Grant Progress Report Total Project Period Frommbrs.uprm.edu/esac/docs/progress.pdf · 2007. 4....

Department of Health and Human Services Public Health Services

Review Group

Type

Activity

Grant Number 5 S06 GM08103-31

Grant Progress Report Total Project Period From: 05-28-79 Through: 04-30-07

Requested Budget Period: From: 05-01-05 Through: 04-03-06

1. TITLE OF PROJECT

MBRS-SCORE Program at University of Puerto Rico at Mayaguez 2a. PRINCIPAL INVESTIGATOR OR PROGRAM DIRECTOR (Name and address, street, city, state, zip code) Nadathur, Govind S. University of Puerto Rico MBRS-SCORE Program P. O. Box 9029 Mayagüez, Puerto Rico 00681-9029

3. APPLICANT ORGANIZATION (Name and address, street, city, state, zip code) UNIVERSITY OF PUERTO RICO MAYAGUEZ CAMPUS MAYAGAUEZ, PUERTO RICO

2b. E-MAIL ADDRESS

[email protected] 4. ENTITY IDENTIFICATION NUMBER

1900002459A1 2c. DEPARTMENT, SERVICE, LABORATORY, OR EQUIVALENT

None 5. TITLE AND ADDRESS OF ADMINISTRATIVE OFFICIAL Finance Officer Research And Development Center University of Puerto Rico Mayaguez, Puerto Rico 00681-9001

2d. MAJOR SUBDIVISION

Research and Development Center

E-MAIL: [email protected] 6. HUMAN SUBJECTS 7. VERTEBRATE ANIMALS

No

Yes

6a. Research Exempt No Yes

6b. Human Subjects Assurance No.

No

Yes

7a. If “Yes,” IACUC approval Date

If Exempt (“Yes” in 6a): Exemption No.

6c. NIH-Defined Phase III Clinical Trial No Yes

7b. Animal Welfare Assurance No.

If Not Exempt (“No” in 6a): Full IRB or

IRB approval date Expedited Review

8. COSTS REQUESTED FOR NEXT BUDGET PERIOD 9. INVENTIONS AND PATENTS

8a. DIRECT $ 8b. TOTAL $ No Yes If “Yes,” Previously Reported Not Previously Reported 11a. PRINCIPAL INVESTIGATOR OR PROGRAM DIRECTOR (Item 2a)

Govind S. Nadathur

TEL 787-834-3165

FAX 787-834-3165

11b. ADMINISTRATIVE OFFICIAL NAME (Item 5) Mayra Borrero

TEL 787-832-4040 Ext. 5209

FAX 787-265-1675

11c. NAME AND TITLE OF OFFICIAL SIGNING FOR APPLICANT ORGANIZATION (Item 14) NAME Dr. Fernando J. Bird TITLE Director Research & Development Center TEL 787-831-2065 FAX 787-831-2060

10. PERFORMANCE SITE(S) (Organizations and addresses) UNIVESITY OF PUERTO RICO MAYAGUEZ CAMPUS MAYAGUEZ, PUERTO RICO

E-MAIL [email protected] 12. Corrections to Face Page 13. PRINCIPAL INVESTIGATOR/PROGRAM DIRECTOR ASSURANCE: I certify that the

statements herein are true, complete and accurate to the best of my knowledge. I am aware that any false, fictitious, or fraudulent statements or claims may subject me to criminal, civil, or administrative penalties. I agree to accept responsibility for the scientific conduct of the project and to provide the required progress reports if a grant is awarded as a result of this application.

SIGNATURE OF PI/PD NAMED IN 2a. (In ink. “Per” signature not acceptable.)

DATE

14. APPLICANT ORGANIZATION CERTIFICATION AND ACCEPTANCE: I certify that the statements herein are true, complete and accurate to the best of my knowledge, and accept the obligation to comply with Public Health Services terms and conditions if a grant is awarded as a result of this application. I am aware that any false, fictitious, or fraudulent statements or claims may subject me to criminal, civil, or administrative penalties.

SIGNATURE OF OFFICIAL NAMED IN 11c. (In ink. “Per” signature not acceptable.)

DATE

Program Director/Principal Investigator (Last, first, middle): Nadathur, Govind S.

2

TABLE OF CONTENTS

SECTION I

PAGE NUMBER

Face Page 1

Table of Contents 2

Tabular Summary PD/PI Reponses to Snap Questions

3

Tabular Summary of Research Project Related Data 4

SCORE PD and PI Other Support 5

SECTION II

Overall Summary Progress Report

6-11

Individual Scientific Subprojects Reports

Juan López-Garriga Subproject # 1

11-14

Gustavo López Subproject # 2

15-20

Enrique Meléndez Subproject # 3

21-25

Govind S. Nadathur Subproject # 4

26-28

Belinda Pastrana Subproject # 5

29-33

Madeline Torres Subproject # 6

34-36

Eduardo Juan Subproject # 7

39-41


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Tabular Summary of PD and PI Responses to SNAP Questions Grant Number: SO6GM08103-31 Reporting Period: From: 05-01-03 To: 04-30-04 Name of Institution: University of Puerto Rico at Mayagüez

PI’s Name (Last Name, First Name, Initials)

Question 1: Has there been a change in the other support of key personnel since the last reporting period?*

Question 2: Will there be in the next budget period, a significant change in the level of effort of key personnel from what was approved for this project?*

Question 3: Is it anticipated that an estimated un-obligated balance including prior year carryover will be greater than 25% of the current year’s total budget?*

Nadathur, Govind S.

Yes 1. Dr. M. Torres 2. Dr. Eduardo Juan 3. Dr. Juan López 4. Dr. Gustavo López

No

No

*If answer is “yes” provide the required explanations on a separate page, (see SNAP instructions).

1. Dr. Madeline Torres, Juan López and Gustavo López had been granted an NIH-INBRE grant with a time commitment of 15% each, Dr. Madeline Torres is the Principal Investigator. However this does not change their time and effort on the SCORE project.

2P20 RR016470-04 - M. Torres PI 09-04 – 09-09 15% NIH-INBRE $180,000/year

2. Dr. Eduardo Juan received a Medtronic, Inc. private grant for $65,363 for the period of January 2004 to September

2004 with a time commitment of 25%. However this does not change his time and effort on the SCORE Project.


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Tabular Summary of Research Project Related Data Grant Number: SO6GM08103-31 Reporting Period: From 05-01-04 To 04-30-05 Name of Institution: University of Puerto Rico at Mayagüez PI’s Name (Last, First, M.I.)

Project Title % Effort on Project

Status of Regulatory Approvals

Number of Publications Acknowledging SCORE Supporta

Typeb and # of Technical Personnel

López-Garriga, Juan

The Unique Hemoglobin I of L. Pectinata: A Study of the Heme (*FeIV=O) Moiety

25% AY 100% Summer

N/A

5 1 submitted

P.T. 1 F.T. 1

López-Quiñones, Gustavo

Squeeze-out and re-uptake of lung surfactant components

25% AY 100% Summer

N/A

2

F.T. 2

Meléndez, Enrique

Design of Ti(IV) and Mo(IV) Complexes with Antitumor Properties: Synthesis and Biochemical Characterization

25% AY 100% Summer

N/A

5

F.T. 1 P.T. 1

Nadathur, Govind S.

MAP Kinase Pathways in the Yeast Debaryomyces hansenni

25% AY 100% Summer

N/A

1

F.T. 2

Pastrana-Ríos, Belinda

Conformational changes in Phosphorylated Protein

25% AY 100% Summer

N/A

2

F.T. 2

Torres-Lugo, Madeline

Mechanisms of Interaction of PEG Rich Matrices with MRP and MDR Transporters

25% AY 100 Summer

N/A

0

F.T. 1

Juan, Eduardo J.

Acoustical Guidance of Liquid-Filled Tubes and Catheters

25%AY 100% Summer

N/A

0

0

a = Published or in press

b= F.T= Full Time, P.T.= Part Time


5

SCORE PD and PI Other Support Grant Number: SO6GM08103-31 Reporting Period: From: 05-01-04 To: 04-30-05 Name of Institution: University of Puerto Rico at Mayagüez PI’s Name (Last, First, M.I.)

Grant Title and Number Funding Per Year

Funding Agency and Grant Period

Percent Effort

Nadathur, Govind S.

-Production of T. reesei mutants that cannot utilize glucose #56-3620-1-12 -Pathogenic bacteria in coastal Reefs #539151

$33,000.00 $5,600.00

USDA 2001-2004 Tropical Ocean Soruces, Inc. 2004-2005

10% 0%

López-Quiñones, Gustavo -Center for Research in Protein Structure, Function and Dynamics #P20 RR16439-01 -Molecular Studies of Proteins Encapsulated in Soft Materials M. Torres - PI 2P20 RR016470-04

$1,444,913/year $180,000/year

NIH-COBRE October 2001-2005 NIH-INBRE September 2004-2009

25% 15%

López-Garriga, Juan (PI)

-Center for Research in Protein Structure, Function and Dynamics #P20-RR16439-01 -Molecular Studies of Proteins Encapsulated in Soft Materials M. Torres - PI 2P20 RR016470-04

PI-$20,000/year Center $1,444,913/year $180,000/year

NIH-COBRE October 2001-2005 NIH-INBRE September 2004-2009

5% 15%

Meléndez, Enrique

Center for Research in Protein Structure, Function and Dynamics #P20-RR16439-01

Co-PI $60,000/year Center $1,444,913/year

NIH-COBRE October 2001-2005

25%


- Center for Research in Protein Structure, Function and Dynamics #P20-RR16439-01 -MBRS-SCORE-Squeeze-out and re-uptake of Pulmonary Surfactant Components. Co-PI # S06GM08103.

Co-PI $137,256/year Center $1,444,913 $122,667/year

NIH-COBRE October 2001-2005 NIGMS 2003-2007

30% 25%


-Àcquisition of a Confocal Laser Microscope for the Enhancement of Biotechnoloy Research at UPRM #0216081 - Molecular Studies of Proteins Encapsulated in Soft Materials 2P20 RR016470-04 -Cytoxic and Transport Studies of Derrite Nanoparticles utilizing the Caco-2 Cell Model

$26,511 PI $180,000/year $20,000

NSF August 02-04 NIH-INBRE September 2004-2009 NSF-EPSCoR

O% 15% 15%

Juan, Eduardo J.

-Bioengineering Research and Educational Experiences at UPRM Co-PI -Development of Technologies for the Manufacture of Cardiac Pacing & Defibrillation Leads: Phase II

$99,722 $65,363

NSF 09-03 – 08-04 Medtronic, Inc.

25% 25%


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PROGRESS REPORT SUMMARY GRANT NUMBER 5 S06GM08103-30

PERIOD COVERED BY THIS REPORT PRINCIPAL INVESTIGATOR OR PROGRAM DIRECTOR Govind S. Nadathur

FROM 05-01-03

THROUGH 04-30-04

APPLICANT ORGANIZATION

University of Puerto Rico at Mayagüez TITLE OF PROJECT (Repeat title shown in Item 1 on first page)

MBRS-SCORE Program at University of Puerto Rico Mayaguez Campus A. Human Subjects (Complete Item 6 on the Face Page)

Involvement of Human Subjects No Change Since Previous Submission Change

B. Vertebrate Animals (Complete Item 7 on the Face Page)

Use of Vertebrate Animals No Change Since Previous Submission Change

SEE PHS 2590 INSTRUCTIONS.

WOMEN AND MINORITY INCLUSION: See PHS 398 Instructions. Use Inclusion Enrollment Report Format Page and, if necessary, Targeted/Planned Enrollment Format Page.

