Mail it, together with your check, money order, or credit card information, before April 27 to: Michigan Avenue Kalamazoo, MI If you would like confirmation of registration, please include a self-addressed, stamped postcard in your mailing. Fax it, including your credit card information, before April 27 to Miller Auditorium at Only checks or money orders in U.
Any checks or money orders sent in currencies other than U. All charges are due at the time of registration. Receipts are issued at the Congress. Checks and money orders made out in an incorrect amount and illegible and incor- rect credit card numbers hold up the registration process. Please sign your check and write in the current date.
Post-dated checks cannot be accepted. All who attend sessions, give papers or preside over sessions, take part in panels, visit the exhibits, or otherwise attend the Congress and participate in its activities must register. On-campus housing assignments are given at that time. Packets may be picked up around the clock from noon on Wednesday until the end of the Congress.
Please note that on-campus housing may no longer be available to on-site registrants. The hours of on-site registration are: Wednesday, noon—midnight Thursday, 8: No refunds are made after that date. Registration for on-campus housing is a part of the Congress registra- tion process. Any rooms booked to on-site registrants will be billed at the single rate, although two attendees who want to share a room may do so. All on-campus rooms will be singles unless specific requests are received for double rooms, with roommate specified at the time of registration.
Please indicate special housing requests at the time of registration. Every effort is made to accommodate timely housing requests, but keep in mind that not every request can be fulfilled. If you and a colleague request sharing a double room, the room assignment will be made only after both registra- tions have been received. If you and a colleague or colleagues request sharing an adjoining bathroom i. Room assignments are indicated on the pre-registration packet, and keys are picked up at registration in the Eldridge-Fox lobby of the Goldsworth Valley III residence halls.
Rooms may be reserved for Wednesday, Thursday, Friday, Saturday, and Sunday nights of the Congress, but neither earlier nor later. Western Michigan University is a tobacco free campus, indoors and out. The campus housing offered through the Congress is designed for undergraduates, i. Those who require hotel amenities such as air-conditioning, refrigerators, and private bathrooms will find them at area hotels.
Those who choose this option will find in the pre-registration packet a ticket to be redeemed at their residence hall desk for the fitted sheet. On-site registration and check in is limited to Wednes- day, noon—midnight; Thursday, 8: See the Congress website for contact information. No hotel on this list offers smoking rooms. The Medieval Institute provides shuttle service to campus and back from the Radis- son Plaza Hotel on Wednesday from 7: The Medieval Institute thanks Discover Kalamazoo for its support of our hotel shuttle service.
Detroit and Minneapolis Delta and Chicago American and United are the major hubs offering air connections. Driving time from Gerald R. On Sunday, bus transportation to the airport is provided from 4: On Wednesday from 7: Kalamazoo Metro Transit bus 16 departing from the transportation center stops near Congress registration limited Sunday service , and taxi service is also available at the transportation center.
Driving from I to Congress registration: Take exit 74B onto US north. Take Stadium Drive east right 2. Turn left onto Howard Street and travel one mile to Valley Drive. Please do not park at meters or in prohibited areas. The first on-campus meal is Wednesday evening dinner, and the last meal is Sunday at noon. Meal tickets all you care to eat may also be purchased at the door cash, Master- Card, Visa, or Discover at these rates: Miscellaneous items such as toilet paper, shampoo, and cleaning supplies are also sold cash, MasterCard, Visa, or Discover. Health and beauty items and sundries are also available.
During the Congress, a complete breakfast and lunch menu is also served: Salads and fresh fruits are also available. Hours during the Congress are: Medieval Institute shuttle buses provide transporta- tion among Congress locations, with buses running continuously from 7: Walking is often the faster option, though, and many veteran Congress attendees recommend wearing comfortable shoes. The lab in the UCC is open 8: The lab in the Bernhard Center is open: Boarding passes, but not longer documents, may be printed at Congress registration Eldridge when on-site registration is open Wednesday, noon—midnight; Thursday, 8: Cash, check, Visa, MasterCard, and Discover are accepted.
The key to the room in the Bernhard Center can be checked out from the Information Desk. The rooms in the Fetzer Center are accessible without a key through an outer door Fetzer and can be locked from the inside. The keys can be checked out from the Eldridge-Fox desk. You must wear your badge to attend sessions, visit the Exhibits Hall, attend the Saturday Night Dance, use the Student Recreation Center for a fee , and use campus computer labs. The facilities and services of the Congress are available only to registered attendees. Wireless access is available throughout the campus, indoors and out.
Please provide a description of the work, the general location, pay, hours, and anything else you would like the hoped-for child care provider to know, as well as your contact information. Those telephones may be used for campus and local calls. A long distance calling card, available for purchase at the Eldridge-Fox desk, must be used for long distance calls. These telephones accept long distance calling cards.
They are available around the clock throughout the Congress. You should be ready to prove that you are 21 before you approach the cash bar. You must have photo ID with you.
You may not bring your own drinks to the dance. All other beverages and snacks are free. The Dance is a social occasion for registered attendees of the Congress only. Please bring your registration badge to the Bernhard Center: Come experience the beauty of medieval manuscripts in a soothing atmosphere and let your soul rest from the hectic world of conferencing. Images of both familiar and little-known manuscripts will be projected in an enhanced, digital slide- show while relaxing music plays. Organizers Mae Kilker Univ. Fox Wayne State Univ.
Laptops and cellphones are permitted as long as the sound is turned off. Adult coloring books or other quiet activities are welcome. Drop in for five minutes or five hours, whatever you need to restore and revitalize before returning to the stimulating, fast-paced world of the Congress. Fetzer Sunday Mass Saturday 7: Fetzer Sunday 7: The information contained in the printed program is available on the Congress website in the months preceding the congress.
In the United States, the Congress program is dispatched beginning in mid-February and extending to early March via the United States Postal Service either bulk mail or, for those who have paid the premium charge, Priority Mail. For delivery outside of the United States, the institute uses a mail service that carries the program air mail to the country of delivery and then deposits the mail in the country system.
If you have forgotten to bring your program to the Congress, you will need to purchase a second copy. Buses depart Valley III at 45 minutes after the hour, starting at 7: Motown shattered barriers, shaped our lives and made us all move to the same beat. Ale and Mead Tasting Saturday: Centre for the Study of the Middle Ages, Univ. Digital Editing and the Medieval Manuscript: The Mostly Medieval Theatre Festival is a biennial performance festival showcasing and invigorating the global heritage of drama, music, dance, and performance styles from late antiquity through the Renaissance.
Cosmic Dance Early Music Michigan A music and dance performance based on the life and music of the twelfth-century mystic and visionary Hildegard of Bingen. The Harlotry Players, Univ. This triple bill features a Tolkien fairy tale staged in a medieval style, a florilegium of fakery from the Harlotry Players, and a filthy French farce. A contemporary reimagining of a pair of plays in Middle Dutch. Additional performance at 3: The deadline for session proposals—including sessions of papers, demonstrations, panel discussions, performances, poster sessions, practica, roundta- bles, and workshops—is June 1.
By the end of June the Committee will have chosen its slate for inclusion in the call for papers posted on the Congress website in July. If you want to give a paper: Send a paper proposal to the contact person as soon as you can, but no later than September 15, OR submit your proposal directly to the Congress Committee for consideration for inclusion in a General Session. The efficient organizer generally tries to line up speakers as soon as possible. The organizer or the person proposing a paper who waits until the last minute may be very disappointed, failing to build a promising session or to place a paper, respec- tively.
Sponsored Sessions are organized by learned societies, associations, and institutions. The organizers set predetermined topics, usually reflecting the considered aims and interests of the organizing group. Special Sessions are organized by individual scholars and ad hoc groups. The orga- nizers set predetermined topics, which are often narrowly focused. Topics include all areas of medieval studies, with individual session topics deter- mined by the topics of abstracts submitted and accepted. All those working in the field of medieval studies, including graduate students and independent scholars and artists, are eligible to give a paper, if accepted, in any session.
