Difference between revisions of "Team:Queens Canada/Description"

Latest revision as of 03:10, 18 October 2018

In The Glow: Luminescent Biosensors for Hormone Detection and Diagnosis

Overview

The human endocrine system is responsible for keeping the body in a delicate balance by controlling essential functions such as thermoregulation, thirst, satiety, sexual drive, and awareness. The body uses chemical messengers called hormones to maintain this delicate balance, by signaling to the cells throughout your body on how to behave. Examples of hormones include Estrogen, Oxytocin, and Cortisol. This year our team sought to produce engineered protein biological sensors for the detection and quantification of hormones present in saliva as tool for diagnosis of hormone disorders, monitoring endocrine function, and determining treatment efficacy. As a starting point for our biological sensors we have focused on detection of the steroid hormone cortisol. We have taken two approaches to the biologic sensors for the measurement of Cortisol. Firstly, we have constructed a glucocorticoid sensor utilizing changes in Fluorescence Resonance Energy Transfer (FRET) to detect hormones. Secondly, we have began developing a novel biological sensor which utilizes intein splicing, producing a signal for hormone quantification. In addition to our work in the laboratory, we developed complimentary hardware and software, which we aim to incorporated into the form of diagnostic pacifier device with a built in luminometer, allowing for portable, quick, and non-invasive collection, and measurement of salivary analytes in infants.

Why Cortisol?

Cortisol is a steroid hormone that is made in the cortex of the adrenal glands. It is released in the bloodstream and transported all around the body. This hormone is necessary for many bodily functions, as indicated by the majority of cells having a receptor for it. Although each individual has his/her own operating range of “normal” cortisol levels, naturally, cortisol levels undergo a diurnal rhythm: they are at a peak when one wakes up, and fall as the day progresses. Determining a normalized range of values for the population can help find abnormalities among individuals. Fluctuating cortisol levels that differ from this diurnal rhythm can serve as an indicator of various disorders. Thus, cortisol can be a very useful biomarker of health and wellness.

Some functions of cortisol include the regulation of blood sugar levels, metabolism, cardiovascular health, controlling inflammation and immune function. [1] Since cortisol enhances the production of glucose from the liver through gluconeogenesis, counterbalancing the effects of insulin, [2] it can potentially be used as a biomarker for conditions like hypoglycemia (low blood sugar) and insulinemia (abnormally elevated blood-insulin levels). In terms of metabolism, cortisol is an overall catabolic hormone which decreases lean body mass, muscle mass, and can increase energy expenditure. [2] Elevated cortisol production and a change in local regulation are associated with obesity, [3] further research on cortisol could be used to help combat this common condition. Using cortisol as a biomarker could potentially have an application in early diagnosis of metabolic disorders such as Cushing’s syndrome and Addison’s disease, which are caused by hyper- and hypo-secretion of cortisol, respectively. Early diagnosis for these disorders would be revolutionary for early treatment options to lessen symptoms including obesity, bone loss, and high blood pressure for Cushing’s, as well as weight loss, muscle/joint pains, and low blood pressure for Addison’s.

Another potential application of cortisol as a biomarker is to monitor fetal wellbeing in pregnant women. Highly stressed pregnant women have elevated glucocorticoid levels which can cause reduced growth, alter the timing of tissue development, and can cause mood disorders later in the child’s life. [4] In a study where maternal cortisol levels were observed during pregnancy and infant development rates were measured following birth, it was found that “exposure to elevated concentrations of cortisol early in gestation were associated with a slower rate of development over the first postnatal year and lower scores on the mental development index of the Bayley Scales of Infant Development (BSID)”. [5] In contrast, higher levels of maternal cortisol late in gestation showed opposite results. Monitoring cortisol levels in pregnant women could help predict developmental problems that may arise following birth.