A. SUMMARY PROGRESS REPORT 1. Narrative Description a. Overall goals and specific objectives of the SCORE program Our SCORE program had six goals when the competitive proposal for renewal was submitted in June 2003. The goals are presented in the following table. All but one of the objectives are quantitative. As we meet these objectives, in each case we will have shown improvement over the baseline established in the initial proposal.

Program Goal #1: Increase the competitiveness of MBRS researchers at UPRM

Measurable Objectives Evaluation Method

1. Researchers will show progress toward completion of specific aims. Aims will be articulated in such a way in SCORE proposals that their completion can be demonstrated qualitatively by completion of specific research benchmarks.

1. Progress toward completion of benchmarks will be demonstrated by researchers in their annual reports. External evaluation of progress and suggestions for corrective action will be provided by the ESAC.

2. New projects will have two peer-reviewed papers published or at least submitted by the end of four years with at least one from the MBRS project. Second cycle projects will have four papers published or submitted with at least two from the MBRS project.

2. Total number of peer-reviewed papers submitted or published and MBRS papers submitted and published will be tabulated from the annual progress reports of each project.

3. New projects will have two national MBRS-related presentations during the four year period. Second cycle projects will average at least one national MBRS-related presentation per year.

3. Numbers of MBRS-related presentations at national research meetings will be tabulated from the annual reports of each project.


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4. Fifty percent of PI’s of first cycle projects will have other support in effect or submitted by the date of the renewal application. All PI’s of second cycle projects will have other support in effect or submitted by the date of the renewal application.

4. Total number of research grants in effect or submitted at the date of the renewal application will be tabulated for each project from the form page describing other support and by interview for projects that are not renewing.

Program Goal #2: Increase the competitiveness of UPRM science and engineering departments through an increase in number of investigators carrying out meritorious biomedical and behavioral research

Measurable Objectives Evaluation Method

1. At least two additional researchers will be recruited to submit supplements during the first 18 months of the award, and at least two researchers new to MBRS will be recruited to submit as part of the next SCORE proposal.

1. Total number of additional researchers submitting supplemental projects will be tabulated. Recruitment of new researchers will be demonstrated by their presence in the next SCORE proposal.

2. At least 50% of the projects in the next SCORE proposal will have an active collaborator or associate investigator.

2. Recruitment of additional collaborators will be demonstrated by their presence in the next SCORE proposal.

b. Progress made toward completing objectives Our current program started on May 1, 2003. It includes seven research subprojects with seven PI’s or CI’s from two Departments in the College of Arts and Sciences and two Departments in the College of Engineering: Seven projects were funded with the initial award.

• Eduardo Juan, Department of Electrical Engineering. Accoustical Guidance of liquid-filled tubes and catheters. This is a new pilot project.

• Gustavo Lopez and Belinda Pastrana, Department of Chemistry. Squeeze-out and re-uptake of lung

surfactant components. This is a new project.

• Juan López-Garriga, Department of Chemistry. The unique Hemoglobin I of L. pectinata: a study of the heme (FeIV=O) moiety. This is a continuation project.

• Enrique Melendez, Department of Chemistry. Design of Ti(IV) and Mo(IV) Complexes with antitumor

properties: synthesis and biochemical characterization. This is a continuation project.

• Govind Nadathur, department of Marine Sciences. MAP kinase pathways in the yeast Debaryomyces hansenii. This is a continuation project.

• Belinda Pastrana-Rios, Department of Chemistry. Changes in Conformation of Phosphorylated Proteins.

This is a continuation project

• Madeline Torres-Lugo Department of Chemical Engineering. Mechanisms of Interaction of PEG rich matrices with MRP and MDR efflux pumps. This is a new project


8

Progress toward completing the measurable objectives of each goal is summarized in the following paragraphs. Goal 1, objective 1. The present report is being written nine months after initiation of funding for this four year cycle. At the time of this writing all of the projects show some scientific progress. Dr. Eduardo Juan, in his project has focused on aim 1 and has utilized time-domain acoustic reflectometry to determine the geometry of sequentially interconnected liquid-filled tubes having rigid walls. He has also done work on determining analytically and experimentally the frequency range over which a liquid filled tube with complaint walls can be assumed to behave acoustically as a rigid conduit.

Drs. Gustavo Lopez and Belinda Pastrana have made progress in all four of their aims. Their research efforts this year has significantly expanded their research since it has allowed them to use experimental and computational techniques to study biomolecular systems.

Dr. Juan López-Garriga has made significant progress in all the aims of the proposal. Their expression techniques for HB1 mutant proteins has been standardized and used to produce six different mutants for analysis.

Dr. Govind Nadathur has made significant progress in both aims of the proposal. He has now analyzed the 5.1 kb fragment containing the LEU2 and the SPL1 gene of Debarymyces hansenii. He is also standardizing techniques for creating knock-out mutations in this organism.

Dr. Enrique Meléndez has developed analytical methodology for the study of metal DNA interactions as well as Ti-transferrin interaction utilizing ICP spectroscopy and NMR. Dr. Belinda Pastrana has made progress in all of the aims and has performed additional studies to understand centrin’s structure/function/stability. Dr. Madeline Torres-Lugo has made progress in all the specific aims of the project. In addition to the synthesis of PEG rich hydrogel matrices, she has also created a PEG and methacrylic acid full interpenetrated networks. The new ESAC committee met in the third week of March 2004 and offered suggestions and recommendation on our program. A number of their recommendations those of the administrative and research area have now been implemented. The significant implementations are given in the program highlights section. Goal 1, objective 2. Fifteen papers have been published or submitted in the current year of funding. If these numbers are calculated for the seven projects then the publication rates are over two papers per investigator per year. With these rates we are on track to exceed the objective for new projects of two papers submitted or published after four years with one paper from the project; and the objective for second cycle projects of four papers after four years with two from the project. Goal 1, objective 3. Thirty six presentations at professional meetings have been made by the seven funded projects. The overall presentation rate is over 2/project/year. Goal 1, objective 4. Six of the seven PIs have other support active or submitted at this time. Goal 2, objective 1. Seven projects were submitted for the 2003 supplemental application. In 2004, we submitted another supplemental with a total of five projects two of which were pilot projects. It was disappointing to note that none of the projects in the supplemental applications were funded. To encourage the submission of good fundable proposals, our program with the help of the University of Puerto Rico, Mayaguez has started a seed funding program to help researchers to initiate research on a project to acquire


9

preliminary data before submitting a subproject to MBRS. Each researcher in the seed funding program has a mentor who is a senior person with funding history in biomedical research. The researchers also make a commitment to submit a proposal in the next cycle. With this new strategy in place we hope to increase the number of younger researchers submitting proposals. Goal 2, objective 2. Of the seven proposals funded one has a co-investigator while nearly all the others have collaborators. 2. Program Highlights Implementation of the research initiatives supported by the MBRS-SCORE grant during the two years of this cycle has resulted in accomplishment of some of the research goals, improvement of the research infrastructure through purchase of new equipment, strengthening collaborations with researchers from outside our campus, and the enrichment of the research environment of the participating departments through the hiring of research associates to work on the projects. Additional highlights of the project are:

1. Establishment of a web site for the NIH-MBRS SCORE program at the Mayaguez Campus (http://mbrs.uprm.edu). This web site highlights the program and gives the latest news, researchers and areas of endeavor.

2. Establishment of seed money research funding for biomedical research to encourage younger researchers to initiate biomedically oriented projects.

3. One student fellowship awarded by the College of Arts and Sciences for students doing research in SCORE funding Laboratories.

Research accomplishments are summarized in the discussion of progress toward meeting objective 1, goal 1. The most significant accomplishment may be the synthesis of the PEG and methacrylic acid full interpenetrated networks. Major equipment purchased during this period include a Dual Xeon 3.06 GZ master node and 7 compute nodes, Dual Xeon 3.2 Ghz EM64T Linux workstation and two Pentium processors (3.5GHz) with HT technology. This P/C cluster is being used for Monte Carlo computer simulations of surfactant molecules at the air/water interface in the Pulmonary Surfactant project. The computer cluster was installed for the Antitumor Properties project to do molecular modeling and analyze NMR data. In the Unique Hemoglobin project the acquisition of a Stopped Flow Cell as part of the Laser Flash Photolysis allows the measurement of kinetic reactions between proteins, which is very useful to study the formation of the ferryl intermediate from the reaction of Hemoglobin I from Luctina pectinata and hydrogen peroxide. Digital Multimeter, Oscilloscope and Wave Form Generator was purchased for measurement, calibration and for testing the components of the experimental setup used in the Acoustical Guidance project. A 2100 Bioanlayzer desktop system was purchased for the Phosphorylated proteins project. This microship technology based instrument is used for analyzing DNA or protein to determine purity and size, and for the analysis of eluted fractions from chromatographic separations of the human centrin protein. These items increase the inventory of research equipment in the participating departments and thereby strengthen infrastructure of the program. The MBRS SCORE program at UPR, Mayaguez seems to have a major impact on the scholarly productivity of its fundees. In general SCORE researchers seem to be the more productive in publishing their results, presentations at national and international meetings as well as securing other support. This is specially significant to our campus since we do not have a medical school and the number of biomedical researchers is relatively small. Since MBRS SCORE is the only program in our campus that targets such researchers specifically it has a large impact on research productivity.


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SCORE Project PI Research Productivity Data Grant Number: SO6GM08103-30 Reporting Period: From: 05-01-03 To: 04-30-04 Name of Institution : Unversity of Puerto Rico at Mayagüez PI’s Name (Last, First, M.I.) Number of

Publications, (SCORE + Other)

Number of Presentations at National Meetings

Number of Grants Submitted

Number of Grants Awarded

Number of Patent Applications pending

Number of Patents Awarded

López-Quiñones, Gustavo

2

2

0

1

0

0

López-Garriga, Juan

5 1 submitted

-13 (7 invited seminars, 6 posters presentations) -13 grad students -4 undergraduate students -3 High school student

1

0

0

0

Meléndez, Enrique

5

1

1

0

0

0

Nadathur, Govind S.

1 + 1

1

1

1

0

0


3

13

1

0

1

0


0

1

3

1

0

0

Juan, Eduardo J.