Agreement to Deliver Papers in Person. Submission of a paper proposal is con- sidered agreement by the author to attend the Congress and to deliver the paper in person if it is accepted. It is a matter of Congress policy that papers are not read in absentia. You are invited to propose one paper for one session. The Congress Committee reserves the right to disallow all participation to those who breach professional courtesy by making multiple submissions. Diversity at Western Michigan University encompasses inclusion, acceptance, respect, and empowerment.
This means understanding that each individual is unique and that our commonalities and differences make the contributions we have to offer all the more valuable. Diversity includes the dimen- sions of race, ethnicity, and, national and regional origins; sex, gender identity, and sexual orientation; socioeconomic status, age, physical attributes, and abilities; and religious, political, cultural, and intellectual ideologies and practices.
The intention of these awards is to draw scholars from regions of the world underrepresented at past Congresses. There are three awards for each Congress: There are two awards for each Congress: Preference is given to Congress participants from central European nations. There is one award for each Congress: Karrer Travel Awards are available to students enrolled in a graduate program in any field at the time of application who are presenting papers in Spon- sored and Special Sessions.
Both awards offer a waiver of registration and room and board fees. See the Congress website for applica- tion requirements and procedures. Named in memory of the founder of the Professorship of Anglo-Saxon at the University of Oxford, Richard Rawlinson — , the Center opened in May , and in it received the endow- ment established by Georgian Rawlinson Tashjian and David Reitler Tashjian to support its mission.
Through the Center, the Medieval Institute offers a Graduate Certificate in the History of Monastic Movements, which is open to students enrolled in a graduate degree program at Western Michigan University. The Center is currently developing two digital projects.
The Monastic Gazetteer is planned as a dataset and interactive map on the geographic scope of monastic movements beginning with Western monasticism over time and provide tools for analysis and scholarly communication. The portal will provide access to unpublished manuscripts by Leopold Janauschek — The Center is sponsoring six sessions at the 52nd Congress on a variety of topics per- taining to the medieval history of the Cistercian order, including one sited at the Lee Honors College.
Program in Medieval Studies While allowing students to pursue specialized interests, the Master of Arts in medieval studies is intended to provide them with a broad interdisciplinary background in medieval history, languages, literature, philosophy, and religion. Thesis writers take 6 hours of thesis credit MDVL Demonstrated proficiency in Latin and a second medieval or a modern language is required. The examination committee will be composed of three members named by the Director in consultation with the student. The student will submit the two Capstone Writing Seminar papers to the committee no less than two weeks prior to the examination date.
If a student fails the examination, the examining faculty will determine whether the student is offered a one-time re-examination to be completed within 12 months of the first examination date. The com- mittee will be composed by the Director in consultation with the student. The deadline for international admissions may vary from those for domestic admissions. See the Medieval Institute website for application procedures. Felkel — Spanish Rand H. Johnson — Classics Paul A. Schulman — English Larry J. Wanner — Comparative Religion Victor C. Seiler — English Paul E.
He returned to the faculty as a Distinguished University Professor in and taught until his retirement in He passed away on October 24, at the age of Offices and dates do not reveal the crucial role Loew played during his career in the promotion and support of early studies at the University. He was present at the creation of both the Medieval Institute and the Institute of Cistercian Studies. He was a strong supporter of what has become the International Congress on Medieval Studies, and his efforts as Dean and as Vice President for Academic Affairs enabled Medieval Institute Publications to develop into the vital enterprise it has become.
His commitment was unflagging. His enthusiasm was infectious. His guidance was firm, generous, and kind. For all his services we thank him, and we remember him by continuing this series of lectures in his name. MIP was established in and now also houses Arc Humanities Press, which specializes in global premodern history, reference books, and public understanding of the past. As a consortium of three publishers MIP, Arc, and AUP currently contract scholarly titles a year in late antique, medieval, and early modern studies, and in related humanities research such as digital humanities and cultural heritage.
Even so, the place of the humanities in education, in popular discourse, in politics, and in business is increasingly in question. Our consortium of presses is proud to take a stand for the humanities. We are committed to the expansion of humanistic study, inquiry, and discourse inside and out- side of the university. We believe that humanities research should progress boldly, keeping pace with technological innovation, globalization, and democratization.
We value a variety of established, new, and diverse voices in humanities research. We provide a platform for high-quality research that explores what it means and has meant to be human across cultures, continents, and eras. The research that we publish: Research into the premodern world offers complex understandings of how cultural ideas, traditions, and practices are constructed, transferred, and disseminated among different agents and regions. Knowledge of the premodern past, in particular, helps us to contextualize contemporary debates about identity, integration, political legitimacy, creativity, and cultural dynamics.
Understanding what it meant to be human in the premodern world is essential to understanding our present moment and our future trajectories. Current innovations in humanities research, employing digital tools for preservation, representation, and analysis, require us to return again to the earliest sources of our shared past, in the media and mentalities of the premodern world. Our publications explore themes in the late antique, medieval, and early modern periods on: Arc Humanities Press publishes research that fosters better public engagement in, and understanding of, the past and of the ways in which the contemporary world is linked to the premodern world.
Many publications focus on late antique, medieval, and early modern periods, especially from a global perspective, while others explore modern applied research. Amsterdam University Press is the largest university press in continental Europe. Over more than twenty years, AUP has built up a catalogue of more than 1, English- and Dutch-language titles. The History list is dominated by medieval and early modern studies and publications on the Dutch Golden Age.
To discuss any current research project please Medieval Institute Publications contact the director and editor-in-chief: Western Michigan University Dr. Simon Forde W. We challenge and engage all members of our community with a university experience that creates skilled, life-long learners. We are committed to pursuing inquiry, disseminating knowledge, and fostering critical thinking that encourages life-long learning. Our scholarship creates new knowledge, forms a basis for innovative solutions, leads to economic development, and makes substantial contributions to society.
We are a community of learners committed to human dignity, sustainability, social responsibility, and justice. Our campus embraces a diverse population of students, faculty, and staff, who devel- op learners and leaders who are locally oriented and globally competent, culturally aware, and ready to contribute to world knowledge and discovery. The synergy of these three pillars enables WMU to be a premier and distinctive university of choice. Western Michigan University offers all students a learning com- munity designed for and dedicated to their success. We are committed to access and affordability and sustaining an environment in which every student can meet the world head-on and triumph.
The Prize, instituted by Dr. The book or monograph may be in any of the standard scholarly languages. To be eligible for the prize the book or mono- graph must have been published in Letters of nomination, 2—4 pages in length, should include sufficient detail and rationale so as to assist the committee in its deliberations. Supporting materials should make the case for the award. Your presence, whether as a plenar- ist, presenter, presider, or auditor contributes to the vitality of the gathering.
GIVING If you would like to contribute to any of these funds, the easiest way to do so is online through our direct giving site: A music and dance performance based on the life and music of the twelfth-century mystic and visionary Hildegard of Bingen. Thursday, May 11 Morning Events 7: Occlusion and Interpretation in the Age of Gerson Sponsor: Jean Gerson Society Organizer: Wendy Love Anderson, Washington Univ. The Hermeneutics of Desire: Denis the Carthusian on 1 Corinthians Houck, Southern Methodist Univ. Felicia Nimue Ackerman, Brown Univ. Stephanie Amsel, Southern Methodist Univ.
