Browsing by Subject "canine"
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Item A Method for Measurement of 3-Bromotyrosine Concentrations in Canine Serum and Its Clinical Application(2014-10-30) Sattasathuchana, PanpichaThe eosinophil count in the peripheral blood does not accurately represent eosinophil activation in tissues, as eosinophils primarily reside in tissues and not in peripheral blood. To date, a non-invasive biomarker for eosinophil activation in dogs has not yet been described. Therefore, the aims of this study were to (1) develop and analytically validate an electron ionization gas chromatography/mass spectrometry (EI-GC/MS) method to identify and measure 3-Bromotyrosine (3-BrY) concentrations in canine serum, (2) establish a reference interval for serum 3-BrY concentrations in healthy pet dogs, (3) determine the short-term stability of canine serum 3-BrY concentrations, and (4) evaluate the clinical usefulness of measuring 3-BrY concentrations in canine serum. Left-over serum samples from dogs were used to analytically validate the assay. Serum samples from 41 healthy dogs were used to establish a reference interval. A stable isotope of 3-BrY, D3-bromotyrosine, was produced in house and added to canine serum samples. 3-BrY in serum samples was extracted and detected by solid-phase extraction and EI-GC/MS, respectively. Limit of blank and limit of detection were 0.33 and 0.63 ?mol/L, respectively. Coefficients of variation for precision and reproducibility of the 3-BrY concentrations were <13.9% and <11.0%, respectively. Linearity and accuracy recoveries ranged from 84.0% to 134.4% and 79.1% to 126.7%, respectively. The reference interval was determined to be ?1.12 ?mol/L. Serum 3-BrY concentrations were stable for ?8, 30, and 180 days at 4?C, -20?C, and -80?C, respectively. To evaluate the clinical usefulness of 3-BrY concentrations in canine serum, samples were collected from healthy dogs, dogs with eosinophilia, dogs with eosinophilic gastroenteritis (EGE), dogs with lymphoplasmacytic enteritis (LPE), dogs with exocrine pancreatic insufficiency, and dogs with pancreatitis. Concentrations of 3-BrY in the serum samples were significantly higher in dogs with eosinophilia, dogs with EGE, dogs with LPE, dogs with pancreatitis compared to healthy control dogs (P<0.0001). In conclusion, the newly developed method for the measurement of 3-BrY concentrations in canine serum samples was precise, reproducible, linear, accurate, and stable. Moreover, serum 3-BrY concentrations may serve as a potential diagnostic marker for dogs with chronic enteropathy after the exclusion of pancreatitis.Item Assessing the repeatability and validity of a questionnaire on pain and lameness in the canine(Texas A&M University, 2004-09-30) Hudson, Jonathan ThomasThe measurement of pain has had a growing importance in animals for both privately owned animals and those animals involved in clinical research. Lameness is considered to be 1 aspect of the pain experience. The ability of a veterinarian to assess lameness during a routine orthopedic examination can be difficult given the short amount of time in which the clinician can observe the animal, and the fact that the animal is in a stressful environment. Thus, the input of the owner concerning the animal's well-being over an extended time period may be extremely useful to the clinician in assessing the degree of lameness of the animal. It was the purpose of this study to establish an instrument that was both repeatable and valid in assessing the degree of lameness. The instrument used was a questionnaire containing 39 questions in a visual analog scale format. A force platform was used as the gold-standard for detecting mechanical lameness. Peak vertical, cranial-caudal, and their associated impulses were forces used to determine lameness, along with maximum slope in some cases. A test-retest measure of repeatability was conducted on a subset of 19 dogs that were confirmed to have less than a 10% change in vertical peak force. Nineteen of the 39 questions were found to be repeatable based on a Spearman rank correlation. These 19 questions were then used as predictor variables in several multiple regression models which predicted force plate measurements. The result was 3 different models each containing 7 independent variables that were thought to be valid representations of the forces measured (vertical peak, vertical impulse, and propulsion peak forces). Each reduced model was found to fit the data as well as the full model containing all 19 of the repeatable questions. The composite of 11 questions from the 3 different models was used to calculate a total score. This total score was found to be significantly correlated with force plate measurements. These 11 questions should be useful to a clinician in detecting the degree of lameness in the dog.Item