Presently, the most common method of measuring cortisol levels is through blood work. Other methods however, such as salivary tests, are generating interest as they are far less invasive and easier for sample collection. One key difference between blood and saliva tests for hormones is that salivary tests are able to measure the amount of free, unbound hormone that is available to act on a given tissue because saliva contains unbound, bioavailable hormones. Since 95-99% of steroid hormones flowing in the blood are bound by carrier proteins rendering them inactive (because steroids are unable to dissolve in water, a large component of blood), blood tests may give a less accurate representation of how much hormone is actually available to target tissues. [6,7] In one study, researchers found a correlation between higher salivary cortisol and increased HbA1c levels (glycosylated hemoglobin, a metabolic factor), as well as high triglyceride and high density lipoprotein (HDL) cholesterol levels. These correlations were not found when cortisol levels were tested through blood. [8] This is where Queen’s Genetically Engineered Machinery (QGEM’s) product can be of great use. We are developing a pacifier that is intended to detect salivary cortisol in infants, and quantify the levels to a mobile application through bioluminescence and Bluetooth technology. This concept is not limited to infants, as the genetically engineered protein could be used to quantify salivary cortisol levels in youth and adults as well. Using saliva as a diagnostic method for cortisol abnormalities has great potential as it is non-invasive, accessible and self-administrable, meaning the general population might be more inclined for regular tests to have more control over their health and wellbeing.

References

[1] McKay LI, Cidlowski JA. Physiologic and Pharmacologic Effects of Corticosteroids. In: Kufe DW, Pollock RE, Weichselbaum RR, et al., editors. Holland-Frei Cancer Medicine. 6th edition. Hamilton (ON): BC Decker; 2003. Available from: https://www.ncbi.nlm.nih.gov/books/NBK13780/
[2] Effects of Cortisol on Carbohydrate, Lipid, and Protein Metabolism: Studies of Acute Cortisol Withdrawal in Adrenocortical Failure. Christiansen J, Djurhuus C, Gravholt C, Iversen P Christiansen J et. al. The Journal of Clinical Endocrinology & Metabolism 2007 vol: 92 (9) pp: 3553-3559
[3] Cortisol dysregulation in obesity-related metabolic disorders. Baudrand R, Vaidya A. Current opinion in endocrinology, diabetes, and obesity. 2015 vol: 22 (3) pp: 143-9
[4] Fetal exposure to excessive stress hormones in the womb linked to adult mood disorders -- ScienceDaily. (n.d.). Retrieved October 8, 2018, from https://www.sciencedaily.com/releases/2013/04/130407090835.htm
[5] Davis, E. P., & Sandman, C. A. (2010). The Timing of Prenatal Exposure to Maternal Cortisol and Psychosocial Stress is Associated with Human Infant Cognitive Development. Child Development, 81(1), 131–148. Read it here.
[6] The choice is clear: saliva vs. blood diagnostics for hormone testing. (n.d.). Retrieved October 8, 2018, from https://www.tecan.com/blog/saliva-hormone-testing-versus-blood-diagnostics
[7] Lewis JG. Steroid analysis in saliva: An overview. Clin Biochem Rev. 2006;27(3):139-146.
[8] Konishi S, O'Connor K. Salivary but not blood cortisol excretion is associated with metabolic biomarkers in healthy young women. Am J Hum Biol 2016; 28(4):539-544.