Sarah Layman, Independent Scholar Presider: Interdisciplinary Graduate Medieval Colloquium, Univ. Philosophy, Logic, and Consolation Sponsor: Center for Thomistic Studies, Univ. Feingold, Ave Maria Univ. One or Many Rationes: Interpreting Summa theologiae 1. Alan Stahl, Princeton Univ. Warszawski; Kiril Myzgin, Univ. Warszawski Barbaric versus Barbarous: Matteo Pace, Columbia Univ. Society of the White Hart Organizer: Mark Arvanigian, California State Univ. Rosenthal, Stony Brook Univ. Makuja, Le Moyne College Presider: Murphy, SJ, Georgetown Univ. Centre for the Study of Christianity and Culture, Univ.
Dee Dyas Power in the Palatinate: Between Babieca and Rocinante: Burningham, Illinois State Univ. Institute for Medieval Studies, Univ. Claussen, California Lutheran Univ. A Tale of Two Magical Cities: Barcelona and Venice Michael A. Autobiography and Auctoritas Sponsor: Christopher Jensen, Florida State Univ. Snow, Michigan State Univ. Marino, Michigan State Univ. Albert Lloret and Nancy F. Marino A roundtable discussion with Charles B. Pretexts, Texts, and Aftertexts Sponsor: Shakespeare at Kalamazoo Organizer: Musicology at Kalamazoo Organizer: Norton, James Madison Univ.
Canterbury Tales Project Organizer: History and Politics in Medieval Archaeology Sponsor: Susan Solway, DePaul Univ. Reflecting the Light of God: Molly Lester, Princeton Univ. Scott de Brestian, Central Michigan Univ. Center for Medieval Studies, Univ. Morris Tichenor Renaissance Reconsidered: A Coincidence of Form: Joey McMullen, Centenary Univ.
Shannon Gayk, Indiana Univ. Shannon Gayk Why Not Nature? Barootes, Centre for Medieval Studies, Univ. Pearl and the Holy Name of Jesus B. Academy of Jewish-Christian Studies Organizer: Lawrence Frizzell, Seton Hall Univ. Sara Lipton, Stony Brook Univ. Preaching to Women Sponsor: Holly Johnson, Mississippi State Univ. Alberto Ferreiro, Seattle Pacific Univ.
Christopher Flavin, Northeastern State Univ. Laughing at the Peasant in the Old French Fabliaux: Behavior Unbecoming a Monk: Spaces in Manuscripts and Printed Books Sponsor: Early Book Society Organizer: Filling in the Blanks: Exploring Carolingian Cultures of Collecting Sponsor: Matthieu van der Meer, Syracuse Univ. Wendy Marie Hoofnagle, Univ. Mercedes Vaquero, Brown Univ. A Tale of Two Bastards: McCormick, Washington and Lee Univ. Frederick Suppe, Ball State Univ. Frederick Suppe Dangerous Foster-Brothers: The Marriage of Llywelyn ap Gruffudd: Visualizing the Global Movement of Manuscripts: Centre for Medieval Studies, Univ.
Nada Zecevic, Central European Univ. Gerhard Jaritz, Central European Univ. Sessions— Thursday, May 11 Lunchtime Events Thursday, May 11 1: Rebecca Barnhouse, Youngstown State Univ. Best, California State Univ. Susan Gottloeber, Maynooth Univ. Status, Space, and Settings Austin C. Lewis and the Middle Ages I: Lewis and Mysticism Sponsor: Center for the Study of C. Lewis and Friends, Taylor Univ. Joe Ricke, Taylor Univ.
Joe Stephenson, Abilene Christian Univ. As Above, So Below: Medieval Echoes in the Underworlds of C. Yearning and Disciplining Joy: Roberts, Abilene Christian Univ. Deliberation and Choice Sponsor: Tamara Bentley Caudill, Tulane Univ. Robinson, Kent State Univ. Vernacular Religious Literature Sponsor: Male and Female Perspectives? Thelma Fenster, Fordham Univ. Johnson, Florida State Univ. Elizabeth Archibald, Durham Univ. Kaufman, Middle Tennessee State Univ. Bower, Pennsylvania State Univ.
Society for Beneventan Studies Organizer: Shaping Pygmalion, Reflecting Narcissus Organizer: Lucas Wood, Indiana Univ. Thomas More College Narcissus and Pygmalion: Rowley, Christopher Newport Univ. Fox, Wayne State Univ. American Cusanus Society Organizer: Adam Knight Gilbert, Univ.
Nancy van Deusen, Claremont Graduate Univ. Cusan Thought in Musical Symbolism and Theory, ca. Ecofeminist Intersections A Roundtable Organizer: Lesley Kordecki, DePaul Univ. Does It Have to Be about Women? The Owl and the Nightingale: Matlock, Kansas State Univ. Feminism and Falconry Sara Petrosillo, Univ. Marcia Smith Marzec, Univ. Francis, Joliet Christ, Creation, and Humanity: Ordering Myths and Men: John Gower Society Organizer: Brian Gastle, Western Carolina Univ. Bioarchaeological Research on Eastern Europe Sponsor: Wien The Anchorite Next Door: Williams, William Paterson Univ.
Societas Johannis Higginsis Organizer: Dempsey, Westfield State Univ. Swain, Bemidji State Univ. International Society of Anglo-Saxonists Organizer: Mary Kate Hurley, Ohio Univ. Jill Hamilton Clements, Univ. Educating the Laity Sponsor: An Education from the Pulpit: Two Sermons by the Franciscan Johannes Sintram d. Medieval and Renaissance Studies, Univ. Kathleen Kennedy, Pennsylvania State Univ. Brooks Hedstrom, Wittenberg Univ. Monastic Landscapes of the Mind: Routes to Survival Sponsor: Welzenbach, Michigan Publishing, Univ.
Erik Gustafson, George Mason Univ. Gerson, Florida State Univ. Frederick Suppe Cut to the Quick: Celtiberian Bear Cult s in Roman Spain: Sierra Lomuto Introductory Remarks: Boyadjian, Michigan State Univ. Definitions and Categories Organizer: Introduction A Workshop Sponsor: Schoenberg Institute for Manuscript Studies Organizer: Dorothy Carr Porter, Univ. No programming experience is required or expected.
Unisciti a Kobo e inizia a leggere oggi stesso
Thursday, May 11 3: Hawley, Lubbock Christian Univ. Christian Raffensperger, Wittenberg Univ. Jane Toswell, Western Univ. Lewis and the Middle Ages II: Natural Law and Natural Love Sponsor: Maeve Callan, Simpson College Presider: Maeve Callan Coming into the Country: History and Literature Sponsor: Mariah Junglan Min, Univ. Robin Norris, Carleton Univ. Damian Fleming, Indiana Univ. Marian Bleeke, Cleveland State Univ. Working as if a Man: Patricia Price, California State Univ.
Kenneth Salzberg, Hamline Univ. Bede and the Virgin Mother Stephen J. Megan Cook, Colby College Presider: Megan Cook Brewing in Hell: Bellitto The Cardinal Grants Indulgences: Cusanus in the Jubilee Year Thomas M. Center for Teaching Excellence, Rice Univ. Joshua Eyler, Rice Univ. Shakespeare and Marlowe, Corrigan—provides an introduction to the Reacting to the Past series of pedagogical role-playing games.
Registration to nlcorrigan gmail. Twomey, Ithaca College Malory and Authorship: Thresholds of Agency Sponsor: The In Articulate Sufferer: Anthony Bale, Birkbeck, Univ. Olivia Holmes, Binghamton Univ. How Important Was the Frame? Bledsoe Translated Bodies and Traveling Souls: Straple, Western Michigan Univ.
Miller Le Jongleur de Notre-Dame: Then and Now Sponsor: Kelly DeVries, Loyola Univ. Networking from North Africa Organizer: Marianne Djuth, Canisius College Presider: Preaching in England Sponsor: Preaching the Word to Women: Renna, Saginaw Valley State Univ. Center for Medieval and Renaissance Studies, St.
