Acceleration of the PanIN Development in Mice Expressing Oncogenic K-Ras Due to a High Fat Diet

Obesity is postulated to be one of the major risk factors for pancreatic cancer, and recently it was indicated that an elevated body mass index (BMI correlates strongly with a decrease in patient survival. Despite the evident relationship, the molecular mechanisms involved are unclear. Oncogenic mutation of K-Ras is found early and is universal in pancreatic cancer. Extensive evidence indicates oncogenic K-Ras is not entirely active and it requires a triggering event to surpass the activity of Ras beyond the threshold necessary for a Ras-inflammation feed-forward loop. We hypothesize that high fat intake induces a persistent low level inflammatory response triggering increased K-Ras activity and that Cox-2 is essential for this inflammatory reaction. To determine this, LSL-K-Ras mice were crossed with Ela-CreER (Acinar-specific) or Pdx-1-Cre (Pancreas-specific) to “knock-in” oncogenic K-Ras. Additionally, these animals were crossed with Cox-2 conditional knockout mice to access the importance of Cox-2 in the inflammatory loop present. The mice were fed isocaloric diets containing 60% energy or 10% energy from fat. We found that a high fat diet increased K-Ras activity, PanIN formation, and fibrotic stroma significantly compared to a control diet. Genetic deletion of Cox-2 prevented high fat diet induced fibrosis and PanIN formation in oncogenic K-Ras expressing mice. Additionally, long term consumption of high fat diet, increased the progression of PanIN lesions leading to invasive cancer and decreased overall survival rate. These findings indicate that a high fat diet can stimulate the activation of oncogenic K-Ras and initiate an inflammatory feed forward loop requiring Cox-2 leading to inflammation, fibrosis, and PanINs. This mechanism could explain the relationship between a high fat diet and elevated risk for pancreatic cancer.

Dynamic Chemical Shift Imaging for Image-Guided Thermal Therapy

Magnetic resonance temperature imaging (MRTI) is recognized as a noninvasive means to provide temperature imaging for guidance in thermal therapies. The most common method of estimating temperature changes in the body using MR is by measuring the water proton resonant frequency (PRF) shift. Calculation of the complex phase difference (CPD) is the method of choice for measuring the PRF indirectly since it facilitates temperature mapping with high spatiotemporal resolution. Chemical shift imaging (CSI) techniques can provide the PRF directly with high sensitivity to temperature changes while minimizing artifacts commonly seen in CPD techniques. However, CSI techniques are currently limited by poor spatiotemporal resolution. This research intends to develop and validate a CSI-based MRTI technique with intentional spectral undersampling which allows relaxed parameters to improve spatiotemporal resolution. An algorithm based on autoregressive moving average (ARMA) modeling is developed and validated to help overcome limitations of Fourier-based analysis allowing highly accurate and precise PRF estimates. From the determined acquisition parameters and ARMA modeling, robust maps of temperature using the k-means algorithm are generated and validated in laser treatments in ex vivo tissue. The use of non-PRF based measurements provided by the technique is also investigated to aid in the validation of thermal damage predicted by an Arrhenius rate dose model.

