Boston University Henry M. Goldman School of Dental Medicine

RESEARCH HANDBOOK 2011-2012

Salivary tissues of individuals diagnosed with SS display significant changes in immunolocalization of E-cadherin, which is frequently diminished at the lateral-apical regions (unfilled white arrows). Size bar: 10 mm. Courtesy of Dr. Maria Kukuruzinska’s laboratory.

1

Research is an integral component of Boston University Henry M. Goldman School of Dental Medicine (GSDM)’s mission, goals, and objectives. The School’s mission statement begins: “The Boston University Henry M. Goldman School of Den tal Medicine will be the premier academic dental institu tion promoting excellence in dental education, research, oral health care, and community service to improve the overall health of the global population”. In addition, the mission states: ”We will shape the future of the profession through scholarship, creating and disseminating new knowl edge, developing and using innovative technologies and educational methodologies, and by promoting critical thinking and lifelong learning.”

What is “dental research”?

Dental research involves the use of scientific analysis, observation, and experimentation to acquire new knowledge in the field of dental medicine.

Deadline to applyFirst-year research 2 x 3 : Jan 2, 2012IREC 1: Feb 1, 2012IREC 2: ongoing during second-yearIREC 3: ongoing during third-year

The benefits of research:

• becometrainedinthedesignandexecutionofscientificstudies;• enhanceanalyticalthinkingabilities;• bringbreadthanddepthtotheirdentaleducation;• haveabetterunderstandingofinnovativedentaltechniques,materials, andtools;• becomemoreinformeddentalclinicians.• contributetothedentalliteraturebypublishingtheresults;and• improveeligibilityforpostgraduatespecialtytrainingprogramsandacademic

appointments.

The research environment at GSDM:

• DepartmentofMolecularandCellBiology,Evans4,72EastConcordSt. http://dentalschool.bu.edu/research/molecular/index.html;

• DepartmentofPeriodontologyandOralBiology,CABRBuilding, 700AlbanyStreet,http://dentalschool.bu.edu/research/perio/index.html;

• ClinicalResearchCenter,100EastNewtonStreet,

2

• DepartmentsofEndodontics,GeneralDentistry,OralandMaxillofacialSurgery,OrthodonticsandDentofacialOrthopedics,andOralDiagnosisandRadiology,100EastNewtonStreet (http://dentalschool.bu.edu/departments/ortho/index.html), (http://dentalschool.bu.edu/treatment-policies/oral-diagnosis.html);

• DepartmentofHealthPolicyandHealthServicesResearch,560HarrisonAvenuehttp://dentalschool.bu.edu/research/health-policy/index.html;

• DepartmentofRestorativeSciences/Biomaterials,801AlbanyStreet, (http://dentalschool.bu.edu/research/biomaterials/index.html);

Other research sites:

• BostonUniversitySchoolofMedicine(BUSM),http://www.bumc.bu.edu/busm/;• anyotherresearchfacilityapprovedbythePredoctoralResearchCommittee. (Early application is necessary to complete the process of executing an affiliation

agreement prior to start of research.)

3

Research faculty mentors:

GENERALDENTISTRY• JudithJones,DDS,MPH,professorandchair.ResearchArea:Healthoutcomes research, oral-systemic relationships• PaulaFriedman,DDS,MSD,MPH,professorassociatedeanforadministration anddirector,GeriatricFellowshipProgram.ResearchArea:Geriatricdentistry• AnitaGohel,BDS,CAGS,PhD,associateprofessor&directoroforaldiagnosis& radiology.ResearchArea:Radiologyimagingandinterpretation• YingWu,DDS,MSD,PhD,assistantprofessor.ResearchArea:Oralmucosal disease, orofacial pain, management of medical compromised patient, oral cancer and salivary gland disease

HEALTHPOLICYANDHEALTHSERVICESRESEARCH• RaulGarcia,DMD,MMS,professorandchair.ResearchArea:Epidemiology• MichelleHenshaw,DDS,MPH,assistantprofessorandassistantdeanfor communitypractice.ResearchArea:Publichealth• ElizabethKaye,MPH,PhD,associateprofessor.ResearchArea:Publichealth• WoosungSohn,DDS,PhD,Dr.PH,associateprofessor.ResearchArea:Cariology and oral health disparities MEDICINE/INFECTIOUSDISEASES• FrankGibson,PhD,associateprofessor.ResearchArea:Microbiology

MOLECULARANDCELLBIOLOGY• DavidLevin,PhD,professorandchair.ResearchArea:Biochemistry/molecular biology • MariaKukuruzinska,PhD,professorandassociatedeanforresearch.Research Area:Molecularandcellbiology/development• CarlosHirschberg,PhD,professor.ResearchArea:Biochemistry/molecular biology • PhillipsRobbins,PhD,professor.ResearchArea:Molecularandcellbiology• MiklosSahin-Toth,MD,PhD,professor.ResearchArea:Biochemistry• JohnSamuelson,MD,PhD,professor.ResearchArea:Microbiology