Monasticisms before and after Benedict of Nursia Sponsor: Gregoriana Beyond the Cloister: Past and Present, Imagined and Real Sponsor: Liana Saif, Oriental Institute, Univ. Where Have Clare and the Sisters Gone? Deirdre Carter, Florida State Univ. Before and After Theresa Coletti, Univ. Apocalypse Bernard McGinn, Univ. Intersections A Roundtable Sponsor: Heide Estes, Monmouth Univ. Montroso, George Washington Univ. Rachel Elizabeth Grabowski, Cornell Univ. Art History That Amy K. Leader, Florida Atlantic Univ.
Identification and Recovery of Fragments Sponsor: Michael Johnston, Purdue Univ. Rethinking the Limits between Urban and Rural Space: Advanced A Workshop Sponsor: Reception in Honor of Richard K. Reception with hosted bar 5: Thursday, May 11 7: Odasso After the Labyrinth: Dreams of Ariadne Jane Beal, Univ. Susan Yager, Iowa State Univ. Susan Yager This workshop is led by Regula M. Evitt, Colorado College, and Elise E. Alan Baragona, Independent Scholar Presider: Bonnie Wheeler, Southern Methodist Univ. A panel discussion with Larry J.
Italians and Italianists at Kalamazoo Organizer: Medieval Textuality in the Digital Domain: Moss, Corpus Christi College, Univ. Constructing Gender in Persianate Literature Sponsor: Great Lakes Adiban Society Organizer: Tabor, Western Michigan Univ. Shifting Our Horizons of Expectation: Christopher Callahan, Illinois Wesleyan Univ. International Anchoritic Society Organizer: Roman, Kent State Univ. Gerry Guest, John Carroll Univ. Tina Boyer, Wake Forest Univ.
Amanda Luyster The Virgin: Moving beyond the Artifacts Sponsor: Neil Peterson, Wilfrid Laurier Univ. Three Points in Time Stevan E. Clint Morrison, Texas Tech Univ. Medieval Studies outside of the Academy Organizer: Julie Polcrack, Western Michigan Univ. Julie Polcrack Marching with Medieval Penguins: Navy Translating Medievalisms on the Regional Stage: A Gawain of Our Own: Writing Her Own Deliverance: Hussey, Simon Fraser Univ. Usha Vishnuvajjala, Indiana Univ.
Usha Vishnuvajjala Near and Sometimes Dear: Ana Oliveira Dias, Durham Univ. Jay Diehl, Long Island Univ. Gelfand, Utah State Univ. Leaf-by-Niggle Gilmore Theatre Univ. Reception with hosted bar 9: Schulman, Western Michigan Univ. Artifacts of the Infidel: Conflict and Resolution Sponsor: Samuel Cohen, Sonoma State Univ. Papers in Honor of Dr. Arthur Northward Sarah M. Jonathan Burgoyne, Ohio State Univ. A panel discussion with Laura Ackerman Smoller, Univ. Oschman Three Scotist Arguments against Averroes: Where Are We Now? Chicago; Thomas Goodmann, Univ. Douglas Morse, New School Presider: Details from Documents Sponsor: Lis Torres, Western Michigan Univ.
Teviotdale, Western Michigan Univ. A Wit-Locker of Sense Full: Medieval Studies Program, Univ. Monasticism and Memory Sponsor: Early Medieval Europe Organizer: The Hagiographic Evidence Nikolas O. Hoel, Northeastern Illinois Univ. Cook, Hartt School, Univ. Teaching a Diverse and Inclusive Middle Ages: Matthew Balensuela, DePauw Univ. Nova de Lisboa Music, Manuscripts, and Materiality: The Origins of Quaestiones in musica T. Rachel May Golden, Univ. Gale Sigal, Wake Forest Univ. Making the Virgin at the Halberstadt Liebfrauenkirche, ca.
Jennifer Schmitt Carnell, Univ. Revisiting the Question Sponsor: Society for Medieval Logic and Metaphysics Organizer: Hall, Clayton State Univ. Robertson A roundtable discussion with William F.
Reward Yourself
Frame versus Core Sponsor: Susanna Fein, Kent State Univ. Tales after Tolkien Society Organizer: Driver The Liber Nemrod de astronomia: Interpretations, Reception, Adaptations, Sources Sponsor: Andrews History and Stories: The Theorized Child Sponsor: Medieval Romance Society Organizer: Eve Salisbury, Western Michigan Univ. Theorizing the Medieval Child: A Journal of Medieval Cultures Organizer: Observed effect of pravastatin and simvastatin on macrophage, SMC, and lipid content in abdominal aortas of adult male cynomolgus monkeys fed an atherogenic diet.
Observed effect of pravastatin and simvastatin on TF expression in abdominal aortas of adult male cynomolgus monkeys fed an atherogenic diet. Predicted effect of microcompetition with foreign N-boxes on skewness and migration distance of macrophages. A photomicrograph of atheroma type IV lesion in proximal left anterior descending coronary artery from a year old man who died of a homicide. Extracellular lipids form a confluent core in the musculoelastic layer of eccentric adaptive thickening.
The region between the core and the endothelial surface contains macrophages and foam cells FC. A photomicrograph of thick part of atheroma type IV lesion in proximal left anterior descending coronary artery from a year-old man who committed suicide. The core of extracellular lipids includes cholesterol crystals. Foam cells FC overlie the core. Macrophages, which are not foam cells arrows , occupy the proteoglycan layer pgc adjacent to endothelium E at lesion surface. Predicted effect of microcompetition with foreign N-boxes on skewness and migration distance of SMC.
Predicted effect of an infection with a GABP virus on the relation between signal intensity and adhesion A and between signal intensity and velocity B. S-shaped curves representing fG-Complex1. SS model of transcription for a signal that is an exclusive suppresser. Structure of a hair follicle. Observed effect of topical treatment with testosterone, DHT, 17b-estradiol, or acetone vehicle alone, on percent of mice with hair regrowth. Experimental configuration in Hodgins ibid and Hibberts ibid.
The following sections present descriptions of elements used in the present invention. Following each definition, one or more exemplary assays are provided to illustrate to one skilled in the art how to use the element. Each assay may include, as its own elements, standard methods in molecular biology, microbiology, cell biology, cell culture, transgenic biology, recombinant DNA, immunology, pharmacology, and toxicology, well known in the art.
Details of the standard methods are available further below. Transcription factors include transcription coactivators. Sharing the same environment, such as cell, or chemical mix, is not required to be regarded microcompetitors. For instance, two genes, which were shown once to bind the same transcription factor are, regarded microcompetitors independent of their actual physical environment.
Such introduction increases the copy number from zero to a positive number. Generally, copy number may be modified by means such as the ones mentioned above, for instance, transfecting the cell with plasmids carrying a DNA sequence of interest, infecting the cell with a virus that includes the DNA sequence of interest, and mutating endogenous DNA to produce a sequence identical to the DNA sequence of interest.
Let L 1 and L 2 be two molecules. Let L 1,s denote L 1 in shape s, and let [L 1,s ] denote concentration of L 1,s. A molecule in a complex is regarded in a different shape relative to the same molecule uncomplexed, or free. Consider, for example, an antibody against L i,j , a specific shape of L 1. Assume the antibody binds L 1,j in the region contacting L 2. By binding L i,j , the antibody changes the shape of L 1 from L 1,j to L 1,k from exposed to hidden contact region.
The decrease in [L 1,j ] is equal to the increase in [L 1,p ], resulting in a fixed sum of [L 1,s ] computed over all s that bind L 2. The following assays identify a change in ma L 1 , following treatment. Assay in a biological system e. Apply a treatment to the system which may change L 1,s.