@@ Line 1: / Line 1: @@
 {{Queens Canada/Header}}
 {{Queens Canada/Navbar}}
 <html>
-<body style="margin:0 auto" max-width:800px;">
+<head>
 <style>
 body {
      background-color: white;
-     max-width: 1200px;
+     font-family: Helvetica, sans-serif;
+    margin-top: 20px;
      position: relative;
      margin-left: auto;
@@ Line 12: / Line 14: @@
 }
 </style>
+</head>
 <body>
-<div class="column full_size">
+<div style="width:70%;margin-left:15%" class="column full_size">
-<h1>Biosensors utilizing ligand dependent intein-splicing: Application in a Diagnostic Pacifier</h1>
+<h2>In The Glow: Luminescent Biosensors for Hormone Detection and Diagnosis</h2>
+<h3>Overview</h3>
+</div>
-<p>This year’s project will produce a novel biochemical assay for the quantification of a given ligand in solution. To achieve this goal, we will be developing an engineered protein construct that consists of three domains, 1) a modular ligand binding domain, 2) an intein splicing domain, 3) and a split NanoLuc® Luciferase domain. While protein constructs such as this can be applied to a variety of substrates and/or ligands, one chosen application for this novel technology is to detect and quantify the amount of cortisol present in saliva. As cortisol is indicative of the human stress response, we would effectively be able to quantify an individual’s stress at a given time. This protein construct may also be used for diagnostic purposes involving the detection of hormone imbalances. A specific application of this technology is the communication of stress in non-verbal individuals, such as infants. Therefore, we are producing a novel protein construct that links the binding of cortisol to the endogenous human glucocorticoid ligand binding domain to intein splicing together two halves of a split NanoLuc® Luciferase. The splicing event would produce a functional luciferase reporter, and the resulting luminescence would allow for a direct quantification of cortisol levels.</p>
+<div style="width:70%;margin-left:15%" class="column full_size">
-<!-- IMAGE IN HERE -->
+<p style="width:1200px; font-size:18px;">The human endocrine system is responsible for keeping the body in a delicate balance by controlling essential functions such as thermoregulation, thirst, satiety, sexual drive, and awareness. The body uses chemical messengers
+called hormones to maintain this delicate balance, by signaling to the cells throughout your body on how to behave. Examples of hormones include
+Estrogen, Oxytocin, and Cortisol. This year our team sought to produce engineered protein biological sensors for the detection and
+quantification of hormones present in saliva as tool for diagnosis of hormone disorders, monitoring endocrine function, and determining treatment efficacy. As a starting point for our biological sensors we have focused on detection of the steroid hormone cortisol. We
+have taken two approaches to the biologic sensors for the measurement of Cortisol. Firstly, we have constructed a glucocorticoid sensor utilizing
+changes in Fluorescence Resonance Energy Transfer (FRET) to detect hormones. Secondly, we have began developing a novel biological sensor which
+utilizes intein splicing, producing a signal for hormone quantification. In addition to our work in the laboratory, we developed complimentary
+hardware and software, which we aim to incorporated into the form of diagnostic pacifier device with a built in luminometer, allowing for
+portable, quick, and non-invasive collection, and measurement of salivary analytes in infants.</p>
 </div>
-<div class="column full_size">
+<div style="width:70%;margin-left:15%">
-<h3>Biosensors</h3>
+<img src="https://static.igem.org/mediawiki/2018/0/04/T--Queens_Canada--Julias_Pacifier.png" width="30%">
-<p>Biosensors are devices that are able to detect the presence of analytes and effectively convert this biological response to an electrical signal. This system consists of three components: a bioreceptor, transducer and detector. The bioreceptor is able to form substrate-specific interactions with the analyte. The transducer is then able to detect the substrate-receptor interaction and transmit this into an electrical signal, which is amplified and processed by the detector. This information is then capable of being sent to a data storage device for quantification and analytical purposes.
+<img src="https://static.igem.org/mediawiki/2018/f/fe/T--Queens_Canada--pac1.jpeg" width="30%">
-<br><em>Our biosensor design will rely on the ability to bind to salivary cortisol and transmit this information to a smart phone application following the production of luminescence. The ability to easily quantify and detect changes in cortisol can reveal critical health information.</em></p>
+<img src="https://static.igem.org/mediawiki/2018/0/0d/T--Queens_Canada--pac4.jpeg" width="30%">
-<!-- IMAGE IN HERE -->
-</div>
-<div class="column full_size">
-<h3>Ligand Binding Domain</h3>
