The Intensity of Tumor Necrosis Factor Alpha and Interferon Gamma

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Immunohistochemistry Studies of Tumor Necrosis Factor Αlpha and
Interferon Gamma Expression in Pseudocapillaria tomentosa Infected
and Uninfected Zebrafish
By
Lalee C. Lo
An Undergraduate Thesis Submitted to
Oregon State University
In partial fulfillment of
the requirements for the
degree of
Baccalaureate of Science in BioResource Research,
Food Science Option and Chemistry Minor
September 02, 2009
1
Signature Page:
APPROVED:
_________________________________
Jan Marie Spitsbergen faculty mentor, department
_______________
Date
_________________________________
Susan Tornquist secondary mentor, department
_______________
Date
_________________________________
Katharine G. Field, BRR Director
_______________
Date
© Copyright by Lalee C. Lo September 02, 2009
All rights reserved
I understand that my project will become part of the permanent collection of the Oregon State
University Library, and will become part of the Scholars Archive collection for BioResource
Research. My signature below authorizes release of my project and thesis to any reader upon
request.
_________________________________
Lalee C. Lo
_______________
Date
2
Immunohistochemistry Studies of Tumor Necrosis Factor Αlpha and
Interferon Gamma Expression in Pseudocapillaria tomentosa Infected
and Uninfected Zebrafish
Lalee Lo
Department of Microbiology, Oregon State University, Corvallis, Oregon, USA
ABSTRACT
Tumor necrosis factor alpha (TNF α) and interferon gamma (IFN γ) were initially recognized as
inflammatory mediators produced by immune cells in response to infectious agents or foreign
material. (1,5) However, over the past decade much research in mammalian species has indicated
that these proteins also play key roles in development and homeostasis of many tissues such as
adipose tissue and endocrine islets of pancreas (2,3,12). Our goal was to determine whether the
levels of these mediators were elevated in tissues of zebrafish following infection with intestinal
nematodes. We examined protein expression for these mediators in whole histologic sections of
zebrafish using immunohistochemistry applied to uninfected fish and fish infected with the
intestinal nematode Pseudocapillaria tomentosa. Fish were sampled for histologic study at
selected time points following infection with the nematodes. We utilized commercially available
antibodies to mammalian TNF α and IFN γ and the DAKO Envision Plus Immunohistochemistry
polymer system for detection of these proteins in fish tissues. Contrary to our initial hypothesis
that parasite infected fish would have increased levels of these inflammatory mediators in tissues,
we found no overall increase in these mediators in parasite infected fish using
immunohistochemistry at the 12 week post-infection sample point. However, the early 5 week
post-infection sample point showed that there was an increase in the levels of these mediators in
parasite infected fish. Similar with observations in mammals that these mediators play important
roles in tissue development and homeostasis, we found consistent, specific, moderate to strong
3
baseline expression of these mediators in several tissues of zebrafish. In assays conducted at 5
weeks following parasite infection we saw increased levels of TNF α and IFN γ in inflamed
skeletal muscle and saw increased TNF α in segments of bowel in which recent inflammation
occurred.
INTRODUCTION
The purpose of these experiments was to investigate the expression of two cytokines, TNF α and
IFN γ, in zebrafish following infection. TNF α and IFN γ are important signaling molecules that
are vital to immune regulation; although they also play roles in many other functions (6,7). The
human body has natural defense mechanisms that protect against invading pathogens such as
bacteria, fungus, and viruses. An important component of these innate defense mechanisms are
white blood cells or leukocytes (10).
The word leukocyte is a broad term that refers to two classes of cells: granulocyte and
agranulocyte. In each class there are different types of cells (Fig. 1); for example, in mammals
there are three types of granulocytes: neutrophils, basophils, and eosinophils. But zebrafish are
different from mammals in having a cell with combined characteristics of both eosinophil and
basophil called the eosinophil/basophil. The agranulocytes include lymphocytes, monocytes, and
macrophages in both mammals and fish (15).
4
Figure 1. Types of Leukocytes (http://www.newworldencyclopedia.org/entry/Leukocyte)
Each type of leukocyte can produce cytokines, including TNF α and IFN γ, which can then be
used as signaling molecules for the autocrine, paracrine, and endocrine communication systems.