Following treatment, assay again the concentrations of all L 1,s , where s is a shape that can bind L 2. Calculate the sum of [L 1,s ] over all s, before and after treatment. An increase or decrease in this sum indicates an increase or decrease in ma L 1. Antibodies specific for L 1,s may be used in immunoprecipitation, Western blot or immunoaffinity to quantify the levels of L 1,s before and after treatment.
See also examples below. Assume the transcription factor F binds DNA. Let G 1 denote a DNA sequence of a certain gene. Assume the transcription factor F binds G 1. An assay can measure changes in G 1 mRNA expression instead of changes in the concentration of bound F. Assume F transactivates G 1. However, an increase in concentration of F bound to G 1 does not necessarily increase transcription if binding of F is necessary but not sufficient for transactivation of G 1. Identify a treatment that decreases ma F by trying different treatments, assaying ma F following each treatment, and choosing a treatment that decreases ma F.
Assay concentration of F bound to DNA 1 in a biological system e. Use the identified treatment to decrease ma F. Following treatment, assay again the concentration of bound F. Transfect a recombinant expression vector carrying the gene expressing F. Expression of this exogenous F will increase the intracellular concentration of F. Contact a cell with antibodies that decrease ma F.
A decrease in G 1 transcription indicates that F is limiting with respect to G 1. This assay is exemplified in a study reported by Kamei ibid. AP-1 interacts with CBP. In this study, G is the gene controlled by the AP-1 promoter. The study showed a close dependent inhibition of gene activation by the transactivation domain of Stat2 following transfection of a RelA expression vector Hottiger , ibid, FIG. Let t 1 and t 2 be two transcription factors that bind F. Assay for expression of the reporter gene. Specifically, assay transactivation of reporter gene following an increase in DNA 2 copy number.
A change in transactivation of the reporter gene indicates microcompetition for a limiting factor. A special case is when DNA 1 , is the entire cellular genome responsible for normal cell morphology and function. If the sequences transcribe mRNA, block translation of proteins with, for instance, an antisense oligonucleotide specific for the exogenous mRNA.
Alternatively, verify that the proteins are not involved in binding of F to either sequence. Also, verify that co-transfection does not mutate the F-boxes in DNA 1 , and DNA 2 , and that the sequences do not change the methylation patterns of their F-boxes. Consider an organism R with standard genome O. Consider O s a segment of O. As example for different organisms, consider the list of standard organisms in the Patentln 3. The list includes organisms such as, homo sapiens human , mus musculus mouse , ovis aries sheep , and gallus gallus chicken.
A standard genome is the genome shared by most representatives of the same organism. A polynucleotide and DNA sequence see above are interchangeable concepts. In multicellular organism, such as humans, the standard genome of the organism is not necessarily found in every cell. The genomes found in sampled cells can vary as a result of somatic mutations, viral integration, etc. Assume Pn expresses the polypeptide Pp.
If Pn is foreign to R, then Pp is foreign to R. Compare the sequence of Pn with the sequence, or sequences of the published, or self sequenced standard genome of R. If the sequence is not a segment of the standard genome, Pn is foreign to R. Isolate DNA from 0 for instance, from a specific cell, or a virus. Try to hybridize Pn to the isolated DNA. If Pn does not hybridize, it is foreign. Pn can still be foreign if it hybridizes with DNA from a specific O specimen.
Consider, for example, the case of integrated viral genomes. Viral sequences integrated into cellular genomes are foreign. Following this definition, integrated sequences, which are only segments of certain O specimens, are identified as foreign. Note that the test is dependent on the N population.
For instance, a colony, which propagates from a single cell, might include a foreign polynucleotide in all daughter cells. Therefore, the N specimens should include genomes or cells from different lineages. A polynucleotide can also be identified as potentially foreign if it is found episomally in the nucleus. If the DNA is found in the cytoplasm, it is most likely foreign. In addition, a large enough polynucleotide can be identified as foreign if many copies of the polynucleotide can be observed in the nucleus.
Finally, if Pn is identical to sequences in genomes of other organisms, such as viruses or bacteria, known to invade R cells, and specifically nuclei of R cells, Pn is likely foreign to R. Consider an organism R. In Definition 1, the comparison between 0, the genome of R, and Pn is performed logically by the observer. In definition 2, the comparison is performed biologically by the immune system of the organism R. Definition 2 can be generalized to any compound or substance. A compound X is called foreign to organism R, if X is immunologically foreign to R. If the test polynucleotide includes a coding region, incorporate the test polynucleotide in an expressing plasmid and transfer the plasmid into organism R, through, for instance, injection see DNA-based immunization protocols.
An immune response against the expressed polypeptide indicates that the polynucleotide is foreign. Inject the test polynucleotide in R. An immune response against the injected polynucleotide indicates that the test polynucleotide is foreign. Many nuclear viruses, such as Epstein-Barr, and cytoplasmic viruses, such as Vaccinia, express proteins that are antigenic and immunogenic in their respective host cells.
Consider O s , a segment of O. In Definition 3, the observer compares O, the genome of the R organism, with Pn using the molecules chemical or physical characteristics. In these assay, let OS be the wild-type polynucleotide and use the assays to identify a foreign polynucleotide. Consider the following examples. Compare the electrophoretic gel mobility of O s , and the test polynucleotide.
If mobility is different, the polynucleotides are different.
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Compare the patterns of restriction enzyme cleavage of O s , and the test polynucleotide. If the patterns are different, the polynucleotides are different. Compare the patterns of methylation of O s , and the test polynucleotide by, for instance, electrophoretic gel mobility. Let [Pn] i denote the copy number of Pn in O.
Consider a cell Cell i. Let [Pn] i denote the copy number of Pn in Cell i. Sequence the genome of Cell i. Count the number of time Pn appears in the genome. Compare the result to the number of times Pn appears in the published standard genome. If the number is greater, Pn is foreign to Cell i. Sequence the genome of Cell i and a group of other cells Cell j ,.
A polynucleotide cannot be both foreign and natural to R. If a polynucleotide is natural, it is not foreign to R, and if a polynucleotide is foreign, it is not natural to R. If Pn is a gene natural to R, then, its gene product is also natural to R. The products of a reaction carried out in a cell between gene products natural to the cell, under normal conditions, are natural to the cell.
For instance, cellular splicing by factors natural to the cell produce splice products natural to the cell. If the sequence is a segment of the standard genome, Pn is natural to R. Isolate DNA from O for instance, from a specific cell, or a virus. If Pn hybridizes, it is natural. Consider the Pn polynucleotide. Consider an organism R with genome O R. A vector is a specific example of a polynucleotide. A vector that includes a non coding polynucleotide natural to R is considered empty with respect to R. A natural polynucleotide means, a polynucleotide natural to at least one organism.
An artificial polynucleotide means a polynucleotide foreign to all known organisms.
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A viral enhancer is a natural polynucleotide. A plasmid with a viral enhancer fused to a human gene is artificial. A vector that includes a coding gene natural to Q, an organism different from R, can still be considered empty with respect to R. For instance, a vector that includes the bacterial chloramphenicol transacetylase CAT , bacterial neomycin phosphotransferase neo , or the firefly luciferase LUC as reporter genes, but no human coding gene is considered empty with respect to humans if it does not express a gene natural to humans.
Identify all gene products encoded by Pn. Compare to the gene products of O R. If all gene products are different, Pn is considered empty with respect to R. See more examples below. Consider Pn, a polynucleotide foreign to organism R. Pn will be called latent in a Cell i of R if over an extended period of time, either:. Pn is undetected by the host immune system.
A virus in a host cell is a foreign polynucleotide. According to the definition, a virus is considered latent if, over an extended period of time, it either shows partial expression of its gene products, no viral mRNA, limited or no replication, is undetected by the host immune system, causes no lytic symptoms in the infected cell, or causes no macroscopic symptoms in the host. The above list of characterizations is not exhaustive.