ORALANDMAXILLOFACIALSURGERY• PushkarMehra,associateprofessorandchair.ResearchArea:Trauma,fracture, maxillofacial management and orthognathic surgery.• RichardD’Innocenzo,associateclinicalprofessor.ResearchArea:Trauma, fracture, maxillofacial management and anesthesia• AndrewSalama,DDS,MD,assistantprofessor.ResearchArea:Evaluatingtonguemotion and speech following reconstructive surgery and developing novel chemo-preventive medications for oral cancer

4

• WaelYouseff,DDS,DMD,CAGS,assistantprofessor.ResearchArea:Inferior alveolar and lingual nerve repair, facial pain syndrome – temporomandibular joint disease

ORTHODONTICS• LeslieWill,DMD,MSD,professorandchair.ResearchArea:Normal&abnormal growth,treatmentoutcomes&diagnostictools• BoHo,DDS,MS,CAGS,PhD,assistantProfessor.ResearchArea:Cellular& molecular mechanisms that control orthodontic tooth movement

ORTHOPEDICSURGERY• LouisGerstenfeld,PhD,professor.ResearchArea:Cellbiology/bone

PEDIATRICDENTISTRY• ChristopherHughes,DDS,PhD,associateprofessorandchair.ResearchArea: Oralmicrobiology

PERIODONTOLOGYANDORALBIOLOGY• SalomonAmar,DMD,PhD,professoranddirectorofinflammatorycenter. ResearchArea:Cellbiology• SergeDibart,DDS,DMD,professorandprogramdirector.ResearchArea: Gingival epithelial cells• RobertGyurko,DDS,PhD,associateprofessor.ResearchArea:Periodontology/ immunology/bone physiology • EvaHelmerhorst,MS,PhD,associateprofessor.ResearchArea:Biochemistry• CataldoLeone,DMD,DMSc,professorandassociatedeanforacademicaffairs. ResearchArea:Biochemistry/periodontology• YoshiyukiMochida,DDS,PhD,associateprofessor.ResearchArea:Molecular biology • FrankOppenheim,DMD,PhD,professor.ResearchArea:Biochemistry• ErdjanSalih,PhD,AssociateProfessor.ResearchArea:BiomedicalSciences/ Biochemistry/Bone Biology and Bone Cancer Interaction/Mass Spectrometry•PhilipTrackman,PhD,professor.ResearchArea:Cellbiology

RESTORATIVESCIENCES/BIOMATERIALS• DanNathanson,DMD,MSD,professorandchair.ResearchArea:Biomaterials• LaishengChou,DMD,PhD,professor.ResearchArea:Cellbiology/oralmedicine• RussellGiordano,DMD,DMSc,associateprofessor.ResearchArea:Biomaterials• GurkanGoktug,DDS,assistantprofessoranddirectorofprosthodontics residents’lab.ResearchArea:Biomechanicsofimplants,immediateloading, soft tissue management around immediate implants, material consideration of implant supported over-dentures

5

6

The following images represent selected research by GSDM faculty.

Maria Kukuruzinska, PhD

Overexpression of DPAGT1 is a feature of a subset of human Oral Squamous Cells Carcinoma (OSCC)

Adhesion in oral cancer

Miklos Sahin-Toth, MD, PhD

The trypsin-dependent pathological pathway in chronic pancreatitis associated with genetic mutations. Ac tivation oftrypsinogen to active trypsin is mitigated by trypsinogen degradati on and ac tive trypsin is inhi bited by pancreaticsecretory trypsin inhibitor (SPINK1). Mutations in PRSS1 s timulate autoac tivation of cationic trypsinogen. Loss-of-functi on mutations in SPINK1 reduce inhibitor expression and compr omise trypsi n inhibition. T he p.G191R variant inPRSS2 stimulates trypsin-mediated degradation of anionic trypsi nogen and thereby protec ts against chronicpancreatitis. Loss- of function mutations in CTRC reduce secreti on or activity of chymotrypsin C and thus impairprotective trypsinogen degradation.

7

Mpk1Swi4

Swi6

SCB

Mpk1Swi4

Swi6Pol II

Paf1

Pol II

Paf1

Pol II

Paf1Sen1Nab3

Nrd1

Swi4Swi6

Pol II Pol II

Paf1 Paf1Mpk1 Mpk1

Attenuation

Unstressed state

Cell wallstress

Elongation

Transcription activation

TerminationcomplexS. cerevisiae

Mpk1 blocks association of termination complex

Cell wall stress-induced genes Use of the yeast Saccharomyces cerevisiae as a model system for understanding stress signaling in eukaryotic cells.

