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Browsing by Author "Coleman, Stephen J., advisor"

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    Calcium signaling genes in association with altitude-induced pulmonary hypertension in Angus cattle
    (Colorado State University. Libraries, 2019) Crawford, Natalie Faye, author; Thomas, Milton G., advisor; Coleman, Stephen J., advisor; Enns, R. Mark, committee member; Speidel, Scott E., committee member; Garry, Franklyn B., committee member
    This research used multi-omics technology (i.e., RNA-seq, qPCR for gene expression, SNP discovery and validation) to understand the influence of a particular subset genes on altitude-induced pulmonary hypertension susceptibility in Angus cattle. Three research aims were established to test the hypothesis that calcium-related genes may be associated with pulmonary hypertension in beef cattle. Data and samples utilized for the research came from the Colorado State University Beef Improvement Center Angus herd managed at 2,150 m of altitude. Transcriptome data from 6 tissues and 14 hypertensive and normotensive Angus steers were utilized for differential expression and pathway analyses. The objectives of the first aim were to: 1) to estimate and identify differentially expressed genes from RNA-Seq and pathway analyses, and 2) select putative candidate genes to analyze with qPCR (gene expression level). The largest number of DE genes was revealed in aorta (n = 631) and right ventricle (n = 2,183) samples. Top canonical pathways related to calcium signaling or utilization included: synaptic long-term depression, signaling by Rho family GTPases, and oxidative phosphorylation. Genes regulating calcium availability and utilization were expressed differently (log2 fold change > 0.589, < -0.589; P < 0.05) in Angus cattle with and without pulmonary hypertension. Isolated RNA from cardiac muscle (n = 9) and control muscle (n = 2) tissues from hypertensive and normotensive Angus steers were utilized to estimate gene expression using quantitative reverse transcription PCR in the candidate genes from Chapter 3. The objectives of this chapter were: 1) to establish the most appropriate reference genes in cardiac muscle tissues, and 2) to estimate and validated relative gene expression of calcium-related genes in cardiac muscle tissues using qPCR methods. Differences (P < 0.0055) among hypertensive and normotensive steers were estimated for right papillary muscle and right cardiac ventricle tissues (top, middle, and bottom) in candidate genes: ASIC2, EDN1, NOX4, PLA2G4A, RCAN1, and THBS4. Results of the current study validate the expression differences previously established of genes that regulate the availability and utilization of calcium with PH status in Angus steers at high altitude. Variant detection and association analyses were completed with 2 sets of available -omics data to identify opportunities for development of selection tools for reduced susceptibility to PH. The objectives of the third aim were to: 1) detect single nucleotide polymorphisms (SNP) in the transcriptome of 6 tissues, and 2) identify functional consequences of those variants associated with validated candidate genes from qPCR analyses. Pooled Angus sample analysis revealed 68 SNP in the 6 candidate genes: ASIC2, EDN1, NOX4, PLA2G4A, RCAN1, and THBS4. Thirty-eight SNP were revealed in the hypertensive group and 8 SNP in the normotensive steer group. Ten of the 68 identified SNP are utilized on large density commercially available bovine SNP chips (Illumina BovineHD BeadChip; GeneSeek Genomic Profiler HD; GeneSeek Genomic Profiler HDv2; Affymetrix Axiom Bovine). Analysis of transcriptome data identified SNP within genes regulating calcium availability and utilization, enhancing our understanding of sequence polymorphisms that may be involved in regulating pulmonary hypertension in Angus cattle raised at high altitude. These SNP are available for additional validation and potential use in genetic improvement programs.
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    Characterization of pulmonary hypertension status and utilization of multi-omics analyses to discover variants that may inform selection against high mean pulmonary arterial pressure in Angus cattle
    (Colorado State University. Libraries, 2020) Jennings, Kaysie Jean, author; Thomas, Milton G., advisor; Coleman, Stephen J., advisor; Enns, R. Mark, committee member; Speidel, Scott E., committee member; Holt, Timothy N., committee member
    This multi-part research characterizes pulmonary hypertension (PH) from a physiologic and genetic point of view using the indicator trait mean pulmonary arterial pressure (PAP). Three aims were designed to address the research objective of investigating the genetics underlying PAP for the purposes of variant discovery. The first aim sought to identify different PAP phenotypes based on altitude and diet during the stocker and finishing phases of production. This longitudinal study evaluated steers with a moderate PAP (41-48 mmHg) from yearling age until harvest, collecting PAP and blood gas parameters throughout the study and carcass data at the conclusion. Through this experimental approach, the role of different finishing systems was able to be evaluated and cattle with increased sensitivity to hypoxic conditions were identified. Results from this study indicated that regardless of finishing system, animals exhibited signs of respiratory alkalosis with renal compensation because of hypoxic conditions. The PAP data from this population corroborated that all steers were hypoxic. However, the only carcass quality differences observed were those between cattle that were grain finished compared to those that were grass finished, regardless of altitude. Aim two was to perform next-generation whole genome sequencing for 30 Angus bulls and steers to compare those with high PAP (HPAP) to those with low PAP (LPAP) measures. All cattle sequenced originated from elevations ≥1,500m and were selected based on their pedigree information, as well as PAP observations. The sequence data from these 30 animals were then compared such that sequence from HPAP cattle was compared to that of LPAP cattle. There were 5,543 variants unique to HPAP cattle and 1,690 variants unique to LPAP cattle. Loci across all 30 chromosomes exhibited variation for PAP phenotype. Evaluation of these variants and validation will be necessary to sift through variants that are in linkage or may be less informative. A multi-omics approach was used to perform variant discovery based on the PAP phenotype in aim three. Through combination of RNA sequence with DNA sequence, the list of variants relevant to PAP phenotype was filtered from millions of variants to hundreds of variants. Transcriptome data was compared for each of six tissues between HPAP and LPAP cattle. These variants were then compared to one another to determine which variants were detected in each PAP category across all six tissue types. Those variants were then compared to the DNA sequence from aim two to elucidate concordant variants for HPAP and LPAP cattle respectively. There were three variants that were unique to LPAP cattle and were concordant between DNA and RNA sequence. However, none of these variants were within 1,000bp of a gene recognized in the ARS-UCD1.2 bovine genome assembly and were therefore considered less informative. There were 523 variants unique to HPAP cattle. Within that population there was a subset that was either near or within a gene. There were six genes that were considered informative for further investigation. Three of those genes were uncharacterized genes on chromosome 16. The other three (U6, SIMC1, CDH23), while not well documented in cattle, had functions in humans that would indicate their function could affect PAP phenotype expression. These genes and the variants within them could be useful for selection if validated in a larger population.