The medical literature includes more aspects of latency that can be added to the definition. Some studies use the terms persistent infection or abortive replication instead of latent infection. Introduce, or identify a foreign polynucleotide in a host cell. Assay the polynucleotide replication, or transcription, or mRNA, or gene products over an extended period of time. If the polynucleotide shows limited replication, no transcription, or a limited set of transcripts, the polynucleotide is latent.
Assay the cell over an extended period of time, if the cell shows no lytic symptoms, the polynucleotide is latent. Based on these observations, the study concluded: A recent review Young 7 discusses the limited sets of Epstein-Barr viral EBV gene products expressed during the period of viral latency. Let c i be a characteristic of a system.
For every c i , assume a non-trivial range of values. Chose any set of characteristics describing the system and assay these characteristics. Assaying blood pressure, blood triglycerides, glucose tolerance, body weight, etc. The set of C characteristics where every characteristic is represented by one value from its respective range of values will be called a state, denoted St C.
The definitions can be modified to accommodate partial descriptions. If the system persists over time in St 1 , the probability that the system is in equilibrium is greater than zero. However, since the system is categorizes based on a subset of C, the probability is less than 1. Overall, an increase in the size of the subset of characteristics increases the probability.
Assay the values of the complete sub set of the system characteristics. Repeat the assays over time. If the values persist, the system is probably in equilibrium. Regular physicals include standard tests, such as blood count, cholesterol levels, HDL, cholesterol, triglycerides, kidney function tests, thyroid function tests, liver function tests, minerals, blood sugar, uric acid, electrolytes, resting electrocardiogram, an exercise treadmill test, vision testing, and audiometry.
When the values in these tests remain within a narrow range over time, the medical condition of the subject can be labeled as a probable equilibrium.
Other tests performed to identify deviations from equilibrium are mammograms and prostate cancer screenings. Consider equilibrium E 0. Take a biological system e. Assay a set of characteristics. Verify that the system is in equilibrium, that is, the values of these characteristics persist over time. Apply treatment to the system and assay the set of characteristics again. Repeat assaying over time.
If the treatment changed the values of the characteristics, and within a reasonable time the values returned to the original levels, the equilibrium is stable. Let a healthy biological system be identified with a certain stable equilibrium. In chronic disease, in contrast to acute disease, the system does not return to the healthy equilibrium on its own. Compare the results with the values of the same characteristics in healthy controls. If some values deviate from the values of healthy controls, and the values continue to deviate over time, the equilibrium of the system can be characterizes as chronic disease.
High blood pressure, high body weight, hyperglycemia, etc. Using the above definitions it can be said that a disruption is an exogenous event that produces a chronic disease. A disruption is a disturbance with a persisting effect.
Verify that the system is in healthy equilibrium. Apply a chosen treatment to the system. Following treatment, assay the same characteristics again. If some values deviate from the values of healthy controls, continue to assay these characteristics over time. If the values continue to deviate over time, the treatment produced a chronic disease, and, therefore, can be considered a disruption. Genetic knockout, carcinogens, infection with persistent viruses e. Let Pp be a polypeptide.
Assume microcompetition with a foreign polynucleotide Pn directly, or indirectly reduces or increases Pp bioactivity. According to the definition, if both microcompetition with a foreign polynucleotide and an exogenous event increase, or both decrease bioactivity of Pp, the exogenous event can be considered as a foreign polynucleotide-type disruption. Microcompetition with a foreign polynucleotide is a special case of foreign polynucleotide-type disruption.
Treatment is a special case of an exogenous event. A foreign polynucleotide-type disruption can first affect a gene or a polypeptide. For instance, a mutation is an effect on a gene.
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Excessive protein phosphorylation is an effect on a polypeptide. Compare the results with the values of the same characteristics in healthy controls to verify that the system is in a healthy equilibrium.
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Modify the copy number of Pn, a polynucleotide of interest by, for instance, transfection, infection, mutation, etc, see above. Identify a gene with modified expression. Assume the assays show decreased expression of G. Take another specimen of the system in healthy equilibrium and apply a chosen treatment to the healthy specimen. Following treatment, assay G expression. Continue to assay G expression over time. If G expression is persistently decreased, the exogenous event can be considered a foreign polynucleotide-type disruption.
A mutation in the leptin receptor, a mutation in the leptin gene, etc see more examples below. Pp can be downstream from G, the microcompeted gene. Modify the copy number of Pn, a polynucleotide of interest, by, from instance, transfection, infection, mutation, etc, see above. Assay bioactivity of genes and polypeptides in the treated system and controls to identify genes and polypeptides with modified bioactivity relative to controls. These genes and polypeptides are disrupted. Let the polypeptide Pp x be disrupted. Apply a treatment to the system that modifies Pp i bioactivity.
Assay Pp x bioactivity. If the bioactivity of Pp x changed, Pp i is in a Pp x disrupted pathway. Apply a treatment to the system that modifies Pp x bioactivity. Assay Pp i bioactivity. If the bioactivity of Pp i changed, Pp i is in a Pp x disrupted pathway. Consider a polypeptide Pp k and a foreign polynucleotide Pn.
Consider, as an example, microcompetition between a cell and a viral polynucleotide, including the entire viral genome. Pp k can be any viral or cellular protein that increase or decreases viral replication. Apply a treatment to the system that modifies Pp k bioactivity, for instance, by increasing expression of a foreign or cellular gene encoding Pp k. Assay Pn copy number. If the copy number changed, Pp k and the gene encoding Pp k , are in a Pn disruptive pathway. Consider a GABP virus. The viral proteins that increase viral replication increase the copy number of viral N-boxes in infected cells.
According to the definition, these proteins belong to a disruptive pathway. See specific examples below. Assume the polynucleotide Pn binds the transcription complex C. Take a cell of interest. Modify the copy number of Pn by, for instance, transfection, infection, mutation, etc, see also above. Assume the transcription factor F binds the complex C.
The two major lists are from reviews by Goodman and Smolik , ibid and Hottiger and Nabel , ibid. Consider the following definition. Assume the gene G is transactivated, or suppressed by the transcription complex C. Preferably, verify that co-transfection did not induce a change in cellular microcompetition, a mutation in the gene promoter, or a change in methylation of gene promoter.
Assay gene expression in the antibody treated cell and in the untreated controls. Select a cell, which expresses a gene of interest. Assay gene expression in both the treated cell and in the untreated controls. Perform chromatin assembly of the gene promoter, for instance, with chromatin assembly extract from Drosophila embryos. Add a transcription factor during the chromatin assembly reactions. Allow time for the interaction of the proteins with the chromatin template. Perform in vitro transcription reaction. Measure the concentration of the RNA products, by for instance, primer extension analysis.
More examples see below. Mantovani 82 provides a list of genes, which include a NF-Y binding site Mantovani , ibid, Table 1. For the listed genes, the table indicates whether the referenced studies report the presence of a proven binding site for a transcription factor close to the NF-Y binding site, whether cross-competition data with bona fide NF-Y binding sites are available, whether EMSA supershift experiments with anti NF-Y antibodies were performed, and whether the studies performed in vitro or in vivo transactivation studies with NF-Y.
Contact a system for instance, organism, cell, cell lysate, chemical mixture with a test molecule L. Contact a system for instance, whole organism, cell, cell lysate, chemical mixture with a test molecule L. See more agents below. Assume Pn is a polynucleotide foreign to organism R. Compare the sequence of Pn with the sequence of the published V genome. If the V genome is not published, its sequence can be determined empirically.
