Laboratory and Basic Science Research Commons^™

Characterization Of The Anti-Apoptotic Function Of The Lysine Demethylase Plant Homeodomain Finger Protein 8 (Phf8), Kimberly Muranko

Electronic Thesis and Dissertation Repository

Apoptosis is an essential process in development and tissue maintenance. The tumor suppressor protein p53 initiates apoptosis through transactivation of pro-apoptotic genes when cellular stress is detected. This study identifies a regulatory role for the lysine demethylase, PHF8, in the p53-mediated apoptosis pathway. We initially suspected PHF8 of demethylating the adaptor protein Numb, however found this to be untrue. PHF8 has been found to have oncogenic properties including an anti-apoptotic effect, however how PHF8 negatively affects apoptosis has not been previously investigated. We found PHF8 inhibits translation of the pro-apoptotic genes TP53, BAX and CASP3. Chromatin immunoprecipitation revealed …

The Future Of Teratology Research Is In Vitro, Jarrod Bailey, Andrew Knight, Jonathan Balcombe

Jonathan Balcombe, Ph.D.

Birth defects induced by maternal exposure to exogenous agents during pregnancy are preventable, if the agents themselves can be identified and avoided. Billions of dollars and manhours have been dedicated to animal-based discovery and characterisation methods over decades. We show here, via a comprehensive systematic review and analysis of this data, that these methods constitute questionable science and pose a hazard to humans. Mean positive and negative predictivities barely exceed 50%; discordance among the species used is substantial; reliable extrapolation from animal data to humans is impossible, and virtually all known human teratogens have so far been identified in spite …

The Future Of Teratology Research Is In Vitro, Jarrod Bailey, Andrew Knight, Jonathan Balcombe

Jonathan Balcombe, PhD

Birth defects induced by maternal exposure to exogenous agents during pregnancy are preventable, if the agents themselves can be identified and avoided. Billions of dollars and manhours have been dedicated to animal-based discovery and characterisation methods over decades. We show here, via a comprehensive systematic review and analysis of this data, that these methods constitute questionable science and pose a hazard to humans. Mean positive and negative predictivities barely exceed 50%; discordance among the species used is substantial; reliable extrapolation from animal data to humans is impossible, and virtually all known human teratogens have so far been identified in spite …

Dissection: The Scientific Case For Alternatives, Jonathan Balcombe

Jonathan Balcombe, PhD

This article presents the scientific argument that learning methods that replace traditional nonhuman animal-consumptive methods in life science education—so-called alternatives to dissection—are pedagogically sound and probably superior to dissection. This article focuses on the pedagogy, a learning method’s effectiveness for conveying knowledge.

Prolonged Pain Research In Mice: Trends In Reference To The 3rs, Jonathan Balcombe, Hope Ferdowsian, Lauren Briese

Jonathan Balcombe, PhD

This literature review documents trends in the use of mice in prolonged pain research, defined herein as research that subjects mice to a source of pain for at least 14 days. The total amount of prolonged pain research on mice has increased dramatically in the past decade for the 3 pain categories examined: neuropathic, inflammatory, and chronic pain. There has also been a significant rise in the number of prolonged mouse pain studies as a proportion of all mouse studies and of all mouse pain studies. The use of transgenic mice has also risen significantly in prolonged pain research, though …

Cancerous Contradictions: The Mis-Regulation Of Human Carcinogens Based On Animal Data, Andrew Knight, Jarrod Bailey, Jonathan Balcombe

Jonathan Balcombe, PhD

The regulation of human exposures to potential carcinogens constitutes society’s most important use of animal carcinogenicity data. However, for environmental contaminants of greatest U.S. concern, we found that in most cases (58.1%; 93/160) the U.S. Environmental Protection Agency (EPA) considered the animal data inadequate to support a classification of probable human carcinogen or noncarcinogen.

The World Health Organisation’s International Agency for Research on Cancer (IARC) is a leading international authority on carcinogenicity assessments. For chemicals lacking human exposure data (the great majority), IARC classifications of identical chemicals were significantly more conservative than EPA classifications (p

Laboratory Rodent Welfare: Thinking Outside The Cage, Jonathan P. Balcombe

Jonathan Balcombe, PhD

This commentary presents the case against housing rats and mice in laboratory cages; the commentary bases its case on their sentience, natural history, and the varied detriments of laboratory conditions. The commentary gives 5 arguments to support this position: (a) rats and mice have a high degree of sentience and can suffer, (b) laboratory environments cause suffering, (c) rats and mice in the wild have discrete behavioral needs, (d) rats and mice bred for many generations in the laboratory retain these needs, and (e) these needs are not met in laboratory cages.