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    Characterizing the microbiota and profiling small non-coding RNAs in the compartments of the equine hindgut
    (Colorado State University. Libraries, 2018) Reed, Kailee Janelle, author; Coleman, Stephen J., advisor; Bruemmer, Jason, committee member; Turk, Phillp, committee member; Bouma, Gerrit, committee member
    Gastrointestinal homeostasis is a complex relationship that encompasses the host's immune response, physiology, gut structure and the microbes residing within the host. Each one of these has pathways of communication in order to keep the host in a 'healthy state' or homeostasis. While each category has been extensively researched independently, interactions that occur between host and microbe are largely still unknown, especially within the equine species. Because horses are extremely prone to various gastrointestinal diseases, understanding the microbial populations and how the horse might communicate with those populations will provide more insight on equine gut homeostasis. The main objectives were to delineate the microbial structures residing within compartment of the hindgut and to begin to profile gene expression patterns of small RNAs within the same areas. Two different populations of animal subjects were used for the two projects in this thesis: a herd from the University of Kentucky (n=6) and a herd from Colorado State University (n=3). The herd from Kentucky was used for the microbiota data set in order to determine the microbial population structure within the cecum, right ventral colon, right dorsal colon and feces. First, we characterized microbial communities present in each of these anatomical sites and then completed a multivariate model to determine similarities of compartments and compared those to the fecal sample. The population of microorganisms observed in the proximal hindgut appeared similar between cecum and ventral colon, while the dorsal colon and fecal samples appeared to be more alike. Interestingly, there is an anatomical structure separating ventral and dorsal portions of the colon called the pelvic flexure. This could possibly be an indication of the host's contribution of determining the microbial communities in each anatomical region. We also demonstrated that while some microbial signatures from the proximal gut were identified in the feces, the distal gut seemed to be more represented in the fecal sample. The herd from Colorado was used to produce the gene expression data for the second project and the main focus was to profile microRNA (miRNA) expression along the hindgut. These small non-coding RNAs have been identified to be involved in gastrointestinal homeostasis within the intestinal epithelium and are host derived molecules. We demonstrated that each tissue (n=8 for each horse) had unique miRNA expression profiles and these miRNAs identified were used to complete a target pathway analysis which shows possible pathways that could be associated with the biological function of each intestinal site. While each project had different objectives, they are both key players of gastrointestinal homeostasis. For future research, we plan to combine these two areas of study by knowing which miRNA could target specific bacteria residing in the gut, which may further the knowledge of how the host contributes to the population structure of the microbes within their gastrointestinal tracts.
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    Gene expression analysis before and after the pelvic flexure in the equine hindgut
    (Colorado State University. Libraries, 2024) Moss, Cameron D., author; Coleman, Stephen J., advisor; Engle, Terry, committee member; Metcalf, Jessica, committee member; Landolt, Gabriele, committee member
    The equine hindgut is the primary site of the horse's nutrient breakdown, absorption, and energy production. More than 60% of the horse's energy comes from hindgut fermentation. In this process, commensal microbes in the hindgut aid in the digestion of plant materials to create volatile fatty acids that can be used by host cells to make energy. Many severe health issues- such as colic, laminitis, or colonic impactions- often occur in the equine hindgut, making it an important site to study to provide better management, treatment, and prevention options for horses suffering from gastrointestinal disease. Although much research exists focusing on the microbiome and overall physiology of the equine hindgut, relatively little addresses the role of gene expression in maintaining a complex yet essential homeostatic balance within the gastrointestinal tract. Previous from our lab found major differences in the microbial content of gastrointestinal compartments of the equine hindgut, separated by the pelvic flexure. The pelvic flexure is a short, narrow, horseshoe-shaped loop in the equine large colon. It defines the ventral and dorsal segments of the colon and is a common site of colonic impaction in horses. Although the pelvic flexure cannot and should not act as a "barrier," something "barrier-like" may be occurring around this region as it pertains to the hindgut microbiome. The mechanism for this action is not defined. As a result, this thesis aims to investigate gene expression in the intestinal epithelial cells of the ventral colon, pelvic flexure, and dorsal colon regions of a healthy hindgut to determine what differences exist. The insight gained from this analysis will provide a baseline for comparison to understand how gene expression patterns in these tissues adapt to changes in the microbiome and external factors like diet. The results of this thesis are the first steps towards a better understanding of homeostasis in the equine hindgut.