David Levin, PhD

8

9

Predoctoral Research Program (PRP)

TheGSDMdevelopedahighlysuccessfulPredoctoralResearchProgramforDMDstudents.ThemissionoftheProgramis:1)toshapethefutureofdentalmedicineanddentaleducationthroughresearch;2)toeducatestudentsfromdiversebackgroundsabouttheimportanceofresearchindentalmedicine;and3)tomentorstudentstomake informed decisions about research career opportunities.

ThePRPattheGSDMbenefitsindividualstudentsandthefieldofdentalmedicine.Through participation in research students enhance their analytical thinking abilities, become trained in the design and execution of scientific studies, gain a better understanding of innovative dental techniques, materials and tools, improve their eligibility for postgraduate specialty training programs and academic appointments, become more informed dental clinicians, and contribute to the dental literature by publishing their research findings.

The GSDM provides state-of-the-art research training resources. Students choose facultymentorsfrom36researchscientistsinvolvedinmorethan100researchprojects that span broad areas of basic and applied biomedical sciences, as well as clinical and public health research. In addition, to direct mentor-student interactions, student trainees are expected to become important contributors to research teams and to participate in the full range of research-related activities, including laboratory/teammeetingsandjournalclubs.Atthecompletionofresearchtraining,studentsare expected to showcase their accomplishments at the School’s Science Day and at the University’s Science and Engineering Day. In addition, students are encouraged to participate in national and international scientific meetings in the areas of their researchtraining.InformationaboutthePRPandtheStudentResearchGroup(SRG)can be obtained at http://www.bu.edu/dental/research/predoctoral/. Information on the GSDM Science Day abstracts and awardees is available at http://www.bu.edu/dental/research/predoctoral/scienceday/ .

Program StructureBecause of its unique curriculum, the GSDM offers formal research training for credit tostudents.Studentswhomaintaina3.0GPAorhigherintheirdidacticandclinicalcourses are considered for research training. Students selected by Committee can participateintheProgram.Thefirst-yeartrainingtakesplacefollowingthecompletionoftheDMDdidacticcoursesduringtheApexrotationfromMaytoJuly.Therotationis based on a five-day week as follows:

a.studentsdedicatetwodaysforresearchtrainingandthreedaysfortheApexclinicalassignment; b. students dedicate three days for research training (30 hours per week) and two daysfortheApexclinicalassignmentundertheIntensiveResearchElectiveCourse(IREC). Students are considered for the IREC 1 if they have participated in research

10

during the second semester of their dental education on a voluntary basis or if they havepriorresearchexperience; c. students can do research on a voluntary basis and are expected to spend no lessthan10hoursperweekinresearchtraining.AdvancedStandingstudentscanstart research during the second semester of their dental education.

Priortoengaginginresearchtraining,thePredoctoralResearchOfficemeetswiththeapplicants to advise them of their assignments and to inform them of the prerequisites toresearchtrainingincludingNIHtrainingintheProtectionofHumanSubjectsinResearch and other regulatory requirements. The students are given a copy of the Research Handbook that contains a detailed description of the program. During researchrotations,studenttraineesareexpectedtoattendmeetingswiththeOfficeofthePredoctoralResearchthatincludepresentationsonscientificwritingskillsandapproaches to better presentations. Trainees are also expected to attend seminars relevanttotheirresearchorganizedbytheGSDM,theSchoolofMedicineandotherresearch institutions in the greater Boston area. In addition, students are required to participate in research competitions. Students have the option to do research rotations outside of Boston University that require the execution of an affiliation agreement that governs the relationship between Boston University and the outside institution.

StudentresearchtrainingisoverseenbythePredoctoralResearchCommittee,chairedbytheDirectorofthePRPandcomposedofmembersoftheGSDMbiomedicalscienceandclinicalfaculty,theAssociateDeanforAcademicAffairs,theAPEXProgramAdministrator,astudentrepresentativeandtheAssistantDirectorofPredoctoralResearch.ThemissionofthiscommitteeistoguideandmonitorresearchactivitiesamongDMDstudents,evaluatetheeffectivenessofthePRPandmakerecommendations for program improvements.

The Intensive Research Elective Course (IREC) The goal of the IREC is to provide intensive and structured research experience throughout the dental school curriculum for students who are interested in careers in oral health research.

The IREC objectives are: 1) to carry out well-defined research projects under the guidanceofresearchmentors;2)toenhancecriticalthinkingskills;3)toparticipateinthe full range of research-related activities, including scientific meetings and journal clubs. Scientific meetings will provide platforms for discussions of research findings, for troubleshooting research strategies and methodologies and for critiquing results andtheirinterpretation;4)totraininthedesignandexecutionofscientificstudies,gain better understanding of innovative dental techniques, materials and tools, develop analytical thinking abilities, contribute to the dental literature by publishing results, showcase accomplishments at local, national and international scientific meetings, become more informed dental clinicians and improve eligibility for academic appointments;and5)tocontributetothediscoveryofnewknowledge.