Assessment of the canine intestinal microflora using molecular methods and serum markers(Texas A&M University, 2007-04-25) Suchodolski, Jan S.Previous studies examining the canine intestinal microflora have focused on cultivation of bacteria from intestinal content. Recently, it has been recognized that the majority of bacteria cannot be identified using standard culture techniques. The aim of this study was to describe the composition and dynamics of the canine intestinal microflora using molecular methods based on identification of the 16S ribosomal DNA (16S rDNA) and to evaluate the clinical use of a 13C-glycocholic acid blood test (13CGCBT) as a serum marker for small intestinal bacterial biomass. Intestinal content was obtained from healthy dogs and the microflora was characterized in different compartments of each dog by denaturing gradient gel electrophoresis (DGGE) and comparative 16S rDNA analysis. A 13C-glycocholic acid blood test (13C-GCBT) was developed as a marker for small intestinal bacterial biomass and the influence of tylosin administration on the 13C-GCBT, serum concentrations of cobalamin, folate, and unconjugated cholic acid (SUCA) was evaluated. There was marked variation in DGGE profiles between individual dogs and also between different intestinal compartments within dogs. DGGE profiles from duodenal juice samples collected endoscopically at different time-points varied within individuals, possibly due to variations over time or a slight variation in sampling location. Direct sequencing revealed 106 individual 16S rDNA sequences. Forty-two sequences showed less than 98% similarity to described sequences in public databases and may constitute previously uncharacterized bacterial species. Serum folate concentrations, SUCA, and the cumulative percent dose/min of 13C administered as 13C-glycocholic acid (CUMPCD) increased significantly following tylosin administration (p<0.01). The results indicate that dogs have a complex intestinal microflora with marked differences between individual dogs. Different intestinal compartments appear to host a unique microflora and the assessment of a fecal sample does not yield accurate information about the composition of the microflora in proximal compartments of the gut. The intestine harbors many previously uncharacterized bacterial species. The clinical significance of these uncharacterized intestinal bacterial species needs to be further investigated in dogs with gastrointestinal disease. Increased serum folate, SUCA, and CUMPCD in the 13C-GCBT suggest that, in the dogs described here, tylosin administration increased the biomass of organisms carrying out these metabolic functions.Item Characterization of Chronic Enteropathies in Dogs by Use of Fecal and Urinary N-methylhistamine Concentrations and Serum Methylmalonic Acid Concentrations(2012-10-19) Berghoff, NoraNon-invasive markers that are clinically useful for the diagnosis and monitoring of canine chronic enteropathies are scarce. The first aim of this study was to investigate the prevalence of cobalamin deficiency on a cellular level in dogs with chronic gastrointestinal disease by measuring serum methylmalonic acid (MMA) concentrations. Hypocobalaminemia has been associated with a negative outcome in dogs with chronic enteropathies, but the prevalence of cellular cobalamin deficiency is unknown. The second aim of this study was to determine the utility of fecal and urinary concentrations of N-methylhistamine (NMH) as a marker of gastrointestinal inflammation and disease activity in dogs with chronic enteropathies. Serum MMA concentrations were measured in healthy control dogs to establish a reference interval, which was calculated to be 415-1,193 nmol/L. Measurement of MMA concentrations in 555 serum samples from dogs with varying cobalamin concentrations showed a significant increase (p<0.05) in dogs with hypocobalaminemia. In a prospective group of 56 dogs with chronic enteropathies, 36% had decreased serum cobalamin concentrations, five of which (9% of 56 dogs) had increased serum MMA concentrations. We conclude that hypocobalaminemia is commonly seen in dogs with chronic gastrointestinal disease, but does not always appear to be associated with cellular cobalamin deficiency. In 47 dogs with chronic enteropathies, fecal and urinary NMH concentrations were increased in 21% and 27%, respectively, indicating that mast cell degranulation plays a role in a subset of dogs with chronic enteropathies. However fecal and urinary NMH concentrations did not correlate with each other, or with the clinical activity index. Urinary NMH concentrations correlated significantly with serum CRP concentrations, and were also significantly associated with severity of duodenal mucosal inflammation (p=0.008). The lack of correlation with the clinical activity index suggests that degranulation of