Laboratory Routines Cause Animal Stress, Jonathan P. Balcombe, Neal D. Barnard, Chad Sandusky

Jonathan Balcombe, PhD

Eighty published studies were appraised to document the potential stress associated with three routine laboratory procedures commonly performed on animals: handling, blood collection, and orogastric gavage. We defined handling as any non-invasive manipulation occurring as part of routine husbandry, including lifting an animal and cleaning or moving an animal's cage. Significant changes in physiologic parameters correlated with stress (e.g., serum or plasma concentrations of corticosterone, glucose, growth hormone or prolactin, heart rate, blood pressure, and behavior) were associated with all three procedures in multiple species in the studies we examined. The results of these studies demonstrated that animals responded with …

Evaluating Colostrum Quality With Niacin Supplementation In The Diet Of Dairy Cattle, Allison Marie Pike

Student Research Projects

Dairy calves are born with a naive immune system, and must obtain passive immunity through ingestion of colostrum. Colostrum provides the newborn with antimicrobial proteins called immunoglobulins, such as immunoglobulin G (IgG), that protect the calf for the first few weeks of life. Calves that receive good quality colostrum (>50 g/L of IgG) are more likely to survive to adulthood. Niacin is a vasodilator that may increase blood flow to the mammary gland during production of colostrum in the mother. This experiment investigated the effect of niacin supplementation at a rate of 48 g/cow/day for 3 weeks before calving …

The Role Of Integrins In Support Of Pancreatic Function, Survival And Maturation, Matthew Riopel

Electronic Thesis and Dissertation Repository

The pancreas is a glandular organ composed of endocrine and exocrine compartments. Integrins are cell adhesion molecules that connect cells to the extracellular matrix (ECM). Integrins modulate a variety of cellular effects, yet their mechanism of action in the developed pancreas is not well understood. Fibrin is a provisional ECM protein that contains ligands for integrin receptors. Fibrin is capable of supporting islet health, but it is unclear how fibrin exerts its effects. The objective of this thesis is to understand the role of integrin receptors on in vivo pancreatic cell function, survival, and proliferation. In addition, this thesis investigates …

Medical Training Using Simulation: Toward Fewer Animals And Safer Patients, Jonathan Balcombe

Jonathan Balcombe, PhD

This paper presents the current status of computer-based simulation in medicine. Recent technological advances have enabled this field to emerge from esoteric explorations in academic laboratories to commercially available simulators designed to train users to perform medical procedures from start to finish. Today, more than a dozen companies are producing virtual reality simulators and interactive manikins for training in endoscopy, laparoscopy, anaesthesia, trauma management, angiography, and needle insertion. For many of these procedures, thousands of animals are still being used in training. Yet simulation has many advantages that can transcend scientific, ethical, economic and logistical problems that arise when using …

Gain-Of-Function Mouse Models To Investigate Biological Roles Of Prmt6, Alessandra Di Lorenzo

Dissertations & Theses (Open Access)

Gain-of-function Mouse Models to Investigate Biological Roles of PRMT6

Alessandra Di Lorenzo, Ph.D. Candidate

Mentor: Dr. Mark T. Bedford

Protein Arginine Methyltransferase 6 (PRMT6) is the histone tail writer that methylates the H3R2 (arginine 2 of histone H3) residue, which counteracts the activating H3K4me3 mark. PRMT6 has been shown to behave both as transcriptional co-repressor (i.e. trhrombospondin-1, p21, p53), and co-activator (nuclear receptors). The co-repressor function of PRMT6 is likely the result of H3K4me3 antagonism, while the mechanism by which PRMT6 exerts its co-activator function has yet to be elucidated. PRMT6 is over-expressed in several types of tumors including small …

Effects Of Heregulin On Muscle: A Biochemical And Histological Analysis, Nicole Justine Moss

Dissertations, Masters Theses, Capstones, and Culminating Projects

Duchenne Muscular Dystrophy, the most common inherited X-linked genetic disease affecting 1 in 5000 boys, results from a dysfunctional dystrophin protein encoded by the DMD gene. Dystrophin interacts with protein complexes linking the extracellular matrix to the cytoskeleton of muscle fibers. Without dystrophin functioning properly, sarcolemmal membrane stabilization is compromised during the mechanical rigors involved with muscle contraction leading to progressive muscular dystrophy. Heregulin/neuregulin-1 (HRG), a member of the epidermal growth factor family has been shown to induce neuromuscular junction (NMJ) gene expression in vitro and improve skeletal muscle function in dystrophic mice. However, it is unclear if the HRG …

Consequences Of Endogenous Mitochondrial Oxidative Stress In The Heart, Kristin Alisha Koenig

Dissertations, Masters Theses, Capstones, and Culminating Projects

For many years, it has been proposed that oxidative stress within the mitochondria causes mutations to the mitochondrial genome, resulting in changes in copy number per single cell. Ultimately, this compensatory increase in mtDNA, coupled with an increase in mutations, has been suggested to play a major role in the pathobiology of aging. However, evidence for this phenomenon is somewhat controversial. Importantly, oxidative stress can vary between individual cells; therefore, the overall goal of this project is to determine if oxidative stress causes both an altered copy number for mtDNA in singlecardiomyocytes and develop a methodology for sequencing mtDNA from …