The autocrine system only affects the cell itself and/or like cells; the paracrine system affects the
cells around the cell regardless of type; the endocrine system affects all cells and functions in
growth, development, metabolism, and function (15,16).
The zebrafish was used in our experiments as it is a powerful model organism to study disease
mechanisms. Zebrafish are ideal for gene knock out studies, as the knocking out of some genes in
mammal embryos will lead to embryo mortality. Because of a whole genome duplication that
occurred in ancestors of bony fish, but not tetrapods, zebrafish often have duplicate genes for
mammalian orthologs. An inactivating mutation of one of these genes may still allow
development of live embryos, while a mutation in the mammalian ortholog is embryonic lethal.
Zebrafish also develop several immune cells homologous to the mammalian counterparts,
including lymphocytes, monocyte/macrophages, and neutrophils; these also produce the cytokines
known as TNF α and IFN γ (11,18). This makes zebrafish ideal, as they are easy to maintain and
keep in high numbers.
The experiment involved the tissue sampling of uninfected zebrafish and Pseudocapillaria
tomentosa infected zebrafish with the use of immunohistochemistry; this was used to aid in the
visualization of the intensity of the TNF α and IFN γ cytokines. Pseudocapillaria tomentosa is a
common gut nematode of fish previously used in Dr. Jan Marie Spitsbergen’s laboratory and was
5
known to cause inflammation in the gut and intestine of zebrafish; infection by this nematode also
leads to the formation of aggressive neoplasm of the intestine.
However, because the narrow sampling window in the original experimental group did not reveal
changes in TNF α and IFN γ expression, a second set of tissue slides with earlier sample times was
also analyzed. This group of zebrafish was exposed to a carcinogen, DMBA (7,12dimethylbenz[a]anthracene) with a DMSO (dimethylsulfoxide 0.1%) carrier in three protocols,
bath exposure of embryos, bath exposure of fry, and dietary exposure of juveniles at a
concentration of 1.0 ppm; the carcinogen also induced neoplasia (14). We also investigated MPO
expression in uninfected and infected zebrafish. Myeloperoxidase (MPO) specifically stains the
cytoplasm of neutrophils of zebrafish and helped determine the sites of inflammation and severity
(4).
Our hypothesis was that Pseudocapillaria tomentosa infected zebrafish would have stronger
staining of tissues with chromogen in immunohistochemistry studies compared to uninfected
zebrafish, indicating elevated tissue levels of TNF α and IFN γ. The study has great potential
importance because it could cast light on current and future research on the function of TNF α and
IFN γ.
MATERIALS AND METHODS
Zebrafish
We maintained a tank of parasite donor fish holding fish infected with Pseudocapillaria
tomentosa. Juvenile fish were used to keep the disease cycling so adults would not develop
immunity and shed the parasites. Zebrafish were raised in flowing well water in 30 gallon tanks at
6
27 Celsius +/- 2 degrees and fed ad libitum daily with Aquatox flake fish feed (Ziegler, Gardners
PA), a diet in which the fish meal component is pretested to ensure minimal levels of natural
carcinogens and nitrosamines. Brine shrimp were also fed daily. The feeding schedule was setup
to prevent contamination to the non-infected zebrafish; this was accomplished by feeding the noninfected zebrafish first and then feeding the infected zebrafish last, and using separate containers
for uninfected and infected fish. Tank algae and waste feed were removed weekly.
Table 1: Experimental Design for Study of Interaction of Gut Nematodes
Date
of
Birth
Date of
Carcinogen
Exposure
Carcinogen
Treatment
Date of
Parasite
Treatment
Parasite
Treatment
Lot
#
1/5/06
2/8/06 (age
30 days)
DMSO
Control
3/20/06
None
ZRN
14-5
1/5/06
2/8/06 (age
30 days)
DMSO
Control
3/20/06
Pseudoca
pillaria
ZRN
145b
1/5/06
2/8/06 (age
30 days)
DMBA 1
ppm
3/20/06
None
ZRN
14-6
1/5/06
2/8/06 (age
30 days)
DMBA 1
ppm
3/20/06
Pseudoca
pillaria
ZRN
146b
ZRN
14-9
ZRN
1410
ZRN
14-
9/10/0
6
9/10/0
6
9/10/0
6
None
None
5/10/2007
Pseudoca
pillaria
None
None
5/10/2007
Pseudoca
pillaria
None
None
5/10/2007
None
Sampling for
Histology and
Immunohistochemistry
5 fish at 3 wk
and 5 wk, 20
fish at 17 wk,
30-50 fish at 29
wk post-parasite
5 fish at 3 wk
and 5 wk, 20
fish at 17 wk,
30-50 fish at 29
wk post-parasite
5 fish at 3 wk
and 5 wk, 20
fish at 17 wk,
30-50 fish at 29
wk post-parasite
5 fish at 3 wk
and 5 wk, 20
fish at 17 wk,
30-50 fish at 29
wk post-parasite
# of
Fish
8/10/2007
20
8/10/2007
20
8/10/2007
20
74
67
74
90
7
9/10/0
6
None
None
5/10/2007
None
11
ZRN
1412
8/10/2007
20
Experimental Tank Setup
Table 1 shows the experimental setup. A total of 8 parasite donors were placed into a plastic mesh
cylinder secured to the side of the tank for each tank for parasite transmission.
