2011-2012 Trainees

Post-Doctoral Fellow Program Trainees

Elizabeth Crane

Faculty Mentor:  Geoffrey Gerstner

Home Department: Biologic and Materials Sciences

Research Title: Rendering and identification of oromandibular movements through quantitative methods.

Research Summary:
Dr. Elizabeth Crane’s post-doctoral research will continue her quantitative work in human biomechanics and movement kinematics. A primary aim of her research is to develop innovative mathematical and statistical models using data obtained with motion analysis systems, magnetic resonance imaging (MRI) and strain/force transducer sensor systems. The modeling work is a continuation of work recently started as a collaboration between the School of Dentistry and the Department of Statistics. The training involves developing further expertise in functional data analysis in combination with principle component analysis, model based clustering, and other statistical methods to identify repertoires of oromandibular movements that are functional or “normal” versus dysfunctional, i.e., those associated with chronic conditions and congenital malformations of the dentoskeletal apparatus. The purpose of this work is to develop automated methods of data collection and analysis to provide a rigorous quantitative means for distinguishing between function and dysfunction, for predicting risks for chronic conditions secondary to tissue damage, fatigue, tissue failure or problematic forces.

Presentations: 
 

1. Introduction to Functional Data Analysis, Statistics 504: Statistical Consulting. (F 2010)
2. How we Communicate Via Facial Expression, Dentistry 509: Introduction to the Function of the Oralfacial Complex. (Su 2010)

Publications:

Peer Reviewed Journal Publications

Crane E., Rothman E., Childers D., Gerstner G., (In Preparation). Automated method for sequence and cycle segmentation of cyclical mastication data.

Crane E., Rothman E., Gerstner G., (In Preparation). Functional data analysis of human mastication: comparison with traditional analytic methods and analysis of intra-cycle phases.

Crane, E., & Gross, M. (Submitted). Kinematic Characteristics of Emotion-Related Body Movement.

 Crane E., Cassidy R., Rothman E., Gerstner G., Effect of registration on cyclical kinematic data. Journal of Biomechanics (2010).PMC2931315

Invited Book Chapters
Crane, E., Childers, D., Rothman, E. D., & Gerstner, G. E.. Functional Data Analysis for Biomechanics. Biomechanics, InTech, (In Preparation, anticipated 2011)

Gerstner, G. E., Crane, E., Kruger, G. Proximate and Ultimate Mechanisms of Mammalian Mastication, Biomechanics, InTech, (In Preparation, anticipated 2011)

 Crane E., Gross M., & Fredrickson B. L. (In Press). Feasibility of Using a Head-Mounted Camera to Capture Dynamic Facial Expressions During Body Movement. Whole Body Interaction, Springer, 2011.

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William King

Faculty Mentor: Paul Krebsbach

Home Department: Biologic and Material Sciences

Research Title: Developing biomaterials to control stem cell behavior.

Research Summary: For my postdoctoral work I am focusing on forming biomaterials to control the activity of master regulatory genes during stem cell differentiation and tissue regeneration. For example, I am forming protein-based hydrogel microspheres that are internalized by stem cells that increase the trafficking of the transcription factor Sox9 into the nucleus to enhance chondrogenesis. In a separate project I am patterning adenoviruses with genes encoding for master transcription factors and/or growth factors onto biomaterials to form tissue interfaces from a single stem cell population. Finally, I am incorporating adenovirus-derived peptides into biomaterials to explore the fundamental mechanisms that control the adenoviral transduction of stem cells.

Presentations:  The development of dynamic materials for biomedical applications. Universityof Toledo Bioengineering Department Seminar. Toledo, OH, Fall, 2010.

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   Christopher Wilson

Faculty Mentor:  Renny Franceschi

Home Department:  Periodontics and Oral Medicine

Research Title: Methods and Mechanisms of Enhancing Regeneration of Bone Via Timely Delivery of Angiogenic and Osteogenic Growth Factors

