David Janz and Lucy Kapronczai Research question Q4 Have changing landscape conditions associated with anthropogenic natural resource extraction activities resulted in changes in habitat selection by ID: 779021
Download The PPT/PDF document "Development and Application of Physiolog..." is the property of its rightful owner. Permission is granted to download and print the materials on this web site for personal, non-commercial use only, and to display it on your personal computer provided you do not modify the materials and that you retain all copyright notices contained in the materials. By downloading content from our website, you accept the terms of this agreement.
Slide1
Development and Application of Physiological Markers of Grizzly Bear Health
David Janz and Lucy Kapronczai
Slide2Research question
Q4: Have changing landscape conditions associated with anthropogenic natural resource extraction activities resulted in changes in habitat selection by,
and the health of, grizzly bears within the study area? 2
Slide3“Conservation Medicine”
3
The application of 21st century technologies used in human medicine (for disease diagnosis, biomarker discovery, and drug development) to free-ranging wildlife species“Omics” technologies (transcriptomics, proteomics, metabolomics)
Technological advances in analytical biochemistry (liquid chromatography coupled with tandem mass spectrometry; LC-MS/MS)
Slide4Rationale
4
Human medicine is focused on individual health, whereas wildlife biology is focused on population health (i.e., population performance and sustainability)Nevertheless, natural selection operates at the level of individual animals to maximize their fitness (Darwin, 1858)In other words, population dynamics (birth rate, mortality rate, immigration, and emigration) is controlled by the fitness of individual animals within populations
Slide5Objectives
5
Determine concentrations of hormones in hair that are associated with reproductive status (testosterone, progesterone, and estradiol) and long-term stress (cortisol)
Develop a novel proteomics technique to quantify expression of proteins in skin that are associated with energetics, reproduction, immune function, and physiological stress
Slide6Objective 1: Hair Hormones
6
Similar to drugs and heavy metals, steroid hormones accumulate in the hair shaft during hair growth and represent a long-term “chronology” of circulating hormone over monthsThe grizzly bear annual hair cycle is ideal for this work, since hair grows from late spring to hibernation and thus represents much of the active season
Slide7Objective 1: Progress
7
Techniques to determine cortisol, testosterone, progesterone and estradiol have been validated in our laboratory and in our work with captive grizzly bearsLab analyses of these hormones is ongoing as we receive hair samples from field personnelApplication of the hair hormone analyses will be integrated with other theme data in the project (environmental, nutritional, and behavioural variables)
Slide8Objective 2: Skin Proteomics
8
Emerging research field of “cutaneous neuroendocrinology”
Slide9Objective 2: Experimental Approach
9
Candidate proteins for the proteomics technique:Markers of energetics (energy metabolism, nutritional status): leptin, ghrelin, adiponectin, apolipoproteinMarkers of reproductive status (sex, pregnancy, maturity) :
relaxin, prolactin, gonadotropins, progesterone receptorMarkers of immune function (immunosuppression, infection): haptoglobin
,
α
2-macroglobulin,
kininogen
, other immunoglobulins
Markers of physiological stress (responses to external stressors)
: glucocorticoid receptor, adrenocorticotropic hormone,
corticotropin
-releasing hormone, HSP70
Slide10Objective 2: Technique
10
Mass spectrometry-based multiple reaction monitoring (MRM)
Steps:Select target proteinsSelect 3-5 unique (“
proteotypic
”) peptides
Select specific peptide fragment ions (“transitions”)
Validate using
stable isotope-labeled
synthetic peptides
Quantitation of proteins and analysis of data
Slide11Objective 2: Progress
11
Developed technique to isolate and purify sufficient protein from small (< 0.1g) skin biopsies to use in MRM assayConducted a “discovery run” using orbitrap LC-MS/MS, which identified amino acid sequences of >250 proteins in grizzly bear skin
Our current work is refining the actual proteins to be included in the MRM assayThe challenge is that very little amino acid sequence information is available for Ursus
arctos
proteins; exact amino acid sequences are ideal for developing a MRM assay
Slide12Objective 2: Progress
12
To inform us on amino acid sequences of grizzly bear proteins, we ran a grizzly bear skin sample on an Illumina NextSeq benchtop sequencerWe performed an “exome run”, which provided us with nucleotide base (A,T,G,C) sequences of all expressed genes in bear skin
Slide13Objective 2: Progress
13
Using available bioinformatics software, these mRNA sequences can then be analyzed (aligned or “mapped”) to determine amino acid sequences of proteins expressed in skinThe challenge in bioinformatics is “big data” – about 400 million alignable
reads were obtained!
Slide14Applications for Resource Managers
14
Key point: the data generated from hair hormone and skin protein analyses should be viewed as tools to assist with identifying the physiological status of individual grizzly bears
These data must be used in combination with other environmental and ecological variables on a larger scale to field validate their utility as physiological markers
For hair cortisol concentration, we have a rich long-term dataset from 1999
For other hair hormones and proteomics, data are just beginning to be collected
Slide15Applications for Resource Managers
15
Caveat: we may not identify a “magic bullet”! That is, in the short term we will likely not identify a single marker that indicates compromised health statusNevertheless, the strength of this work is that by determining a range of markers indicative of important physiological functions, greater insight into bear health can be obtained
Slide16Thanks for Listening!
16
Questions?David M. Janz, Ph.D.Professor, Veterinary Biomedical Sciences University of Saskatchewan
david.janz@usask.ca306-966-7434http://www.usask.ca/toxicology/people/faculty/david-janz.php
https://www.usask.ca/wcvm/wcvm_people/profiles/Janz_David.php