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Description
Induced pluripotent stem cells (iPSCs) are an intriguing approach for neurological disease modeling, because neural lineage-specific cell types that retain the donors' complex genetics can be established in vitro. The statistical power of these iPSC-based models, however, is dependent on accurate diagnoses of the somatic cell donors; unfortunately, many neurodegenerative

Induced pluripotent stem cells (iPSCs) are an intriguing approach for neurological disease modeling, because neural lineage-specific cell types that retain the donors' complex genetics can be established in vitro. The statistical power of these iPSC-based models, however, is dependent on accurate diagnoses of the somatic cell donors; unfortunately, many neurodegenerative diseases are commonly misdiagnosed in live human subjects. Postmortem histopathological examination of a donor's brain, combined with premortem clinical criteria, is often the most robust approach to correctly classify an individual as a disease-specific case or unaffected control. We describe the establishment of primary dermal fibroblasts cells lines from 28 autopsy donors. These fibroblasts were used to examine the proliferative effects of establishment protocol, tissue amount, biopsy site, and donor age. As proof-of-principle, iPSCs were generated from fibroblasts from a 75-year-old male, whole body donor, defined as an unaffected neurological control by both clinical and histopathological criteria. To our knowledge, this is the first study describing autopsy donor-derived somatic cells being used for iPSC generation and subsequent neural differentiation. This unique approach also enables us to compare iPSC-derived cell cultures to endogenous tissues from the same donor. We utilized RNA sequencing (RNA-Seq) to evaluate the transcriptional progression of in vitro-differentiated neural cells (over a timecourse of 0, 35, 70, 105 and 140 days), and compared this with donor-identical temporal lobe tissue. We observed in vitro progression towards the reference brain tissue, supported by (i) a significant increasing monotonic correlation between the days of our timecourse and the number of actively transcribed protein-coding genes and long intergenic non-coding RNAs (lincRNAs) (P < 0.05), consistent with the transcriptional complexity of the brain, (ii) an increase in CpG methylation after neural differentiation that resembled the epigenomic signature of the endogenous tissue, and (iii) a significant decreasing monotonic correlation between the days of our timecourse and the percent of in vitro to brain-tissue differences (P < 0.05) for tissue-specific protein-coding genes and all putative lincRNAs. These studies support the utility of autopsy donors' somatic cells for iPSC-based neurological disease models, and provide evidence that in vitro neural differentiation can result in physiologically progression.
ContributorsHjelm, Brooke E (Author) / Craig, David W. (Thesis advisor) / Wilson-Rawls, Norma J. (Thesis advisor) / Huentelman, Matthew J. (Committee member) / Mason, Hugh S. (Committee member) / Kusumi, Kenro (Committee member) / Arizona State University (Publisher)
Created2013
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Description
Bats (order Chiroptera) are the longest lived mammals for their size, with particularly extreme longevity evolving in the family Vespertilionidae, or vesper bats. Because of this, researchers have proposed using bats to study ageing and cancer suppression. Here, we study gene duplications across mammalian genomes and show that, similar to

Bats (order Chiroptera) are the longest lived mammals for their size, with particularly extreme longevity evolving in the family Vespertilionidae, or vesper bats. Because of this, researchers have proposed using bats to study ageing and cancer suppression. Here, we study gene duplications across mammalian genomes and show that, similar to previous findings in elephants, bats have experienced duplications of the tumor suppressor gene TP53, including five genomic copies in the genome of the little brown bat (Myotis lucifugus) and two copies in Brandt's bat (Myotis brandtii). These species can live 37 and 41 years, respectively, despite having an adult body mass of only ~7 grams. We use evolutionary genetics and next generation sequencing approaches to show that positive selection has acted on the TP53 locus across bats, and two recently duplicated TP53 gene copies in the little brown bat are both highly conserved and expressed, suggesting they are functional. We also report an extraordinary genomic copy number expansion of the tumor suppressor gene FBXO31 in the common ancestor of vesper bats which accelerated in the Myotis lineage, leading to 34\u201457 copies and the expression of 20 functional FBXO31 homologs in Brandt's bat. As FBXO31 directs the degradation of MDM2, which is a negative regulator of TP53, we suggest that increased expression of both FBXO31 and TP53 may be related to an enhanced DNA-damage response to genotoxic stress brought on by long lifespans and rapid metabolic rates in bats.
ContributorsSchneider-Utaka, Aika Kunigunda (Author) / Maley, Carlo (Thesis director) / Wilson Sayres, Melissa (Committee member) / Tollis, Marc (Committee member) / School of Life Sciences (Contributor, Contributor) / Barrett, The Honors College (Contributor)
Created2018-12
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Description
Damage to the central nervous system due to spinal cord or traumatic brain injury, as well as degenerative musculoskeletal disorders such as arthritis, drastically impact the quality of life. Regeneration of complex structures is quite limited in mammals, though other vertebrates possess this ability. Lizards are the most closely related

