATTGTTGATCATCACCCCCAGCACCTAGCACCATGCCTGGCTCAGAGCAGGCACTCAATAAATATCCTTCCTTGGAAATATATGTGAGATCCTTTTCTGAGGGGCTCAAGGATCATTTGCTTTGTTGTATTTACTCAGTTATCTGTTTCCTAGAACAACAATGCTGTTATGGTGGAAATGTCACATAGCTCTGCTGGCAGTAGAGTAGCCACTAGCCATGTGGCACTGTTGAGTGGTTGGAATGTGGCTAGTACAACTGAGGGGTTACATTAATTTAATTTAATTTAATAACCATATGTGGCACTAGTGGCTGTCATATCAGCCAGCACAGTTCAAGGGCCAGAGGTTTAATTTTTTTTTTCAGGAGCCATAACTCTAGGAACTTTCGTGTTTTCAAGGAAATAGAATTGCCACAAAATCCAGAAGTTTGTCAGTAATACTCATGTAGTTAATGTTTGGAACTGGGTTCCCAAATGACACGTATATCATGTCACTTGTTGGGTGATGCTGAGCATCTCAGTTTTAGGCAATGGAGGGCAGTGCCTTGATTAGCATGTCTGCTCTGACACCTTCTGGTATGTGCCCTTAACATTTTGTGTCTCAGTTTTTCAAGTATAAAATGGAGCTGATAATAGTTTGTATCTCAAATAATTGTGTTAAATGGGAGTGTGTGTGTGTGTGTGTGTGTGTGTGTCTATGTGTGTGTGATAAAGTACTTAGTACAGTGCCTGGTATGTAGTATGCTCTCAAACATGCTAATTGCCATGATTAATCATTCCATTCTTCCATGTTTCTTCATCAGCATGGCATTTGGATACATGTGCATACATCCCAGTATACCCAGTAAATATGCTTCACAAGTTTTGGTCAGGCAAAATGAAGGTGAATGTGAATGTAAAGTACTATGAAAAGGTCAACAGATAGGTGTAATAGACTATTAAACTGGACCATTATTAGGGAAGGTAACTCATTTTACAAAGATAAAGTATCTTGAAATGTTATTTTTAAAAGGCAACCATATTTGAAATGGGAAGTAGAGTTCCTGCTTGCAGGAAGGCTTTGCTGCACACATTTCTATACCTCTTTTCTCTTTGTGTCACCTGTCCAGGATGCTCTTTTGCCATCCTTTCAATAGGCAAAATGTTTAGGGGGCAGCTGCCCTGTGCCAAGCACCACAGGGACTGATATGGGCTGAAGCAAGGAGTCTAAAATCAACCTTTGAATTTTACTATTCAAATCAATATGCTGTACAAAGTGTGTGTTCTTTGTTGCTCCAAAGCCCCATCAGGAAACTGTGCGAGCCAGGGAGGGAGATTGGGAGACGGTTCCCAGAATCAGTAGCTGATTTGCAAGCCTGAGGAGTTTGCATGAAAGGCAGCAGTAAAAGTCAGTCATCAGCTCTGATATATATAGTGAGAGGGAGGAAAGAAAGAAGAGAAAGCTGTGAAATGTTATTAGGAAGTCAGGTAAATAGAGAGCTACTCAATATAACTCATACCTTACTACCTGTTTTTCATTTTTTTTAATGTGTGATGTGTATGTGGGATCTGTTTTTAGAAATGGAAGCAAGAAAGTATCCTCTCTCTGATCAATGGTATAAAGTAAGTTACTATTTTTTGAAATACAAAATTGAAATGATCTAAACAAATTCAGGCAGATGAGATCTAAAAAGCAATATGAGGTTTTCTCTTCCCTTTTTGTATGTCAGAAATTGGGGAATCATACTGCCTATTCCCAATACAAAGTGTTGCATATTTTGCAAGTTTAGATACATTTTTAAAGTATTTTATAAAATGTATTTGTATAGTGAGAAGATAGAAGAAATAAAGCAATTCTCACATGATATTAGAAGAGTATCTAATGGTTTGCATATAAGTAACAATACAATATCGTACAAACAGAAAGCATTGATTTTATTGTGAATACAAATAAAGAAATAGCAGTAGCAGTGAGGAACAAACACTGAAAACTATTGGAAATACTGAAATGCCACAGAATTAGGGAGTTTTAATGTCCCCTGATGAATTGTCACTATTGTAGATTCAGTATTATTTGGCTATGTAGCTTGTTTCTGTAACTCTGATTTATTATTATCCTGGGAAGATTAGATCATGATGCTTGGAATTCTAATTGACCCTCAAGCTCGGAGATTAATTCCTATACAACCTAGCATCTGGGAGGTAGGACAAGCGTTTGGGGGGAGAATAGCTCAAATATGCAGTAAAGTGAAAAACATATCTACGCATACCCCTTCTTTAAAAGTCTTCTTGGGTGTTTGTAAAAGCGTAAGTCCTAGCACTAAAACTACCCCTTGGATTAGAATTAGGACGCTTTAGGAGGGCTTATTAAAGCATTCATCTTTGTGAGGCCACTGTGTTTCTACTGCACTGAGTTCACTAAACCAGAAGCAATCACAGAATATGAGAGACTGAACAGCAGCTCTCCTCTCCCTCCCCTTATTTTTTAGGTTAGAAAACTGGAGTCCCAGAGATTGATGGCCTTGCCCAGGCTGCACACTGGTCAGAAGTAAGGCTGATTCTAAAGTGGCACCTTTCAAGCCTCATCCAGTGCTCCTCACCACAGCATGCTCCCTGGCAGTCCATGGTACCTAGCACCTAATCCAATGCCTGGCACATAGTAGGCTCACAATAAATGTCTATGGACTGACTGAATGGCCTAAGAGCCTTACTATTTTTATAAATGATTTTAAAAAAGCACCAAACAAATCTGGTTTTAGGAGAGGCTAGCCAGTTGTTAACAAGGTCATTATGAACACGAAGAGAAGTTACTGAATAACATAAACAGAAGGAAAGAATTCAGCTGTAGCCTATACTAAAAACGCTGAAACTTATGGTAAGGGGCTGAAAAAGCTGGTAAAACTGTGACTGTGTCAAAAGGGATAAAACATCCTTGGCTATGATGGAAAAAATGTGGTGTGAAATTCCTTTGCTTTCTGCTGCTATTTAAACAGCAGGTGAATGTTCAGAATGCTTCTTTTAGAGAAAACAGTGAAATTTTGACATCAAAAGGTCTTGAGATAACCATAGTAATGAGAATGACCACTGGGGGAAGAGCATCCCTTTTTGAAAGTACTGATTTAGGATACAGCTATTTTGAGCATTTTTATTCAAAAAAATGGAAATTTTGCTTACTTTCCATTTATCTTGTTCTTAGTCTGCAAAGCAGAGGCATGATTGATCTTAAAATGTCAATCTCTCACCTAAAGAGAGGAAGAAAAGATTTATTTAAAGAATAAAATGTCATAGGGGTTCTTAGGGGAAAAGCTCCGGGCTTGGATTTTCTCTTGACAGCCAGGCTTCTAGGTGAGCTACTCTCCCTTTCCCTCCATCTCATAGTAGCTCCCCAGATAGCTCTGAGCGAGACATCCAGGCTGGCAAATGTTCCCTTGTTTTAGTTTCCTTTTAGGCAGCTCATCTACCTGAGCACTGGTTAGTTTCAGTAAAGAGTTCTTGATACTGAAAGAAAAAAGTCCTCAGTATTTTGACTTTGAGGAGTTGATAAAATCATGCTCCTGGAGACACTCATCCCAATGAAAATGTGTCCACTAGGCTGTTGTCTTTGGATTAGTCCTGGAGGATTTGGGAGGCAGCTGAATTCCACGATTTGGAGAACTTACCTCAGTACAGTTTAATGTATAAGTAGGTACAGAAAATATGAACGGTTTATCAGAAGGTCCCAAGAACTCTCAGCGTCTAGATGCAGTCTGCACACTGCAGGTTTAATAAACGCACTATGCAGTATTTTCAGCATCTACAGCAGGGCCCACCTGGCACGTAATTAGGCATTTGAGAATCATTTGTTAAAATAATTCAGTGTTAAAATAAGAGAAAATCTCCCACTGGCTAAGGTATGAGCATAAATGTATATTGTTTATGAAAGTTTTAGCTGTTATTTTATGACCCTGTGAATTAAAAAACAAAACAAATAAAACATGAATTTAAAGACGCCTTGAGGTGTTGTGTAAATTGGTCCCCTGTTTGAAAAAGAATCTTACTCAAATCACCCTAGATAAATGGGATCTATTCTAAAGATCTTCAGATGGAGAATAGCTGTATATAAAAGTCCACTGACTGGTGCCTTGGTTTTCTTCGTATAAGGGTGCATAATCCTTGTTCTTCAGGTTTTCTGTGCTTTTCTATGGTGGGCAGGATTTATGGGGTTTGTTGCCAATGCTTTGGACTAGCTGGAGGGACCGCTCATCCTCCTGACAAATAAGGTTAAAAACTCAACCTCTATCTTCACAGTCTTTACAGAGGAGCAAAAGGCAGAGAGGAGATTCCCCATAGTCGGAGAAAACTGGACTAAAGAAGAATCACTCTACGTAAGCGAATCTGTGGTCTTCTCTCTCACTCAGAGACCTCAGGGGAGCATGAGAAATGAGTCAGTCTCTCTGAGGACCCACCAAGCTGCCAGAGCTAAACAAGAGTCTTGTTTGAACTCTGAAGGGGAGAAGGGACAGAAGGCATGGATGAGAACACAGCTGCAGTTGACTTCCGTGAAACCATCTGGAGAGCATGGCCCAAGAAAGAGAAAAGGGCACGGGGACCAAATTAAGAATGTTCTTAGTTAATTTTCTAAAGAATGAGTCCACTGAAAAACCACCCACTCCTGGGTTCCTGATAATGCAAACTTCATTCATTTGTTTATTCATTCAACAAATGGAGTAGCTCCGACCAATATTATGGATTGGCTTGGAAAATACCTCTGGCTTCTTGGCAGAGAATCAAGCAAGAAACTTACATGTGATTTTGGTCTATTCTAAATAACTTCTCTAGGTTACATACTTACACGCTGATGGTTCACACATAAGGAGGACAAAAGGAATGCTACCAAAACAAAAATCACCCCTCAAAATTATTTAAAAGGCAGAAGATTTTAAATTGAAGAAATATCTTTAGCATCAAGTCAGGGTATGAATGTACCAGAAAGGCTTTGTTTTAGCAAATTTACTTCCCAAAGGGCAGAATAATATATATGAAATGAAAAATGCACAGGCCTTTTGTTCTTTAATGACCTTGGGGGAATATTCTAATTTATCAGGAATCTGCCAAGAAAACAGTTTACCTCTTCAGTTGTCTCATTTTGTGTTTCTGAATTTTTTTTTTTTTTTTTTTTTTAGCCAGGGTCTCAATCTGTTGTCCAGGATGGAGTGCAGTGGTGCAATCATGGCTCACTGTAGCCTCGACCTCCTGGGATCAAGTGATCCTCCCACTATCAGCCTCCTGAGTTGCTGGGACTACAGCTATGTGCCACCATACCCAGCAAATTTTTGTATTTTTTGTAGAGACGAGGTCTCACCATGTTGCCCAGGCTGGTCTTGAACTCCTGAGCTCAAGTAATTTGCCCATTTCAGCCTCTCAAAGTCTGGGATTATAGGTGTGAGCCACTGTGCCCTGCCCATGTTTCTGAATTTTTAATACATTTTATTAATTCTTGCTGATCTGTGAAACACTAAGAAAAGTAAATTGTTTTATAATTTTGTCTCTTAATATCCTCCAAAAAATTAATTCTATTGCTTTTTCTTTGCATACTTCTTGGATACTATTTTCCTCCCTTTGTGTCAGTGACTTATCACAGGAAGAAAAAAAAAATGTTCCCCATCCATTCCTTACTTGGTATCTATAGGGCTGTAAGCGAAGACATTGGCTTTATCTACAAACTGCTGCAGTCAGATTGTTATGGAAATAGGAATTAATTATTGTAGAAGCAGTATAATGATCTTCTTTGGAATCAGAGAATTAAAACCAGGATTCAAAGCAATACCCGTAAGGGAAAGTGGTTGGGGTTTATATCGGAATCATGGGGGTATATTATATTAAGGTTAAAGAGGGAGGATGAGATGGAAAGACAGAAAATCTCGCCACAGCATGTGGAAGTTTCTCCTTAGGAGAGCCTTGTGGCTCATTGTGCCTGTCAAGTAGGCTAATGTGCCTGTAAATTTCCTCTGACAGAAAACACATCACAGCAGCAGCACCAGAAAACAACACAACCAAACTGTGGAAGACTGAAAACCCAGTGCTTTCAAGGGTGATCAGAATATGCCAGAGTAAAAAACAAAAACAAAAACCAAAAAAAAAGCAAGGCATTGAAAATAAACAAAAGCTAGGGAGAAATGAGATCACCTTCCAAGAAATAAGCATTTATCATTCATTCATAGGCGAAAGGAAATGCTTCTCCCACAGTCATTTTTTAATGTATTATTGGGCAGTCTCAGTTCAAGGTCAAAAATGTCAGAGGGCAAAAGGAATAGGACTCAAGATTTAATGCTGTATTTATCATAACTGGCTTTGTAATTGCATAAATATTAAATATGTAGAACTAATTGCAAGCACATTATCTCCATATCTCCACACATTGCTTTAATTTTTAATGTAATCTTTTATCTGCCTTTTCCTCAGCTGGCTGTGGGTTTGTAATTTGCATAGCTAGAATGATCCGGTGCCATCAATTTAGTAATGAAGTCACTCATTGAGCAGAAGAGTTTTCTAAAGATTATACAAAGCTGTAGGAAAGAAAAGAACAGAAATGAAATTTTATGTTTCAAATAAATGGGAAGTTTATTTTAGAACATATTTTCCATTGTAAATTTAATGCAACCACTTCATAACAAATTTGGAAAACAGAGAAGATTGGGAAAAAGCACCCATATTTCATCATCCTAACTTATCAGTGTTTTGTGGTCTTTTATACCTAGGCACATATATTTTTAACATCGATGTAATCATAATTATGTGTGTGTATATGTGTGTAATTTTACATCCTATTTTTTGTATTATTACTATGGTTTTCATAACTATAACTTTTATGACTATAAAATACTAATAAAAAGAGGTGTCTATAATTTTCTCACTTACTAAATTTCTTGTGGCTAGCCGAGCAGAGGGGTGGAATCAGATGGATTAAATTTCAGCTCTGCTATTGAACTGTGTGGATTTGGGCAAGTTAGTAAAAAGCTGGGCGTTAGTCTTCTCATCTACAAAATGAGGATAGTGATAGTGCTTTTATTAGAGTGTTCTTGTTAGAGTTAAAGGAAGTAATCCAGCATTTAGCCTGGCTCTGTCATATAATAGGTACTAAAACTAAGTATTAGTAAAGGCAAAAATGTCAAATACATTACATTGGGTTTCAAAACTTAGGAAAAAAAACTTAAGTCCTACAAAACTGAATTTCCTGGGATATTTCCATCACCCCCTTTAGAATTTTTTTAAAAATTTCTATTTAAAGATGGGGTCTTGCTCTGTTTCTCAAGCTGGACTCAAATTTTTGGGCTCCAGTGATTCTCCTGCCTCAGCCTCCCATGCAGCTGGGACTATAGGCATGCATCAGTGAGCCCTGCTTGCCCTCACCCTTGAAGTTCCCATGTGCCCTTTCCCAGTCACAACTCCTTTCTTCCACCGCGAACTAGACATTGATGCTTAGTGCTTTGTTGCTTTCCTTTATGCTTTACATGTAAGTATGCTTCTCTAAAGATACAGCTTATTCCCTGTTATTGAACTTGATATAAGTGGAGTCACACACACACACATGCACACACACACACATCATTCTGTATTTAGATAGCATGCAATCTAAGGCTTGGTTTTGTGATGAAAAAGCTTTAGACGTGGAACCAGAGAACTGGGCTTCTAGTCTAGTCTCCTTGAGACCTCGGGCAAATCTTTTTACCTTTCTCCATGTAAAATGTTTGGATGTTCTAAGTGTCTTTGAAGCTTTAGAGTACAGAGTTGTTAAGCCTTTCCTATTTGAGTATTGTGTGGCATATGAAAATCAGAGTCAAAGAGAATCCATAGAAAGGCCTAGTCTGGCTGGAAATAGACCTCCTCTTCCCCCAAATTAGGCCAATTGTAGTGACAGCAAACAAAGGCTTGTAGAAAAATGAAAGTGATGATCCTACATCACAAATAATTTTCTCATCTCTGATAAGTGCACATTTAGACATATTATTAGCCCTAATTATACTGTACATAGAGGATATTTTCCCCAGGATAAATCATAGCCATAAAGATTGTAATTTCAAGAGTTCAGAGTTCAAATAAGTAATCATATATTAGATAATATAGGTTTTTCTGGAAAGAGACAGTTTTAAACATTTTATTCCATTCTCTCTGTAGATGTATTTGTATTTCTACAAATACTGCCTATGGTAAGTACTGCAGGCTGGCTCACTCACTGTCCATTCTCCTCTCTTCTTATGGTGGCTTTTGTACTTCAGAGGCTGGAAAATTAAAACATTTCCTAGACTTCTTTGCACGTAGAGCTATGATATGAATTAGGTCTACAATTCGATACACTCATGCAAAACTTAAGAGAAAAGAAGTTAAGGCATCCTCCTGACCACTTTTGGTGGCATTTCTCTTAGAATTAAGGTCATAGAAACATTTTTCTGCAGCAGTGTTCCATGGTCCATTATTCAGCCTCCTGGAAGACAATATGCAGCAGAGGCCGTTGACAGCAGTAGGTTTTTGACTCCATCTTCTGTCATAGGGTTATGTTCTTGAACTCAGTGTTCCAGTAGTCTTCAGAGGTACTAGCTTCACTGATAGGTCACTTGGTATTATACGGGGAATCACTTTTGGAAGCTTGGTCTACAGCCCATTTCTTCAATCCTGACATGGATTTTTGTAAGCATCCTCTATTAAATAACTTCTTGCTTAAAATACCTAGAATGATTACTAATTTTTACACTTAATCTGAGCAATACACTTCCTAAATGTTATTTTCAAAAATCTATTCGTTGTAATACGAGGTATAAAGTATCATAATTCTATAGAATGCTGCATCAAAAGAGGAGTTTTGAAACCAGAAAAACGAAGCTATACCATTTAGTAGCTAACTATTCTAAAGCAAGTTATTTAACTTCTCTGTATCTAAGTTTCTTTATATGTAAAATAGATGTTAATATAATAGTCTTGCAGAGTTATGATAATTAAATGAGGCAACATAAATAAAGCACTGAGCATAGATGCATTATATGGTGTTCATTTCTTTCTATCCCTATATTGGACTTCCTTTCGTGATCGAGATGATTAGTAAAATAATTTGGGGGAGAAATTATCTTTAAAGCGTTTTTTATTAAAGAAACGCTCACTTTATAGGAATATAAAGATTTAGTGCTGACTCACAAAGACAAAAGGAATAGCTTTAAATGCAGCTCAAAAGCAAACAGACATCTATTACGAGCCAGGGCAACTCATAATGGGCACCACCAATCCACATCAACTGGTAGCTCAGGACCAAGATTAGTATTCACTCATTCAACAAATATTTATTAGGCCTCAATATTGCTAGACACTATTTTACTCACTAAGATACAATGTTGAATCACAAGGTCTCTGACTTGTAGCAGCTTGCATTCTATTGGGAGGATAGACATAAAATAAGTCAATACATAATTTCAAATAGTGAGAAGGGTTATAAAGAATAAAGTCCTTCTCCAGCTCTCCTTCCCTTACCTTCTTATGTCTCTCTCAGCAGTGTATTATGCTACTTGACTCATCCTCTCTTGCAGCTTCCTTTCATGGGAAGAAAGGCACAGGGAGGTAAAGGCTATGCCAACAATATGAAGAGTCTCTGAGGTAGGTGGGTAATTTCAAGATCTGTAAAAGGTCGAGGGGTCACTACTGTTTCGTGGATGTCAGTGGAAAACACAAGACACCCTGATTAGAGACAAAGGAAGATGTTAAAGAGACAGGAAGATGTTGAGCCTGATGTCGGTGCTAGTTTCTCATGTCCCCCAGATCCCACAGGGTCTGAGGGAAAGGGTCCAGGTGGATGCAACATACACAGTGGATTTGTCACAGCAAAGGAATATTGAGCTTGAGGAATTTACTGCTTTACAGTGTGTGGTAAGTCAGTCTGCTCATTGTTTGGGAGAAGACATTACTCTGTTGCTCAAGGCTGCTCCCTGCAAACACAACCCTGAGAGGACACAAGTAAATAATGGTCAGGGCCTTGCATTCTTAGCATACCCAGAAAGAATGTGTAAGGATGCTCAGGGCTTTGACAGACTGATTCTCACAATAGGTTGAGTGGAGAAAAGGATTAAACCCGCATTCATTTATAGTTTTATGCAACCACAATTATAAAATTTTCTCAAGTTTTTAGGCTTTAGAACTTTGTCCTACTCCTTTCCAATTTGTCCCCCTTAGATATGATCTATTTAGGCCTTTGAGGAGCTCAGACAGACTTAGAGTATCAGTTGACAGAAAAGATAGATTTTCCTATTGTTAGCAAATATAAGCTGAGGCTCTTGTGCACATACAGACTTATCACCAGAAGCATGGGGCTAGACCTCATGCCTTCCAGAAACCTCACCCCAAGCACTCCTTCGAGACCTTTCCTATTCAAAATTACATTGAGTGCTCACTTTAACTTAATCTTCACTGCAAAAATGTATATCAGTGGGTAGGAAAAATGACAGATATCTATGATATGCTCCCAACCTGGAGCATTCCTTGGGTGGCCGTTGTTCTTGCATCCTGGTAGAAGGGGTTCTTCCTGGATTATGCTCACAGTACACTTTCATTGAGCTCTCAAGCCATAGATCGATGACAAGGGAGAAAATGGTGAACTTGCTCCATGAGAAATCAATGCTCTCCAATTATCCACATGGACTTCCTTCTCCATGTCCCATAGCAGCTGTTTCCAATGTTCTCTCCCCTACTCACCTCTCAATTGCACCCATCTTTCCCTTTCCCAGAGTAAATGGAAGTATCTGGTGAATGCTCTCCCAATATGTCACTACCTGTGGCAATCATACAGCAAGGCTGAAAGATCACTGTTACTACCCCAATATAATATGTTGGAAAATGGAGGCTTAGAGATATTATGTAACTTTCCCAAAGTCACAAAAGCTTTATGTACTGGAGAAGCAATCAAATTTGAACCTTTATGATATGGGTTCTGCCCTCATGTACCTTCCCTTCCCTCTATTTTCAGTGTTCCTACAAAAAGCTCTAGCCTCCTGAGCCCCAGAAACATGTACACTATAGAGAATGTTTGCTTTCACTTTTGCTAGAAAATTTTCTCATAGCATAAGGATTAAATCTGTAGCAATTTGTTTCCCTAATGACAGAGATAATTGCTCTGATCCAAAGGGAGGATAAAATTTCTCATATCTTTCCCAAATGAAAAGATTGTATCCCAGGATAGGCACCTAGGTTTGTTCTGAAGGTCCTGGGGAAACCAGAATGTCTGTCTGTCTGTCTATCTATCTATCTATCTATCTATCTATCTATCTATCTATCTATCTATCATCTATCTATCTTTCTATCTGTCATCTATGTCATCTCATCTATTTATCCATCCTATGTATTTATCATCTAATTTATCTGTATATCTCTATCATCAATTTATCAATTATCTATCAATCTATTTTCCAGCTTTATTGAGGTATTATTGGTAAATAAAAATTGTATATATTTATGGTGTACAACATGAAGTTCTGATACATGTATCCATTATAAAGTGATTACCACAAGTAAGCTTATTAACGTATCCATCACCTCACATAATTACCATTATTTTGTGGATGATGAGAACGCTTAAGATCTATCCTCTTAGCAAATTTTCAGTACACAATACAGTATCATTAACCATAGTCACCATGCTGTTTGTTAGCTCTCCAGAATTTATTCATCTAATAATTGAAGGTTTATACCCTTAGACCAAAATCCCCCCATTTCCTCCACTTCCCAGCCTCTGGCAACCACCATACAACTTTCTATTTCTATGAATTTGACTTCTTTAGATTCCACATACCAGTATAAGTGAGATCATGAGGTATTTGTCTTCCTGAGTCTGGCTTATGTCACTTAACATAATGTTCCTCAGGTTCATCTAAGTTGTTGTACATGATAGGATTTCCTTCCTTTTTAAAAGCTGAATAGTATTCCATTGTTTATATATACCACATTTTCTTTATTCATTTGTCTGTTGATGGACACTTAGGTTTATTCCATATCTTGGCTATTGTGAATAATTCTGCAATGAAAATGGGAGTGCAGCTATCTAATGAGATAACACCTCACATCTCTTGGAATGGCTATTGTCAGCTTTTGGAGACCTAAAGACTCATATGTTAGTAACAATAGGAAAACATACAGCTTTTTCATAAGTGATAGTAAAATCTTATTTCAGTAAAGTAAATAAGTGATCAAGATCATCCAAATTAATTTTCTGAAAAAGAGAAGTTGCTTTGGATATATACACAGGAGTGGGATTGCTGGATAATGTGGTAGTTCTATTTTTAAGTTTTTAAGAACTTCATACTGTTTTCCACAATGGCCTACCAGTCTATAATCCTATAAATAGTGCACAAGGGTTCCTTTTCTCCATTTCTTGGTCAACTCTTATTATTATTTGACATGTTTCTAGTAGCCATCCTAACAGGTATGAGGTAATATCTCATTGTGGCTTTCATTTACATTTGCCTGATGATTAGAAAACCTTTTTTTTTTTTTTTATATGGAGTCTTGCTCTGTCACCCAAGCTGGAGTGCAGTGGTGCAACCTCTGCTCACTGCAACTTCTGCTTCCTGGTTTCAAGCGATTCTCCTGCCTCAGCCTCCACAGTAGGTGGGATTATAGGCACCTCCCACTACACATGGCTAATTTTTGTATTTTTAATAGAGAACGGATTTCACCAGTGATCTGCCCGCCTCAGCCTCTCAAAGTGCTGGAAGTACAGGCAGAAGCCACAGTGCCCGACCTAGAGCACCTTTTAATATTAGCTGTGGGCCATTTGTATGTCTTCTTTGAAAAAATGTTTATCCAGGTGACTTGTTCATTTTAAGATTGGGCTATTTATTTATTTACTATTGAATTATGTGGGTTCATTATATATTTTGGATATTAGTCCCTTAACAAATATGTGGTTTGCAAATATTAGGTTGGTGCAAAAGTAATTGCGGTTTTTTTGCCATTACCTTTAAAAAATCACAGTTGCCATTACCTGCAAAAACCGCAAATACTTTTGCACCTACCTAGTATTTTCTATCAGCTGGGTGGTTGCCTTCTGTATTTGTTTGCTAGGGATGGCATAACAAAATACTAAGGACTGGCTAACTTAGACAACAGAAATTTATTTTCTCAGTCTGGAGGCTAGAAGTCCAAGATCAAAGTGTCAGCGGGTTTGGTTTCTCCTGAGGCCTCTCTCCTTGGCTTGCAGATGGCTGCCATCCCAATCTGTCCTCACATGGTCTTTTCTCTGTGTGCATGTATCCCTGGTCTCTCTCTCTTCTTATAAGGACACCAGTCAGATTGGATTAGGGCCTTACTAACAGCTTCATTTTAACTTAATCGTCTTTTTAACTTAATCTCCTCTTTAAAGACCTTATCTCTAAATACTATTACACTCTGAGGTACTGGGGGTTGGGACTTCAACACGTGTATTTTAAAGAGAAACATTCAGCCTATACCACTTTCCTTCCATGGCCACGTGATTGGTATGACCAGTCCCTGCAACAATCACTTTGCCCCTTTTCCCCCAAATCCTAACCATTGCTAACCCACACCATCTGTTTGGATTCATCTCTCAGTGACTCAGTGGTTTTGTCCTCATATAGCTTTACCATCCTAGAACATATCCCACATCCGAAGTGACCACTTTAGAGTTGTCATCTCATACTCAAGATCATTATCTCCTTCTCTCTGGTTCAAATATATATTATTACAGCACCTAAAATACTTACTAATTCCTGCTCATCAGGTCCAATCAGCATAATGTTGTCAATGTAGTGGACCAGTGTGATATTTGATGGTATGTCAAGACAATCAATATCTCTGTGGACTATATTAAGGTGAATTGACATACCCCCAAGACAATACTGTGAAGGTATCCTGTTGGCCCTAACATGAGAAAGCCAACTGCTTCTGGTGGTTTTACAAACAGATATAGAGAAAACAGCATTAGCCACATCAGTAGCTATATACCAAGTGCCAAGTGCTATGTTGATTTGTTTCAGTAAAAATAATACATTTGAGACAGCAGCAGCAATTGGAATCACTTCCTGATTAAGTTTATGAGTCCACAGTTATTCTACTAGATGTATCCTGTACTGGTGAATTAAATGAGAGTAAGTATCACCACTCCTACTCCTTTTAAGACTTTTATGATAGCACTGATTCCTGCAATTCCTGCCAGCCATGCAGTGTTGTGTTTAGTGAGCTAAGTGGAAGCTCTAAGAGCTCCTGGAGCTTCCATTTAGCTCTCCCTAATAATAGCCCTCATTCTATGGCTAAATCCCAATGTGGGGATCCTGTCAGTTGCTAAGTATGTCTGTTACAATTATTAGATTGGTGCAAAAGTAATTGCTGTTTTTGCCATTAAAATTATGCTGTTAATACATATTCCAATTACACTGGGAAAGTAGCCACATAGTTGGTATGTGGACTAACAGTGGTGATTTCTGTCCCCAGGAATTAGCATCAACTCAGAGCCAGTGTTTAGTAAACCTCAAATGATCTGTGTATTTCCTTTTCGCCAATGCAGTTTCACTAGTAAATGGCCACATGTCGTTTTGGGGAAAGCTTGAAAGAATTTGCCATGTGTACTGTAGCCAAGGTGCAAGATCCTTCTTAAAGGGAACACAGATTCTCTTTCAACCAAGGGACTATGGGTCTGTGAATTGACTTAGGTCTGAGAACTGAGAGACTGTGACTATCCACTGTGGTGACTCAAGTCAGGTTTTGGCTACCTGACTTAGATCTTTTTACCTGTTATAAATATCAAGCAATAATCTATTTCAACTGCCTATCTATTTTGTTCTCAGAGCAGCTCCGTAAGCCAAGGCAGTCTGATTTCCTGCTGCCCTTTAAGGTACATGTGCGCCCACCTTGTCTTTGTTGGTTATGTGCTGCCTGGTTTCTGCTATTCTGAGATCGTGTCATCCCCATTGAAATCAGGGAGCCCATCTAGATAGTGGCATCCTCTATAGTCAAGGTTTTCAAGCAAGGCTAGTCTCTTTAGACACTGGAGGGCAACCTACTTCCACTGATAAGAGAAAGAGTTGGCGTGTTTAAGATTGTCGGTTTCATCTGGGTCCAACCCATTTCAAGTATTGAGATTCTATTATTAAAAAATAACACCATTTTACACGAAAAACTCAGAATATGTGTGAATTTAATAGCAGTTATAATTCAGTAACATGAAAATTAAAATTTTGTTTGATTCTCAGCATAATTCTTGTTGCTACAATAAACCAGAGCTTTTGGAATTCTGCAGAATATCTCTGGTTCTCATACCATAACTCTGAGAGTTAAAAAACCTGTGCTTGTCATTTCTCTTTATAACCACTCAAAAGCACGCAAAAGAATCCATCCCACACCAGTCTTTCTAGTCATCTTTATAACCATAGCAGTCAAGTGTAGCAGCCACTTGGGGCCCCAAGGCACAGGCTTCAGTTGGCACTTCATCAGGATCAACCACAGGTAATTGCTTAATTGAGATGCCCAGCATGTCATGAATTACATTTCCCTTTGGCAAGGAGCTAAAGCTCAAAACCTCAATCAAACTGACCTTTAATCCCATCTTTGCAGGTCTATTTCTTGGGACTACTCCCGGGACAGGGAAGGCTTAATAAAAAGAATTGTTAACTATAATAGAGGATTAGAGTAATGAAGGATTCACTAGCAAGAAGAGAACTTTAAAGAATATAGCAACAGCAGCTATAAGGAGCTAACCTTATAACTAACTCTTGGGTTGAGAGACAGAGTGCCCAATGAAGAACCTCCCCAATTCCTCCCAGGGCTAAGATCTAGACGTTTTTGGAGAGGGCATGACCTGGCTCACTGAATGGCAGAGAAGTCACCATTGGGCTACGTCTGCAAAGCTTGTGGAAGATGTGCCCTCTGCAACTTGCTGCAAATGCCTCTAGGGTGCCAGGGAAGTTATTCATGGGGTAGTGTCTCACCTCCTCTACAAAGCTCCTAGAGTTGGGCATGTGAGAGGAAATTGCTAGTTACTGAGCACTATAGGACCTTGGCAGTGGAGAAAGCCAGGTATGCTGCAAAAGCTTATCAATACACAGAAATCAGGAAGCAAAATCCTTTTTTTCTGTAATGTCTCTTCAGCGCCCTCTGCTGATAAAGCTTCAGTCCCAGAAGGCAAACGAAAAACTGTTTAAAAGGCTCAGATCCGTTTTTACAGTGCAGTCAAAAAGGGCGAATTTGGAGCTGAGAGGCAATAAATCAATAACTGGCAGAAGCATCACTTCTCAGATTACACTCATGGATTTCGTAGTCAGATGTTTCTACATGTGCCTGGACGGCCCTTTGTCAGTCTCCTTTCCCCCAGGTTTTAACAGGCTGTCACCAAAGGAGGAAATAGTGAGCCCGAAGAGAATGTGCAAGCCCTCTTCTCTTGCATTTCCTCCAGCCCGTCACCAAGTACTCTTCCAGGCACGCCAGGCCCGAGTGTCCTAGGATAGGAAACCTGAGCAACAAAGGGAAGTGGGGTGTAAGCTCAACCCCTGTTGGACGTGGGGGTGCTGGCGCCTGAACGCCCCCGAAGGCCTCAACTCCCAGAAAGCTGCCTGCCAGGTCACAAGAGCCCTGTGAGGTCTTCCTATCGCCGGGACACTGGGAGGCAGGCGCGGGGCGGGTCCGCCGGGCGACGCGCTGTGATTGGGCGCCGTGGGGCCCGCCCCCTCATCTCGGAGCGTCGGGAGGGGGCGGGGCCAGCCGCCCGCTCGGCTCAGGCGCTGCGGGCGCCTATTGACCCAGCGGCTGCTGCGCCGCCGCTGTCTCCTCCTGCTCGTGGCGGGCGGTGCTGGAGCGCCAAGTGGCGCTGGAGAACCGGCGCTTCCTTTCGCCGCTTCCGCCGCCATCTCCGCGTTTGTGGGGCGGGAAAGAGGGAGGGGGCTAGCGGCTGCAGCTGGAGCGGGCTTCTCTCCGGGGACGGTCCTTTCCTCCCTGCTCTCCTTTTCCTTCTTTCCGCGTTGCCGCCGCCCGCCCCCTGCGCCTCCCCGCGGAGCCTGGGTCCGGGAGGGGGAAGGTAGGGGCGGCGGGGGGCGGGAGAGTCTGGCGAGCGGACGCTAGCTCTGAGGAAACTCATCAATCCGTGAGCCCCGGAGTCCGGGGTGCACATCGGCCCAGCCGCAGCGTCGGCGGCGGCGGCGGCAGCAGCACGAAGGGGCTCAGTCGGGGTAGGCGGGGGCGGTGCCGGTGCCGCGGGGGCGGGCCCGACCG