0

1

1

1

0

0


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MBRS SCORE Grant Annual Progress Report Face Page for Investigator–Initiated Research Project Face Page for Investigator–Initiated Research Project Grant Number: S06 GM 08103-31 __ Project Number____1____. Institution: University of Puerto Rico Mayagüez Campus ________________________________________________________________________ 1. Descriptive Title: (56 characters or less, including spaces) The Unique Hemoglobin I of L. Pectinata: A Study of the Heme (FeIV=O) Moiety ________________________________________________________________________ 2. Investigator (s) Last Name: First M.I. Degree Department Lopez-Garriga Juan PhD Chemistry 3a. Supplemental Project : Yes:_____ No___X__. If yes, award date:______ 3b. Are the SNAP procedures followed? Yes:__X__ No:____. If no, Budget pages submitted: Yes:____ No:____. 4a. Regular Research Project ____X___ Pilot Research Project _____. 4b. Inventions and Patents Yes:____ No:_ __X__ If “yes”, include details in progress report narrative. 5. Total number of research workers employed on the project: Full time___1_ Part time___1__ 6. Protection Against Research Risks: A. Were human subjects involved in the project? Yes:____ No:__X__ If yes, were the procedures approved by your IRB? Yes:____ No:_____ IRB approval termination date:________ B. Were vertebrate animals used? Yes::____ No: __X___ If yes, were the procedures approved by your IACUC? Yes:____ No:_____ IACUC approval termination date:_________ C. Are there potential hazards (including biohazards) to laboratory workers (carcinogens, pathogens, ionizing radiation, etc.) engaged in the research? Yes: __X__ No: ______ If yes, was training in biohazards conducted? Yes: __X__ No:_____


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PROGRESS REPORT Grant number: S 06 GM 08103 Period: March 2004 - March 2005

Title: “The Unique Hemoglobin I of L. Pectinata: A Study of the Heme (FeIV=O) Moiety Researcher: Juan Lopez-Garriga Introduction:

Hemoglobin I (HbI) from Lucina pectinata is part of a symbiotic interaction between an anaerobic bacteria and the clam. This unique hemoglobin binds and transports H2S from the clam environment to the bacteria. The unusual function of this hemeprotein is accomplished when the heme ferric high spin species binds H2S to form a six coordinated low spin ferric sulfide derivatives and then delivers the ligand to the bacteria. HbI has Phe(68) instead of Val at position E11, Phe(29) in place of Leu at position B10 and Gln(64) as a substitute of His at position E7 or distal position. Our results on the function of Gln64 strengthened the role of distal face functional groups on heterolytic O-O bond cleavage of H2O2. (De Jesús-Bonilla W., Cortes, López-Garriga J., (2001). Arch. Biochem.Biophys., 390, 304-308. Stopped-flow kinetics and resonance Raman showed the formation of both, met-aquo HbI and compound II ferryl species in the cyclic reaction of HbI with H2O2. The ferryl vibration frequency appears for compound II at 805 cm-1 and 769 cm -1 for HbIFeIV=16O and HbIFeIV=18O species, respectively. This ferryl mode indicates the absence of a hydrogen bonding between the carbonyl group of the distal Gln64 and the HbIFeIV=O ferryl moiety. The observation suggests that both the trans-ligand effect and the polarizabilty of the HbI heme pocket are responsible for the observed ferryl oxo vibrational energy. The results also suggested that the carbonyl group of the distal Q64 is oriented toward the iron of the heme group increasing the distal pocket electron density. (De Jesús, W., Ramírez, E., J, López-Garriga, J. Biopolymers (Biospectroscopy) 2002,62,176-185. a. Specific Aims of the Proposal.

Regarding this, the proposal focus to clarify the interplay of these factors and the stability of the HbIFeIV=O ferryl group. To understand the factors that control the heme FeIV=O structure is very important since this ferryl intermediate is present also in the reaction path of H2O2 with other heme-proteins like hemoglobin, myoglobin, peroxidases and catalases. The proposed work to address the issue by following the formation of HbI mutants their heme-ferryl derivative by stopped flow kinetics and time resolved resonance Raman. The aims of the original proposal have NOT been modified to achieve the proposed goals. b. Studies and Results.

Aim 1. Mutated HbI protein will be expressed in large quantities and purified from E. coli using the same techniques as for the recombinant HbI species. In collaboration with Dr. Carmen Cadilla from UPR-Medical School, the cDNA-derived amino acid sequence was obtained from overlapping clones containing the cDNA that codes for HbI. The Reverse Transcriptase-Polymerase Chain Reaction and Rapid Amplification of cDNA Ends methods were employed to synthesize the cDNA fragments. An initial 354 bp cDNA clone encoding 118 amino acid residues of HbI was amplified from total RNA by RT-PCR using degenerate oligonucleotides. The coding region of the full-length cDNA translates for 143 amino acid residues. Expression studies of this protein were performed in E.coli . Hemoglobin I cDNA was amplified and cloned into the pBAD-TOPO expression vector, which contains a fusion tag of 6 histidine residues. The highest yield of recombinant HbI (up to 70mg of pure HbI from 1L of TB culture medium, adding glucose to a final concentration of 5%) was obtained when the HbI cDNA was incorporated in the pET28 plasmid and expressed in BLi5 cells. This result also allowed to produce all the mutants proposed, these are presented in Table 1.

Aim 3. Determine the kinetics of the transient ferryl species upon reaction of HbI from L. pectinata and its mutants with H2O2. Transient kinetic measurement for the reaction between HbI and H2O2 (at different concentrations) will be also determined upon photolysis of the HbICO moiety. This will establish the kinetic constant for the formation and disappearance of the transient species. This aim has been 50% achieved by nanosecond flash photolysis experiment upon measuring the association and dissociation


13

constants of the HbICO and HbIO2 mutants presented in table 1. The results show a clear dependence in the amino acid size and polarity to determine HbI conformation changes and ligand dynamics.

Aim 4. Correlate the vibrational frequencies and kinetics corresponding to HbIFeIV=O mutated species with the Fe-His vibrational frequency of the same heme-protein. This will help to elucidate the ligand trans-effect role on the stability of HbI ferryl intermediate. This aim has been 50% achieved since we have determined by resonance Raman the Fe-His vibrational frequencies of the mutants presented in table 1. The results show that the amino acids in the heme pocket do not influence the Fe-His vibrational mode.

Aim 5. Pursue research cooperation with experts in particular fields of 1HNMR and single crystal X-ray crystallography in the time period of the proposed research. This is a long term study, but during this period has been obtained NMR spectra of the HbISH2, HbICN, and HbIPheB10Tyr mutant, which reveled the presence of hydrogen bonding between H2S in the HbISH2 complex and the carbonyl group of GlnE7. Single cristals has been obtained for the HbI, HbII, and HbIPheB10Tyr mutant but until now they have not been suitable for X-ray structure determination.

Table 1. HbI Proposed mutants Position 64

Position 29

Position 68

Nature of change

Gln Phe Phe Wild type HbI heme pocket His Phe Phe Single change in position 64 as in SWMb (sperm whale

myoglobin) Val Phe Phe Single amino acid change in position 64that changes

the HbI polar environment Gln Phe Tyr Single amino acid change in position 68 as in HbII His Leu Val Changes in positions 29, 64 and 68 as in SWMb His Phe Val Change in position 68 from one nonpolar amino acid to

another, in addition to change in position 64 to His His Leu Phe Change in position 29 from one nonpolar amino acid to

another, in addition to change in position 64 to His I n this period of the proposal we published five manuscripts in the literature manuscript have been submitted for publication, and five extended abstracts also have been published. Publications

1. E. Collazo, R. Pietri, W. De Jesus, C. Ramos, A. Del Toro, R., Leon, C., Cadilla, J. Lopez-Garriga. Functional Characterization of the Purified Holo form of Hemoglobin I from Lucina pectinata Over-Expressed in Escherichia coli. The Protein Journal. 2004, 23, 239-245. 2. E. Ramirez, C. Ramos, M. Rodríguez, R. Pietri, M. Chergui, J.Lopez-Garriga. Ultrafast Studies of Ferric Complexes of Hemoglobin I from Lucina pectinata. Femtochemistry and Femtobiology 2004 (Ed. M. M. Martin, J. T. Hynes) Elsevier N.Y pag. 395-398. 3. Helbing, J., Bredenbeck, J., Hamm, P. Bonacina, L., van Mouririk, F., Chaussard, F., Gonzalez, A., Chergui, M., Pietry, R., Ramos, C., Ruiz, C., Lopez-Garriga, J. Time-resolved visible and infrared study of the cyano complexes of Myoglobin and Hemoglobin I from Lucina Pectinata Biophysical Journal, 2004, 87, 1-11. 4. Ruth Gretchen León, Hélène Munier-Lehmann, Octavian Barzu, Véronique Baudin Creuza, Ruth Pietri, Juan López-Garriga and Carmen L. Cadilla. High-level production of recombinant wild type and mutant sulfide-reactive hemoglobin I (HbI) from Lucina pectinata in Escherichia coli. Protein Expression and Purification, 2004, 38, 184-195. 5. Tyrosine B10 and Heme-ligand Interactions of Lucina pectinata Hemeglobin II: Control of Heme Reactivity. Ruth Pietri, Laura Granell, Anthony Cruz, Walleska De Jesús, Ariel Lewis, Ruth Leon, Carmen Cadilla, Juan López Garriga. BBA - Proteins and Proteomics 2004 Accepted for publication.


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Submitted Publications

1. NMR Investigation of the influence of Distal Gln E7 on the Structural Properties of Lucina pectinata. B. J. Ramos, R. Pietri, J.López-Garriga, Biochemistry 2005. C. Signicance.

The results presented here indicate that the Aim 1 has fully been achieved, and the problem of producing recombinant and mutants forms of HbI in relatively large volume and concentration, that in the past it was a main limitation for the research progress, has been solved. Similarly Aims 3 and 4 have been 50% accomplished and Aims 5 has produced interesting NMR results confirning the presence of hydrogen bonding between Gln64 and the hemeSH2 moiety. Thus, a windows has been open to used HbI and its mutated analogs to understand the stability of the HbIFeIV=O derivative and its peroxidative reactions that are important for the mechanism cell function decline. D. Plans.

We will continue the progress of the proposed research by pursuing the original establish aims without any change in the following order. Aim 2. Determine the structure of the ferryl stable intermediates upon reaction of HbI from L. pectinata and its mutants with H2O2. Time-resolved resonance Raman spectroscopy coupled to isotopic ligand substitution will allow detection of the ferryl-heme moiety. The study will clarify interactions between the ferryl structure and heme pocket amino acids (i.e. hydrogen bonding or multipole interactions). Aim 3. Determine the kinetics of the transient ferryl species upon reaction of HbI from L. pectinata and its mutants with H2O2. Transient kinetic measurement for the reaction between HbI and H2O2 (at different concentrations) will be also determined upon photolysis of the HbICO moiety. This will establish the kinetic constant for the formation and disappearance of the transient species. Aim 4. Correlate the vibrational frequencies and kinetics corresponding to HbIFeIV=O mutated species with the Fe-His vibrational frequency of the same heme-protein. This will help to elucidate the ligand trans-effect role on the stability of HbI ferryl intermediate. Aim 5. Pursue research cooperation with experts in particular fields of 1HNMR and single crystal X-ray crystallography in the time period of the proposed research. We will continue trying to obtain single crystals for the HbI and mutants suitable X-ray structure determination. Also we will continue the determination of the complexes by NMR spectroscopy.


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MBRS SCORE Grant Annual Progress Report Face Page for Investigator–Initiated Research Project Grant Number: S06 GM 08103-31 Project Number___________2__________. Institution: University of Puerto Rico at Mayagüez 1. Descriptive Title: (56 characters or less, including spaces) Squeeze out and re-uptake of Pulmonary Surfactant Components 2. Investigator (s) Last Name: First M.I. Degree Department López Gustavo Ph.D Chemistry 3a. Supplemental Project : Yes:_____ No__X__. If yes, award date:______ 3b. Are the SNAP procedures followed? Yes:_ X___ No:____. If no, Budget pages submitted: Yes:____ No:____. 4a. Regular Research Projec t ___X___ Pilot Research Project _____. 4b. Inventions and Patents Yes: ____ No: __X__ If “yes”, include details in progress report narrative. 5. Total number of research workers employed on the project: Full time__1__ Part time_____ 6. Protection Against Research Risks: A. Were human subjects involved in the project? Yes:__ __ No: _X____ If yes, were the procedures approved by your IRB? Yes:____ No:_____ IRB approval termination date:__ ______ B. Were vertebrate animals used? Yes::____ No: _X_ If yes, were the procedures approved by your IACUC? Yes:____ No:_____ IACUC approval termination date:_________ C. Are there potential hazards (including biohazards) to laboratory workers (carcinogens, pathogens, ionizing radiation, etc.) engaged in the research? Yes:_____ No: X If yes, was training in biohazards conducted? Yes:____ No:_____


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NIH-SCORE Individual Subproject Progress Report 2004 Title: Squeeze-out and re-uptake of pulmonary surfactant components Summary: Spectroscopic infrared techniques and computational models have been used to describe the components of pulmonary surfactants. A computational model based on statistical thermodynamics and coarse grain potential was developed that permit the proper description of model peptides and lipid molecules forming a Langmuir monolayer and bilayers. Infrared spectra have been taken of model peptides. SPECIFIC AIMS Specific Aim 1: To study via computational techniques the effects of SP-C on the squeeze-out and re-uptake of phospholipids. Simulate the squeeze-out and re-uptake process for binary and ternary mixtures of phospholipids.