Each treatment group, ZRN 14-9 to 14-12, contained 20 zebrafish; all were sampled at 11 months
after the date of birth and 12 weeks post infection. The sampling was performed by using a lethal
dose of MS-222 which was obtained through the Argent Chemical Company; the fish were then
transferred to the veterinary diagnostic laboratory of the OSU school of Veterinary Medicine and
placed in paraffin blocks.
Immunohistochemistry Protocol Procedures
Table 2: Immunohistochemistry chemicals used in the experiment.
Immunohistochemistry
Chemical
Description
ABCam TNF α
Monocolonally derived in rabbits immunized with human TNF
alpha. ABCam, Cambridge, MA. The following concentrations
using the ABCam antibody diluent: 1/50, 1/100, 1/1000,
1/5000, 1/10000, 1/50,000, 1/100,000.
Polycolonally derived in rabbit, ABCam, Cambridge, MA. The
following concentrations using the ABCam diluent: 1/100,
1/200, 1/400, 1/1000.
Polyclonal antibody derived in rabbits immunized with human
neutrophil myeloperoxidase. ABCam, Cambridge, MA.
Specifically stains the cytoplasm of neutrophils of zebrafish
and helped determine the sites of inflammation and severity.
Dako North America, Carpinteria, CA. Used between the
binding of the primary and secondary antibodies and also after
a peroxidase block to reduce the risk of unspecific binding; the
slides were then rinsed 6 times after each binding process.
ABCam IFN γ
MPO (Myeloperoxidase)
A Dako wash buffer
(PBST; phosphate buffered
saline, pH 7.6, containing
0.01% Tween 20)
8
The zebrafish tissue was obtained through a paraffin sectioning method; this procedure was
accomplished by the veterinary diagnostic laboratory of the OSU School of Veterinary Medicine.
In brief, the whole zebrafish was set in a block of paraffin wax and then 4 micrometer thick slices
were cut and placed onto microscope slides.
Immunohistochemistry Slide Preparation
The next step was to expose the slides to the immunohistochemistry chemicals (see Table 2); we
used an immunohistochemistry (IHC) protocol which was developed in a previous experiment
utilizing the Dako Envision Plus immunohistochemistry kit. The slide with the tissue was heated
on a hot plate at 37 degrees Celsius for 15-20 minutes. It was then immersed for two treatments in
P-xylene for 10 minutes; then placed in 2 treatments of 100% EtOH for 3 minutes each; then
washed in 95% EtOH for 3 minutes; and then 3 minutes in 70% EtOH. The slides were dipped 10
times in fresh deionized water and then 10 times in PBST. The slides were then left in the PBST
if other slides were to be prepared.
Antigen Retrieval
For antigen retrieval, we used a steam and enzymatic protocol developed in a previous experiment.
After the slides were prepared, they were placed in a plastic slide holder filled with PBST. The
container was then heated in a rice cooker filled with water for 20 minutes. The slides were then
removed and rinsed in PBST and then set in a Microprobe (a slide holder for specialized Probe On
slides which allow solution wicking.) and set in blocking solution (BSA 1%, DMSO 1%, normal
goat serum 2% in PBS, pH 7.6) for 30 minutes. The blocking solution prevents nonspecific
binding of primary and secondary antibodies to the tissue sections.