mast cells only plays a role in some dogs with chronic enteropathies. Also, these results suggest that NMH alone may not be a good marker for clinical disease activity in dogs with chronic enteropathies. Due to its linear association with serum CRP and severity of mucosal inflammation, urinary NMH concentrations may be a better marker of intestinal inflammation than fecal NMH concentrations.Item Characterization of the Fecal Microbiota in Dogs with Chronic Enteropathies and Acute Hemorrhagic Diarrhea(2012-10-19) Markel, MelissaRecent 16S rRNA gene sequencing studies of the duodenal and fecal microbiota have revealed alterations in the abundance of specific bacterial groups in dogs with gastrointestinal (GI) disorders. The aim of this study was to establish a panel of quantitative real-time PCR (qPCR) assays for the evaluation of specific bacterial groups in fecal samples of healthy dogs, dogs with chronic enteropathies (CE), and dogs with acute hemorrhagic diarrhea (AHD). Fecal samples from 242 healthy dogs, 118 dogs with CE, and 57 dogs with AHD were analyzed using qPCR assays targeting Faecalibacterium spp., Turicibacter spp., Bifidobacterium spp., Lactobacillus spp., Streptococcus spp., Ruminococcaceae, C. perfringens, E. coli, gamma-Proteobacteria, Bacteroidetes, and Firmicutes). Differences in bacterial abundance among the three groups were evaluated using a Kruskal-Wallis test followed by a Dunn's post-test. A Bonferroni correction was used to correct for multiple comparisons and an adjusted p<0.05 was considered for statistical significance. Faecalibacterium spp., Turicibacter spp., and Ruminococcaceae were significantly decreased in CE and AHD compared to healthy dogs (p<0.001 for all). Lactobacillus spp. and Streptococcus spp. were significantly increased in dogs with CE (p<0.001 for both) when compared to the healthy dogs. In contrast, Lactobacillus spp. and Streptococcus spp. were significantly decreased in dogs with AHD compared to healthy dogs (p<0.01 and p<0.05, respectively) and also when compared to the dogs with CE (p<0.001 for both). C. perfringens and E. coli were significantly increased in dogs with AHD (p<0.001 and p<0.01, respectively), when compared to healthy dogs. E. coli was also significantly increased in dogs with CE when compared to the healthy dogs (p<0.001). Bacteroidetes were significantly lower in dogs with CE compared to healthy dogs (<0.001). Firmicutes were significantly higher in healthy dogs in comparison to dogs with AHD (p<0.05). Bifidobacterium spp. and gamma-Proteobacteria were not significantly different among all three groups of dogs. In conclusion, the qPCR panel employed here revealed a fecal dysbiosis in dogs with CE and AHD when compared to healthy dogs. These results are similar to recently reported findings using molecular sequencing approaches. Quantification of these bacterial groups by qPCR may be a useful adjunct for the diagnosis or monitoring of gastrointestinal disease in dogs.Item Characterization of the mutation causative for autosomal recessive hereditary nephropathy in the english cocker spaniel and analysis of gene expression in multiple models of hereditary nephropathy(2009-05-15) Davidson, Ashley GreeneThe domestic dog, Canis familiaris, has over 450 naturally occurring inherited diseases. Over half of these diseases are clinically similar to human diseases making the dog an excellent model in which to study human hereditary diseases. Alport syndrome (AS), a group of heterogeneous, hereditary renal diseases, is one example of such a human disease. The disease is transmitted in three fashions: X-linked, autosomal recessive, and autosomal dominant. AS is caused by mutations in COL4?3, COL4?4 or COL4?5, all members of the type IV collagen family. The proteins products of these genes along with those of the other type IV collagen family members (COL4?1, COL4?2, and COL4?6) are structural components of basement membranes throughout the body. This dissertation describes the measurement of mRNA transcripts in two canine models of AS: a mixed breed model of X-linked AS (XLAS) and the English Cocker Spaniel (ECS) model of autosomal recessive AS (ARAS). The work done revealed a decrease in COL4?4 transcripts. The similarity between the decrease of COL4?5 in the XLAS model and that for COL4?4 in the ARAS model lead to the investigation of COL4?4 as the gene harboring the mutation causative for ARAS in the ECS. Upon sequencing COL4?4, the causative mutation was determined to be an A to T transversion in exon 3. To provide an in vitro model to study type IV collagens, a protocol was designed and experimentally validated to isolate and culture canine Sertoli cells. Canine testes cells were isolated and cultured. Cells were verified as Sertoli cells through positive identification of both SOX9 and Clusterin B proteins, along with sequence verification