-<p>The glucocorticoid receptor is an evolutionarily conserved nuclear receptor protein that functions as a ligand-dependent transcription factor [1]. After binding to the ligand of interest, the protein is shuttled between the cytoplasm and the nucleus which then alters gene transcription [1]. Circulating glucocorticoids, which includes the steroid hormone, cortisol, is able to activate the receptor and mediate processes such as stress response, energy metabolism and immune responses [2]. The ligand binding domain consists of eleven alpha-helices and two beta-sheets that enable the formation of a three-layered protein structure [2]. There also exists a regulatory C-terminal helix that is essential for hormone binding. There are conserved residues within these helices which form critical interactions with carbon atoms of cortisol and allow for specificity within the interaction [2].
-<br><em>Although our team is starting with the glucocorticoid receptor, the modular design of this protein construct allows for flexibility and the ability to switch out the receptor in order to quantify a vast array of different analytes.</em></p>
 </div>
-<div class="column full_size">
+<div style="width:70%;margin-left:15%" class="column full_size">
-<h3>Intein Splicing Domain</h3>
-<p>Found in organisms from all domains of life, inteins (intervening proteins) are auto-processing proteins that function both in endogenous and exogenous contexts [3]. These proteins are involved in the cleavage and formation of peptide bonds during a unique process where they excise themselves from a polypeptide and ligate the flanking extein (external protein) [3]. This spontaneous splicing process occurs post-translationally and is most commonly observed in proteins involved in DNA transcription, replication and maintenance processes within a cell [3].</p>
-</div>
-<div class="column full_size">
+<h3 style="width:1200px;">Why Cortisol?</h3>
-<h3>Intein Splicing Mechanisms</h3>
+<p style="width:1200px;font-size:18px;">
-<p>The Class 1 Splicing Mechanism: Step 1<br>
+Cortisol is a steroid hormone that is made in the cortex of the adrenal glands. It is released in the bloodstream and transported all around the
-The intein splicing mechanism consists of a series of acyl-transfer reactions that results in peptide bond cleavage at the junction between the intein and extein, followed by the formation of a new peptide bond between the N and C termini of the exteins [3]. Either a cysteine or serine residue is almost always present at the N terminus of the intein, which attacks the carbonyl carbon of the N-extein residue to produce an intermediate [3]. Next the C-extein residue undergoes nucleophilic attack, followed by cyclization at the C-terminal of the intein [3].</p>
+body. This hormone is necessary for many bodily functions, as indicated by the majority of cells having a receptor for it. Although each
+individual has his/her own operating range of “normal” cortisol levels, naturally, cortisol levels undergo a diurnal rhythm: they are at a peak
+when one wakes up, and fall as the day progresses. Determining a normalized range of values for the population can help find abnormalities among
+individuals. Fluctuating cortisol levels that differ from this diurnal rhythm can serve as an indicator of various disorders. Thus, cortisol can
+be a very useful biomarker of health and wellness.</p>
+<p style="width:1200px;font-size:18px;">Some functions of cortisol include the regulation of blood sugar levels, metabolism, cardiovascular health, controlling inflammation and
+immune function. [1] Since cortisol enhances the production of glucose from the liver through gluconeogenesis, counterbalancing the effects of
+insulin, [2] it can potentially be used as a biomarker for conditions like hypoglycemia (low blood sugar) and insulinemia (abnormally elevated
+blood-insulin levels). In terms of metabolism, cortisol is an overall catabolic hormone which decreases lean body mass, muscle mass, and can
+increase energy expenditure. [2] Elevated cortisol production and a change in local regulation are associated with obesity, [3] further
+research on cortisol could be used to help combat this common condition. Using cortisol as a biomarker could potentially have an application in
+early diagnosis of metabolic disorders such as Cushing’s syndrome and Addison’s disease, which are caused by hyper- and hypo-secretion of
+cortisol, respectively. Early diagnosis for these disorders would be revolutionary for early treatment options to lessen symptoms including
+obesity, bone loss, and high blood pressure for Cushing’s, as well as weight loss, muscle/joint pains, and low blood pressure for Addison’s.</p>
+<p style="width:1200px;font-size:18px;">Another potential application of cortisol as a biomarker is to monitor fetal wellbeing in pregnant women. Highly stressed pregnant women have
+elevated glucocorticoid levels which can cause reduced growth, alter the timing of tissue development, and can cause mood disorders later in the
+child’s life. [4] In a study where maternal cortisol levels were observed during pregnancy and infant development rates were measured following