FUNCTIONS OF DEADENYLATION FACTORS IN MRNA DECAY AND MRNA PROCESSING BODY FORMATION

Most newly synthesized messenger RNAs possess a 5’ cap and a 3’ poly(A) tail. The process of poly(A) tail shortening, also termed deadenylation, is important for post-transcriptional gene regulation, because deadenylation not only leads to mRNA translational inhibition but also is the first step of major mRNA degradation. Translationally inhibited mRNAs can be stored and/or degraded in dynamic cytoplasmic foci termed mRNA processing bodies, or P bodies, which are conserved in eukaryotes. To shed new light on the mechanisms of P body formation and P body functions, I focused on the link between deadenylation factors and P bodies. I found that the two major deadenylation complexes, Pan3-Pan2 and Ccr4-Caf1, can both be enriched in P bodies. The deadenylase activity of the Ccr4-Caf1 complex is prerequisite for P body formation. Pan3, but not the deadenylase Pan2, is essential for P body formation. While the C-terminal domain of Pan3 is important for interaction with Pan2, Pan3 N-terminal domain is important for Pan3 to form cytoplasmic foci colocalizing with P bodies and to promote mRNA decay. Interestingly, Pan3 N-terminal domain may be phosphorylated to regulate Pan3 localization and functions. Aside from the functions of the two deadenylation complexes in P bodies, I also studied all reported human P body proteins as a whole using bioinformatics. This effort not only has generated a comprehensive picture of the functions of and interactions among human P body proteins, but also has predicted proteins that may regulate P body formation and/or functions. In summary, my study has established a direct link between mRNA deadenylation and P body formation and has also led to new hypotheses to guide future research on how P body dynamics are controlled.

Dynamic Remodeling of the Stressed Heart: Role of Protein Degradation Pathways

The heart is a remarkable organ. In order to maintain its function, it remodels in response to a variety of environmental stresses, including pressure overload, volume overload, mechanical or pharmacological unloading and hormonal or metabolic disturbances. All these responses are linked to the inherent capacity of the heart to rebuild itself. Particularly, cardiac pressure overload activates signaling pathways of both protein synthesis and degradation. While much is known about regulators of protein synthesis, little is known about regulators of protein degradation in hypertrophy. The ubiquitin-proteasome system (UPS) selectively degrades unused and abnormal intracellular proteins. I speculated that the UPS may play an important role in both qualitative and quantitative changes in the composition of heart muscle during hypertrophic remodeling. My study hypothesized that cardiac remodeling in response to hypertrophic stimuli is a dynamic process that requires activation of highly regulated mechanisms of protein degradation as much as it requires protein synthesis. My first aim was to adopt a model of left ventricular hypertrophy and determine its gene expression and structural changes. Male Sprague-Dawley rats were submitted to ascending aortic banding and sacrificed at 7 and 14 days after surgery. Sham operated animals served as controls. Effective aortic banding was confirmed by hemodynamic assessment by Doppler flow measurements in vivo. Banded rats showed a four-fold increase in peak stenotic jet velocities. Histomorphometric analysis revealed a significant increase in myocyte size as well as fibrosis in the banded animals. Transcript analysis showed that banded animals had reverted to the fetal gene program. My second aim was to assess if the UPS is increased and transcriptionally regulated in hypertrophic left ventricular remodeling. Protein extracts from the left ventricles of the banded and control animals were used to perform an in vitro peptidase assay to assess the overall catalytic activity of the UPS. The results showed no difference between hypertrophied and control animals. Transcript analysis revealed decreases in transcript levels of candidate UPS genes in the hypertrophied hearts at 7 days post-banding but not at 14 days. However, protein expression analysis showed no difference at either time point compared to controls. These findings indicate that elements of the UPS are downregulated in the early phase of hypertrophic remodeling and normalizes in a later phase. The results provide evidence in support of a dynamic transcriptional regulation of a major pathway of intracellular protein degradation in the heart. The discrepancy between transcript levels on the one hand and protein levels on the other hand supports post-transcriptional regulation of the UPS pathway in the hypertrophied heart. The exact mechanisms and the functional consequences remain to be elucidated.

Deadenylation is prerequisite for P-body formation and mRNA decay in mammalian cells.

Deadenylation is the major step triggering mammalian mRNA decay. One consequence of deadenylation is the formation of nontranslatable messenger RNA (mRNA) protein complexes (messenger ribonucleoproteins [mRNPs]). Nontranslatable mRNPs may accumulate in P-bodies, which contain factors involved in translation repression, decapping, and 5'-to-3' degradation. We demonstrate that deadenylation is required for mammalian P-body formation and mRNA decay. We identify Pan2, Pan3, and Caf1 deadenylases as new P-body components and show that Pan3 helps recruit Pan2, Ccr4, and Caf1 to P-bodies. Pan3 knockdown causes a reduction of P-bodies and has differential effects on mRNA decay. Knocking down Caf1 or overexpressing a Caf1 catalytically inactive mutant impairs deadenylation and mRNA decay. P-bodies are not detected when deadenylation is blocked and are restored when the blockage is released. When deadenylation is impaired, P-body formation is not restorable, even when mRNAs exit the translating pool. These results support a dynamic interplay among deadenylation, mRNP remodeling, and P-body formation in selective decay of mammalian mRNA.