Primary and Secondary Antibody Application
After the antigen retrieval, the slides were then put into the primary antibody and left for 30
minutes. The slides were then washed with PBST and a secondary antibody was applied for a
9
minimum of 30 minutes. The secondary antibody was obtained with the primary antibody in the
ABCam kit. Table 2 shows the specific types of antibodies used, their sources and dilutions in the
experiment.
Negative Controls
The negative control slides were prepared identically to the experimental slides, but were not
exposed to the primary and secondary antibody; instead they were set in normal rabbit serum,
normal mouse serum, and PBST. Negative controls indicate whether there is significant
nonspecific tissue binding of non-immune serum of the same species as the primary antibody.
Slide Staining and Visualization
The slides were then washed with PBST, incubated with chromogen for 20 minutes; then rinsed
with deionized water and transferred to deionized water. The slide were briefly stained with
hematoxylin in one dip and quickly thoroughly rinsed in water. Slides were then placed on the
heater plate set to 80 degrees F and the water allowed toevaporate; afterwards 3 drops of crystal
mount were used to cover and protect the stained tissue. The slides were then analyzed using a
visual scoring from a scale of 1-3, 1 being light staining and 3 being very heavy staining.
RESULTS AND DISCUSSION
At the beginning of any immunohistochemistry study, one must optimize the protocol for the
procedure and for the specific antibodies to be used. Therefore we tested various factors during
the optimization process. We first determined the optimal concentration of antibody to use for
each antibody tested, by evaluating a range of dilutions of each antibody. The TNF α and IFN γ
antibodies at full concentration caused the antigen to bind too strongly and created too much
background. The best result with the ABCam TNF α was with a dilution of 1/10000 since this
10
produced minimal background and optimal specific staining of positive tissues. The best result
with the ABCam IFN γ antibody was at a dilution of 1/1000.
We then evaluated whether antigen retrieval was required for optimal assay results. Enzymatic
antigen retrieval was not helpful with these tissues and antibodies. We compared two methods of
antigen retrieval, heat treatment and enzyme treatment of histological sections (19). Steam heat
treatment resulted in better staining, so we included it in the final protocol. When we compared
various buffers for the steam heat treatment, we found that there was little to no difference in the
results; therefore, we decided to use the Dako antigen retrieval buffer.
We included a positive control to ensure that the reagents were working. We first did an
immunohistochemistry assay for proliferating cell nuclear antibody (PCNA), using a mouse
monoclonal antibody to mammal PCNA. This antibody had been validated as reacting strongly
with proliferating zebrafish cells in previous assays in Dr. Spitsbergen’s laboratory. We also tried
various antibody concentrations to optimize the dilutions.
We also investigated MPO expression in uninfected and infected zebrafish. Because the
concentration of the prediluted MPO antibody was optimized for mammal tissues, we found that
the optimal concentration for use on zebrafish tissue actually was a dilution of 1:2 of this
prediluted antibody.
After the sampling time, MPO expression was used to determine the severity of the inflammation
of the infected and uninfected zebrafish tissue. MPO specifically stains the cytoplasm of
neutrophils of zebrafish which are the first responders to these sites of inflammation (10, 15). A
11
comparison of MPO expression in uninfected and infected zebrafish revealed that the intestine of
zebrafish chronically infected with Pseudocapillaria tomentosa (12 weeks post infection) showed
increased numbers of neutrophils compared to intestine of uninfected fish (Figures 1,2). Reactive
oxygen from inflammatory cells such as neutrophils is known to cause damage to proteins and
nucleic acid in tissues experiencing chronic infection (4). Specific types of DNA damage are
associated with this chronic inflammation and this DNA damage likely plays a role in the
neoplasia associated with chronic inflammation.
Figure 1: MPO negative in uninfected zebrafish (left photo) and MPO positive in infected
zebrafish (right photo).
Figure 2: MPO in neutrophils in impression smear from kidney.
All negative controls (tissues with no added antibody) showed no staining (Figure 3 and Figure 4)
12
Figure 3: 1.25x mouse serum negative control.
Figure 4: 1.25x rabbit serum negative control.
