Transdifferentiation of fibroblasts to neural stem cells

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2013.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Cataloged from student-submitted PDF version of thesis.Includes bibliographical references.The developmental process is carefully controlled by transcriptional and epigenetic changes that occur as a zygote transforms into an adult organism. This process can be reversed by the overexpression of transcription factors Oct4, Sox2, Klf4, and c-Myc, which reprogram a differentiated cell!s nucleus to one that is transcriptionally and epigenetically indistinguishable from an embryonic stem (ES) cell. However, it is still unclear if transcription factors can completely convert the nucleus of a differentiated cell into that of a distantly related somatic cell type with complete transcriptional and epigenetic reprogramming maintained in the absence of exogenous factor expression. To test this idea, we generated doxycyline (dox)-inducible vectors encoding neural stem cell-expressed factors. We found that stable, self-maintaining NSC-like cells could be induced under defined growth conditions. These cells were characterized in the absence of exogenous factor induction and were shown to be transcriptionally, epigenetically, and functionally similar to endogenous embryonic cortical NSCs. Additionally, a cellular system was created for reproducible generation of doxindependent iNSCs without additional factor transduction. Our results show that a transcriptionally and epigenetically reprogrammed somatic nucleus can be stabilized in vitro and provides a tool to study the mechanism of somatic cell conversion.by John P. Cassady.Ph.D

Genetic engineering of human ES and iPS cells using TALE nucleases

Targeted genetic engineering of human pluripotent cells is a prerequisite for exploiting their full potential. Such genetic manipulations can be achieved using site-specific nucleases. Here we engineered transcription activator–like effector nucleases (TALENs) for five distinct genomic loci. At all loci tested we obtained human embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) clones carrying transgenic cassettes solely at the TALEN-specified location. Our data suggest that TALENs employing the specific architectures described here mediate site-specific genome modification in human pluripotent cells with similar efficiency and precision as do zinc-finger nucleases (ZFNs).National Institutes of Health (U.S.) (Grant R37-CA084198)National Institutes of Health (U.S.) (Grant RO1-CA087869)National Institutes of Health (U.S.) (Grant RO1-HD045022)Howard Hughes Medical Institut

First insights into the vertical habitat use of the whitespotted eagle ray Aetobatus narinari revealed by pop‐up satellite archival tags

The whitespotted eagle ray Aetobatus narinari is a tropical to warm‐temperate benthopelagic batoid that ranges widely throughout the western Atlantic Ocean. Despite conservation concerns for the species, its vertical habitat use and diving behaviour remain unknown. Patterns and drivers in the depth distribution of A. narinari were investigated at two separate locations, the western North Atlantic (Islands of Bermuda) and the eastern Gulf of Mexico (Sarasota, Florida, U.S.A.). Between 2010 and 2014, seven pop‐up satellite archival tags were attached to A. narinari using three methods: a through‐tail suture, an external tail‐band and through‐wing attachment. Retention time ranged from 0 to 180 days, with tags attached via the through‐tail method retained longest. Tagged rays spent the majority of time (82.85 ± 12.17% S.D.) within the upper 10 m of the water column and, with one exception, no rays travelled deeper than ~26 m. One Bermuda ray recorded a maximum depth of 50.5 m, suggesting that these animals make excursions off the fore‐reef slope of the Bermuda Platform. Individuals occupied deeper depths (7.42 ± 3.99 m S.D.) during the day versus night (4.90 ± 2.89 m S.D.), which may be explained by foraging and/or predator avoidance. Each individual experienced a significant difference in depth and temperature distributions over the diel cycle. There was evidence that mean hourly depth was best described by location and individual variation using a generalized additive mixed model approach. This is the first study to compare depth distributions of A. narinari from different locations and describe the thermal habitat for this species. Our study highlights the importance of region in describing A. narinari depth use, which may be relevant when developing management plans, whilst demonstrating that diel patterns appear to hold across individuals

An Analysis of the Legal, Social, and Political Issues Raised by Asbestos Litigation

This Special Project examines the most important issues of the asbestos problem and advocates a congressional solution (1) to relieve the courts of the thousands of present and potential asbestos cases, (2) to protect future claimants\u27 rights to adequate compensation, and (3) to provide for equitable participation by all responsible parties, which, in addition to asbestos manufacturers,include the federal government, insurance companies, and the tobacco industry. The first six parts of the Special Project examine the various issues of asbestos litigation: theories of liability in products liability suits against asbestos manufacturers, causation,defenses, statutory limitations on actions, collateral estoppel, and punitive damages. The Special Project then discusses in parts VIII,IX, and X the methods used by asbestos manufacturers to attempt to spread their liability through asserting insurer liability, the exclusive remedy of workers\u27 compensation, and indemnity and contribution from the United States. Finally, the Special Project evaluates and analyzes recent developments in the asbestos litigation area, including proposals for federal legislative compensation programs and business alternatives available to asbestos manufacturers facing enormous asbestos-related liabilities... This Special Project critically has examined the most important issues concerning the asbestos problem. It has considered the complex legal, legislative, and social questions that society must confront in order to resolve this predicament. Only swift action by Congress in the form of a fair and comprehensive compensation scheme for victims of asbestos-related disabilities will initiate a solution to this difficult and pervasive problem