Deadenylation is prerequisite for P-body formation and mRNA decay in mammalian cells.

Deadenylation is the major step triggering mammalian mRNA decay. One consequence of deadenylation is the formation of nontranslatable messenger RNA (mRNA) protein complexes (messenger ribonucleoproteins [mRNPs]). Nontranslatable mRNPs may accumulate in P-bodies, which contain factors involved in translation repression, decapping, and 5'-to-3' degradation. We demonstrate that deadenylation is required for mammalian P-body formation and mRNA decay. We identify Pan2, Pan3, and Caf1 deadenylases as new P-body components and show that Pan3 helps recruit Pan2, Ccr4, and Caf1 to P-bodies. Pan3 knockdown causes a reduction of P-bodies and has differential effects on mRNA decay. Knocking down Caf1 or overexpressing a Caf1 catalytically inactive mutant impairs deadenylation and mRNA decay. P-bodies are not detected when deadenylation is blocked and are restored when the blockage is released. When deadenylation is impaired, P-body formation is not restorable, even when mRNAs exit the translating pool. These results support a dynamic interplay among deadenylation, mRNP remodeling, and P-body formation in selective decay of mammalian mRNA.

Changes in blood lipids and their associations with changes in measures of height, weight and body mass index from age 8 to 18 years: Project Heartbeat!

A variety of studies indicate that the process of athrosclerosis begins in childhood. There was limited information on the association of the changes in anthropometric variables to blood lipids in school age children and adolescents. Previous longitudinal studies of children typically with insufficient frequency of observation could not provide sound inference on the dynamics of change in blood lipids. The aims of this analysis are (1) to document the sex- and ethnic-specific trajectory and velocity curves of blood lipids (TC, LDL-C, HDL-C and TG); (2) to evaluate the relationship of changes in anthropometric variables, such as height, weight and BMI, to blood lipids from age 8 to 18 years. ^ Project HeartBeat! is a longitudinal study designed to examine the patterns of serial change in major cardiovascular risk factors. Cohort of three different age levels, 8, 11 and 14 years at baseline, with a total of 678 participants were enrolled. Each member of these cohorts was examined three times per year for up to four years. ^ Sex- and ethnic-specific trajectory and velocity curves of blood lipids; demonstrated the complex and polyphasic changes in TC, LDL-C, HDL-C and TG longitudinally. The trajectory curves of TC, LDL-C and HDL-C with age showed curvilinear patterns of change. The velocity change in TC, HDL-C and LDL-C showed U-shaped curves for non-Blacks, and nearly linear lines in velocity of TG for both Blacks and non-Blacks. ^ The relationship of changes in anthropometric variables to blood lipids was evaulated by adding height, weight, or BMI and associated interaction terms separately to the basic age-sex models. Height or height gain had a significant negative association with changes in TC, LDL-C and HDL-C. Weight or BMI gain showed positive associations with TC, LDL-C and TC, and a negative relationship with HDL-C. ^ Dynamic changes of blood lipids in school age children and adolescents observed from this analysis suggested that using fixed screening criteria under the current NCEP guidelines for all ages 2–19 may not be appropriate for this age group. The association of increasing BMI or weight to an adverse blood lipid profile found in this analysis also indicated that weight or BMI monitoring could be a future intervention to be implemented in the pediatric population. ^