Research Summary: The aims of our work are to 1) implement cutting edge gene therapy techniques for the tight spatiotemporal control over expression of multiple growth factors for enhanced regeneration of bone; 2) examine the mechanisms by which sequential induction of angiogenic and osteogenic growth factors enhance the regeneration of bone. With respect to aim 1, I am working with synthetic genetic circuits that provide stringent control over expression of target genes. These circuits drive expression of an angiogenic factor (basic FGF) or an osteogenic factor (BMP2) under the control of a heat shock promoter and a transactivator sensitive to the presence of rapamycin or mifepristone. In parallel with development of these circuits, we are collaborating with faculty and staff in the department of radiology to apply high intensity focused ultrasound for spatially-restricted activation of gene expression by localized tissue heating. With respect to aim 2, I am evaluating the immunophenotype and differentiation potential of progenitor cell populations following stimulation with basic FGF in vivo. We are using flow cytometry to characterize the abundance of hematopoietic, endothelial, and mesenchymal cells within these populations, and we are interested in the abundance and recruitment of “pericytes,” progenitor cells found adjacent to blood vessels throughout the body and thought to hold a particularly potent regenerative capacity.

Presentations: 

University of Michigan School of Dentistry Research Day 2010: “Temperature- and Ligand-Inducible Gene Expression Systems for Bone Regeneration” Feb 2, 2010.

“Engineering the Sequential Delivery of FGF2 and BMP2 for Regeneration of Bone.” 2011 Annual Meeting of the Orthopaedic Research Society, Long Beach, CA.

Publications:

WilsonCG, Sisco PN, Gadala-Maria F, Murphy CJ, Goldsmith EC. (2009) “Polyelectrolyte-Coated Gold Nanorods and their Interactions with Type I Collagen” Biomaterials 30(29):5639-48. PMC2754819. (Epub 2009 Jul 30)

WilsonCG, Vanderploeg, EJ, Zuo F, Sandy JD, Levenston ME. (2009) “Aggrecanolysis and In Vitro Degradation in the Immature Bovine Meniscus: Mechanisms and Functional Implications” Arthritis Research & Therapy 11(6):R173. PMC - In Process. (Epub 2009 Nov 17)

Connelly JT, Vanderploeg EJ, Mouw JK, Wilson CG, Levenston ML. (2010) “Tensile Loading Modulates BMSC Differentiation and the Development of Engineered Fibrocartilage Constructs” Accepted, Tissue Engineering. (Epub 2010 Jan 20)

Franceschi RT, Ge C, Wilson CG. (2011) “Osteoblasts of Craniofacial Bone,” in Tissues in Oral and Craniofacial Science: Biological Principles and Clinical Correlates. (Book Chapter)

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  PhD Program

 Jeremy Holzwarth

Faculty Mentor:  Peter Ma

Home Department: Biomedical Engineering

 Research Title: Material architecture effects on embryonic stem cell differentiation

Research Summary:  Embryonic stem cells are a promising cell source for tissue engineering due to their high proliferative capacity and pluripotency. However, that pluripotency also makes their fate difficult to control with a high level of efficiency. Recent findings in both our lab and others have shown that the fiber size of the matrix the cells are seeded on can have a significant effect on their differentiation. My work delves into this further to elucidate the effect of the material architecture on stem cell fate. I will focus on the fiber size, but also look at fiber density and orientation.

One of the applications of this work will be to develop a scaffold for neuronal tissue engineering. Currently, there are no truly effective and reliable treatment strategies to repair serious nerve damage. Combining an optimized scaffold designed to guide embryonic stem cells along the neuronal cell line with controlled drug delivery will hopefully provide a means for functional recovery.

Presentations: 

J. M. Holzwarth & M. Gupte. Polymeric biomaterials and tissue engineering. Biomedical Engineering Laboratory Expo, University of Michigan, Ann Arbor, MI (March 2010)
 

Publications:

J. M. Holzwarth & P. X. Ma. 3D nanofibrous scaffolds for tissue engineering. (In submission process)

X. Liu, J. M. Holzwarth, & P. X. Ma. Functionalized biodegradable polymer scaffolds for tissue engineering. (In submission process)

C. Sun, X. Jin, J. M. Holzwarth, X. Liu, J. Hu, M. Gupte, Y. Zhao, & P. X. Ma. Nanofibrous thin-walled single and multichannel poly(L-lactic acid) conduits for neural tissue engineering. (In submission process)

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 Megan Michalski

 Home Department: Oral Health Sciences PhD Program, School of Dentistry 

Research Mentor: Dr. Vesa Kaartinen, PhD       

 Research Title: Summer/Fall 2010 Lab Rotation: Understanding the Role of TGF-beta Type I Receptor/ALK5 in Palatal Development; Exploring Cardiomyoctye and Craniofacial Muscle Development

 Research Summary: I am involved in two projects in Dr. Kaartinen’s lab.  The first project will look at TGF-beta type I receptor/ALK5 and its role in palatal development.  Mutant mice lacking the Alk5 gene develop cleft palate; therefore, understanding TGF-beta signaling via ALK5 may provide insight into abnormal palate development.  We will culture mesenchymal cells from the palate of E14 Alk5^fx/fx mice and look at levels of different proteins in the TGF-beta signaling pathway.  The second project will look at cardiomyocyte and craniofacial muscle development and factors associated with differentiation of embryonic stem cells.
 