11

The IREC components include mentored research and a completed project. Studentsneed to complete the mentored project for the section and report the results at Science Day and at other scientific events. The project could be ongoing throughout the IREC training.

There will be three options to IREC: IREC1-IntensiveResearchDMDyear1underApex;IREC2-IntensiveResearchDMDyear2(2credits);IREC 3 - Intensive Research DMD year 3 (2 credits).

Research Training Eligibility Studentswhomaintaina3.0GPAorhigherintheirdidacticandclinicalcoursesareconsidered for research training. Students selected by Committee can participate in the IREC.

1- The IREC 1 course takes place during the first-year following the completion of the DMD didactic courses from May to July. The rotation is based on a five-day week as follows:

a. Students dedicate three days for research training (30 hours per week) and two daysfortheApexclinicalassignmentundertheIntensiveResearchElectiveCourse(IREC). Students are considered for the IREC 1 if they have participated in research during the second semester of their dental education on a voluntary basis or if they have prior research experience. b.IREC1traineesaregradedbytheendoftheApexrotation.

2- The IREC 2 course takes place during DMD year 2. Students who completed IREC 1 training or those with prior research experience can apply.

a. The expected number of hours is 100 contact hours minimum in the laboratory or in the clinical setting. The activities outlined below need to be accomplished outside the contact hours. b. Students need to complete the mentored project and present it at Science Day and at other scientific events. The project could be an ongoing product carried from IREC 1. c. IREC 2 trainees are graded by the end of July.

3- The IREC 3 course takes place during DMD year 3. Students who completed IREC 1 and/or IREC 2 training or those with prior research experience can apply.

a. The expected number of hours is 100 contact hours minimum in the laboratory or in the clinical setting. The activities outlined below need to be accomplished outside the contact hours. b. Students need to complete the mentored project and report the results at Science Day and at other scientific events. The project could be an ongoing product

12

throughout the IREC training. c. IREC 3 trainees are graded by the end of DMD year 3.

Activities during the IRECProject Development IRPtraineesworktogetherwiththeirmentorsonthepreparationofresearchproposals through literature reviews, analyses of preliminary data and pilot studies. Projectdescriptionincludeconceptdefinition,formulationofspecifichypotheses,aims and timelines, as well as expected outcomes. Mentors assigned to train IREC students assume the responsibility for supporting the students through the selection, designandexecutionofaproject.Oncetheprojectiscompleted,studentsareexpected to present at local, national and international meetings.

Seminar Series ThePredoctoralResearchProgram(PRP)officeorganizeaseminarseriesthroughwhich IREC trainees learn about different scientific methodologies and approaches. These seminars enrich the trainees’ research experience by exposing them to the latest scientific findings and facilitate development of personal relationships among peers.

Journal ClubEach trainee is required to attend a journal club directed at developing skills in the critical evaluation of literature by critiquing research papers.

Scientific Writing and Presentation SkillsThePRPOfficeassiststheIRPtraineesinthepresentationoftheresearchaccomplishmentsatscientificmeetings.Anemphasisismadeonimprovingoralpresentation and writing skills.

Scientific EventsThePRPOfficesupportstheIRPtraineestopresenttheirresearchprojectsattheIADR/AADRmeetings,theHinmanSymposium,theYankeeDentalSymposium,theannual GSDM Science Day and Boston University Science and Engineering Day.

Instructions in the Responsible Conduct of Research (RCR)PriortoApextraining,ThePRPOfficeinformstraineesoftheirresponsibilitiesthatincludeasessionontheNIHtrainingintheProtectionofHumanSubjectsinResearch.DuringtheIRECtraining,thePRPofficefacilitatestrainingattendanceinRCR.Theactivities include discussion of standards of good practice and policies for handling misconduct allegations. The training program on RCR consists of a series of lectures, seminars and workshops on several major issues that include Human Subjects, ResearchNotebooks,AuthorshipResponsibility,InstitutionalPoliciesonScientificMisconduct,ProperApplicationofStatisticalAnalysisandConflictofInterest.

13

Training and AssessmentEach research mentor expects to provide guidance and supervision to the trainee. Each mentor formally meets with the IREC trainee on a regular basis to review progress. In addition, mentors are expected to interact informally with the IREC trainees on a regular basis during the elective course in years two and three. The IREC trainee’s progress is determined by an evaluation questionnaire completed by the research mentor to provide an assessment of the trainee’s degree of research progress and knowledge of the specific subject area. In addition, the IREC trainee’s research experience is evaluated in relation to subsequent research activities and his/herfuturecareerplans.Afinalgradeisissuedandanassessmentsummaryuponcompletion of training is provided to the trainee with a comprehensive overview of his/her performance.