Contraceptive use in women by body mass index categories

The prevalence of obesity has increased sharply in the United States since the mid 1970's. Obese women who become pregnant are at increased risk of pregnancy complications for both mother and fetus. This study assessed whether women in higher body mass index (BMI) categories engage in the preventive behaviors of contraception more frequently than normal weight women. It also evaluated the type of contraception used by both obese and normal weight women. The study used cross-sectional data from 7 states participating in the Family Planning Module of the 2006 Behavioral Risk Factor Surveillance System (BRFSS). The Behavioral Risk Factor Surveillance System survey is an annual random digit dialed telephone survey of the non-institutionalized civilian population aged 18 years and older. The Family Planning Module was administered by Arizona, Kentucky, Minnesota, Missouri, Montana, Oregon, and Wisconsin. Of the 4,757 women who participated in the Family Planning Module, 2,244 (53.2%) were normal weight, 1,202 (25.6%) were overweight, and 1,072 (21.2%) were obese. The majority of these women 4,115 (86.2%) reported using some type of contraception to prevent pregnancy. Six hundred forty two women (13.8%) stated they did not use any type of contraception to prevent pregnancy. Within body mass index categories, 14% of normal weight women, 13% of overweight women, and 13.4% of obese women did not use any type of contraception. Neither the bivariate analysis nor the logistic regressions found body mass index categories to be statistically associated with contraceptive use. The relationship between body mass index categories and contraceptive method was found to be statistically significant. The predictive probability graph found that women at all levels of BMI have a lower probability of using barrier contraception methods as compared to procedural and hormonal methods. Hormonal contraception methods have the highest probability of use for women with a BMI of 15 to 25. In contrast, the probability of using procedural contraception methods is relatively flat and less than hormonal methods for BMI between 15 and 25. However, the probability of using procedural contraception increases dramatically with a BMI greater than 25. At a BMI greater than 42, women have a greater than 50% probability of using procedural contraception. Although a relationship between body mass index and contraception use was not found, contraception method was found to be associated with body mass index. The reasons why normal weight women prefer hormonal contraception while overweight/obese women are more likely to use procedural methods needs to be explored. By understanding the relationship between obesity and contraception, we can hopefully decrease unintended pregnancies and overall improve pregnancy related health outcomes. To determine if relationships between contraception use/type and body mass index exist, further research needs to be conducted on a national level. ^

The association of peer victimization and/or bullying perpetration and body dissatisfaction in elementary students from the healthy passages baseline study

This is the first U.S. study to investigate the association between peer victimization and/or bullying perpetration and body image dissatisfaction in American elementary school students. The study used data collected from the Healthy Passages baseline cohort of 5152 ethnically diverse fifth grade students from Alabama, Texas, and California. The overall prevalence of body dissatisfaction in our fifth grade sample was 50%. Students who were dissatisfied with their bodies were 23% more likely to be involved in peer victimization than students who were satisfied with their bodies (OR = 1.23; 95% CI = 1.02, 1.48). No significant associations were found between bullying status or bully-victim status and body dissatisfaction. Other factors that contributed significantly to a student's body image status were BMI, level of self-worth, level of household income, gender, pubertal timing and support received from adults (other than parents). It is noteworthy that those who were dissatisfied were heavier, came from lower income homes and were more likely to be male. Additional longitudinal studies are needed to confirm these associations, especially among large ethnically and socio-economically diverse samples. ^

Selection of body fat distribution indices in adults and associations with plasma lipid metabolism variables