+birth, it was found that “exposure to elevated concentrations of cortisol early in gestation were associated with a slower rate of development
+over the first postnatal year and lower scores on the mental development index of the Bayley Scales of Infant Development (BSID)”. [5] In
+contrast, higher levels of maternal cortisol late in gestation showed opposite results. Monitoring cortisol levels in pregnant women could help
+predict developmental problems that may arise following birth.</p>
+<p style="width:1200px;font-size:18px;">Presently, the most common method of measuring cortisol levels is through blood work. Other methods however, such as salivary tests, are
+generating interest as they are far less invasive and easier for sample collection. One key difference between blood and saliva tests for
+hormones is that salivary tests are able to measure the amount of free, unbound hormone that is available to act on a given tissue because saliva
+contains unbound, bioavailable hormones. Since 95-99% of steroid hormones flowing in the blood are bound by carrier proteins rendering them
+inactive (because steroids are unable to dissolve in water, a large component of blood), blood tests may give a less accurate representation of
+how much hormone is actually available to target tissues. [6,7] In one study, researchers found a correlation between higher salivary cortisol
+and increased HbA1c levels (glycosylated hemoglobin, a metabolic factor), as well as high triglyceride and high density lipoprotein (HDL)
+cholesterol levels. These correlations were not found when cortisol levels were tested through blood. [8] This is where Queen’s Genetically
+Engineered Machinery (QGEM’s) product can be of great use. We are developing a pacifier that is intended to detect salivary cortisol in infants,
+and quantify the levels to a mobile application through bioluminescence and Bluetooth technology. This concept is not limited to
+infants, as the genetically engineered protein could be used to quantify salivary cortisol levels in youth and adults as well. Using saliva as a
+diagnostic method for cortisol abnormalities has great potential as it is non-invasive, accessible and self-administrable, meaning the general
+population might be more inclined for regular tests to have more control over their health and wellbeing.</p>
 </div>
-<div class="column full_size">
-<h3>Small Molecule Triggered Intein Splicing</h3>
-<p>The <em>mycobacterium tuberculosis</em> RecA intein was selected for use as it has been shown to splice in a wide variety of protein contexts [6]. Small molecule triggered intein splicing allows the production of a “molecular switch” which is only activated in the presence of the designated ligand. To function, this system requires that the intein is able to bind with a high affinity to its specific ligand, and that the resulting conformational change initiates the process of protein splicing [6]. For the initial application of our technology, we will be using the binding of cortisol to the human glucocorticoid receptor as the initiating reaction that triggers intein splicing.</p>
-</div>
-<div class="column full_size">
+<div style="width:70%;margin-left:15%" class="column full_size">
-<h3>Previous Part: 4HT Dependent Intein</h3>
-<p>The 4HT Dependent Intein splices in the presence of 4-hydroxytamoxifen which allows for the ability to control the engineered protein in a dose-dependent manner.
-<!-- IMAGE IN HERE -->
-<br><em>Our team will utilize cortisol binding to the ligand binding domain of the glucocorticoid receptor as a signal to trigger the intein excision and extein ligation process.</em></p>
-<!-- IMAGE IN HERE -->
-</div>
-<div class="column full_size">
+<h3>References</h3>
-<h3>Split NanoLuc Luciferase Domain</h3>
+<p style="width:1200px;font-size:14px;">
-<p>NanoLuc® is an engineered protein produced by Promega following directed evolution of luciferase derived from Oplophorus gracilirostris [4]. This enzyme obtained from deep-sea shrimp was optimized following the discovery of a novel substrate, furimazine, which allows for the production of visible light with less background activity [4,5]. NanoLuc® is a 19.1 kDa monomeric protein that is both soluble and ATP-independent [4]. Compared to Firefly and Renilla luciferases, this novel protein offers many advantages reflected by its increased stability, smaller size and >150-fold increase in in luminescence [5]. The unique characteristics of this enzyme construct combined with its high luminescence activity allow for the production of a very sensitive diagnostic assay.
+[1]	McKay LI, Cidlowski JA. Physiologic and Pharmacologic Effects of Corticosteroids. In: Kufe DW, Pollock RE, Weichselbaum RR, et al., editors. Holland-Frei Cancer Medicine. 6th edition. Hamilton (ON): BC Decker; 2003. Available from:  <a href="https://www.ncbi.nlm.nih.gov/books/NBK13780/" target="_blank">https://www.ncbi.nlm.nih.gov/books/NBK13780/</a><br>