We have implemented the NAMD software1 on a PC-cluster where all atom calculation can be performed on biomolecular systems. Specifically, the all-atom potentials developed for SP-C systems are used to describe the forces in the system and classical molecular dynamics were used to generate trajectories. The temperature was fixed to 298 K and solvation of the system was performed so the protein will not see the image in the periodic boundary conditions. The simulation was run for 10 with one, three, and seven SP-C molecules solvated in water. Figures 1a and 1b show the initial and final structures for SP-C, respectively, after 10 ns of simulation. A significant change in the amino terminal is observed which can be confirmed by the Ramachandra plots (Figure 1c and 1d) for these structures. The first seven amino acids form a random coil

with angles that favors the beta sheet secondary structures. The other portion retains the alpha helix structure. Figure 2 shows the structure for the trimer and heptamer where the random coil portions of the proteins interact with each other.

Using the all-atom MD calculations, a lipid bilayer composed of DPPC has been constructed and simulated for 2 ns (Figure 3). A total of 128 DPPC molecules where modeled at a temperature of 323 K and a pressure of 1 atm. The bilayer shows a typical fluid-like ordered structure where the tails interact with each other and the head interact with water. After stabilization of the bilayer, one SP-C molecule

was incorporated in the system where structural parameters will be calculated.

A second group of software named GROMAC2 is being implemented in our laboratory which permits more flexible and efficient modeling of macromolecular systems. Specific Aim 2. To study via infrared reflection-absorption FT-IR spectroscopy (IRRAS) the effect of SP-C on the squeeze-out and reuptake of phospholipids.

A second generation ribbon trough has been designed and is currently being tested to allow for the study of squeeze-out and re-uptake of phospholipids.


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Specific Aim 3: To simulate the squeeze-out and re-uptake process for binary and ternary mixtures of phospholipids.

A coarse grain model (CGM) was used to describe the thermodynamic properties of surfactant molecules forming an air-water interphase (Langmuir monolayer). The various liquid phases and the liquid-liquid equilibrium were characterized for surfactant molecules using this model. An article was published3 in the Journal of Chemical Physics associated to this portion of the study. We have implemented a more sophisticated CGM4 that uses stiffness potential and multipolar moments to describe the interaction between lipids. At present, the various phases of lipidic monolayers and bilayers are being considered using Monte Carlo simulations in the isothermal-isobaric ensemble. Figure 4 show a snapshot of a 500 DPPC monolayer interacting with 9000 water molecules. Variations in the heat capacity as a function of temperature are being used to characterize lipidic phases. Specific Aim 4. To compare the secondary structure of SP-C with peptide analogs using FT-IR spectroscopy.

Two peptide analogs shown in Scheme 1, have been compared with native SP-C in model lipidic environment. (1) modified SP-C is comprised of random coil and helical structure while, (2) T11 peptoid has a random coil structure. Thus, the T11 peptoid is not a candidate for native SP-C substitution.

(1) 1 10 20 30 35 FGIPFFPVHLKRLLILLLLLLLILLLILGALLMGL Scheme 1. Amino acid sequence and structure of (1) modified SP-C (MW 3,881 Da) and (2) T11 peptoid

(MW 3,309 Da). Specific Aim 5. To study the lipid-peptide interactions using ATR-FTIR and transmission FT-IR spectroscopy.

Modified SP-C peptide and T11 peptoid were reconstituted into vesicles with binary mixtures of DPPC-d62/DPPG (7:1 mol/mol) at a level of 0.5 mol % and 1 mol %, respectively. Temperature dependence of the CH2, CD2 and lipid carbonyl stretching modes in both samples were monitored. Figure 5, shows the transition temperature for CH2 symmetric stretch modes in lipid/Modified SP-C ternary mixture. The study of the C-D and C-H stretching modes permits the simultaneous evaluation of acyl chain conformational order in deuterated (DPPC-d62) and proteated (DPPG), respectively. FT-IR

melting curves constructed from the symmetric C-H (~2852 cm-1), symmetric C-D (~2092 cm-1) and lipid carbonyl (~1735 cm-1) from the DPPC-d62 and DPPG components studied. The lipid mixture undergoes its phase transition at about 38 oC, whereas in the presence of T11 (Tm ~37.0 oC) and modified SP-C (Tm ~38.5 oC) an alteration on this value is observed. T11 reduce the phase

(2)


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transition by about 1 oC, suggesting a destabilizing of the vesicles. However, a slight ordering may be inferred from the Modified SP-C data, as in native SP-C5. FT-IR spectra for T11, Modified SP-C, and binary lipid mixture are shown in Figure 6, panels A, B and C, respectively. The spectrum of T11 in the range of 1400-1800 cm-1

revealed amide I vibration near 1672 cm-1 which is characteristic of random coil. No amide II vibration was observed as was expected. Nevertheless, the Modified SP-C spectrum revealed amide I and II vibrations, typically of peptides and proteins. Amide I vibration was observed near 1655 cm-1, which is characteristic of proteins with α-helical secondary structure, with a component of random coil (~1672 cm-1). Thus, suggesting modified SP-C to be the better candidate as native SP-C substitute. The thermodynamic data (∆HvH) also confirm the relative stability of these mixtures, Table 1. (Manuscript is in preparation).

Table 1. Summary of thermal transition temperature and thermodynamic data.

Sample Lipid:protein Tm /oC ∆HvH mol ratio CH2 sym CD2 sym Lipid C=O /(cal/mol) DPPC-d62/DPPG N/A* 38.3 38.1 38.1 -98.25 Lipid/T11 100:1 37.0 36.9 37.0 -187.04 Lipid/Mod. SP-C 200:1 38.5 37.6 38.5 -296.57

*Lipid mol ratio was 7:1. Specific Aim 6. To study the lipid-peptide interactions at the A/W interface by IRRAS and determine if these peptides enhance the squeeze-out and the re-uptake process. Trough design is still being optimized for these experiments. Specific Aim 7. To simulate the peptides at an A/W interface using the experimental results obtained. This phase of the project will begin once preliminary data is obtained from the IRRAS set up (Specific Aims 2 and 6). In Summary: The research performed during this year has considerably expanded our research capabilities because it has allowed us to combine experimental and computational techniques in the study of biomolecular systems. References cited

1. Kale L., Skeel R., Bhandarkar, M, Brunner, R., Gursoy, A., Krawetz, N., Phillips, J., Shinozaki, A., Varadarajan, K., and Schulten, K.., NAMD2: Greater scalability for parallel molecular dynamics, 1999, J. Comp. Chem. 151, 283.

2. ,Berendsen, HJC, Spoel D van der , Drumen R van, GROMACS: a message-passing parallel molecular dynamics implementation 1995, Comput. Phys. Comm. 95, 43.

3. Liquid-liquid equilibrium in Langmuir monolayers; López-Álvarez, Y., Villalobos, L., Pastrana-Ríos, B., López, G.E., J. Chem. Phys. 2005 (In Press).

4. Marrink, S.J., de Vries, A.H., Mark, A.E., Coarse grained model for semiquantitative lipid simulations, J. Chem. B 2004, 108, 750.

5. Pastrana, B., Mautone, A. J., Mendelsohn, R., "Fourier Transform Infrared Studies of Secondary Structure and Orientation of Pulmonary Surfactant SP-C and Its Effect on the Dynamic Surface Properties of Phospholipids"1991 Biochemistry, 30, 10058 -10064.

Publications in Peer-reviewed Journals 1. Maury-Evertsz, Estévez, A. and López, G. E., “Effect of branching and confinement on star-branched

polymeric systems”, J. Chem. Phys. Vol 121, no. 14. 2004 2. Villalobos, L., López-Álvarez, Y., Pastrana-Ríos, B., and López, G.E., “Thermodynamics of the liquid

states of Langmuir monolayers”; J. Chem. Phys. 2005 (In Press)


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Abstracts Presented in Meetings 3. E. Rámirez, I. Plasencia, B. Pastrana-Ríos and G. López “Molecular dynamics of surfactant protein c in a

hydrophilic media” Current Trends in Biotechnology & Pharmaceutical Engineering, October 1, 2004. Mayagüez, Puerto Rico.

4. Sindia Ramos, Muriel Campbell, Gustavo López, and Belinda Pastrana-Rios “Surfactant protein C analogues as exogenous therapeutics for severe respiratory disorders” 2nd Colloquim in Protein Structure, Function and Dynamics, February 15-19, 2005, San Juan, Puerto Rico. (accepted)

Awards None


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BIOGRAPHICAL SKETCH Provide the following information for the key personnel in the order listed for Form Page 2. Follow the sample format for each person. DO NOT EXCEED FOUR PAGES. NAME Anthony Cruz Balberdi

POSITION TITLE Research Assistant

EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, and include postdoctoral training.)

INSTITUTION AND LOCATION DEGREE (if applicable)

YEAR(s) FIELD OF STUDY

University of Puerto Rico, Mayagüez, PR B.S. 1995 Chemistry University of Puerto Rico, Mayagüez, PR M.S. 1999 Biophysics

Professional Experience 2000-2003 Research Assistant, Chemistry Department, University of Puerto Rico, Mayagüez, PR 2005-present Scientific Research Specialist, Chemistry Department, University of Puerto Rico, Mayagüez, PR Publications – 2004:

1. Pietri R, Granell L, Cruz A, De Jesús W, Lewis A, Leon R, Cadilla L C and López-Garriga J. “Tyrosine B10 and heme–ligand interactions of Lucina pectinata hemoglobin II: control of heme reactivity”. 2004 Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics In Press


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MBRS SCORE Grant Annual Progress Report Face Page for Investigator–Initiated Research Project Grant Number: S06 GM 08103-31 Project Number____3_____. Institution: University of Puerto Rico at Mayagüez 1. Descriptive Title: (56 characters or less, including spaces) Design of Ti(IV) and Mo(IV) Complexes with Antitumor Properties: Synthesis and Biochemical Characterization 2. Investigator (s) Last Name: First M.I. Degree Department Meléndez, Enrique Ph.D. Chemistry Department 3a. Supplemental Project : Yes:_____ No X . If yes, award date:______ 3b. Are the SNAP procedures followed? Yes: X No: ___. If no, Budget pages submitted: Yes:____ No:____. 4a. Regular Research Project X Pilot Research Project _____. 4b. Inventions and Patents Yes :____ No: X If “yes”, include details in progress report narrative. 5. Total number of research workers employed on the project: Full time 2 Part time_____ 6. Protection Against Research Risks: A. Were human subjects involved in the project? Yes ____ No X If yes, were the procedures approved by your IRB? Yes: ____ No:_____ IRB approval termination date: ________ B. Were vertebrate animals used? Yes::____ No: __X__ If yes, were the procedures approved by your IACUC? Yes:____ No:_____ IACUC approval termination date:_________ C. Are there potential hazards (including biohazards) to laboratory workers (carcinogens, pathogens, ionizing radiation, etc.) engaged in the research? Yes: X No: ______ If yes, was training in biohazards conducted? Yes: X No: _____


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PROGRESS REPORT Our research project involves the Design of Titanium (IV) and Mo(IV) complexes with Antitumor Properties and their Physicochemical and Biochemical Characterizations. Our major goal is to determine the structure-activity relationship of these complexes in order to develop a rational design of metallic antitumor agents. Toward this goal, we published our research results on titanocene-aminoacid and molybdenocene-thionucleobase complexes (from last year report) and we continue to study some of the remaining biophysical aspects of these complexes such as the characterization of the Mo-Oligonucleotide and Ti-Protein interactions by NMR and ICP Spectroscopies. In addition, we have initiated the synthesis of bis-aminoacid ligands to coordinate Ti(IV), V(IV) and Mo(IV) centers in order to develop new inorganic antittumor agents.