Body fat distribution is a cardiovascular health risk factor in adults. Body fat distribution can be measured through various methods including anthropometry. It is not clear which anthropometric index is suitable for epidemiologic studies of fat distribution and cardiovascular disease. The purpose of the present study was to select a measure of body fat distribution from among a series of indices (those traditionally used in the literature and others constructed from the analysis) that is most highly correlated with lipid-related variables and is independent of overall fatness. Subjects were Mexican-American men and women (N = 1004) from a study of gallbladder disease in Starr County, Texas. Multivariate associations were sought between lipid profile measures (lipids, lipoproteins, and apolipoproteins) and two sets of anthropometric variables (4 circumferences and 6 skinfolds). This was done to assess the association between lipid-related measures and the two sets of anthropometric variables and guide the construction of indices.^ Two indices emerged from the analysis that seemed to be highly correlated with lipid profile measures independent of obesity. These indices are: 2*arm circumference-thigh skinfold in pre- and post-menopausal women and arm/thigh circumference ratio in men. Next, using the sum of all skinfolds to represent obesity and the selected body fat distribution indices, the following hypotheses were tested: (1) state of obesity and centrally/upper distributed body fat are equally predictive of lipids, lipoproteins and apolipoproteins, and (2) the correlation among the lipid-related measures is not altered by obesity and body fat distribution.^ With respect to the first hypothesis, the present study found that most lipids, lipoproteins and apolipoproteins were significantly associated with both overall fatness and anatomical location of body fat in both sex and menopausal groups. However, within men and post-menopausal women, certain lipid profile measures (triglyceride and HDLT among post-menopausal women and apos C-II, CIII, and E among men) had substantially higher correlation with body fat distribution as compared with overall fatness.^ With respect to the second hypothesis, both obesity and body fat distribution were found to alter the association among plasma lipid variables in men and women. There was a suggestion from the data that the pattern of correlations among men and post-menopausal women are more comparable. Among men correlations involving apo A-I, HDLT, and HDL$\sb2$ seemed greatly influenced by obesity, and A-II by fat distribution; among post-menopausal women correlations involving apos A-I and A-II were highly affected by the location of body fat.^ Thus, these data point out that not only can obesity and fat distribution affect levels of single measures, they also can markedly influence the pattern of relationship among measures. The fact that such changes are seen for both obesity and fat distribution is significant, since the indices employed were chosen because they were independent of one another. ^

Mind-body therapy use among U.S. children and adolescents

The use of complementary and alternative medicine (CAM) has increased dramatically in the United States in the past several decades. While botanicals and dietary supplements have received the majority of attention in the popular and scholarly literature on alternative therapies, mind-body therapies, such as biofeedback, meditation, hypnosis, massage and chiropractic care presently constitute a large portion of the American public's use of CAM. The present study explores the patterns and prevalence of such therapy use among an under-studied population of CAM users: children and adolescents. Using data from the 2007 National Health Interview Survey, this paper describes the prevalence, patterns, and predictors of mind-body therapy use among a nationally representative sample of children (n=9,417) using a multidimensional model of health care behavior. The contribution of predisposing, enabling, and medical factors to mind-body therapy use among children will be also examined. Results provide additional evidence to a growing body of literature documenting that children and adolescents increasingly turn to mind-body therapies to alleviate symptoms, cope with chronic or life-threatening diseases, and to promote overall well-being. ^

Examining the Relationship Between Media Use and Aggression, Sexuality, and Body Image

Objectives: The primary purpose of this research is to understand the media's impact on individual attitudes and behaviors related to aggression, sexuality, and body image. This research is of particular importance because it uses up-to-date data reflecting effects based on the current media environment. Additionally, it includes a racially diverse sample. Methods: A survey of 407 students at a large, public university was conducted. The survey instrument contained general measures related to media consumption, including overall television, video game, and internet use, as well as more specific questions related to particular types of media, such as pornography. For the dependent variables, questions were included that measured both attitudes and behaviors related to aggression, sexuality, and body image. Results: Consistent with predictions, media use impacted both attitudes and behaviors related to aggression, sexuality, and body image. Specifically, overall television consumption led to increased levels of aggression (r=.18, pr=.20, pr=.24, pr=.42, pr=.40, p Conclusions: The media continue to play an important role in the development of attitudes and behaviors. It is warranted, therefore, to continue to investigate what media can cause negative outcomes, as well as to determine how those outcomes vary based on race and gender.