-<br>Based on <a href="https://www.sciencedirect.com/science/article/pii/S0167488915004152?via%3Dihub">these findings</a>. A Split sites for NanoLuc Luciferase was chosen between the following amino acids: 52/53;  The N- and C-termini were inverted and reconnected through a flexible GGGGS–GGGGS linker.</p>
+[2]	Effects of Cortisol on Carbohydrate, Lipid, and Protein Metabolism: Studies of Acute Cortisol Withdrawal in Adrenocortical Failure. Christiansen J, Djurhuus C, Gravholt C, Iversen P Christiansen J et. al. The Journal of Clinical Endocrinology & Metabolism 2007 vol: 92 (9) pp: 3553-3559<br>
+[3]	Cortisol dysregulation in obesity-related metabolic disorders. Baudrand R, Vaidya A. Current opinion in endocrinology, diabetes, and obesity. 2015 vol: 22 (3) pp: 143-9<br>
+[4]	Fetal exposure to excessive stress hormones in the womb linked to adult mood disorders -- ScienceDaily. (n.d.). Retrieved October 8, 2018, from <a href="https://www.sciencedaily.com/releases/2013/04/130407090835.htm" target="_blank">https://www.sciencedaily.com/releases/2013/04/130407090835.htm</a><br>
+[5]	Davis, E. P., & Sandman, C. A. (2010). The Timing of Prenatal Exposure to Maternal Cortisol and Psychosocial Stress is Associated with Human Infant Cognitive Development. Child Development, 81(1), 131–148. <a href="https://doi.org/10.1111/j.1467-8624.2009.01385.x" target="_blank">Read it here.</a><br>
+[6]	The choice is clear: saliva vs. blood diagnostics for hormone testing. (n.d.). Retrieved October 8, 2018, from <a href="https://www.tecan.com/blog/saliva-hormone-testing-versus-blood-diagnostics" target="_blank">https://www.tecan.com/blog/saliva-hormone-testing-versus-blood-diagnostics</a><br>
+[7]	Lewis JG. Steroid analysis in saliva: An overview. Clin Biochem Rev. 2006;27(3):139-146.<br>
+[8]	Konishi S, O'Connor K. Salivary but not blood cortisol excretion is associated with metabolic biomarkers in healthy young women. Am J Hum Biol 2016; 28(4):539-544.<br>
+</p>
 </div>
+<footer style="background-color: #212121;height:90px ">
+            <div class="container">
+                <div class="row">
+                    <div class="col-md-4">
+                        <ul class="list-inline quicklinks">
+                            <li><a href="https://igem.org/Main_Page" style="color:white; font-size:16pt">iGEM Headquarters</a>
+                            </li>
+                        </ul>
+                    </div>
+                    <div class="col-md-4">
+                        <ul class="list-inline social-buttons" style="margin-left:20%">
+                            <li><a href="https://twitter.com/iGEMQueens"style="    display: block;
+    background-color: #fff;
+    height: 40px;
+    width: 40px;
+    border-radius: 100%;
+    font-size: 20px;
+    line-height: 40px;
+    color: #000;
+    outline: 0;
+    -webkit-transition: all .3s;
+    -moz-transition: all .3s;
+    transition: all .3;"><i class="fa fa-twitter"style="color:black; font-family:FontAwesome;margin-left:10.75px;"></i></a>
+                            </li>
+                            <li><a href="https://www.facebook.com/iGEMQueens/?fref=ts"style="    display: block;
+    background-color: #fff;
+    height: 40px;
+    width: 40px;
+    border-radius: 100%;
+    font-size: 20px;
+    line-height: 40px;
+    color: #000;
+    outline: 0;
+    -webkit-transition: all .3s;
+    -moz-transition: all .3s;
+    transition: all .3;"><i class="fa fa-facebook"style="color:black;font-family:FontAwesome;margin-left:10.75px;"></i></a>
+                            </li>
+                            <li><a href="mailto:director@qgemteam.com" style="    display: block;
+    background-color: #fff;
+    height: 40px;
+    width: 40px;
+    border-radius: 100%;
+    font-size: 20px;
+    line-height: 40px;
+    color: #000;
+    outline: 0;
+    -webkit-transition: all .3s;
+    -moz-transition: all .3s;
+    transition: all .3;"><i class="fa fa-envelope"style="color:black;font-family:FontAwesome;margin-left:10.75px;"></i></a>
+                            </li>
+                            <li><a href="https://www.linkedin.com/company/1633448?trk=tyah&amp;trkInfo=clickedVertical%3Acompany%2CclickedEntityId%3A1633448%2Cidx%3A1-1-1%2CtarId%3A1464110668532%2Ctas%3Aqgem" style="    display: block;
+    background-color: #fff;
+    height: 40px;
+    width: 40px;
+    border-radius: 100%;
+    font-size: 20px;
+    line-height: 40px;
+    color: #000;
+    outline: 0;
+    -webkit-transition: all .3s;
+    -moz-transition: all .3s;
+    transition: all .3;"><i class="fa fa-linkedin"style="color:black; font-family:FontAwesome;margin-left:10.75px;"></i></a>
+                            </li>
+                        </ul>
+                    </div>
+                    <div class="col-md-4">
+                        <ul class="list-inline quicklinks" style="color: white;font-size:16pt;">
+                            <li><a href="http://www.queensu.ca/" style="color:white;margin-left:100px;">Queen's University</a>
+                            </li>
+                        </ul>
+                    </div>
+                </div>
+            </div>
+        </footer>
 </body>
 </html>