of SOX9 transcripts. This in vitro model provides a tool to further study the type IV collagens. Overall, the research described herein lead to the identification of the mutation causative for ARAS in the ECS. With this knowledge a genetic test was developed to test for the disease. This research also provided valuable information about the transcript levels of type IV collagens in two models of AS, and provided a novel model in which to study the type IV collagens further.Item Development of a multiplexing strategy for whole genome scans of the domestic dog and analysis of hereditary deafness in the Dalmatian(Texas A&M University, 2005-08-29) Cargill, Edward JamesThe Dalmatian is affected by deafness more than any other breed of domestic dog, with 30% of the United States population suffering from unilateral or bilateral deafness. The genetic origin of deafness in the Dalmatian is unknown. The objective of this work was to identify, using linkage analysis, any chromosomal region(s) in which the gene(s) responsible for deafness in the Dalmatian may be located. To achieve this objective it was necessary to 1) develop multiplexed microsatellite markers for an efficient whole genome scan, 2) assemble a multigenerational Dalmatian kindred segregating deafness, 3) estimate the heritability of deafness and perform complex segregation analysis, and 4) perform linkage analysis of deafness, and other phenotypic traits, in the Dalmatian kindred. A set of 172 microsatellite markers, termed Minimal Screening Set 1 (MSS1), was characterized, prior to this work, for whole genome scans of the domestic dog. 155 of the MSS1 markers were multiplexed into 48 multiplex sets. Amplification of the multiplex sets was achieved using a single thermal cycling program. The markers were labeled with fluorescent dyes and optimized for resolution on an ABI 310 Genetic Analyzer or ABI 377 Sequencer. A kindred of 266 Dalmatians was assembled, of which 199 had been diagnosed using the brainstem auditory evoked response to determine auditory status. Of these, 74.4% (N = 148) had normal hearing, 18.1% (N = 36) were unilaterally deaf, and 7.5% (N = 15) were bilaterally deaf. A heritability of 0.73 was estimated considering deafness a dichotomous trait and 0.75 as a trichotomous trait. Although deafness in the Dalmatian is clearly heritable, the evidence for the presence of a major gene affecting the disorder was not persuasive. Dalmatians (N = 117) from the assembled kindred were genotyped for the MSS1 markers (149 were polymorphic). Linkage analysis was performed for deafness, eye color, and spot color. The maximum LOD scores for deafness were found with markers Cos15 on CFA17 (LOD = 1.69) and FH2585 on CFA28 (LOD = 1.34). No significant linkage was found with eye color. Significant linkage for spot color was found with marker FH2319 (LOD = 9.7) on CFA11.Item Fecal Microbiome in Dogs with Acute Diarrhea(2013-11-07) Guard, Blake CrosbyRecent molecular studies have revealed that the canine gastrointestinal tract (GIT) harbors a highly complex microbial ecosystem. Gut microbes play a very important role in the development and regulation of the immune system of the host, mediated in-part through the production of immunomodulatory metabolites (e.g., butyrate, propionate, indole). Limited information is available about potential changes in the predominant bacterial groups in dogs with acute diarrhea, and characterizing the functional gene content of the microbiome may help to understand relationships between microbiota, endogenous metabolites, and gastrointestinal disease. Therefore, the aim of this study was (1) to characterize the fecal microbiome in healthy dogs, dogs with acute non-hemorrhagic diarrhea (NHD), and dogs with acute hemorrhagic diarrhea (AHD) using 16S rRNA gene sequencing and qPCR analysis; (2) to measure fecal concentrations of short-chain fatty acids (SCFAs) and branched-chain fatty acids (BCFAs); and (3) to describe the functional gene content of the fecal microbiome. Fecal samples were collected from healthy dogs (n=13), dogs with NHD (n=5), and dogs with AHD (n=6). The fecal microbiota were analyzed by 454-pyrosequencing of 16S rRNA genes and qPCR assays. SCFAs were quantified by gas chromatography/mass spectrometry (GC/MS). Functional genes present in the microbiome were predicted from the 16S rRNA gene data using the software PICRUSt. The Shannon Index for bacterial diversity was significantly decreased in dogs with acute diarrhea (AD; both NHD and AHD groups combined) compared to healthy dogs (p=0.0020). Sequences belonging to Bacteroidetes were significantly decreased in dogs with AD compared to healthy dogs (p=0.0280). Sequences belonging to the genus Faecalibacterium and an unclassified genus within the family Ruminococcaceae were both significantly decreased in dogs with AD compared to healthy dogs (p=0.0319 and 0.0368, respectively). Also, a significant decrease in Blautia spp. were observed in dogs with AD compared to healthy dogs (p=0.0472). The proportions of butyric acid were significantly increased and proportions of propionic acid were significantly decreased in dogs with AD compared to healthy dogs (p<0.05 for both). Significant differences were not observed in functional categories among all dogs after adjustment for multiple comparisons. Results of this study revealed a bacterial dysbiosis in fecal samples of dogs with NHD and dogs with AHD compared to healthy dogs. The bacterial groups that were commonly decreased during acute diarrhea are considered to be important SCFA producers and may be important for canine intestinal health. Future studies to evaluate broader metabolomic profiles in dogs with acute diarrhea are indicated.Item Genetic analysis of canine hip dysplasia(Texas A&M University, 2007-04-25) Tsai, Kate LeanneThe morphologic variability seen in the domestic dog, Canis lupus familiaris, is unique among mammals. Selective pressures imposed by humans have divided dogs into almost 400 separate breeds. Selection has also led to the development of approximately 450 hereditary diseases, many of which are limited to specific breeds. Over half of these diseases present with similar clinical characteristics to those of many human hereditary diseases, making the dog an ideal model for study of the genetic bases of such diseases. Many diseases do not have candidate genes or have too many candidates to characterize. This is exacerbated in complex diseases that are caused by several genes. Whole-genome scans can provide insight into diseases by identifying marker(s) that co-segregate with a disease phenotype. The Minimal Screening Set - 2 (MSS-2) is the most recent set of microsatellites suitable for whole-genome screens. The first objective of this work was to streamline genomic screens in order to efficiently analyze large numbers of animals. To this end, chromosome-specific microsatellite panels were developed for the MSS-2. Canine hip dysplasia (CHD) is the most common orthopedic disease of the dog. CHD primarily affects medium and large breed dogs, but is found in almost every breed. The major objective of this work was to use linkage analysis to identify chromosomal regions that contain genes that are involved in CHD. Two populations were screened using the MSS-2. The first was a small family of Boykin Spaniels, though no markers were statistically significant in a whole-genome screen. An outcrossed pedigree of Greyhound/Labrador Retrievers was created for quantitative trait loci (QTL) mapping of CHD. The informativeness of markers in the F2 and backcrossed generations were calculated to show the utility of using such a population. Other factors that affect the power of this pedigree to identify QTL were also highlighted. Chromosomes that were identified in a previous screen as harboring putative QTLs were examined using the chromosome-specific panels to further define and confirm the regions of interest. Although no markers reached statistical significance, several areas of interest were identified.Item Genomic analyses of induced hypercholesterolemia and atherosclerosis in a mixed breed colony of dogs and developmental abnormalities in the Havanese(2009-05-15) Starr, Alison NicoleThe domestic dog, Canis lupus familiaris, is a unique model system for the dissection of hereditary diseases. Selective breeding practices have created more than 300 distinct breeds of dogs, born from a desire to create specific physical and behavioral characteristics. Breeds represent closed breeding populations and the extensive records maintained for members of each breed (e.g., multi-generational pedigrees, veterinary medical records) present an incredible tool for genetic research. Two closed populations were used in the work presented here: a colony of mixed-breed dogs segregating resistance and sensitivity to cholesterol feeding, and a purebred pet population of Havanese experiencing a high frequency of developmental abnormalities. Estimates of heritability were calculated for each disease to evaluate the degree of phenotypic variation attributable to genetics among dogs in the populations used. A heritability of 0.55 (? 0.16) was identified for cholesterol resistance and sensitivity in the mixed-breed colony. The small sample size prevented the use of complex segregation analyses to examine mode of transmission. A heritability of 0.36 (? 0.26) was calculated for the composite phenotype in the Havanese, encompassing the spectrum of abnormalities in the breed. Polygenic inheritance was identified for the composite phenotype, but the action of a major gene was identified by complex segregation analyses in the Havanese. Complex diseases preclude the use of a candidate gene approach, owing to the multitude of genes involved in the disease process. Whole genome screens provide a practical approach to the identification of chromosomal region(s) associated with a disease phenotype by narrowing the search for candidate gene(s). The Minimal Screening Set ? 