Microrna Expression Signature Of Retinal Pigmented Epithelial Cells Associated With The Phenotype Of Age-Related Macular Degeneration, Mark Anthony Gutierrez

Dissertations, Masters Theses, Capstones, and Culminating Projects

Age-related macular degeneration is a debilitating condition that manifests as a loss of the central portion of an individual’s field of vision, affecting 1 in 3 people over the age of 80. With regards to the retina, the organ primarily responsible for an individual’s sense of vision, this condition is associated with the degeneration of the central point in the retina, known as the macula, which contains the highest concentration of light-sensitive photoreceptors. It is currently known that the human retina exhibits a gradual decrease in protective detoxifying factors such as glutathione-S-transferase-1 and catalase as well as increased lipid peroxidation, …

Apolipoprotein E4 And Sirt1 Interaction In Alzheimer’S Disease, Brittany Ann Philpot

Dissertations, Masters Theses, Capstones, and Culminating Projects

Alzheimer’s disease (AD) is a neurodegenerative disorder whose exact cause(s) are still unknown. Εpsilon 4, an allele of apolipoprotein E (ApoE4), is currently the most important risk factor for Alzheimer’s disease. Individuals that inherit two copies of the ApoE ε4 allele have an approximately ten to thirty times increased risk of developing AD in comparison to persons not carrying this allele. This association may relate to ApoE4’s susceptibility to proteolysis and neurotoxicity. Amyloid precursor protein (APP) is involved with neurite extension and neurite retraction, and has been shown to function as a molecular switch with two separate cleavage patterns. Cleavage …

Alendronate Alters Single Cell Gene Expression Of Cortical Osteoblast Lineage Cells During Bone Loss, Ryan Jakob Murphy

Dissertations, Masters Theses, Capstones, and Culminating Projects

The mineralized matrix of bone makes it difficult to examine specific populations of cells which are integral to the tissue using traditional molecular methods. For this study we examined single cell cortical osteoblasts derived from the femurs of C57BL/6J mice. After isolating single cells from bone, we were able to individually analyze their gene expression profiles using the quantitative polymerase chain reaction (qPCR). The mice used for the study were divided into 4 treatment groups, including ovariectomized mice (OVX) and sham surgical controls (SHAM), treated with or without the anti-resorptive bisphosphonate drug Alendronate, an effective FDA approved therapeutic for slowing …

Application Of High-Performance Liquid-Chromatography Mass-Spectrometry Platform To Study Metabolism And Epigenetic Control Of Metabolism, Kylie Patricia Mitchell

Dissertations, Masters Theses, Capstones, and Culminating Projects

Naturally occurring small molecules (metabolites, signaling intermediates) are a critical component of the information flow in biology, along with DNA, RNA, and proteins. Metabolomics is an analytical approach that seeks to comprehensively analyze naturally occurring small molecules and quantify their dynamic changes in biological systems. In recent years metabolomics has begun to provide understanding of the metabolic basis of different diseases, such as heart disease, cancer, and diabetes. Our lab built a High-Performance Liquid-Chromatography Mass-Spectrometry (HPLC-MS) based metabolomics platform to analyze metabolites from mammalian cells, spent cellular media, and model organisms such as C. elegans. We used C. elegans to …

Evaluation Of Select Publicly Available In Silico Methods For Predicting Functional Effects Of Missense Mutations In The Galns Gene, Kathryn Davidson

Dissertations, Masters Theses, Capstones, and Culminating Projects

The ability to sequence patient DNA has led to an explosion in the reports of mutations for a number of diseases. Frequently, published reports include in silico predictions of the probability that the mutations are disease-associated. The question asked here is how well these in silico methods predict the effects of new mutations, i.e., mutations not included in the dataset(s) used for training and testing the in silico method. To address this question, we examined mutations associated, or potentially associated, with Morquio A (MPS IVA), a rare, autosomal recessive lysosomal storage disorder (LSD) caused by a deficiency of lysosomal enzyme …

Investigations Into Aldefluor As A Novel Method For Identifying Leukemia In Soft-Shell Clams, Katherine F. Norwood

Student Research Projects

The clam species Mya arenaria is a common model organism in leukemia research. The current method for classifying the degree of cancer progression is by examining cell morphology with light microscopy. This approach is highly qualitative, which makes differentiation of pre-leukemic and semi-leukemic individuals difficult. One quantitative approach that may differentiate individuals is based on levels of aldehyde dehydrogenase (ALDH) expression. The enzyme assay ALDEFLUOR® can actively measure ALDH expression in viable cells, but the effectiveness of certain protocol conditions is dependent upon the cell type.