A. Titanocene-Aminoacid Complexes Three titanocene complexes, [Cp2Ti(L)2]Cl2 where L = L-cysteine, L-methionine and D-penicillamine have

been studied by cyclic voltammetry and by spectroscopic methods. Interaction of the Ti complexes with apo-Transferrin (apo-Tr), the protein involved in the metal ion transport in the blood, has been pursued using UV-Vis spectroscopy. Our studies suggest that the aminoacid ligand competes with apo-transferrin for the binding of Ti(IV) at physiological pH. This study is currently being published in Journal of Biological Inorganic Chemistry 2005 in press.

B. Molybdenocene-thionucleobase and –amino acid Complexes The synthesis, characterization and electrochemical characterization of the Mo-thionulceobase complexes

finally came out in Inorganica Chimica Acta 2004. Three new molybdenocene –aminoacid complexes, [Cp2Mo(L)2]Cl2 have been prepared and characterized. Although, carboxylate coordination is presumed, additional isomers exist probably coordination through the sulfur atom. We need to establish the structure by x-ray crystallography. In any event, we have analyzed the interaction of these species as well as the molybdenocene-thionucelobase complexes with biomolecules such as DNA using electrochemistry and ICP Spectroscopy. Our studies demonstrated that Cp2MoCl2 and [Cp2Mo(L)n]Cl2 (n=1, L = thiopurines; n=2, L= aminoacids) bind DNA in a range of 3.1-10% as measured by electrochemical methods and corroborated by ICP Spectroscopy while for L = thiopyrimidines, no binding to DNA was detected. This study was accepted for publication and is currently in press: J. Electroanalytical Chemistry 2005.

C. Development of Analytical Methodology for Metal-DNA Interactions 1. We have developed an analytical methodology where combines sample dialysis and ICP-AES to

determine Ti-DNA and Mo-DNA interactions at pH of 7.4. Our results demonstrated that titanium binds DNA irreversible over 95% while molybdenum only binds 5%. These results support previous investigations where reported that molybdenocene does not bind strongly DNA at physiological pH. This study was published in Analytical and BioAnalytical Chemistry 2004.

2. We are currently developing an analytical methodology to determine Ti-Transferrin (protein) interaction using ICP Spectroscopy. The currently methodology involves titration of Ti2-Tr with Fe3+ which it is not a reproducible procedure, yielding erratic results. We are planning to use ICP to measure simultaneously Ti and S (protein) to determine the loading ratio.

3. We are also pursuing the characterization of the Ti2-Tr interactions by NMR methods.

D. Synthesis of Bis-aminoacid ligands

We are currently developing the synthesis of bis-L-cysteine and bis-D-pencillamine ligands, which possess

six(6) coordinating atoms to chelate Ti(IV), V(IV) and Mo(IV). The synthesis of these ligands have been pursued in low yields and with substantial amount of impurities. We are in the process of improving their synthesis.


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E. Biological Testing

We have established collaboration with Professor Madeline Torres (UPRM-Bio-Chemical Engineer) to

elucidate the biological activity of these complexes in CACO cells (colon cancer cells). Thus far, Cp2TiCl2 (as a baseline) and [Cp2Ti(L)2]Cl2 where L = L-cysteine, L-methionine and D-penicillamine have been studied. Our initial studies revealed that [Cp2Ti(L-cysteine)2]Cl2 and [Cp2Ti(D-penicillamine)2]Cl2 are slightly more active than Cp2TiCl2. Cp2TiCl2 and [Cp2Ti(L-methionine)2]Cl2 have about the same activity.

Presentations 1. “Molybdenocene-DNA Interaction Studies using Electrochemical Analysis” Presented at the 28th Technical Meeting, Isabela, PR, November 19, 2004. Publications: 1. Water Soluble Titanocene Complexes with Sulfur Containing Aminoacids: Synthesis, Spectroscopic, Electrochemical and Ti(IV)-Transferrin Interaction Studies J. Biological Inorganic Chemistry 2005 in press.

2. Molybdenocene-DNA Interaction Studies using Electrochemical Analysis J. Electroanalytical Chemistry 2005 in press. Proceeding from previous report 3. Synthesis, spectroscopic and electrochemical characterization of water soluble Cp2Mo(thionucleobase/thionucleoside)Cl2 Complexes Inorganica Chimica Acta 2004, 357, 1092-1102. 4. Electrochemical and spectroscopic analysis of the interaction of molybdenocene dichloride with nitrogen bases J. Elecetroanalytical Chemistry 2004, 565, 77-83 5. Determination of the Titanocene- and Molybdenocene-DNA Interaction via Inductive Coupled Plasma-Atomic Emission Spectroscopy Analytical and Bioanalytical Chemistry 2004, 379, 399-403.


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BIOGRAPHICAL SKETCH Provide the following information for the key personnel in the order listed for Form Page 2. Follow the sample format for each person. DO NOT EXCEED FOUR PAGES. NAME Fan Daming

POSITION TITLE Postdoctoral Research Associate




Shanghai Normal Univ., China B.Sc. 08/1986-07/1990

Chemistry

Fudan Univ. China M.Sc. 08/1993-07/1996

Chemistry

National Univ. of Singapore, Singapore Ph.D. 03/1997-12/2000

Chemistry

Profesional Experiences: Jan. 2001- Sept. 2003: R & D Chemist, Oculus Limited, Singapore July 1997- Jan. 2001: Research Scholar (Ph.D.), Majored in Bioinorganic Chemistry Sept. 1993- July 1996: Research Scholar (M.Sc.), Majored in Organometallic August 1990- July 1993: Research Assistant, Shanghai Institute of Planned Publications: 1. Daming Fan, John David Ranford, and Jagadese J. Vittal, “ Study on Carbon –Hydrogen Bond Activation

byGold (III) Complexes”, Inorg. Chim. Acta, Submitted. 2. Daming Fan, Changtong Yang, John David Ranford, and Jagadese J. Vittal, “ Chemical and Biological

Studiesof Gold (III) Complexes with Uninegative Bidentate N-N Ligands” J. Chem. Soc., Dalton Trans., In press.

3. Daming Fan, Changtong Yang, John David Ranford, and Jagadese J. Vittal, “ Synthesis, Characterization, and Biological Activities of 2-Phenylpyridine Gold (III) complexes with Thiolate Ligands”, J. Chem. Soc., Dalton Trans., 3376-3381, 2003.

4. Daming Fan, Changtong Yang, John David Ranford, Peter Lee, and Jagadese J. Vittal, “ Chemical and Biological Studies of Dichloro-(2-phenylpyidine Gold (III) Complex and Its Derivatives”, J. Chem. Soc., Dalton Trans., 2680-2685, 2003.

5. Petter Lee, Changtong Yang, Daming Fan, and John David Ranford, “ Synthesis, Characterization and Properties of Copper (II) Complexes Containing Salicylaldehyde Semicarbazone”, Polyhedron, 2781-2786, 22,2003.

6. William Henderson, Brian K. Nicholson, Suzanne J. Faville, Daming Fan, and John David Ranford, Gold (III) Thiosalicylate Complexes Containing Cycloaurated 2-Arylpyridine, 2-Anilinopyridine and 2-Benzylpyridine Ligands”, J. Organomet. Chem ., 41-46, 31,2001.

7. Daming Fan, and John David Ranford, “ Chemical and Biological Studies of Dichloro (2-Phenylpiridine)Gold (III) Complexes”, Poster, M32, IC/99 Conference, Wellington, Jan. 31-Feb. 4, 1999.

8. Daming Fan, and John David Ranford, “ Chemical and Biological Properties of Gold (III) Analogues of Platinum (II) Anticancer Drugs”, Oral Presentation, T81, SCC-1 Conference Singapore, Dec, 7-9, 1998.


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9. Daming Fan, Zuen Huang, and Ruifang Cai, “ Synthesis and Chemical Properties of Binuclear Organolanthanide Alkoxides”, Fundan Xuebao (Science, Chinese character), 56-62, 1997.

10. Zhongzhi Wu, Daming Fan, Zuen Huang, and Ruifang Cai , “ Synthesis and Characterization of Binuclear Biscyclopentadienyl Lanthanide Acetoximate Derivates”, Polyhedron, 127-133, 15, 1996.

11. Daming Fan, Zuen Huang, Zhongzhi Wu, Ruifang Cai, and Yu Chen, “ Synthesis and Characterization of Binuclear Organolanthanide Alkoxides”, Organic Chemistry (Chinese character), 524-527,16,1996.

12. Yu Chen, Zuen Huang, Ruifang Cai, Daming Fan, and Shiming Chen, “ Photoconductivity and Paramagnetism of Fullerene Chemically Modified Polymers”, J. of Applied Science, 2185-2190,61,1996.


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MBRS SCORE Grant Annual Progress Report Face Page for Investigator–Initiated Research Project Grant Number: S06 GM 08103-31 Project Number___________4__________. Institution: University of Puerto Rico, Mayaguez 1. Descriptive Title: (56 characters or less, including spaces) MAP kinase pathways in the yeast Debaryomyces hansenii. 2. Investigator (s) Last Name: First M.I. Degree Department Nadathur Govind S. Ph.D. Marine Sciences 3a. Supplemental Project : Yes:_____ No__X___. If yes, award date:______ 3b. Are the SNAP procedures followed? Yes:_X__ No:____. If no, Budget pages submitted: Yes:____ No:____. 4a. Regular Research Project ____X___ Pilot Research Project _____. 4b. Inventions and Patents Yes:____ No:__X__ If “yes”, include details in progress report narrative. 5. Total number of research workers employed on the project: Full time__2__ Part time___1__ 6. Protection Against Research Risks: A. Were human subjects involved in the project? Yes:____ No:___X__ If yes, were the procedures approved by your IRB? Yes:____ No:_____ IRB approval termination date:________ B. Were vertebrate animals used? Yes::____ No: ___X___ If yes, were the procedures approved by your IACUC? Yes:____ No:_____ IACUC approval termination date:_________ C. Are there potential hazards (including biohazards) to laboratory workers (carcinogens, pathogens, ionizing radiation, etc.) engaged in the research? Yes:__X___ No: ______ If yes, was training in biohazards conducted? Yes:__X__ No:_____


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A. Specific Aims: The aims as stated in the original funded proposal are as follows:

1. Isolation of the genes involved in the osmoregulatory response in Debaryomyces upstream of the PBS2 MAPKK gene. There were three methods described in this strategy: construction of the ssk1∆, ste11∆, double mutants in Saccharomyces, construction of a gene library of Debaryomyces, isolate the two genes and use them to construct deletion mutants in Debaryomyces. Also the isolation of auxotrophs from Debaryomyces was proposed

2. Isolation of genes downstream of the HOG 1 MAP kinase : Two strategies for the these genes was proposed, one utilizing the yeast two hybrid system and the other was the isolation of transcription factors utilizing the GPD1 regulatory sequences(already isolated in our laboratory) as a bait.

Aim 1 of the original proposal has been modified. In the last year the genome of Debaryomyces hansenii has been sequenced and was shown to have a size of 1.2 X 107 base pairs(Dujon, et al. 2004. Nature. 430:35-44). Based on sequence homology with other yeasts we have now identified the sequences related to the MAP kinase genes in this organism. We are, thus modifying our approach to standardize techniques for the isolation of mutants in one or more of the genes in the pathway. The isolation of auxotrophs for the organism remains unchanged. B. Studies and Results

We cloned and sequenced a 5.1 kb DNA fragment from a D. hansenii strain NRRL Y-7426 genomic library that complemented the Leu2 phenotype of S. cerevisiae strain YPH500. We found two ORFs. The deduced aa sequence from the first ORF closely matched sequences in GenBank for �-isopropylmalate dehydrogenase genes (LEU2) Genbank accession number for this isolated sequence is AY683206. The deduced aa sequence from the second ORF matched sequences in GenBank for SPL1 genes of several yeast species (GenBank accession # AY682470).