But there are two copies of the “story” in every cell • Human genome is a LONG EPIC STORY (3 billion characters) • Divided into 23 “BIG BOOKS” (chromosomes - 50-250 million characters / book). • Each of the “BIG BOOKS” has a volume number: volume 1, volume 2, volume 3… (chromosome number) • Each sperm and each egg cell has ONE complete copy of the STORY (one copy of each BOOK (23 chromosomes)). • Fertilization: one copy of the “STORY” donated from the sperm, one copy of the “STORY” from the egg -> fertilized egg (final: 2 STORIES, 46 BOOKS). • Fertilized egg -> cell division -> embryo -> adult organism. All of the cells in the organism contain the “identical” 2 copies of the STORY (2 copies of every volume, 2 copies of every sentence in every cell).

But the “stories” are not identical • Two copies of the “STORY” each cell are not absolutely the same. Some sentences are not identical. • Compare the two STORIES, >99.999999% of the sentences will be identical, butNOT ALL ARE IDENTICAL: because one copy came from the mother, other copy came from the father. • Most sentences in a single STORY are fine, some rare sentences have garbled words. • OK if one copy of a sentence makes no sense, if the same sentence in the other STORY is OK. • As long as you have one good sentence that makes sense between the two STORIES, then you are OK. But how can one good version of the story be enough?