Presentations: 

M.N. Michalski, F.Q. Pirih, A.J. Koh, J.E. Berry, C.W. Ross, and L.K. McCauley. Interleukin-6 is a critical mediator of parathyroid hormone expansion of hematopoietic Cells, 3rd International Conference on Osteoimmunology, June 2010.
 

Publications:
Pirih FQ, Michalski MN, Cho SW, Koh AJ, Berry JE, et al. (2010) Parathyroid Hormone Mediates Hematopoietic Cell Expansion through Interleukin-6. PLoS ONE 5(10): e13657. PMCID: PMC2965090

 Awards:

3rd International Conference on Osteoimmunology Young Investigator Travel Award

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Stephanie Nunez

Home Department: Oral Health Sciences PhD Program, School of Dentistry 

Research Mentor:  Dr. James Simmer  

Research Title: Identification of the gene and protein product responsible for Golgi casein kinase activity. 

Research Summary: Phosphorylation is an important and ubiquitous way for cells to regulate a multitude of functions.  The protein Golgi casein kinase (GCK) has been implicated in the phosphorylation of numerous secreted proteins at S-X-E/pS motifs (Duncan et al., 2000; Lasa et al., 1997; Tibaldi et al., 2006).  Several enamel extracellular matrix (ECM) proteins including amelogenin (AMEL), enamelin (ENAM), and ameloblastin (AMBN) are members of the secretory calcium-binding phospho-protein (SCPP) family of proteins that contain phosphorylation sites at such SXE motifs which suggests that GCK could potentially be the kinase responsible for phosphorylating these proteins.  To date, GCK has only been characterized biochemically.  Therefore, the purpose of this study is to identify the primary protein sequence and gene responsible for GCK.

Current evidence suggests that GCK is localized to the Golgi membrane.  Studies have shown that GCK activity is present on the luminal side of the Golgi and that its activity increases after detergent solubilization of the membrane (Bingham and Farrel, 1974; Vegh and Varro, 1994; West and Clegg, 1984).  In addition, after a series of detergent and salt washes to separate luminal from membrane proteins, kinase activity was highest in the membrane fraction (Lamkin and Lindhe, 2001).  A previous study found GCK activity in purified bovine milk extracts and suggested that GCK might instead be a soluble enzyme.  However, the study was not able to differentiate whether the activity was due to an entirely soluble enzyme or whether it resulted from a cleaved active fragment (Duncan et al., 2000).  Therefore, this study will also try to determine whether GCK activity preferentially correlates with Golgi membrane components as this will affect how the protein could potentially function. 

Evidence that GCK potentially plays a crucial role in the regulation of many secreted proteins that are involved in a multitude of functions underlines the need to determine the structural and genetic basis for this kinase.  Therefore, I hypothesized that GCK activity is localized to the Golgi membrane and that it is responsible for phosphorylating amelogenin.

 Specific Aim 1:Identify the primary protein sequence and gene responsible for GCK.

Freshly prepared pig liver homogenates will be ultracentrifuged to separate the Golgi from other subcellular compartments.  The Golgi fraction will be identified by western blot and confirmed with transmission electron microscopy (TEM).  This fraction will then be injected into a C18 column for separation of proteins based on hydrophobicity.  Samples will be collected every five minutes and tested for GCK activity.  Positive samples will be reapplied to the C-18 column and each peak will then be tested for GCK activity.  Active peaks will be further fractionated using the PF2D system.  Each resulting fraction will be tested for GCK activity. 

GCK activity will be determined using the non-phosphorylated recombinant AMEL and a β-casein peptide fragment which only GCK can phosphorylate but not other casein kinases (CKI or CKII) (Lasa et al., 1997).  Both peptides will be radiolabeled with P-32 to track phosphorylation by the kinase.  The β-casein fragment will be used to outcompete AMEL phosphorylation to confirm that GCK is responsible for phosphorylation of both peptides.  Radiolabeling will occur in the presence of staurosporine, a general inhibitor of protein kinases to which GCK is uniquely resistant to (Tibaldi et al., 2006). Radiolabeling results will be quantified using a liquid scintillation counter (LS 6500, Beckman Coulter). 