Damage to the central nervous system due to spinal cord or traumatic brain injury, as well as degenerative musculoskeletal disorders such as arthritis, drastically impact the quality of life. Regeneration of complex structures is quite limited in mammals, though other vertebrates possess this ability. Lizards are the most closely related organism to humans that can regenerate de novo skeletal muscle, hyaline cartilage, spinal cord, vasculature, and skin. Progress in studying the cellular and molecular mechanisms of lizard regeneration has previously been limited by a lack of genomic resources. Building on the release of the genome of the green anole, Anolis carolinensis, we developed a second generation, robust RNA-Seq-based genome annotation, and performed the first transcriptomic analysis of tail regeneration in this species. In order to investigate gene expression in regenerating tissue, we performed whole transcriptome and microRNA transcriptome analysis of regenerating tail tip and base and associated tissues, identifying key genetic targets in the regenerative process. These studies have identified components of a genetic program for regeneration in the lizard that includes both developmental and adult repair mechanisms shared with mammals, indicating value in the translation of these findings to future regenerative therapies.
ContributorsHutchins, Elizabeth (Author) / Kusumi, Kenro (Thesis advisor) / Rawls, Jeffrey A. (Committee member) / Denardo, Dale F. (Committee member) / Huentelman, Matthew J. (Committee member) / Arizona State University (Publisher)
Created2015
Description

Agassiz’s desert tortoise (Gopherus agassizii) is a long-lived species native to the Mojave Desert and is listed as threatened under the US Endangered Species Act. To aid conservation efforts for preserving the genetic diversity of this species, we generated a whole genome reference sequence with an annotation based on dee

Agassiz’s desert tortoise (Gopherus agassizii) is a long-lived species native to the Mojave Desert and is listed as threatened under the US Endangered Species Act. To aid conservation efforts for preserving the genetic diversity of this species, we generated a whole genome reference sequence with an annotation based on deep transcriptome sequences of adult skeletal muscle, lung, brain, and blood. The draft genome assembly for G. agassizii has a scaffold N50 length of 252 kbp and a total length of 2.4 Gbp. Genome annotation reveals 20,172 protein-coding genes in the G. agassizii assembly, and that gene structure is more similar to chicken than other turtles. We provide a series of comparative analyses demonstrating (1) that turtles are among the slowest-evolving genome-enabled reptiles, (2) amino acid changes in genes controlling desert tortoise traits such as shell development, longevity and osmoregulation, and (3) fixed variants across the Gopherus species complex in genes related to desert adaptations, including circadian rhythm and innate immune response. This G. agassizii genome reference and annotation is the first such resource for any tortoise, and will serve as a foundation for future analysis of the genetic basis of adaptations to the desert environment, allow for investigation into genomic factors affecting tortoise health, disease and longevity, and serve as a valuable resource for additional studies in this species complex.

Data Availability: All genomic and transcriptomic sequence files are available from the NIH-NCBI BioProject database (accession numbers PRJNA352725, PRJNA352726, and PRJNA281763). All genome assembly, transcriptome assembly, predicted protein, transcript, genome annotation, repeatmasker, phylogenetic trees, .vcf and GO enrichment files are available on Harvard Dataverse (doi:10.7910/DVN/EH2S9K).

ContributorsTollis, Marc (Author) / DeNardo, Dale F (Author) / Cornelius, John A (Author) / Dolby, Greer A (Author) / Edwards, Taylor (Author) / Henen, Brian T. (Author) / Karl, Alice E. (Author) / Murphy, Robert W. (Author) / Kusumi, Kenro (Author)
Created2017-05-31
Description
Cancer is a disease which can affect all animals across the tree of life. Certain species have undergone natural selection to reduce or prevent cancer. Mechanisms to block cancer may include, among others, a species possessing additional paralogues of tumor suppressor genes, or decreasing the number of oncogenes within their

Cancer is a disease which can affect all animals across the tree of life. Certain species have undergone natural selection to reduce or prevent cancer. Mechanisms to block cancer may include, among others, a species possessing additional paralogues of tumor suppressor genes, or decreasing the number of oncogenes within their genome. To understand cancer prevention patterns across species, I developed a bioinformatic pipeline to identify copies of 545 known tumor suppressor genes and oncogenes across 63 species of mammals. I used phylogenetic regressions to test for associations between cancer gene copy numbers and a species’ life history. I found a significant association between cancer gene copies and species’ longevity quotient. Additional paralogues of tumor suppressor genes and oncogenes is not solely dependent on body size, but rather the balance between body size and longevity. Additionally, there is a significance association between life history traits and genes that are both germline and somatic tumor suppressor genes. The bioinformatic pipeline identified large tumor suppressor gene and oncogene copy numbers in the naked mole rat (Heterocephalus glaber), armadillo (Dasypus novemcinctus), and the two-fingered sloth (Choloepus hoffmanni). These results suggest that increased paralogues of tumor suppressor genes and oncogenes are these species’ modes of cancer resistance.
ContributorsSchneider-Utaka, Aika Kunigunda (Author) / Maley, Carlo C (Thesis advisor) / Wilson, Melissa A. (Committee member) / Tollis, Marc (Committee member) / Arizona State University (Publisher)
Created2019