The answer is linked to how the cell “reads” the story • Cells don’t read STORIES like people: they DO NOT read one story from beginning to end. • In a living cell: • Thousands of sentences are read from BOTH STORIES at the same time. • The same sentences are read in both books at the same time. • Read some sentences over and over again, other sentences relatively infrequently, and others not at all. • Some cells read different combinations of sentences with different frequencies. • Differences between cell types reflect which sentences are read, how often they are read, and when they are read. • Differences between individuals are determined by: • which sentences make sense and which don’t make sense • which sentences are read, when they are read, and how often they were read in different cells during development.

How can a “blueprint” (gene) be defective? • Genes are read like sentences with 3 letter words (always 3 letters!!!). • If a letter is changed, added, or removed -> words don’t make sense. • You have 2 copies of each book -> 2 copies of each sentence. • Chances are good that one copy of the sentence will be OK. • One good copy is usually enough: both copies don’t need to be “normal” • It is very rare to get 2 bad copies of the same sentence. Normal sentence: The fat cat ate the wee rat. Change one letter: The fat hat ate the wee rat. Delete a letter: The fat ata tet hew eer at. Add a letter: The fat rca tat eth ewe era t. Delete a word: The fat ate the wee rat. Sentence in one book: The fat cat ate the wee rat. Sentence in the other book: The fat hat ate the wee rat. Sentence in one book: The fat ata tet hew eer at. Sentence in the other book: The fat hat ate the wee rat.