 Active fractions isolated by the PF2D system will be subjected to 2D-gel analysis.  All resulting spots on the 2D-gel will be isolated and sequenced.  Sequences will be checked using BLAST search to identify similar protein and nucleotide sequences among various species, and then the percent sequence identity will be determined.  Protein functional domain analyses will be conducted and the GCK unique sequence will be used to generate two anti-peptide antibodies. 

Specific Aim 2:Determine if GCK activity correlates with Golgi membrane

The Golgi fraction that is isolated according to the above described method will be further centrifuged at higher speeds and increased lengths to separate out the Golgi membrane portion from soluble components.  The Golgi membrane fraction will additionally be subjected to a series of NaCl washes and detergents to remove peripheral and membrane bound proteins.  Each of the fractions (soluble and membrane) will be applied to the C-18 and PF2D systems separately and resulting fractions will be tested for GCK activity. 

            GCK expression pattern in liver sections will be determined using immunohistochemistry and its subcellular localization will be defined using immunogold labeling of the anti-peptide antibodies at the TEM level. 

Publications:

Nunez S, Lee JS, Zhang Y, Bai G, Ro JY. Role of peripheral mu-opioid receptors in inflammatory orofacial muscle pain. Neuroscience. 2007 May 25;146(3):1346-54. Epub 2007 Mar 26. PMID: 17379421 (published before being on training grant)

Sahu SN, Nunez S, Bai G, Gupta A.INTERACTION OF PYK2 AND PTP-PEST WITH LEUPAXIN IN PROSTATE CANCER CELLS. Am J Physiol Cell Physiol. 2007 Feb 28; PMID: 17329398 (published before being on training grant)

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Kathryn Ritchie

Research Mentor:  Dr. Yvonne Kapila

Home Department:  Oral Health Sciences PhD Program, School of Dentistry

Research Summary:  Oral squamous cell carcinoma (OSCC) accounts for 90% of oral malignancies and 5-year survival rates remain poor. Resistance to apoptosis contributes to development and progression of oral cancer; therefore, finding therapies promoting selective OSCC apoptosis remains crucial for mitigating oral cancer. Antimicrobial peptides affect many biological functions including induction of tumor cell apoptosis. Antimicrobial peptides have been investigated as potential therapeutic drug candidates, and function, in part, by increasing intracellular calcium. Such peptides include nisin, a common food additive
 

We hypothesized that nisin induces apoptosis in OSCC cells and tumors by increasing intracellular calcium.    In vitro experiments to examine the effect of nisin on OSCC cells included: cell proliferation and apoptosis assays, caspase-3 immunoblotting, cell cycle status via flow cytometry, and calcium influx assays and affymetrix arrays to examine nisin’s mechanistic effects.  An orthotopic oral cancer mouse model was used to study the inhibitory effect of nisin on OSCC tumor growth in vivo.  
Nisin induced preferential apoptosis of OSCC cells compared to primary oral keratinocytes, which did not undergo apoptosis at the same concentrations of nisin. High levels of active caspase-3 in OSCC cells confirmed nisin-induced apoptosis. Nisin inhibited OSCC cell proliferation in a time- and dose-dependent manner, unlike in keratinocytes. A concomitant influx of extracellular calcium and cell cycle arrest followed nisin treatment. Affymetrix arrays identified potential nisin-modified genes, including calcium-transport genes. Mice tolerated high nisin doses without compromising survival. Nisin administration reduced OSCC tumor burden in mice compared to controls.  
These studies demonstrate that nisin suppresses OSCC cell and tumor growth in vitro and in vivo by promoting OSCC cell apoptosis and cell cycle arrest, while inhibiting cell proliferation. Increases in intracellular calcium potentially mediate this mechanism. These findings help establish baseline information regarding potential therapeutic use of nisin for OSCC treatment. 

Presentations:

Nisin, a bacteriocin and food-additive, suppresses OSCC in vitro/in vivo

K. Ritchie, N.E. Joo, P. Kamarajan, Y. Kapila

University of Michigan School of Dentistry Research Day, 2010.

AADR Hatton Award Competition, March 2010

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