In previous experiments, the nematode Pseudocapillaria tomentosa caused the production of
tumors in the intestine, and so we expected there to be a higher concentration of TNF α in these
areas of inflamed intestinal tissue. Figures 5 and 6, show strong chromogen staining for both
inflammatory mediators in several tissues of the zebrafish; these results are summarized in Table
3,4,5. We found the immunity cytokines TNF α and IFN γ to be present in several of the tissues
derived from ectoderm, mesoderm, and endoderm. (Figures 7-9)
13
Figure 5: TNF α stain of ZRN 14-5 at 5x magnification. Image of immunohistochemistry with
antibody used at a dilution of 1:1000. TNF α reactivity was present in several tissues. Specific
positive staining was seen in certain layers of the eye and skeletal muscle of the head.
Figure 6: TNF α stain of ZRN 14-5 at 5x magnification. Presence of TNF α was seen in selected
tissues in meso and endoderm.
Figure 7: Post 5 week infection of TNF α stain of ZRN 5 uninfected intestine (left photo) and
TNF α stain of ZRN 5 infected intestine (right photo). TNF α was present at higher levels in the
infected tissue.
14
Figure 8: IFN γ revealed by immunohistochemistry of ZRN 14-9 heart at 10x magnification. The
red indicated that the cells in the heart contained TNF α.
Figure 9: Post 5 week infection of IFNγ in inflamed skeletal muscle of trunk (left photo) and
IFNγ in skeletal muscle near optic nerve (right photo).
According to our initial hypothesis, the uninfected group should have shown lower concentrations
of TNF α and IFN γ due to less gut inflammation; however the visual intensity of inflammatory
mediators as indicated by immunohistochemistry was the same for all the treatment groups at the
12 week post infection sample point (Table 3,4,5). We were unable to establish a link between
the intensity levels of TNF α and IFN γ in parasite infection at this with the later sampling point.
We expected that TNF α and IFN γ production would increase in parasite infected fish as previous
studies showed them to be immune response signaling proteins. However, even uninfected
zebrafish expressed relatively high levels of these cytokines in certain tissues. (Figure 5)
15
Finally, an earlier experiment treated zebrafish with carcinogen (Table 1), then infected two of the
four groups with Pseudocapillaria tomentosa. These slides included earlier sample points (Figure
7) and the results were as expected. The 5 week post-infection TNF α stains indicated that the
infected zebrafish inflamed intestine showed elevated levels of TNF α, while the uninfected
showed very low levels of TNF α; this also supports the hypothesis. IFN γ at the 5 week postinfection sample point also displayed an increase in the inflamed skeletal muscle on the trunk and
near the optic nerve (Figure 9) when compared to the uninfected zebrafish. This also supports the
hypothesis that parasite infection does increase the production of IFN γ.
Table 3: Summary of Tissues Expressing Protein for Inflammatory Mediators
Intensity of Staining: 1+=low; 2+=medium; 3+=high
Proteins Expressed
Tissue
Category
Body
Region
Organ
System
Organ
Tissue
Ectoderm
Head
Skin
Sensory organ
Eye
CNS
Ear
Nose
Taste bud
Brain
Neuroepithelium
Lens
Cornea
Vestibular neuroepithelium
Sensory neuroepithelium
Neurons in midbrain and
cerebellum
Neurons in diencephalon,
midbrain, hindbrain
Pituitary
Optic nerve
Trunk
Mesoderm
Tail
Head
Gill
Pseudobranch
Skin
Fin
Skin
Vascular
Lymphohemopoietic
Surface epithelium
Epithelium
Skin epithelium
Blood vessel
Thymus
Anterior
kidney
Abcam
TNF α
0
2+
0
0
Abcam
IFN γ
0
2+
1+
0
0
0
0
0
2+
2+
2+
1+
2+
1+
0
2+
1+
0
0
0
NA
NA
2+
0
0
1+
0
0
0
NA
NA
NA
NA
0
16
Muscular
Skeletal
Trunk
Gill
Cardiovascular
Heart
Urinary
Trunk
Kidney
Lymphohemopoietic
Muscular
Trunk
Kidney
Skeletal
Fin
Tail
Endoderm
Head
Gastrointestinal
Trunk
Gastrointestinal
Oropharynx
Pharyngeal
mill
Esophagus
Pneumatic
duct
Intestine
Gas Bladder
Liver
Neural
Crest
Head
Skeletal muscle
Cartilage
Bone
Cartilage
Bulbus
Atrium
Ventricle
Renal tubules
3+
3+
1+
1+
1+
1+
2+
2+
1+
0
0
0
3+
3+
3+
0
Mesonephric duct
Hemopoietic tissue
1+
1+
0
2+
Red skeletal muscle
White skeletal muscle
Cartilage
Bone
Skeletal muscle
Cartilage
Skeletal muscle
Cartilage
Mucosal epithelium
3+
3+
1+
1+
3+
1+
3+
1+
1+
1+
0
Mucosal epithelium
1+
1+
0
0
Anterior intestine near
esophagus, mucosal
epithelium
1+
0
Middle mucosal epithelium
Posterior mucosal epithelium
1+
1+
1+
2+
2+
NA
0
0
0
0
0
2+
Hepatocyte
Bile duct
8th cranial nerve
Cranial nerves
0
0
0
0
0
0
0
0
0
Table 4: Tumor Necrosis Factor Alpha Histochemical Staining Summary.