Disease inheritance: which sentence did you get? • Example: a-iduronidase sentence • Each parent has one “good” sentence (I) and one “bad” sentence (i) • Sentences from the STORY are inherited randomly from each parent Daughter (normal) Sentence 1: I Sentence 2: I Iduronidase protein levels: 100% 25% Dad (normal) Sentence 1: I Sentence 2: i Iduronidase protein levels: 50% Son (normal) Sentence 1: I Sentence 2: i Iduronidase protein levels: 50% 50% Daughter (normal) Sentence 1: i Sentence 2: I Iduronidase protein levels: 50% Mom (normal) Sentence 1: I Sentence 2: i Iduronidase protein levels: 50% Son (Hurler Syndrome) Sentence 1: i Sentence 2: i Iduronidase protein levels: 0% 25%

ACTGA CTAGA GCG… ACTGA CTAGA GCG… ACTGA CTAGA GCG… ACTGA CTAGA GCG… ACTGA CTAGA GCG… How are the sentences read? • Sentences are “read” in one language but must be translated into a different language before “use”. • Each sentence is like a single line of an instruction written in Greek. • Instruction are read or copied in Greek -> handed over to a “translator” -> converted into English so that it can be “used”. “Master” Blueprint ACTGA CTAGA GCG… Sentence is copied (in Greek) Sentence is carried to translator Translation from Greek to English Use sentence in English “Story” (in Greek) Genome (DNA) Transcribe RNA RNA copy Translate RNA into protein Use the protein

What do you make from the sentences and why is it important? • TRANSLATION: conversion from the language of genetic information (DNA and RNA) to the language of proteins. • Proteins do a lot of things (most things) in a cell: • Establish structure and movement to cells • Carry out chemical reactions: chemical work • Regulate various processes:transmit signals • Transport molecules: Carrier proteins • Huge number of other things!!!

What are “Proteins”? • Proteins: “random” sequences strung together like “beads on a string”. • Different representations of protein sequences. • BUT: Proteins are not linear, they fold into spherical “blobs”

Change in the sequence of “beads” can block folding • Proteins sequences are NOT random. Defined by the sentence in the blueprint. Change in even one “bead” can stop the folding of the “blob”. Normal protein sequence Normal folded protein Defective protein sequence Defective protein folding

It is only the correctly folded “blobs” that have a function • Proteins (“enzymes”) accelerate chemical reactions • Act like “Pac-Man” to chew away or glue together molecules Enzyme molecules Mantra: folded protein = functional protein

Secretory pathway as an assembly line for protein production • Analogous to a factory production process: • Factory model: • Blueprint as a “plan” to convert raw materials into products. • “Quality control” checks in the assembly line. • Package products for shipment out of the factory. • Use of assembly line based on a blueprint: • Blueprint housed in the “corporate office”. • Workers copy the blueprint. • Copies taken to the factory floor. • Products are created in an assembly line from raw materials. • Quality control checks made before packaging of the product. • Products are packaged, shipped out of the factory to the “consumer”.