We have used partial sequences from the isolated LEU 2 gene along with a hygromycin resistance gene under the control of the yeast cytochrome C1 promoter to construct a knock out cassette. The purpose of this was to create a double auxotroph in our strain (ura 3-, ∆leu2). We are also in the process of creating a HIS 3 knockout cassette.

The Spl1 polypeptide from D. hansenii is similar to other Spl1p in that the first 60 to 70 aa residues are

not conserved (Plant, et al. 1998 Yeast. 14:287-295). The most notable difference between the polypeptide from D. hansenii and SPL1 from other yeast species is that the D. hansenii polypeptide is shorter on the carboxyl terminal by 99 aa residues. Since our sequence matches 100% the sequence from a different strain of D. hansenii (from the D. hansenii genome sequencing project)we can confidently say that the shorter ORF is not a product of sequencing error. These missing 99 aa residues, show an 80% conservation in other yeast species suggesting their importance in the function of the protein. We found that the deduced aa sequence of a partial ORF downstream of the SPL1 stop codon matches the missing 99 amino acids Kolman and Soll (1993. J. Bact. 175:1433-1442) report that SPL1 from S. cerevisiae is an essential gene. Therefore, we are currently determining whether the mRNA for SPL1 from D. hansenii matches in size to the gene we have isolated. For aim 2 we have now constructed two “bait” plasmids—one with the Debaryomyces habsebii HOG1 gene and one with the PBS2 gene. cDNA synthesis and prey plasmid construction is now underway. Supplements: none C. Significance This project was originally aimed at the isolation of the MAP kinase genes involved in salt-induced expression. Since the genome of the organism is now sequenced, we will now use this information to take the study one step further—to create tools for the study of environmentally controlled gene expression. Even though we have information ion the genome of the organism, techniques for creating knock-out mutations and over-expression


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systems are yet to be developed. Our plans for the next two years will create such tools and also study the MAP kinase pathway utilizing these tools. The creation of multiple auxotrophs for the organism will help eventually help in creation of mutants of the pathway. d. Plans: In the next year, we plan to create auxotrphic mutation in Debaryomyces and create a ssk1 as well as a ste11 mutation. In fact once the technique for creating deletion mutants is standardized we will systematically mutate all the genes of the pathway and study the phenotypic manifestation of such mutations. In aim 2 we hope to finish the construction of a cDNA library in the “prey” plasmid of the yeast two hybrid system initiate the screening of the library for the isolation of the transcription factors. In a subproject that is related we have already initiated the analysis of cis-acting regions of the GPD1 promoter that was previously isolated in our laboratory. We will continue this analysis.


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MBRS SCORE Grant Annual Progress Report Face Page for Investigator–Initiated Research Project Grant Number: 5 S06GM08103-31 Project Number________5________. Institution: University of Puerto Rico Mayagüez Campus ________________________________________________________________________ 1. Descriptive Title: (56 characters or less, including spaces)

Conformational Changes in Phosphorylated Proteins 2. Investigator (s) Last Name: First M.I. Degree Department Pastrana-Ríos Belinda Ph.D. Chemistry 3a. Supplemental Project : Yes:_____ No X If yes, award date:______ 3b. Are the SNAP procedures followed? Yes: X No:____. If no, Budget pages submitted: Yes:____ No:____. 4a. Regular Research Project X Pilot Research Project _____. 4b. Inventions and Patents Yes:____ No: X If “yes”, include details in progress report narrative. 5. Total number of research workers employed on the project: Full time___1_ Part time___1__ 6. Protection Against Research Risks: A. Were human subjects involved in the project? Yes: ____ No: X If yes, were the procedures approved by your IRB? Yes:____ No:_____ IRB approval termination date:________ B. Were vertebrate animals used? Yes::____ No: X If yes, were the procedures approved by your IACUC? Yes:____ No:_____ IACUC approval termination date:_________ C. Are there potential hazards (including biohazards) to laboratory workers (carcinogens, pathogens, ionizing radiation, etc.) engaged in the research? Yes:____ No: X If yes, was training in biohazards conducted? Yes:____ No: X


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B. NIH-SCORE Individual Subproject Progress Report Title: Conformational changes of phosphorylated proteins Summary: All of the proposed specific aims associated to this proposal application are being accomplished and additional work has been carried-out which has contributed significantly towards the understanding of centrin’s structure/function/stability relationship. Several manuscripts have been submitted and accepted several more are in preparation. Students and key personnel have attended national meetings to comply with dissemination of scientific knowledge.

SPECIFIC AIMS: Specific Aim 1: To determine the calcium binding mechanism and relative affinity between the terminal domains of centrin. Calcium titration experiments have been successfully carried-out for Chlamydomonas centrin, results of which have led to a patent application (in preparation). Specific Aim 2: To investigate the structural changes that occur upon phosphorylation of a calcium binding protein. Chlamydomonas centrin and the carboxy terminal end peptide have been phosphorylated in vitro using protein kinase A (PKA) and adenosine triphosphate (ATP) in sufficient quantities for biophysical characterization. The extent of phosphorylation was determined by MALDI mass spectrometry. In addition, we have determined the relative stability due to phosphorylation for the full length and terminal peptide by thermal dependence study using FT-IR spectroscopy, differential scanning calorimetry (DSC) and circular dichroism (CD). Phosphorylated holo-centrin was found not to self-associate and the arginine modes were also affected due to band broadening possibly due to ionic interaction with the nearby phosphate. Although the phosphorylated form of the protein has a short shelf life the thermal denaturation experiment revealed little change in the spectral features observed Amide I’, Amide II and side chain modes. Upon increase in temperature the turns were less stable in the phosphorylated form of the protein as shown in Figure 1B. (manuscript submitted to Journal Biol. Chem.). Specific Aim 3: To understand the protein/protein interactions and to understand the structural and dynamic changes that occur during this interaction. 13C recombinant Chlamydomonas centrin, human centrin 1, and human centrin 2 have also been expressed using enriched 13C minimal media, isolated and purified as described in Pastrana et al.,[1]. This method would isotopically (13C=O) label the backbone of the protein of interest, causing a shift to lower wavenumbers of the amide I band (primarily comprised of C=O stretch). These protein samples have been sent for partial amino acid sequencing and MALDI Mass Spectrometry before continuing with the biophysical studies.

We have developed an FT-IR spectral analysis methodology that has proven useful in the study of Hydrogen/Deuterium exchange dynamics of Chlamydomonas centrin and terminal domain peptides[2]. Fast H/D exchange allows for the study of exchangeable protons in proteins. Typical spectra acquired for 13C recombinant Chlamydomonas centrin in the spectral region of 1725 –


31

1525 cm-1 (data not shown). Thermal denaturation studies were also carried out for 13C-centrin and Mellitin, a model helical peptide from bee venom, Figure 2, panel A and B, respectively. In panel A (13C-centrin ) the amide I band shifts to higher wavenumbers from 1590.5 cm-1 to 1630.6 cm-1, thus from helical to random coil conformation. Intensity decrease is also observed for the helical component while a concomitant intensity increase is observed for the random coil component. While, for Mellitin, panel B, amide I band is also comprised of two subbands: 1645 cm-1 for the alpha-helix and 1660 cm-1 for the random coil structure. The alpha-helix also shifts to higher wavenumbers from 1645 cm-1 to

1660 cm-1, thus from helical to random coil conformation. Two dimensional correlational analysis was done

for these two proteins within the spectral region of 1725 – 1550 cm-1 (data not shown). The spectral features for 13C-centrin and that of Mellitin observed are in different spectral regions thus, the results shown for the individual proteins demonstrate the feasibility of this binary study. A patent application is in preparation. Specific Aim 4: To perform molecular modeling of the proposed structures. A tentative model of centrin can be developed by combining the results obtained from the Biophysical techniques used in this proposal with the available structural information of similar proteins (calmodulin’s x-ray structure). We have begun this project using holo-centrin and we are minimizing the proposed structures using CHARMM and annealing method. We have also carried-out a homology study, based on the sequence identity of centrin to calmodulin an arginine mapping on the CAM structure Figure 3. We have also begun the molecular modeling and simulation of the amino terminal fragment of centrin. In Summary: We have been able to express and Isolate both recombinant and 13C-labeled Chlamydomonas Centrin, Human centrin 1 and Human centrin 2 proteins. A detailed dynamics paper on full-length Chlamydomonas Centrin, terminal peptides and Calmodulin is in press in Biochemistry. The phosphorylated form of Chlamydomonas centrin has been studied and results compared with the unphosphorylated form of the protein, giving rise to molecular level information as to the proteins putative function. Finally, continued molecular modeling of centrin terminal fragments and holo-full length protein are underway. 1 Pastrana-Rios, B., Wanda Ocaña, Michelle Rios, German Lorenzo Vargas, Ginny Ysa, Greg Poynter, Javier Tapia, and Salisbury, J. L., "Centrin: Its Secondary Structure in the Presence and Absence of Cations" 2002 Biochemistry 41 (22), 6911-6919

“Publications in Peer-reviewed Journals”

1. Orrtiz, M., Sanoguet, Z., Hu, H., Chazin, W.J., McMurray, C., Salisbury, J.L. and Pastrana-Rios, B. “Dynamics of Hydrogen/Deuterium Exchange in Chlamydomonas Centrin” 2005 Biochemistry (In Press).

2. Sanoguet, Z., Negrón V., Campbell, M., Seda, C. and Pastrana-Rios, B. “Relative thermal stablity of phosphorylated and unphosphorylated chlamydomonas centrin” (submitted to Journal Biological Chemistry)

“Published Abstracts”

1. Belinda Pastrana-Rios, Mildred Ortiz, Haitao Hu, Walter Chazin, “ATR/FT-IR Spectroscopic Study of Chlamydomonas Centrin During H/D Exchange” 2004 (Biophysical Journal)


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2. Sanoguet-Carrero, Z., Campbell, M., Lebrón-Rodriguez, R., and Pastrana-Rios, B. “Characterization of Phosphorylated Recombinant Chlamydomonas Centrin” 2004 (Biophysical Journal).

“Abstracts Presented in Meetings” 1.Jessica De Orbeta, Elmar Schiebel, and Belinda Pastrana-Rios “Expression of recombinant calcium binding human protein” Summer Conference in Protein Research, at the Wyndham Hotel August 2003. San Juan, Puerto Rico. 2.Verónica Meza, José Fernández and Belinda Pastrana-Rios “Construction of Recombinant Clones of a Human Calcium Binding Protein” Summer Conference in Protein Research, at the Wyndham Hotel August 2003. San Juan, Puerto Rico. 3.Michelle Vázquez, Gisselle Román, Mariena Silvestry, Zuleika Sanoguet and Belinda Pastrana-Rios “Fermentor Expression and Subsequent Isolation of Recombinant 13C labeled and native Chlamydomonas centrin” Summer Conference in Protein Research, at the Wyndham Hotel August 2003. San Juan, Puerto Rico. 4.Muriel Campbell, Walter Chazin and Belinda Pastrana “Phosphorylation provides an additional mechanism for target regulation by centrin” Summer Conference in Protein Research, at the Wyndham Hotel August 2003. San Juan, Puerto Rico.