For instance, Flory, et al. Other studies demonstrate competition between these viral enhancers and enhancers of other viruses. Mantovani ibid provides a list of viruses that include a NF-Y binding site Table 1. Modify the copy number of Pn in the cell by, for instance, transfection, infection, mutation, etc, see also above. Modify the copy number of Pn in the cell. Assay expression of the reporter gene and compare to cells with unmodified copy number of Pn. A polynucleotide of at least 18 nucleotides should be sufficient to ensure specificity and validate alignment.
Hybridization conditions should be sufficiently stringent to permit specific, but not promiscuous, hybridization. Such conditions are well known in the art. The literature lists five subunits of GABP: Control of this ratio within the cell regulates transcription of genes with binding sites for GABP Suzuki GABP may be identified using antibodies in binding assays, oligonucleotide probes in hybridization assays, etc.
Select a cell that endogenously expresses the gene of interest and transfect it with a vector expressing GABP. Preferably, verify that co-transfection did not induce a change in cellular microcompetition, a mutation in the gene promoter, or a change in methylation of the gene promoter. Contact the cell with an antibody against GABP. Assay gene expression in the antibody treated cell and untreated controls.
If the reporter gene expression is higher or lower in the antibody treated cell compared to the untreated controls, the gene is GABP regulated. Select a cell that expresses a gene of interest. Assay gene expression in both the treated cell and untreated controls. If gene expression is higher or lower in the antibody treated cell compared to the untreated controls, the gene is GABP regulated.
GABP binds promoters and enhancers of many cellular genes including see above. See details and more agents below. Combine assays in the GABP polynucleotide and foreign polynucleotide sections above. If the V genome is not published, determine the sequence empirically and compare. Preferably, identify two N-boxes separated by multiples of 0. Modify the copy number of a second polynucleotide, Pn2, in the cell. Transfect the cell with a vector that expresses a reporter gene under the control of a promoter of a GABP regulated gene.
Take a cell of interest, which expresses an endogenous GABP, regulated gene. Infect the cell with a GABP virus. Assay viral replication and compare to cells with unmodified copy number of Pn for instance, in cells infected with a non GABP virus. Hybridization conditions should be sufficiently stringent to permit specific, but not promiscuous hybridization. Consider a polynucleotide Pn.
A treatment, such as irradiation, can also be a modulator. In principle, according to the definition, any foreign polynucleotide-type disruption is a modulator. Assay the effect of an agent on Pn copy number. Specifically, take a biological system e. Modify the copy number of Pn by, for instance, transfection, infection, mutation, etc, see above.
Call this cell the Pn cell. Assay the Pn copy number in the Pn cell see above. Contact the biological system with an agent of interest. Assay again the Pn copy number. If the Pn copy number is higher or lower compared to the copy number in Pn cells not contacted with the agent, the agent is a modulator. If the binding is higher or lower compared to binding in Pn cells not contacted with the agent, the agent is a modulator.
If binding is higher or lower compared to binding in Pn cells not contacted with the agent, the agent is a modulator. Consider a gene suppressed by microcompetition with a foreign polynucleotide. Consider such a gene in a cell without a foreign polynucleotide. Now consider a mutation, which reduces the gene bioactivity. An agent that stimulates expression of such mutated gene will also be called constructive.
If, on the other hand, the mutation stimulates the gene bioactivity, an agent that suppresses its bioactivity will also be called constructive. A constructive agent can be an agonist, if it stimulates expression of a gene suppressed by microcompetition with a foreign polynucleotide, or if is stimulates bioactivity of a polypeptide encoded by such a gene. A constructive agent can also be an antagonist if it inhibits expression of a gene stimulated by microcompetition with a foreign polynucleotide, or inhibits the bioactivity of a polypeptide encoded by such a gene.
A foreign polynucleotide-type disruption can be constructive. See assays in Modulator section above. In these assay if either;. If the effect is in the opposite direction, the agent is disruptive. Antiviral drugs, sodium butyrate, garlic, etc. See more examples in Treatment section below. The elements of the present invention may include, as their own elements, standard methods in molecular biology, microbiology, cell biology, transgenic biology, recombinant DNA, immunology, cell culture, pharmacology, and toxicology, well known in the art.
The following sections provide details for some standard methods. Complete descriptions are available in the literature. The following list provides a sample of books in the series: Current Protocols in Cell Biology, edited by: The following lists include more books with standard methods. A more extensive list of books with detailed description of standard methods is available at the Promega web site: The Promega list includes books. For each element, one or more exemplary protocols are presented. All examples included in the application should be considered as illustrations, and, therefore, should not be construed as limiting the invention in any way.
More details regarding the presented exemplary protocols, and details of other protocols that can be used instead of the presented protocols, are available in the cited references, and in the books listed above. The contents of all references cited in the application, including, but not limited to, abstracts, papers, books, published patent applications, issued patents, available in paper format or electronically, are hereby expressly and entirely incorporated by reference.
The following sections first present protocols for formulation of a drug candidate, then protocols, that as elements of above assays, can be used to test a drug candidate for a desired biological activity during drug discovery, development and clinical trials. The assays can also be used for diagnostic purposes.
Finally, the following sections also present protocols for effective use of a drug as treatment. One aspect of the invention pertains to administration of a molecule of interest, equivalent molecules, or homologous molecules, isolated from, or substantially free of contaminating molecules, as treatment of a chronic disease. In one exemplary embodiment, homologous molecules may be synthesized by chemical modification of a molecule of interest, for instance, by adding any of a number of chemical groups, including but not limited to, sugars i.
In one exemplary embodiment, homologous polypeptides or homologous polynucleotides include polypeptides or polynucleotides that differ by one or more amino acid, or nucleotides, respectively, from the polypeptide or polynucleotide of interest. The differences may arise from substitutions, deletions, or insertions into the initial sequence, naturally occurring or artificially formulated, in vivo or in vitro.
Techniques well known in the art may be applied to introduce mutations, such as point mutations, insertions or deletion, or introduction of premature translational stops, leading to the synthesis of truncated polypeptides. In every case, homologs may show attenuated activities compared to the original molecules, exaggerated activities, or may express a subset or superset of the total activities elicited by the original molecule. In these ways, homologs of constructive or disruptive polypeptides or polynucleotides have biological activities either diminished or expanded compared to the original molecule.
In every case, a homolog may, or may not prove more effective in achieving a desired therapeutic effect. Methods for identifying homologous polypeptides or polynucleotides are well known in the art, for instance, molecular hybridization techniques, including, but not limited to, Northern and Southern blot analysis, performed under variable conditions of temperature and salt, can formulate nucleic acid sequences with different levels of stringency. Suitable protocols for identifying homologous polypeptides or polynucleotides are well known in the art see, for instance, Sambrook and above listed books of standard protocols.
Homologous polypeptides or polynucleotides can also be generated, for instance by a suitable combinatorial approach. It is well known in the art that the ribonucleotide triplets, termed codons, encoding each amino acid, comprise a set of similar sequences typically differing in their third position. Variations, known as degeneracy, occur naturally, and in practice mean that any given amino acid may be encoded by more than one codon.
For instance, the amino acids arginine, serine, and leucine can be encoded by 6 codons. As a result, in one exemplary embodiment, homologous DNA and RNA polynucleotides can be produced which encode the same polypeptide of interest. In another exemplary embodiment, a set of homologous polypeptides may be generated by incorporating a population of synthetic oligodeoxyribonucleotides into expression vectors already carrying additional portions of the polypeptide of interest.
The site into which the oligonucleotide-gene fusion is incorporated must include appropriate transcriptional and translational regulatory sequences flanking the inserted oligonucleotides to permit expression in host cells. Once introduced into an appropriate host cell, the resulting collection of gene-oligonucleotide recombinant vectors expresses polypeptide variants of the polypeptide of interest. The expressed polypeptide may be separately purified by cloning the vector bearing host cells, or by employing appropriate bacteriophage vectors, such as gt or its derivatives, and screening plaques with antibodies against the polypeptide of interest, or against an immunological tag included in the recombinants.