ACTGA CTAGA GCG… ACTGA CTAGA GCG… ACTGA CTAGA GCG… ACTGA CTAGA GCG… Factory production as a model for cell secretion Raw materials “Master” Blueprint Copy ACTGA CTAGA GCG… ACTGA CTAGA GCG… ACTGA CTAGA GCG… Translator Assembly line Corporate Office Final Shipped Products Assembly line

Comparison between a factory and a cell: ACTGA CTAGA GCG… ACTGA CTAGA GCG… ACTGA CTAGA GCG… ACTGA CTAGA GCG… ACTGA CTAGA GCG… ACTGA CTAGA GCG… ACTGA CTAGA GCG… ACTGA CTAGA GCG… ACTGA CTAGA GCG… Raw materials “Master” Blueprint Copy ACTGA CTAGA GCG… Factory ACTGA CTAGA GCG… ACTGA CTAGA GCG… Translator Assembly line Corporate Office Amino acids Chromosomes (genes, DNA) ACTGA CTAGA GCG… ACTGA CTAGA GCG… Cell ACTGA CTAGA GCG… ACTGA CTAGA GCG… RNA copy carried to the cytoplasm Conversion into proteins (translation by ribosomes) Copying DNA -> RNA Nucleus Cytoplasm

Cells have a variety of internal structures • Separation of function in different compartments • Nucleus:contains the genetic information for the organism • Mitochondria:generates cellular energy from breakdown of carbon compounds • Cytoskeleton:fibers that give cells shape and allow them to move • Secretory pathway (endoplasmic reticulum, Golgi, secretory vesicles): compartments that that allow the transport of products out of the cell • Endosomal/Lysosomal system:compartments that import materials into the cell, break them down, transport building blocks to the cytosol • Cytosol:space inside of the cell but not inside of any other organelle, where many chemical reactions in the cell occur. http://www.DennisKunkel.com/ http://www.biosci.uga.edu/almanac/bio_103/notes/may_15.html

Assembly line processing and packaging Final Shipped Products Assembly line Factory Cell Secretory vesicles Final Secreted Products Secretory Pathway (Endoplasmic Reticulum, Golgi complex)

What happens on the assembly line? • Branched carbohydrates are attached to newly made proteins • Carbohydrates are the “handles” that helpers use to make the proteins fold • Helpers have a limited time to make proteins fold Plant Manager amino acids Ribosome ACTG TGCA GTGC TRASH What is the human analogy?

“Normal” assembly line and quality control

BUT not all corporate blueprints have good plans: defects in the plans stop the shipment of “good” products ACTGA CTAGA GCG… ACTGA CTAGA GCG… ACTGA CTAGA GCG… ACTGA CTAGA GCG… “Bad” products are thrown away during assembly Raw materials “Master” Blueprint Copy Trash can ACTGA CTAGA GCG… x x x x x ACTGA CTAGA GCG… ACTGA CTAGA GCG… x x Translator Assembly line Corporate Office Final Shipped Products Assembly line

Defective genes make proteins that can not fold • “Bad” blueprints result in strings of beads that will not fold • Carbohydrates determine the time limit for folding • Carbohydrates are the “handles” used to drag defective proteins to the trash Plant Manager amino acids Ribosome ACTG TGCA GTGC X TRASH What is the human analogy?

When the assembly line is overloaded with proteins that will not fold….

Just a few of the diseases that result in proteins that fold slowly (or not at all) Adapted from Brodsky and McCracken (1999)

Endoplasmic Reticulum Lysosomes: the flip-side of secretion • Uptake of molecules into a cell is similar to secretion (but in the opposite direction). • Molecules bind to the cell surface, the membrane buds inward, pinches off, becomes an “ENDOSOME”. Fuses with an compartment called a “LYSOSOME”. • All classes of molecules are degraded in LYSOSOMES to smaller building blocks. • Analogy: Recycling Center that brings in materials, sorts them, breaks them into smaller pieces, sends them out for re-use as materials Protein secretion Protein uptake/degradation Lysosomal Enzyme synthesis Endosome Golgi Complex Lysosome Nucleus

Lysosomes contain a collection of enzymes that degrade all classes of molecules • 4 broad classes of molecules in a cell • All 4 classes of molecules brought into the cell for degradation • Enzymes in lysosomes can cleave all 4 classes of molecules into small building blocks • Only the small building blocks can escape from lysosomes into the cytosol for use in making new molecules • RECYCLING CENTER -> reuse of the molecule building blocks

Lysosome Hydrolases Late Endosome Hydrolases Including lysosomal a-iduronidase Hydrolases Including lysosomal a-iduronidase Lysosome Early Endosome Plasma Membrane Outside of the cell Lysosomal storage diseases: absence of a lysosomal enzyme • Both “sentences” used to make a lysosomal enzyme are defective -> protein is defective -> no degradation of the molecules that are normally cleaved Normal Lysosomal degradation  Lysosomes Hydrolases Late Endosome Lysosome Early Endosome Plasma Membrane Outside of the cell

MPS ML Many different lysosomal storage diseases: absence of a lysosomal enzyme • Each lysosomal storage disease results in the accumulation of a different type of molecule. Why does accumulation of these molecules cause a disease?

Recycling center model for lysosome function • Materials are brought into cell for “recycling”. • Disassembly of materials to building blocks -> removal from the recycling center to make room for more material to be recycled. • The process must be complete -> there is no room for storage. http://www.epa.gov/recyclecity/recovery.htm

Lysosomal storage diseases: landfill model • No degradation of molecules -> they accumulate, and accumulate, and accumulate… • Lysosomes proliferate, the cell is occupied with landfills. • Accumulation causes loss of function of lysosomes (the recycling centers are full !!!). • Normal cell functionis compromised, burden of too manyfull lysosomes is choking out normal cell function. • Disease ensues!!! http://www.epa.gov/recyclecity/landfill.htm

Lysosomal storage diseases: take home message • DNA in the genome is an EPIC STORY. Read as “sentences”. Sentences are translated from genetic language to the language of proteins. • Proteins are the “machines” that do all of the work inside cells. • Defects in genome -> defective “sentences” -> defective proteins (enzymes) -> don’t do their job in chewing up carbohydrates in lysosomes. • If a lysosomal enzyme is defective, then molecules that normally should be degraded will accumulate. • Accumulation is progressive, defects in the recycling system back up to hurt overall cell function -> progressive onset of disease symptoms!!!

Lysosomal Storage Diseases …an overview for the non-scientist