“Abstracts Presented in Meetings” (cont.) 1.Zuleika Sanoguet and Belinda Pastrana-Rios “Studies of phosphorylated recombinant Chlamydomonas centrin” Summer Conference in Protein Research, at the Wyndham Hotel August 2003. San Juan, Puerto Rico. 2.Zuleika Sanoguet, Muriel Campbell, Rebecca Lebrón and Belinda Pastrana “Characterization of Phosphorylated Recombinant Chlamydomonas centrin” 48th Annual Meeting of the Biophyscal Society, February 14-18, 2004. Baltimore, Maryland. 3.Muriel Campbell, Z. Sanoguet, H. Hu, W. Chazin, B. Pastrana “Characterization of Phosphorylated Chlamydomonas centrin” 39th ACS Junior Technical Meeting, March 8th, 2004. Humacao, Puerto Rico. 4.Pastrana-Rios, B. “Domain Specific Dynamics in a Calcium Binding Protein Leading Towards Biological Function” Spring Conference in Protein Research, April 16th, 2004. San Juan, PR 5.Muriel M. Campbell Ocasio, Zuleika Sanoguet, Daniel Narváez, Nancy Guillén, Jessica Colón, Haitao Hu, Walter Chazin, and Belinda Pastrana, “Evidence of Aggregation using Differential Scanning Calorimetry for: A Calcium binding protein Chlamydomonas centrin, and EGFR ligands” Spring Conference in Protein Research, April 16th, 2004. San Juan, PR 6.Zuleika Sanoguet-Carrero Christina Seda, Muriel Campbell, Belinda Pastrana-Ríos “Understanding the Effect of Phosphorylation in Recombinant Chlamydomonas Centrin” Spring Conference in Protein Research, April 16th, 2004. San Juan, PR 7.Pastrana-Rios, B. “A Calcium Binding Protein and its Role in Cell Division and Cancer” Biotechnology Meeting, October 1-2, 2004, Mayagüez, PR. 8.Christina Seda, Muriel Campbell, Kevin Weiss, Walter Chazin, and Belinda Pastrana-Ríos “Techniques suitable for verifying phosphorylation of Chlamydomonas centrin” Biotechnology Meeting, October 1-2, 2004, Mayagüez, PR. 9.Zuleika Sanoguet-Carrero, Christina Seda, Muriel Campbell, R. Lebrón, Belinda Pastrana-Ríos “Fourier transform infrared spectroscopy and MALDI mass spectrometry of phosphorylated Chlamydomonas centrin” Biotechnology Meeting, October 1-2, 2004, Mayagüez, PR. 10.Liliana del Valle Sosa2 and Belinda Pastrana-Ríos “Structural changes in 13C-centrin and mellitin upon binding” Biotechnology Meeting, October 1-2, 2004, Mayagüez, PR. 11.Z. Sanoguet-Carrero, C. Seda, B. Pastrana-Rios “Relative thermal stablity of phosphorylated and unphosphorylated chlamydomonas centrin” AACR Meeting December 5-12, 2004, Fort Lauderdale, Florida 12. M. M. Campbell-Ocasio, Z. Sanoguet-Carrero, W. Chazin and B. Pastrana-Rios “Calorimetric studies of a centrosomal associated protein: centrin” AACR Meeting December 5-12, 2004, Fort Lauderdale, Florida.


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BIOGRAPHICAL SKETCH NAME POSITION TITLE

Ana María Gómez-Buitrago Research Assistant


INSTITUTION AND LOCATION DEGREE (if


Universidad del Valle, Cali, Colombia B.S. 1993 Biology (Genetics) Pennstate University, State College, PA, USA ABD in Ph.D. 2002 Molecular Biology

Professional Experience 1993-1995 Research Assistant, Biotechnology Unit, Centro Internacional de Agricultura Tropical, Cali,

Colombia 1997-2001 Teaching Assistant, Biology Department, Pennstate University, State College, PA, USA 1997-2002 Research Assistant, Biology Department, Pennstate University, State College, PA, USA 2004-present Research Specialist, Chemistry Department, University of Puerto Rico, Mayagüez, PR Honors and Awards 1986-1989 Univalle Merit Scholarship for academic merit, Universidad del Valle, Cali, Colombia Publications:

Mahalingam R., Gomez-Buitrago A.M., Eckard N., Shah N., Raina R. and Fedoroff N.V. Characterizing the stress/defense transcriptome of Arabidopsis. 2003 Genome Biology, Volume 4, Issue 3, Article R20.

Posters and Seminars

1. Identification and analysis of genes differentially expressed in response to the bacterial pathogen Pseudomonas syringae in Arabidopsis thaliana. Seminar. October 2001. Defense Advanced Research Project Agency (DARPA) meeting. PennState University.

2. Molecular analysis of hypersensitive response, disease and systemic acquired resistance in Arabidopsis using cDNA microarrays. Poster. June 2001. University of Wisconsin, Madison.

3. Global expression analysis of genes differentially expressed during activation of HR and SAR in Arabidopsis thaliana. Poster. June 2000. University of Wisconsin, Madison.

4. Identification of genes induced by Salicylic Acid, avirulent and virulent bacteria in Arabidopsis thaliana. Poster. May 2000. First Symposium in Plant signalling. Penn State University.

5. Identification of salicylic acid induced genes in Arabidopsis thaliana. Poster. August 1999. The annual meeting of the American Phytopathological Society. Montreal, Canada.

6. Hunt for genes involved in disease resistance in plants. Poster. March 1999. Graduate student exhibition. Penn State University.

7. Identification of salicylic acid induced genes in Arabidopsis thaliana. Poster. June 1998. International conference on Arabidopsis reseach. University of Wisconsin, Madison.

8. The characterization of a programmed cell death mutant in Arabidopsis. Poster. October 1997. BMB/Biology joint research forum. Penn State University.

9. Salicylic Acid (SA) and the Systemic Acquired Resistance (SAR). Seminar. March 1997. Plant Physiology weekly seminars. Penn State University.


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MBRS SCORE Grant Annual Progress Report Face Page for Investigator–Initiated Research Project Grant Number: S06 GM08103-30 Project Number__________6___________. Institution: University of Puerto Rico at Mayagüez 1. Descriptive Title: (56 characters or less, including spaces) Examination of the Mechanisms of Interaction of PEG rich Matrices with the MRP and MDR Transporters 2. Investigator (s) Last Name: First: M.I. Degree Department Torres-Lugo Madeline Ph.D. Chemical Engineering 3a. Supplemental Project : Yes:_____ No X . If yes, award date:______ 3b. Are the SNAP procedures followed? Yes: X No:____. If no, Budget pages submitted: Yes:____ No:____. 4a. Regular Research Project X Pilot Research Project _____. 4b. Inventions and Patents Yes:____ No X If “yes”, include details in progress report narrative. 5. Total number of research workers employed on the project: Full time 1 Part time _______ 6. Protection Against Research Risks: A. Were human subjects involved in the project? Yes:____ No X If yes, were the procedures approved by your IRB? Yes:____ No:_____ IRB approval termination date:________ B. Were vertebrate animals used? Yes::____ No: X If yes, were the procedures approved by your IACUC? Yes:____ No:_____ IACUC approval termination date:_________ C. Are there potential hazards (including biohazards) to laboratory workers (carcinogens, pathogens, ionizing radiation, etc.) engaged in the research? Yes:_____ No: ___X___ If yes, was training in biohazards conducted? Yes:____ No:_____


35

Progress Report Summary Mechanisms of Interaction of PEG rich Matrices with MRP and MDR Efflux Pumps Summary:

This progress report describes the activities performed during the second year of the award period. The year started with the appointment of a postdoctoral associate and her training. Efforts were placed on the continuation of experiments related to Specific Aim I and the initiation of the experiments related to Specific Aims II and III. Specific Aims:

Specific Aim III was modified to include an additional sub aim. This aim should state: “Optimization of sample preparation for cell culture experiments.” This optimization process included the protocol development for sample preparation and the investigation of the possible sample cytotoxic effects and its relationship with preparation. Studies and Results:

Throughout last year focus was placed on the continuation and/or initiation of experiments related Specific Aims I, II and III: • Synthesize PEG rich hydrogel networks with various copolymers with tethered PEG of various chain lengths,

including PEG-star copolymers. • Investigate the fundamental mechanisms of diffusion of MRP and p-glycoprotein transporter substrates

through the hydrogel membranes; and • Examine the physicochemical interactions between the various tailored hydrogel networks and the

epithelial cell monolayer using Caco-2 cells. The synthesis of poly(ethylene glycol) hydrogels with various morphologies was performed. These included

the synthesis of poly(ethylene glycol) monomethyl ether monomethacrylate (PEGMA) with PEG molecular weights of 1000, 600, and 400 with poly(ethylene glycol) dimethacrylate (PEGDMA) with PEG molecular weights of 400, 600, and 1000. Results indicated that to obtain a polymerized network the molecular weight of the crosslinker (i.e PEGDMA) must be similar in size or larger than the molecular weight of the PEG tether chain (i.e PEGMA). Those networks that were successfully synthesized were characterized by swelling studies and by the determination of the partition coefficient of rhodamine B. The equilibrium swelling demonstrated to be independent of crosslinker for those matrices where the tether chain molecular weight was 1000, whereas for the tether chain molecular weight of 600 a significant difference in the equilibrium swelling was observed. This could be related to the fact that for tether chains of higher molecular weight the crosslinker length is smaller or equivalent in size, thus impeding further incorporation of water. The partition coefficient of rhodamine B was found to be independent of crosslinker and tether chain length. This indicated that the affinity of the molecule was independent of morphology as expected.

Efforts were also focused on the creation of a poly(ethylene glycol) and methacrylic acid full interpenetrated networks (IPN). IPN’s are noteworthy because each network maintains its own physical characteristics. For this case, a poly(ethylene glycol) and methacrylic acid (MAA) were the selected polymers. Poly(ethylene glycol) was incorporated for the reasons described herein, whereas methacrylic acid will provide mechanical stability and it is well known to be capable of opening tight junctions between epithelial cells. The creation of an IPN rather than a copolymer provides the flexibility of manipulating the morphology without the limitation of monomer reactivity. Two morphologies were successfully synthesized, PEG as the primary network with MAA as the secondary network, and viceversa. Examination of the swelling characteristics of PEG-MAA IPN’s demonstrated pH sensitivity although not to the same extent as the pure MAA network. This was a first indication that interpenetration may have occurred. Similar experiments were conducted with the MAA-PEG IPN. Results illustrated that although pH sensitivity was maintained, the network swelled to a lesser extent when compared to the PEG-MAA IPN. This could also be an indication that interpenetration may have occurred. The behavior of the networks suggested that the swelling behavior was controlled by the secondary


36

network. Further characterization included the utilization of a fluorescent crosslinker for the second network. For this purpose, a Confocal Laser Microscope (CLSM) was utilized. The presence of fluorescence throughout the thickness of the IPN network indicated that interpenetration occurred homogeneously throughout the mesh.

Cell studies were initiated during this second year after the installation of the new clean room (June

2004). Although the cytotoxicity of the networks was not envisioned in the original goals of this project, several viability experiments were conducted. They demonstrated that the sample preparation method was instrumental. Samples were prepared by crushing the polymer membranes, autoclaving, and re-suspending in culture media. Viability experiments indicated that crushed particles were cytotoxic to the cells independently of polymer composition. Also, the particles were filtered and the media analyzed as well. The filtered media from the particles also demonstrated to be cytotoxic. These results demonstrated that particles must be washed to remove any unreacted monomer and/or olygomers that may have not been incorporated. Washed samples were analyzed and showed that the cell monolayers remained viable. Significance:

These findings will provide the starting point for the future development of novel PEG networks that could be employed as drug delivery carriers for anticancer agents. In particular, they provided us with information regarding the morphologies that could be successfully synthesized and employed for cell studies. Of particular importance, is the synthesis of the PEG-IPN. This network will allow a higher density of both PEG and MAA that cannot be obtained from the copolymerization of both monomers. The presence of a high concentration of PEG may provide the maximization of the transport of anti-cancerous drugs, whereas the high concentration of MAA could provide the maximization of the opening of tight junctions, which could increase the penetration of drugs further inside the tumor. Plans: Plans for the following year include the continuation of cell studies. These will include the examination of the transport behavior of various model fluorescent molecules in the presence and absence of various PEG hydrogel morphologies. Also, the CLSM will be utilized to visualize the mechanisms of transport. Other studies will include the further characterization of the PEG morphologies herein described. Publications: No peer reviewed publications were submitted this year. However, the described work was presented as a poster in the 2004 AiChE meeting, Austin, TX, presented at Princeton University during an invited seminar (November 2004), and during the 11th Chemical Engineering Symposium, Mayagüez PR (November 2004). Project-Generated Resources: None


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NOTE: The Biographical Sketch may not exceed four pages. Items A and B may not exceed two of the four-page limit.