2 (MSS-2) was used in the present studies to evaluate the segregation of microsatellite markers in pedigrees for both the mixed-breed colony and the Havanese. No significant LOD scores were identified, though suggestive LOD scores were obtained in both analyses. A canine-specific oligonucleotide microarray was used to create gene expression profiles for developmental abnormalities in the Havanese and for cholesterol sensitivity in the mixed-breed colony dogs. Distinct expression profiles were generated for each group, and several genes of interest were identified as being both differentially expressed (>?2-fold change) and statistically significant (p-value<0.05).Item Identification of a mutation in COL4A5 causative for X-linked Alport syndrome in the domestic dog and analysis of gene expression in the kidneys of affected and nonaffected siblings(Texas A&M University, 2004-09-30) Cox, Melissa LuanneThe domestic dog, Canis lupus familiaris, plays many roles in the lives of humans. Additionally, the dog is recognized for its potential as a model for many human hereditary diseases. Thus, the genetics and genomics of the dog are being studied extensively in order to facilitate its use as a model, as well as to help the dog for its own sake. As part of this research effort, our laboratory has added type I markers (i.e., the acidic and basic keratins, c-kit, type I and IV collagens, and the gene encoding uromodulin) to the emerging map of the canine genome. The mapping of genes, particularly those in large gene families such as the collagens, is valuable because it rapidly increases the density of gene loci on the map and provides insight regarding conservation of synteny between the dog and other mammals. The major focus of work reported here is the genetics of X-linked Alport syndrome (XLAS), a terminal renal disease that affects the human and the dog. The disease results from mutations in COL4A5, a type IV collagen gene. Reported here are the 1) sequencing and mapping of the canine cDNA encoding uromodulin, 2) mapping of the type I and type IV collagen genes, 3) sequencing of the full-length cDNA of canine COL4A5, 4) identification of a 10 bp deletion in COL4A5, causative for XLAS in our colony of mixed breed dogs, 5) development of a genetic test for identification of affected and carrier dogs in the colony and 6) assessment of gene expression in the kidneys of normal and XLAS-dogs. This assessment was performed using a canine-specific oligonucleotide microarray. XLAS dogs demonstrated up-regulation of many genes involved in extracellular matrix reorganization, cell structure, and immune response, as expected in a glomerulopathy with tubulointerstitial nephritis. Trends were verified by quantitative RT-PCR. A review of the current status of canine genetics research, and current understanding of hereditary diseases in the dog, concludes this dissertation.Item In vitro and in vivo analysis of differential gene expression between normal norfolk terrier dogs and those with an autosomal recessive mutation in KRT10(Texas A&M University, 2005-11-01) Barnhart, Kirstin FayeNatural diseases caused by keratin mutations are rare and have only been reported in humans. We have recently identified a heritable skin disorder in Norfolk terriers caused by a mutation in KRT10. Affected dogs have a tendency to form shallow erosions or blisters following mild trauma, which is first noted after the birthing process. As the dogs age, they display generalized hyperpigmentation and scaling that is most severe in the axillary and inguinal regions. The main histologic and ultrastructural features include: marked hyperkeratosis, epidermal hyperplasia, prominent vacuolation of the upper suprabasal layers, eosinophilic intracytoplasmic aggregates (keratin bundles), numerous and frequently enlarged keratohyaline granules, and epidermal hyperplasia. Analysis of an extended pedigree through seven generations confirmed an autosomal recessive mode of inheritance. The keratin 10 mutation was defined as a G-T point mutation in intron 5 that affected splicing at the boundary of exon 4 and intron 5. The primary outcome of the mutation was a 35 bp deletion in exon 4 caused by use of a cryptic splice site. Real-time PCR quantitation of KRT10 confirmed that this mutation led to premature mRNA decay and an average 35-fold decrease in KRT10 message. Organotypic cell culture techniques were used to establish in vitro models for normal and affected Norfolk terriers. After 21 days of culture, normal epidermis was cornified with a compact and multifocally parakeratotic stratum