Program EvaluationAssessmentoftheeducationaloutcomeisusedbymeasuringtheinitialbaselinethroughapre-programquestionnaire.Apost-programquestionnaireisusedtoquantify changes in knowledge, skills and career choices. Feedback gathered through evaluationisdocumentedandusedtoimprovethequalityoftheProgram.Thestudent’s self-assessment is triangulated with the actual assessment by the mentor. The evaluation helps in the adjustment of goals and objectives of the research training toimprovetheProgramoutcome.

Benefits while in the PRP• AADRmembership;• IADR/AADRannualmeeting;• Poster/OralPresentationsatScienceDay,ScienceandEngineeringResearch Symposium,Hinnmansymposium,YankeeDentalCongress,etc;• Publishingopportunity;• SigmaXimembership;• Regulatoryandethicalconductofresearchtraining;• FogartyInternationalClinicalResearchScholarsProgramopportunity;• HowardHughesMedicalInstituteResearchTrainingProgramsopportunity.

Expectations during first-year research rotationMeeting I (during the second week of rotation)• Studentsareadvisedonprinciplesinconductingresearch.Expectationsareemphasizedregardingpresentationoftheirworkatscientificmeetingsandinparticular GSDM Science Day and BU Science and Engineering Research Symposium Day (during March of every year). Students are expected to report on their project andresearchexperience.Informationonendofrotationpresentations,AmericanAssociationforDentalResearch(AADR)membershipsandAADRmeetingattendance are discussed. Information on mentor end of rotation assessment is given.

14

Meeting II (during the second month of rotation)• Studentsattendaseminaron“ScientificWritingSkills.”Informationonoptional seminarson“ApproachedtoBetterPresentations”isdiscussed.Meeting III (end of rotation)• presentorallyasummaryoftheirresearchtotheircolleagues(first-yearrotation) andpresentorallyorasaposterduringGSDMScienceDay;• completeaprogramevaluationandadetailedreportoftheresearchexperience (disk or email).

Evaluation criteria:• Mentorevaluation:50%(mentor)• Otherassignments:30%(mentor)• Presentations:10%(PRPoffice)• Meetingattendance:10%(PRPoffice) Mentor evaluation criteria include research science aptitudes, report writing, research skills, and interpersonal/communication skills.

Rotation prerequisites•ResearchRotationApprovalForm;•Researchoutline;•NIHtutorialontheprotectionofhumansubjectsinresearchcertificate;Youcangoonlinehttp://phrp.nihtraining.com/users/login.phptotakeacourseandquiz.Oncethetwo-hourtutorialiscompleted,acompletioncertificatecanbedownloaded.Onsitetrainingsessionsareavailableandinformationcanbeobtainedathttp://www.bumc.bu.edu/ocr;

In addition:

•Laboratorysafetytrainingforlabsettings.Informationonupcomingsessionsat:http://www.bu.edu/orctraining/ehs/research-safety-training/lab-safety-training-schedule/

•Studentsworkingwithanimals,mentorneedstoaddthetraineetotheprotocol.Coursetrainingscheduleshttp://www.bumc.bu.edu/IACUC.Theanimalrequirementsare:1- InstitutionalAnimalCareandUseCommittee(IACUC)Orientation;2- LaboratoryAnimalScienceCenter(LASC)NewResearcherOrientation;3- MedicalSurveillanceClearancebyOEMResearchOccupationalHealthProgram.Toscheduleanappointment(ROHP)617-414-7647orrohp@bu.edu

•Studentswhowillbeworkingindirectcontactwiththesubjectsand/oridentifiabledatamustbeaddedtotheIRBprotocol;

•StudentswhowillbehandlingHuman-Derivedsamples(includingcelllines)orrecombinantDNA,PIneedstofileanamendmenttoaddthestudent’snameto

15

theformfoundat:http://www.bumc.bu.edu/Dept/Home.aspx?DepartmentID=357•Additionalrequirementsasperindividualmentor.

The Student Research Group (SRG)

Interested students are encouraged to participate in the School's SRG, a local chapter oftheAmericanAssociationforDentalResearch(AADR)StudentResearchGroup.ThenationalSRGwasestablishedin1980asameansbywhichtheAADRcouldfoster a major source of future researchers from the ranks of dental students.TheSRGatGSDMwasestablishedin1992andisacomponentoftheAADRintheBoston/Connecticut Region. This region includes: Boston University, Tufts University, HarvardSchoolofDentalMedicine,andtheUniversityofConnecticut.TheAADRstrongly urges schools within a region to work together to promote student research activity, and to share experiences inclusive of: competitions, conferences and interactionwithresearchfaculty.Intercampuseventshavebeenestablished.Allstudents are invited to attend.