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For example, separating a preparation of nucleic acids by gel electrophoresis, by itself, does not constitute purification unless the individual molecular species are subsequently isolated from the gel matrix. In one exemplary embodiment, a polynucleotide encoding a polypeptide of interest is ligated into a fusion polynucleotide encoding another polypeptide which facilitates purification, for instance, a polypeptide with readily available antibodies, such as VP6 rotavirus capsid protein, a vaccinia virus capsid protein, or the bacterial GST protein.
When expressed, the facilitator polypeptide enables purification of the polypeptide of interest and immunological identification of host cells that express it. In the case of GST-fusion proteins, purification may be achieved by use of glutathione-conjugated sepharose beads in affinity chromatographic techniques well known in the art see, for instance, Ausubel In this example, purification may be achieved through nickel metal affinity chromatography. Once purified, the polyhistidine tract included to enable purification can be removed by treatment with enterokinase in vitro to release the polypeptide fragment of interest.
For molecules synthesized by an organism, for instance, polypeptides or polynucleotides synthesized by human subjects, in a preferred exemplary embodiment, a purified polynucleotide or polypeptide is free of other molecules synthesized by same organism, accomplished, for example, by expression of a human gene in a non-human host cell. The following sections present standard protocols for the formulation of certain types of agents. One aspect of the invention pertains to administration of a small molecule of interest, equivalent small molecules, or homologous small molecules, isolated from, or substantially free of contaminating molecules, as treatment of a chronic disease.
The following sections present standard protocols for formulation of small molecules. Small molecules, organic or inorganic, may be synthesized in vitro by any of a number of methods well known in the art. Those small molecules, and others synthesized in vivo, may by purified by, for instance, liquid or thin layer chromatography, high performance liquid chromatography HPLC , electrophoresis, or some other suitable technique.
Another aspect of the invention pertains to administration of a polypeptide of interest, equivalent polypeptides, or homologous polypeptides, isolated from, or substantially free of contaminating molecules, as treatment of a chronic disease. The following sections present standard protocols for the formulation of polypeptides. In one exemplary embodiment, a polypeptide of interest is produced in vitro by introducing into a host cell by any of a number of means well known in the art see protocols below a recombinant expression vector carrying a polynucleotide, preferably obtained from vertebrates, especially mammals, encoding a polypeptide of interest, equivalents of such polypeptide, or homologous polypeptides.
The recombinant polypeptide is engineered to include a tag to facilitate purification. Such tags include fragments of the GST protein, or polyamino acid tracts either recognized by specific antibodies, or which convey physical properties facilitating purification see also below. Following culture under suitable conditions, the cells are lysed and the expressed polypeptide purified.
Typical culture conditions include appropriate host cells, growth medium, antibiotics, nutrients, and other metabolic byproducts. The expressed polypeptide may be isolated from a host cell lysate, culture medium, or both depending on the expressed polypeptide. Purification may involve any of many techniques well known in the art, including but not limited to, gel filtration, affinity chromatography, gel electrophoresis, ion-exchange chromatography, and others.
Polynucleotides, both mRNA and DNA, can be extracted from prokaryotic or eukaryotic cells, or whole animals, at any developmental stage, for instance, adults, juveniles, or embryos. Polynucleotides may be isolated, or cloned from a genomic library, cDNA library, or freshly isolated nucleic acids, using protocols well known in the art.
Alternatively, a polynucleotide of interest may be amplified using PCR. The resulting DNA is inserted into an appropriate vector, for instance, bacterial plasmid, recombinant virus, cosmid, or bacteriophage, using procedures well known in the art. Nucleotide sequences are considered functionally linked if one sequence regulates expression of the other. To facilitate expression of a polypeptide of interest, the cloning vector should include suitable transcriptional regulatory sequences well known in the art, for instance, promoter, enhancer, polyadenylation site, etc. In one exemplary embodiment, an expression vector is constructed to carry a polynucleotide, a naturally occurring sequence, a gene, a fusion of two or more genes, or some other synthetic variant, under control of a regulatory sequence, such that when introduced into a cell expresses a polypeptide of interest.
Both viral and nonviral gene transfer methods may be used to introduce desirable polynucleotides into cells. Viral methods exploit natural mechanisms for viral attachment and entry into target cells. Nonviral methods take advantage of normal mammalian transmembrane transport mechanisms, for example, endocytosis. Exemplary protocols employ packaging of deliverable polynucleotides in liposomes, encasement in synthetic viral envelopes or poly-lysine, and precipitation with calcium phosphate see also below.
The variety of suitable expression vectors is vast and growing. For example, mammalian expression vectors typically include prokaryotic elements, which facilitate propagation in the laboratory, eukaryotic elements which promote and regulate expression in mammalian cells, and genes encoding selectable markers. The use of mammalian expression vectors is well known in the art see, for example, Sambrook , ibid, chapters 15 and The use of expression vectors in yeast is well known in the art.
In addition to mammalian and yeast expression systems, a system of vectors is available which permits expression in insect cells. In another exemplary embodiment, a polypeptide of interest is expressed in situ by administering to an animal or human subject by any of a number of means well known in the art see protocols below a recombinant expression vector carrying a polynucleotide encoding the polypeptide of interest, equivalent polypeptides, or homologous polypeptides. In the present invention, such vectors may be used as therapeutic agents to introduce polynucleotides into cells that express constructive or disruptive polypeptides for exemplary applications see, for instance, Friedmann It is critical that the potential effects of microcompetition between the enhancer, or other polynucleotide sequences carried in the delivery vector, and cellular genes be considered and manipulated where needed.
As an example consider a case where the polypeptide of interest binds an enhancer carried by the vector, for instance, a delivery vector that expresses GABP under control of a promoter that includes an N-box. In one exemplary embodiment, the vector expresses, in situ, a high enough concentration of the polypeptide of interest such that any binding of the polypeptide to the enhancer sequences within the vector itself is negligible.
In other words, the vector expresses enough free polypeptides to produce the desired biological activity in treated cells. In another example, the polypeptide is not a transcription factor, but the delivery vector carries a polynucleotide that microcompetes with cellular genes for a cellular transcription factor, for instance, a vector that expresses Rb and microcompetes with cellular genes for GABP. In an exemplary embodiment, the delivery vector also includes a polynucleotide sequence that expresses the microcompeted transcription factor, or is delivered in conjunction with another vector that expresses the microcompeted transcription factor.
The following sections present standard protocols for the formulation of such polynucleotides. In consideration of these common issues, the general methods for the formulation and delivery, as well as caveats regarding the use of nucleic agents, described first, apply similarly to each subsequent agent. The techniques and conditions for achieving such interactions are well known in the art. For instance, the antisense preferred target is the translational initiation site of a gene of interest, from approximately 10 nucleotides upstream to approximately 10 nucleotides downstream of the translational initiation site.
Antisense agents targeting the coding region are less effective inhibitors of translation but may be used when appropriate. Effective synthetic agents are typically between 20 and 30 nucleotides in length. However, to be effective, a complementary sequence must be sufficiently complementary to bind tightly and uniquely to the polynucleotide of interest. In other words, three bases of mismatch in a 20 base oligonucleotide have a more profoundly detrimental effect than three bases of mismatch in a base oligonucleotide. Inadequate complementarity results in ineffective inhibition, or unwanted binding to sequences other than the polynucleotide of interest.
In the latter case, inadvertent effects may include unwanted inhibition of genes other than a gene of interest. Specificity and binding avidity are easily determined empirically by methods known in the art. In one exemplary embodiment, a recombinant expression plasmid is engineered to express antisense RNA following introduction into host cells. Such oligonucleotides may contain modified nucleotides to attain increased stability once exposed to cellular nucleases.