Modeling Techniques for the High-Resolution Interpretation of Cryo-Electron Microscopy Reconstructions

Essential biological processes are governed by organized, dynamic interactions between multiple biomolecular systems. Complexes are thus formed to enable the biological function and get dissembled as the process is completed. Examples of such processes include the translation of the messenger RNA into protein by the ribosome, the folding of proteins by chaperonins or the entry of viruses in host cells. Understanding these fundamental processes by characterizing the molecular mechanisms that enable then, would allow the (better) design of therapies and drugs. Such molecular mechanisms may be revealed trough the structural elucidation of the biomolecular assemblies at the core of these processes. Various experimental techniques may be applied to investigate the molecular architecture of biomolecular assemblies. High-resolution techniques, such as X-ray crystallography, may solve the atomic structure of the system, but are typically constrained to biomolecules of reduced flexibility and dimensions. In particular, X-ray crystallography requires the sample to form a three dimensional (3D) crystal lattice which is technically di‑cult, if not impossible, to obtain, especially for large, dynamic systems. Often these techniques solve the structure of the different constituent components within the assembly, but encounter difficulties when investigating the entire system. On the other hand, imaging techniques, such as cryo-electron microscopy (cryo-EM), are able to depict large systems in near-native environment, without requiring the formation of crystals. The structures solved by cryo-EM cover a wide range of resolutions, from very low level of detail where only the overall shape of the system is visible, to high-resolution that approach, but not yet reach, atomic level of detail. In this dissertation, several modeling methods are introduced to either integrate cryo-EM datasets with structural data from X-ray crystallography, or to directly interpret the cryo-EM reconstruction. Such computational techniques were developed with the goal of creating an atomic model for the cryo-EM data. The low-resolution reconstructions lack the level of detail to permit a direct atomic interpretation, i.e. one cannot reliably locate the atoms or amino-acid residues within the structure obtained by cryo-EM. Thereby one needs to consider additional information, for example, structural data from other sources such as X-ray crystallography, in order to enable such a high-resolution interpretation. Modeling techniques are thus developed to integrate the structural data from the different biophysical sources, examples including the work described in the manuscript I and II of this dissertation. At intermediate and high-resolution, cryo-EM reconstructions depict consistent 3D folds such as tubular features which in general correspond to alpha-helices. Such features can be annotated and later on used to build the atomic model of the system, see manuscript III as alternative. Three manuscripts are presented as part of the PhD dissertation, each introducing a computational technique that facilitates the interpretation of cryo-EM reconstructions. The first manuscript is an application paper that describes a heuristics to generate the atomic model for the protein envelope of the Rift Valley fever virus. The second manuscript introduces the evolutionary tabu search strategies to enable the integration of multiple component atomic structures with the cryo-EM map of their assembly. Finally, the third manuscript develops further the latter technique and apply it to annotate consistent 3D patterns in intermediate-resolution cryo-EM reconstructions. The first manuscript, titled An assembly model for Rift Valley fever virus, was submitted for publication in the Journal of Molecular Biology. The cryo-EM structure of the Rift Valley fever virus was previously solved at 27Å-resolution by Dr. Freiberg and collaborators. Such reconstruction shows the overall shape of the virus envelope, yet the reduced level of detail prevents the direct atomic interpretation. High-resolution structures are not yet available for the entire virus nor for the two different component glycoproteins that form its envelope. However, homology models may be generated for these glycoproteins based on similar structures that are available at atomic resolutions. The manuscript presents the steps required to identify an atomic model of the entire virus envelope, based on the low-resolution cryo-EM map of the envelope and the homology models of the two glycoproteins. Starting with the results of the exhaustive search to place the two glycoproteins, the model is built iterative by running multiple multi-body refinements to hierarchically generate models for the different regions of the envelope. The generated atomic model is supported by prior knowledge regarding virus biology and contains valuable information about the molecular architecture of the system. It provides the basis for further investigations seeking to reveal different processes in which the virus is involved such as assembly or fusion. The second manuscript was recently published in the of Journal of Structural Biology (doi:10.1016/j.jsb.2009.12.028) under the title Evolutionary tabu search strategies for the simultaneous registration of multiple atomic structures in cryo-EM reconstructions. This manuscript introduces the evolutionary tabu search strategies applied to enable a multi-body registration. This technique is a hybrid approach that combines a genetic algorithm with a tabu search strategy to promote the proper exploration of the high-dimensional search space. Similar to the Rift Valley fever virus, it is common that the structure of a large multi-component assembly is available at low-resolution from cryo-EM, while high-resolution structures are solved for the different components but lack for the entire system. Evolutionary tabu search strategies enable the building of an atomic model for the entire system by considering simultaneously the different components. Such registration indirectly introduces spatial constrains as all components need to be placed within the assembly, enabling the proper docked in the low-resolution map of the entire assembly. Along with the method description, the manuscript covers the validation, presenting the benefit of the technique in both synthetic and experimental test cases. Such approach successfully docked multiple components up to resolutions of 40Å. The third manuscript is entitled Evolutionary Bidirectional Expansion for the Annotation of Alpha Helices in Electron Cryo-Microscopy Reconstructions and was submitted for publication in the Journal of Structural Biology. The modeling approach described in this manuscript applies the evolutionary tabu search strategies in combination with the bidirectional expansion to annotate secondary structure elements in intermediate resolution cryo-EM reconstructions. In particular, secondary structure elements such as alpha helices show consistent patterns in cryo-EM data, and are visible as rod-like patterns of high density. The evolutionary tabu search strategy is applied to identify the placement of the different alpha helices, while the bidirectional expansion characterizes their length and curvature. The manuscript presents the validation of the approach at resolutions ranging between 6 and 14Å, a level of detail where alpha helices are visible. Up to resolution of 12 Å, the method measures sensitivities between 70-100% as estimated in experimental test cases, i.e. 70-100% of the alpha-helices were correctly predicted in an automatic manner in the experimental data. The three manuscripts presented in this PhD dissertation cover different computation methods for the integration and interpretation of cryo-EM reconstructions. The methods were developed in the molecular modeling software Sculptor (http://sculptor.biomachina.org) and are available for the scientific community interested in the multi-resolution modeling of cryo-EM data. The work spans a wide range of resolution covering multi-body refinement and registration at low-resolution along with annotation of consistent patterns at high-resolution. Such methods are essential for the modeling of cryo-EM data, and may be applied in other fields where similar spatial problems are encountered, such as medical imaging.