The VASIMR[registered trademark] VF-200-1 ISS Experiment as a Laboratory for Astrophysics

The VASIMR[R] Flight Experiment (VF-200-1) will be tested in space aboard the International Space Station (ISS) in about four years. It will consist of two 100 kW parallel plasma engines with opposite magnetic dipoles, resulting in a near zero-torque magnetic system. Electrical energy will come from ISS at low power level, be stored in batteries and used to fire the engine at 200 kW. The VF-200-1 project will provide a unique opportunity on the ISS National Laboratory for astrophysicists and space physicists to study the dynamic evolution of an expanding and reconnecting plasma loop. Here, we review the status of the project and discuss our current plans for computational modeling and in situ observation of a dynamic plasma loop on an experimental platform in low-Earth orbit. The VF-200-1 project is still in the early stages of development and we welcome new collaborators

Association between neighborhood need and spatial access to food stores and fast food restaurants in neighborhoods of Colonias

Objective To determine the extent to which neighborhood needs (socioeconomic deprivation and vehicle availability) are associated with two criteria of food environment access: 1) distance to the nearest food store and fast food restaurant and 2) coverage (number) of food stores and fast food restaurants within a specified network distance of neighborhood areas of colonias, using ground-truthed methods. Methods Data included locational points for 315 food stores and 204 fast food restaurants, and neighborhood characteristics from the 2000 U.S. Census for the 197 census block group (CBG) study area. Neighborhood deprivation and vehicle availability were calculated for each CBG. Minimum distance was determined by calculating network distance from the population-weighted center of each CBG to the nearest supercenter, supermarket, grocery, convenience store, dollar store, mass merchandiser, and fast food restaurant. Coverage was determined by calculating the number of each type of food store and fast food restaurant within a network distance of 1, 3, and 5 miles of each population-weighted CBG center. Neighborhood need and access were examined using Spearman ranked correlations, spatial autocorrelation, and multivariate regression models that adjusted for population density. Results Overall, neighborhoods had best access to convenience stores, fast food restaurants, and dollar stores. After adjusting for population density, residents in neighborhoods with increased deprivation had to travel a significantly greater distance to the nearest supercenter or supermarket, grocery store, mass merchandiser, dollar store, and pharmacy for food items. The results were quite different for association of need with the number of stores within 1 mile. Deprivation was only associated with fast food restaurants; greater deprivation was associated with fewer fast food restaurants within 1 mile. CBG with greater lack of vehicle availability had slightly better access to more supercenters or supermarkets, grocery stores, or fast food restaurants. Increasing deprivation was associated with decreasing numbers of grocery stores, mass merchandisers, dollar stores, and fast food restaurants within 3 miles. Conclusion It is important to understand not only the distance that people must travel to the nearest store to make a purchase, but also how many shopping opportunities they have in order to compare price, quality, and selection. Future research should examine how spatial access to the food environment influences the utilization of food stores and fast food restaurants, and the strategies used by low-income families to obtain food for the household

c-Myc Is Dispensable for Direct Reprogramming of Mouse Fibroblasts

SummaryRetroviral transduction of the four transcription factors Oct4, Sox2, Klf4, and c-Myc has been shown to initiate a reprogramming process that results in the transformation of mouse fibroblasts into embryonic stem (ES)-like cells designated as induced pluripotent stem (iPS) cells (Maherali et al., 2007; Meissner et al., 2007; Okita et al., 2007; Takahashi and Yamanaka, 2006; Wernig et al., 2007). The promise of somatic reprogramming is the possibility to generate pluripotent stem cells that are patient specific and can be used as a unique source for autologous cell types for transplantation therapy (Jaenisch, 2004; Yamanaka, 2007). Many iPS cell-derived animals develop tumors due to the reactivation of the c-Myc virus (Okita et al., 2007), and this represents a major safety concern if we want to translate this approach to humans. It is thus of great importance to achieve reprogramming without this particular oncogene in the future. Here we show that fibroblasts can be reprogrammed to a pluripotent state by Oct4, Sox2, and Klf4 in the absence of c-Myc

Stable isotopes reveal rail-associated behavior in a threatened carnivore

Human–wildlife conflict is a leading cause of adult mortality for large carnivores worldwide. Train collision is the primary cause of mortality for threatened grizzly bears (Ursus arctos) in Banff National Park. We investigated the use of stable isotope analysis as a tool for identifying bears that use the railway in Banff. Rail-associated bears had higher δ(15)N and δ(34)S values than bears sampled away from the rail, but similar δ(13)C values. Because elevated δ(15)N values are indicative of higher animal protein consumption, rail-associated bears likely preyed on ungulates that foraged along the rail or scavenged on train-killed animals. The higher δ(34)S values in bear hair could have resulted from bears consuming sulfur pellets spilled on the rail or through the uptake of sulfur in the plants bears or animals consumed. Similar δ(13)C values suggest that the two types of bears had generally similar plant-based diets. Results from this study suggest that stable isotopes analysis could be used as a non-invasive, affordable, and efficient technique to identify and monitor bears that forage on the railway in Banff and potentially other transportation corridors worldwide