SRG Officer’s Duties

Motivated students involved with the SRG are encouraged to run for officer’s positions. Democratic elections are held annually.President• runsSRGmeetingsandofficers’meetings;• conductselections;• assuresthatotherofficers’dutiesarecarriedout;• organizesrequiredtasksoftheSRG,includingofficialschoolrecognition;• directsSRG-dentalschoolrelationsandvisibility;• writesthewelcomelettertoincomingfreshmanstudentsbeforeschool• starts;andactsasAADRcontactwithhelpfromthefacultyadvisor.Vice President• organizesbig-brother/sisterassignmentfornewstudentresearchers;• assiststhepresidentwhennecessary;• assiststhesecretaryincompletingmembershipapplications;and• assiststhetreasurerwhennecessary.Secretary• recordsminutesfromallofficers’meetingsanddistributesthem;and• ensuresthatallstudentshavecompletedAADRmembershipapplications.Treasurer• authorizesandrecordsallmonetarytransactionsoftheSRG;and• ensuresthatSRGbudgetisbalancedandappropriatelydistributed.

16

Timeline of events

May21–July13,2012APEXRotationAugust2012 RotationoralpresentationsOctober10-212012 ADA/DentsplyStudentClinicianProgram,SanFranciscoOctober26-282012 HinmanSymposiumJanuary1,2013 DeadlineforAPEXIrotationapplicationsFebruary2,2013 YankeeDentalCongress(YDC38)StudentTableClinicsFebruary4,2013 DeadlineforIREC1March14,2013 GSDMScienceDay2013March 2013 BU Science and Engineering Day 2013March20-23,2013 AmericanAssociationforDentalResearch(AADR) Meeting, Seattle, WashingtonApril2013 OrientationDMDIandASI(11a.m.-12p.m.)April2013April1,2013 OrientationtoIREC1May2013 ADADentalStudentConferenceonResearch, Bethesda, MD

Information about research• http://www.bu.edu/dental-research•http://blackboard.bu.edu/

Sample Outline Goal:Todeterminetheroleofmonocytechemoattractantprotein-1(MCP-1)intheformation of lesions of endodontic origin.Rationale:Inflammation resulting from tissue injury or exposure to pathogenic stimuli are known to cause release of inflammatory mediators. The release of one of these mediators,IL-1,subsequentlystimulatestheosteoblaststoexpressthemonocytechemoattractantprotein-1(MCP-1)gene.Inseveralstudies,MCP-1hasbeendocumented to attract monocytes, memory T-lymphocytes, and natural killer cells. Inmodelsofinflammation,MCP-1deficientmicewereunabletorecruitmonocytes,suggestingthatMCP-1playsanintegralanduniqueroleinattractingmonocytesto the sites of inflammation. The expression of this gene has been documented in severaldisorderscharacterizedbymononuclearinfiltrates,andhasbeenshowntocontribute to the inflammatory component of these diseases. In this study, we will determinethefunctionalsignificanceoftheexpressionofMCP-1asrelatedtothelesions of endodontic origin.

17

Specific Aims:Inthisstudy,wewillexaminetheeffectofMCP-1deletionintransgenicmiceonendodontic lesion as measured by the following factors:1) Thesizeofthelesion2) The recruitment of monocytes

Sample Abstract

Role of E-cadherin Junctions in Sjogren's DiseaseD.M.AFSHAR1,S.KHALIL1,L.BAN2,D.FAUSTMAN2,andM.KUKURUZINSKA1,1BostonUniversity,Boston,MA,USA,2HarvardUniversity,Charlestown,MA,USA

Objectives: Sjogren disease is an autoimmune systemic inflammatory disorder that affects a number of organs including salivary glands. Current understanding is that altered cell-cell adhesion of autoimmune target organs occurs prior to the establishment of lymphocytic infiltrates. The goal of this study was to gain insights into the cell biology of the developing submandibular glands (SMG) from aNODmouse,amodelfordiabetesandSjogren-likedisease.Ourhypothesisisthat dysfunctional cell-cell adhesion in the developing SMG renders it a target for lymphocytic infiltration. Here, we investigated E-cadherin, the major salivary cell-cell adhesionreceptor,intheembryonicstagedSMGsfromtheNODmousetoassesstheir functional status and effect on branching morphogenesis and cytodifferentiation. Methods:SubmandibularglandrudimentsweredissectedfromE13.5andE18NODmiceandcultured.Isolatedglandswerefixed,permeabilizedandblockedovernight.The glands were then stained for E-cadherin and actin cytoskeleton using the indirect immunofluorescencestainingmethod.PrimaryantibodytoE-cadherinwasobtainedfromBDTransductionandthesecondaryantibody,AfiiniPureFabfragmentgoatanti-mouseIgGfromJacksonImmunoReseachLaboratories.Phallodin,astainforfilamentousactin(F-actin),waspurchasedfromMolecularProbes.Theslideswereanalyzedusingconfocalmicroscopy.Results:E13.5SMGsfromNODmicedisplayedaltered morphology. Indirect immunofluorescence staining of E-cadherin showed mislocalizeddistributionofE-cadherinjunctionalcomplexeswithapronouncedlackoftargetingtotheapicallateralcell-cellborders.PhalloidinstainingforF-actinrevealeddisorganizationoftheactincytoskeletonandthiscorrelatedwiththelossofsalivarycellpolarity.Similarly,apopulationofSMGsatE18displayeddiscohesivemorphology, altered acinar structures and an apparent collapse of ductal structures. Conclusion:Ourstudiesshowthatimpairedcell-celladhesionintheembryonicallydeveloping SMG may explain the susceptibility of this tissue to autoimmunity. SupportedbygrantsPHSRO1DE10183andRO1DE14437.