Tissues Staining Strongly
Skeletal muscle of head and at
bases of fins (derived from
neural crest)
Intestine mucosal surface
(entire intestine)
Tissues Staining Moderately
Tissues Staining Lightly
Neurosensory epithelium of
nose
Bile duct mucosal surface
Taste Buds
Exocrine pancreas acinar cell
Ultimobranchial gland
Chloride cell of gill filament
and lamellae
Outer plexiform layer of retina
of eye
17
Heart ventricular myocardium
Heart atrial myocardium
Table 5: Interferon Gamma Histochemical Staining Summary.
Tissues Staining Strongly
Tissues Staining Moderately
Endocardium of heart
Neurosensory epithelium of
nose
Taste Buds
Macrophages in inflammation
in skeletal muscle
Inflammatory cells in
connective tissue
Tissues Staining Lightly
Pseudobranch epithelium
Ameloblastic epithelium of
tooth
Hemopoietic tissue in kidney
(multifocal)
Meninges and ventricles of
brain
Chloride cells of gill filament
Ganglia of cranial nerves
Developing oocytes
(perinucleolar and
vitellogenic)
Tooth pulp
Our hypothesis, that Pseudocapillaria tomentosa infected zebrafish would have stronger staining
of tissues with chromogen in immunohischemistry studies, indicating elevated tissue levels of
TNF α and IFN γ, is supported with this research project. However, this was only observed at the
5 weeks post infection sample time. At the later sample time points, uninfected and infected
zebrafish showed similar levels of TNF α and IFN γ. This suggests that they may play other roles
besides immune cell signaling such as homeostasis. Mammalian data also indicate that these
mediators do play essential roles in development and homeostasis of many tissues (12, 13, 17).
Improvements to the experiment
18
In the future, the experiment could be improved upon by incorporating a Western blot to clarify
the specificity of both of the antibodies. Because TNF α and IFN γ are part of large super-families
of related proteins in both mammals and fish, (21) we need to show that the ABCam antibodies
bind specifically to the TNF α and IFN γ antigens of zebrafish. In zebrafish there are duplicate
IFN γ genes and proteins, (11) so it is important to clarify if the antibody to IFN γ binds to both
IFN γ1 and IFN γ2 of zebrafish. The antibodies purchased from ABCam were shown to bind to
mouse, guinea pig, human, cynomolgus monkey, and rhesus monkey TNF α and human and
rhesus monkey IFN γ, but it is not yet known whether it binds specifically to zebrafish TNF α and
IFN γ. Another way to validate the immunohistochemsitry studies would be to look at expression
of TNF α and IFN γ in zebrafish tissues using situ hybridization to evaluate RNA expression in the
tissues.
ACKNOWLEDGEMENTS
This work was supported by a National Institutes of Health (NIH) grant from Dr. Jan Marie
Spitsbergen, the facilities at the John Fryer Salmon Disease Lab, the Marine and Freshwater
Biomedical Science Center, and the Environmental Health Science Center at Oregon State
University. I am also truly grateful for the extensive support from my mentor, Jan Marie
Spitsbergen for it would not have been possible without her help; she taught me
immunohistochemistry, zebrafish maintenance, ethical necropsy procedures, project
proposal/thesis writing and revising, and most importantly her ability to motivate me to complete
my project. I would also like to acknowledge my project advisor, Kate Field and secondary
mentor, Susan Tornquist for their added insight on the project. And finally, thanks to Wanda
Crannell for her support as the Bioresource Research advisor.
19
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