Dynamic contrast agent -enhanced magnetic resonance imaging assessment of tumor microvasculature

Dynamic contrast agent-enhanced magnetic resonance imaging (DCE MRI) data, when analyzed with the appropriate pharmacokinetic models, have been shown to provide quantitative estimates of microvascular parameters important in characterizing the angiogenic activity of malignant tissue. These parameters consist of the whole blood volume per unit volume of tissue, v b, transport constant from the plasma to the extravascular, extracellular space (EES), k1 and the transport constant from the EES to the plasma, k2. Parameters vb and k1 are expected to correlate with microvascular density (MVD) and vascular permeability, respectively, which have been suggested to serve as surrogate markers for angiogenesis. In addition to being a marker for angiogenesis, vascular permeability is also useful in estimating tumor penetration potential of chemotherapeutic agents. ^ Histological measurements of the intratumoral microvascular environment are limited by their invasiveness and susceptibility to sampling errors. Also, MVD and vascular permeability, while useful for characterizing tumors at a single time point, have shown less utility in longitudinal studies, particularly when used to monitor the efficacy of antiangiogenic and traditional chemotherapeutic agents. These limitations led to a search for a non-invasive means of characterizing the microvascular environment of an entire tumor. ^ The overall goal of this project was to determine the utility of DCE MRI for monitoring the effect of antiangiogenic agents. Further applications of a validated DCE MRI technique include in vivo measurements of tumor microvascular characteristics to aid in determining prognosis at presentation and in estimating drug penetration. DCE MRI data were generated using single- and dual-tracer pharmacokinetic models with different molecular-weight contrast agents. The resulting pharmacokinetic parameters were compared to immunohistochemical measurements. The model and contrast agent combination yielding the best correlation between the pharmacokinetic parameters and histological measures was further evaluated in a longitudinal study to evaluate the efficacy of DCE MRI in monitoring the intratumoral microvascular environment following antiangiogenic treatment. ^