18

Sample Research Report

FunctionalSignificanceofFoxO1AinOsteoblasticDifferentiationJason Conn, Camille Siquiera, and Dana GravesBoston University Henry M. Goldman School of Dental Medicine

INTRODUCTION

TheFOXO,orForkheadBox-containingprotein,family(FoxO1/FKHR,FoxO3a/FKHRL1,FoxO4/AFX,andFoxO6)isastructurallyrelatedgroupofwingedhelixtranscriptionalfactors. These factors have been shown to regulate a variety of critically important cellularprocessesincludingapoptosis,metabolism,DNArepair,cellcyclearrest,differentiation,anddefenseagainstoxidativestress.FortheseeventstooccurFOXOrequires dephosphorylation and translocation to the nucleus, where it acts by binding tospecificDNAsequenceregions,therebyfunctioningasatranscriptionalactivator.FOXOisdeactivatedviaphosphorylationbytheAktfamilyofproteinsinresponsetoinsulin, by this means being retained in the cytosol unable to act on its target.WhileFOXO’smechanismandmethodisemerginginrecentliterature,itisnotyetcleartheroleitplaysinassortedcelllines.Thisfact,teamedwithFOXO’sapparent encompassing and dynamic regulatory mechanism, was the foundation of thisproject.TheaimwastodetermineFOXO’ssignificanceinmesenchymalcells,specificallyinthedifferentiationofosteoblasts.Aclonalmurinecalvarial-derivedcell line (MC3T3-E1) was used which performs a developmental progression similar to osteoblasts. It exhibits early proliferation without differentiation followed by osteoblastic differentiation with the expression of alkaline phosphatase, formation of extra cellular matrix and the production of osteocalcin.

METHODS

TodeterminetheroleofFOXO1inthedifferentiationofosteoblasts,MC3T3cellswere cultured and treated with ascorbic acid and beta-glycerolphosphate (as detailed below)for14daystotriggerosteoblasticdifferentiation.72hoursintothistreatment,smallinterruptingRNA(siRNA)wasintroducedaseitherFOXO1orscrambled.RNAwascollectedat7and14daysandRealTimePCRofFOXO1andotherdifferentiationrelated factors was performed. Cell Culture.Pre-osteoblast(MC3T3)mousecellswereculturedusingAlphaMEMmediawith10%filteredFetalBovineSerum(FBS),1%penicillin/streptomycinand1%non-essentialaminoacidsina37oChumidifiedatmospherewith5%CO2.Passageswereperformedatorbelow80%confluence.Transfection.72hourspriortotransfection20Lof0.03MAscorbicacidwasintroducedtocultures.TransfectionwasperformedutilizingHiperfect(12μL),silencingRNA(1μLofeach881&746)orscrambledRNA(2L)in400μL of0.5%FBS.Transfectionwascontinuedfor24hours.Followingtransfection,100μL/10mLof1.08g/5mLbeta-glycerolphosphatealongwiththeascorbicacidin10%FBS

19

wascontinueduntilcollection.CollectionpointswereDay7andDay14followingtransfection.RNAextraction.Mediawasremovedatcollectiontimeandcultureswererapidlyfrozenutilizingliquidnitrogen.RNAextractionwasperformedutilizingtheQiagenmiRNeasykitandprotocol.Reverse Transcription. 2μgofRNAwascombinedwith6.61μLMgCl2,6μLdNTPs,3μLRTbuffer1.5μLrandomhexomers,1.89μLreversetranscriptaseand0.6μL RNAseinhibitor.RT-PCR.4μLofcDNAwasdilutedin196μLDNAse-freewater.9μLofthedilutewascombined with 10μLofPCRMasterMix(concentratedsolutionofDNAPolymerase,dNTPsandallthecomponentsneededforPCRexceptprimers)and1μLofTaqManFOXO1probe.