corneum. Affected epidermis largely reproduced the expected morphologic alterations. Immunoblotting and immunohistochemistry for keratin 10 protein and real-time PCR quantitation of KRT10 message showed significantly less keratin expression in vitro than in vivo suggesting that the differentiation program in vitro underwent significant alterations. A diagnostic PCR assay was established for detection of the carrier state. Global analysis of gene expression between normal, carrier and affected dogs was performed with DermArray cDNA microarrays. Affected and carrier dogs showed differential regulation of 320 and 298 genes, respectively, between normal dogs. In affected dogs, 217 were upregulated and 103 were downregulated. In carrier dogs, 222 were upregulated and 76 were downregulated. 72 genes (65 upregulated, 7 downregulated) were altered in both affected and heterozygous dogs.Item MicroRNA expression in canine mammary cancer(Texas A&M University, 2008-10-10) Boggs, Rene' MichelleMicroRNAs (miRNAs) play a vital role in differentiation, proliferation and tumorigenesis by binding to messenger RNAs (mRNA) and inhibiting translation. To initiate an investigation into the identification of miRNAs in the domestic dog, an emerging model for human disease, a comparison of the human and canine genetic databases was conducted. The bioinformatics work revealed significant conservation of miRNA genes between the two species. Proof of principle experiments, including serial dilutions and sequencing, were performed to verify that primers made to amplify human mature miRNAs can be used to amplify canine miRNAs, providing that the mature sequences are conserved. TaqMan? Real-time RT-PCR, a sensitive and specific method, was used to isolate the first miRNA mature products from canine tissues. The expression levels of miR-17-3p, miR-17-5p, miR-18, miR-19a, miR-19b, miR-20, and miR-92 were evaluated in five canine tissues (heart, lung, brain, kidney, and liver). Because miRNAs have been found to act as both tumor suppressors and oncogenes in several different cancers, expression patterns of ten miRNAs (miR-15a, miR-16, miR-17-5p, miR-21, miR-29b, miR-125b, miR-145, miR-155, miR-181b, let-7f) known to be associated with human breast cancer were compared between malignant canine mammary tumors (n=6) and normal canine mammary tissue (n=10). Resulting data revealed miR-29b and miR-21 to have a statistically significant (p<0.05) up-regulation in cancerous samples. Overall expression patterns showed nine of the ten miRNAs follow the same pattern of expression in the domestic dog as the human, while the miR-145 expression does not show a difference between the normal and cancerous samples.Item Understanding the genetics of aging: a canine model(Texas A&M University, 2007-04-25) Canterberry, Sarah ChristineAs life expectancy in the United States increases each year, the percentage of the population that is comprised of aged individuals rises also. Researchers expect the largest increase in population to occur in the segment consisting of individuals 85 and older. Thus, investigations of the aging process, with the goals of further extending average life expectancy and improving the quality of life for aged individuals, have become increasingly important to our society. To better understand the genetics of aging, we elected to utilize another model organism, the domestic dog. The benefit to this work is that breeds exhibit extreme, natural variation in life expectancies. Here I report my contributions towards establishing the dog as another model organism for investigations of the aging process. Multiple linear regression analysis was carried out to determine the association between life spans and breed size in the dog, based upon data derived from the American pet population. A negative correlation was observed between both height and longevity and between weight and longevity with weight being the significant predictor of life span. Fifty-four genes implicated in the aging process were mapped to the canine genome. These genes were selected because of their demonstrated contribution to longevity in other organisms or based upon their proximity to a marker, D4S1564, on human chromosome 4. Four genes that are associated with dwarf mice and extended life span were analyzed in nine dog breeds of varying sizes and life expectancies. Fifty-three polymorphisms were discovered in Ghr, Ghrhr, Pit1, and Prop1. Thirteen ancestral SNPs were discovered in which both alleles were found in every breed. In Ghrhr, a transition mutation was found that changes the amino acid sequence as well as the function of the protein and is statistically significant (p=4.8 x 10-6) when large dogs are compared to medium-sized breeds, but not when they are compared to small breeds (p=0.001). This SNP warrants further investigation in additional dogs and breeds.