A. Positions and Honors. List in chronological order previous positions, concluding with your present

position. List any honors. Include present membership on any Federal Government public advisory committee.

Positions

1979-1983 Research Assistant, Department of Crop Protection, University of Puerto Rico. 1983-85 Graduate Assistant, Plant Pathology Department, University of Florida, FL. 2/90-6/91 Research Assistant, Department of Crop Protection, University of Puerto Rico, Mayaguez, P.R.

7/92-1995 Physical Science Technician, USDA-ARS-Soil-Plant-Nutrient Research, Ft. Collins, Colorado. 1996-2000 Graduate Assistant, Dept. of Plant Pathology, Fifield Hall, University of Florida 2000-2001 Senior Biochemist, Nanosphere, Inc., Sid Martin Biotechnology Development Institute, 12850

Research Drive, Suite N, Alachua, Florida 32615. 2001-2003 Research Plant Pathologist, GS-12, USDA-ARS-TARS May 04-Present Post Doctoral Research Associate-Department of Chemical Engineering, University of Puerto Rico, Mayaguez. P. R. Honors 1976 Dean Ralph Garwood Award-Best Student, Dept. of Horticulture, University of Puerto Rico, Mayaguez. 1997 Presidential Recognition Award -Outstanding Student, University of Florida, Gainesville, Florida. 1997 International Honorary for Leaders in University Apartment Communities, University of Florida. 1997 Multicultural Recognition Award, University of Florida, Gainesville, Florida. 2001 Ad Honorem Position UPR, Mayaguez, Crop Protection Department.

BIOGRAPHICAL SKETCH Provide the following information for the key personnel in the order listed for Form Page 2. Follow this format for each person. DO NOT EXCEED FOUR PAGES. NAME Margarita Licha

POSITION TITLE Post Doctoral Research Associate




University of Puerto Rico, Mayaguez, Puerto Rico

B. Sc. 1976 Horticulture

University of Puerto Rico, Mayaguez, Puerto Rico

M. Sc. 1979 Agronomy

University of Florida, Gainesville, Florida Ph.D. 1982 Plant Pathology-Virology


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B. Selected peer-reviewed publications (in chronological order). Do not include publications submitted or in preparation.

1. Ferwerda-Licha, M. and R. Pingel. 2003. Ongoing survey on the incidence of papaya ringspot virus in weeds found in a papaya field in northern Puerto Rico. Phytopathology. June v. 93(6) p. S25. Abstr. 2. Ferwerda-Licha, M. 2003. Rapid in vitro propagation of mangosteen through shoot division. JAUPR .Vol 87 (1-2). 3. Ferwerda-Licha, M. 2003. New method for long term storage of mangosteen seed. JAUPR Vol 87 (1-2). 4. Ferwerda-Licha, M. 2002. Mixed infection of papaya ringspot virus, zucchini yellow mosaic virus and papaya bunchy top affecting papaya (Carica papaya L.) in Puerto Rico. Phytopathology . June. v. 92(6), p. S25. Presented at APS Meeting July 27-31. Milwaukee, Wisconsin. Abstr. 5. Ferwerda-Licha. M. 2002. First report of algal leaf spot caused by Cephaleuros virescens Kunze on longan trees in Puerto Rico. JAUPR. Vol 86 (1-2). 6. Ferwerda-Licha, M., M. J. Davis, E. Hiebert, C. Ramadugu and D. E. Purcifull. Development of Infectious Papaya Mosaic Virus Using a cDNA Clone Fused to the 35S CaMV Promoter PMV. JAUPR (National Agricultural Library). 7. Ferwerda-Licha, M., E. Hiebert, and M. J. Davis. 2000. Full-length infectious clone of papaya mosaic virus using the 35S promoter of CaMV. Submitted to the American Phytopathological Society Meetings, August 12-16, 2000, New Orleans, LA. (Abstr.). 8. Liu, L. J., E. Rosa-Marques, M. Licha, and M.L. Biascoechea. 1988. Tanier (Xanthosoma spp.) propagation in vitro. J. Agri. Univ. P.R. Vol 72 (3):413-425. 9. Liu, L.J., M. Licha, E. Rosa Marquez and M.L. Biascoechea. 1983. Isolation and culture of callus and protoplasts of tanier (Xanthosoma spp.) in Puerto Rico. Phytopathology 73 (5): 791. 10. Liu, L.J., M. Licha , and M.L. Biascoechea. 1983. Tanier tissue culture in Puerto Rico. Proc. XIX Caribbean Food Crops Society, Mayaguez, Puerto Rico. p. 280. 11.Liu, L. J., M. Licha, D. Baella, and E. Rosa Marquez. 1982. Variation in morphology and mosaic virus resistance in plantlets of tanier (Xanthosoma spp.) via tissue culture. Phytopathology 71 (1): 990. 12. Licha, M., D. Baella, and L.J. Liu. 1980. Effect of various media in callus formation proliferation and plantlet development in taniers. Phytopathology 72 (1): 171. 13. Licha, M. 1980. Thesis. The Witches' Broom Disease of Pigeon Peas (Cajanus cajan L. Millsp.) in Puerto Rico. J. Agric. Univ. of Puerto Rico p. 424-441.


39

MBRS SCORE Grant Annual Progress Report Face Page for Investigator–Initiated Research Project Grant Number: S06 GM 08103 Project Number______7________ Institution: University of Puerto Rico at Mayaguez 1. Descriptive Title: (56 characters or less, including spaces) Acoustical Guidance of Liquid-Filled tubes and Catheters 2. Investigator (s) Last Name: First M.I. Degree Department Juan, Eduardo .J. Ph.D. Electrical Engineering 3a. Supplemental Project : Yes:_____ No X . If yes, award date:______ 3b. Are the SNAP procedures followed? Yes: X No:____. If no, Budget pages submitted: Yes:____ No:____. 4a. Regular Research Project Pilot Research Project __X__. 4b. Inventions and Patents Yes:____ No: X If “yes”, include details in progress report narrative. 5. Total number of research workers employed on the project: Full time Part time_____ 6. Protection Against Research Risks: A. Were human subjects involved in the project? Yes:____ No X If yes, were the procedures approved by your IRB? Yes:____ No:_____ IRB approval termination date:________ B. Were vertebrate animals used? Yes::____ No: __X__ If yes, were the procedures approved by your IACUC? Yes:____ No:_____ IACUC approval termination date:_________ C. Are there potential hazards (including biohazards) to laboratory workers (carcinogens, pathogens, ionizing radiation, etc.) engaged in the research? Yes: No: X If yes, was training in biohazards conducted? Yes:____ No:_____


40

Progress Report Summary Acoustical Guidance of Liquid-Filled Tubes and Catheters Summary:

This progress report describes the activities performed during the second year of the award period.

During this period, efforts were focused on achieving Specific Aim I. Significant progress was achieved during this year and in collaboration with biomedical engineering researchers from Purdue University. Specific Aims:

The specific aims of the project remain as actually funded. Studies and Results:

During the last year, efforts focused on achieving Specific Aim I, which seeks to develop and evaluate a method to acoustically determine the position of liquid-filled catheters. Specific Aim I is divided in two sub-aims:

a) Utilize time-domain acoustic reflectometry to determine the geometry of sequentially interconnected liquid-filled tubes having rigid walls.

b) Determine analytically and experimentally the frequency range over which a liquid-filled tube with compliant walls can be assumed to behave acoustically as a rigid conduit.

Acoustic measurements were performed on water-filled copper pipes (1/2 inch diameter) using two

hydrophones. One transducer was used to generate sound while the other transducer was used to record sound signals inside the tube. The goal was to generate an acoustic pulse that traveled inside the tube and to record any reflections that occurred due to changes in acoustic impedance within the tube. The time delay between reflections and the assumed speed of sound in water (1,480 m/s) was used to determine the location of acoustic impedance discontinuities. However, these experiments were not as straight forward as it was expected considering that a rigid conduit (copper) was used. Lower sound speeds were observed, with values ranging between 1,100 – 1,250 m/s. Also, distortion of the sound signals due to attenuation and dispersion was observed. These findings made us change strategies and forced us to consider a more fundamental approach to understand the results we were obtaining. The acoustical transmission line model described in the proposal was employed to model sound propagation in liquid filled tubes, even for the rigid copper tube.

The model was specifically used to determine the attenuation coefficient α(ω) and the phase velocity vp(ω). Both of these parameters are functions of frequency and can cause significant distortion of sound signals traveling in a liquid-filled tube. The experimental setup was modified so that sound signals could be recorded at two different points within the copper tube. All the physical parameters of the experimental setup (density of the medium, wall thickness, tube diameter, etc.) were included in the simulations to obtain the attenuation coefficient and the phase velocity of the system. A MATLAB script was developed to make an animation that shows the shape of acoustic pulses as they travel down the simulated tube system, illustrating the effects of attenuation and dispersion. Experimental results were compared with the simulations in order to validate model predictions. Specifically, group velocity values and energy loss per unit length are used as a basis of comparison. At the time of preparation of this progress report, the results are being analyzed and we expect to publish the results before the summer of 2005. A limiting factor in this project has been the lack of appropriate acoustic transducers (hydrophones). We have been limited by the size and frequency response of the current transducers, which have a diameter of 1 cm and a frequency range of 40-100 kHz. The size of the hydrophone prevents us from using a smaller copper tube, while the diameter of the tube prevents us from operating at frequencies higher than 60 kHz because non-planar sound propagation would result.


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Significance:

Probably, the most significant achievement of this budget period was getting involved with the simulations, which has allowed us to understand the results that we were obtaining from the experimental setup. Understanding and further developing the acoustical transmission line model (or other type of model) will allow predicting the way in which sound propagates in liquid-filled conduits of several types, including drug delivery catheters of implantable infusion pumps and blood vessels such as the coronary arteries. Plans:

There are several plans for the upcoming year. We plan to submit an article to a peer reviewed journal within the next six months. The article will essentially be about the acoustical transmission line model for liquid-filled tubes and its validation using experimental results. Another plan for next year is to perform a thorough search of underwater acoustic transducers, in order to develop an acoustic reflectometer of adequate size and frequency range. This is a critical point in order to complete Specific Aim I. Finally, we will start working on Specific Aim II. Since we do not have transducers to perform the work on fluid-filled tubes, we will initially work on air-filled tubes. The methods and algorithms that will be developed will be adapted to the liquid-filled case when appropriate transducers are found. Publications:

No peer reviewed publications were submitted this year. However, the described work was presented at the 11th Chemical Engineering Symposium, Mayagüez PR (November 2004). Project-Generated Resources: None

Grant Progress Report Total Project Period Frommbrs.uprm.edu/esac/docs/progress.pdf · 2007. 4....

Documents

Transcript of Grant Progress Report Total Project Period Frommbrs.uprm.edu/esac/docs/progress.pdf · 2007. 4....