RESULTS

TheRT-PCRofFOXO1mRNAshowsthatthesmallinterferingRNAdecreasedgeneexpressionofFOXO1whencomparedtoscrambledsiRNAorthedifferentiatedosteoblasts,thoughonlybyasignificantlevelafter14days.After7daysoftreatment,resultsshowlittlechangeinFOXOexpressioninanyofthegroups.Thereisapproximatelya4-foldincreaseinFOXOexpressioncomparingthescrambledwiththezerodaycontrolwhilea2folddecreasecomparingtheFOXOsiRNAtoscrambled.

20

ThesecondgeneexaminedbyRT-PCRwasRunt-RelatedTranscriptionFactor2(RUNX2)whichisafactorknowntobeessentialforosteoblastdifferentiation.The14dayscrambledandcontrolgroupsshowa9-foldincreaseinRUNX2mRNAexpression,confirmingdifferentiation,whiletheFOXO1siRNAshowsa3-folddecreaseinRUNX2expressionwhencomparedtothescrambledandcontrol.

21

Anotherindicatorfordifferentiationwastested.Osteocalcin,anoncollagenousprotein manufactured by osteoblasts and found in bone, is a common biochemical markerforboneformation.ThiswasalsotestedbyRT-PCRshowinga35-foldincreaseinthescrambledandcontrolledgroupswhencomparedtothezeropointwitha7-folddecreasewhentheFOXOsiRNAwascomparedtothescrambled.

22

Type-1 Collagen represents an organic component of bone matrix. The gene Col1 (A1&A2)wasalsotestedbyRT-PCR.Therewasa3-foldincreaseinthescrambledandcontrolgroupswhencomparedtothezerotimepoint,whilea2-folddecreasewhenFOXO1siRNAwascomparedwithscrambled.

SMAD5,amemberoftheTGFβ family of modulators for bone morphogenetic proteins(BMPs),wasalsoexamined.Thescrambledandcontrolshoweda3-foldincreasecomparedwiththezeropoint,whiletheFOXOsiRNAdisplayeda2-folddecrease compared with the scrambled.

LastlyMatrixmetalloproteinase13(Collaginase3),asecretedpeptideknowntodegradecollageninnormalprocesseslikeremodeling,wasalsoexamined.The14dayscrambledandcontrolshoweda40-foldincreasewhencomparedwiththezerodaypointwhiletheFOXOsiRNAshoweda2-folddecreasewhencomparedwiththescrambled.

DISCUSSION

TheaimofthisprojectwastodeterminethesignificanceofFOXO1inthedifferentiation of osteoblasts. To achieve this, three successive goals were required. ThefirstwastodemonstratethesuccessfulabilitytoadequatelysilenceFOXO’stranslationbytheintroductionofsiRNAtonearconfluentpre-osteoblastsattheonsetofdifferentiation.ThiswasshownwiththeupregulationofFOXOmRNAin14dayscrambledandcontrolgroupswitha2-folddecreaseintheintendedsilencegroup. The second objective was to confirm MC3T3 differentiation to osteoblasts. This was achieved by showing up regulation in several common osteoblast genes (RUNX2,Osteocalcin,Col1,SMAD5,MMP13)frompre-treatmentto14dayspost

23

differentiationtreatment.Lastly,byachievingtheprevioustwo,itwasshownthatFOXO1holdsaproportionalrelationshiptoseveralknownosteoblastdifferentiationfactorsbyshowingthatimpairingFOXO1translationresultedinadownregulationoftheaforementionedgenes.Withthat,ithasbecomeevidentthatFOXOplaysanimportant regulatory role in the differentiation of osteoblasts.

REFERENCES

1. DominiqueAGlauserandWernerSchlegel,TheemergingroleofFOXOtranscriptionfactorsinpancreaticbcells,JournalofEndocrinology(2007)193,195–207.

2. P. Bialek, B. Kern, X. Yang, M. Schrock, D. Sosic, N. Hong, H. Wu, K. Yu, D.Ornitz, E. Olson, A Twist CodeDetermines theOnset ofOsteoblast Differentiation.DevelopmentalCell(2004),Volume6,Issue3,423-435

3. MorimichiMizunoandYoshinoriKuboki,Osteoblast-RelatedGeneExpressionofBoneMarrowCellsduringtheOsteoblasticDifferentiationInducedbyTypeICollagen.J.Biochem,2001,Vol.129,No.1133-138

4. Lu H. Kraut D. Gerstenfeld LC. Graves DT. Diabetes interferes with the boneformation by affecting the expression of transcription factors that regulate osteoblast differentiation.Endocrinology.144(1):346-52,2003Jan.

RELATED LINKS

NationalInstituteofDentalandCraniofacialResearch:http://www.nidcr.nih.gov/

Loanrepaymentprogram:http://www.lrp.nih.gov/

Dentalresearchorganizations:

http://www.dentalresearch.org/

http://www.aadronline.org/

Fellowships:

http://www.bu.edu/dental-research