Office of Undergraduate Research

Title: Social Interaction and social preference in DAT +/+ and DAT +/- mice
Faculty Mentor: Frank Hall

Background: Social preference and social play are fundamental behavioral characteristics of mammals. Social preference indicates motivation directed toward social stimuli and social play influences the development of communication, cognitive, and emotional functions. Deficits in these behaviors are observed in autistic patients, and deficits in social motivation and social play are central to the disorder. The primary symptoms of Autism Spectrum Disorder (ASD) are reduced social preference and reduced social play, which are particularly observed during early adolescence. Since dopaminergic systems in the brain are involved in motivated behavior, altered dopaminergic activity may affect social motivation and preference. To address the potential involvement of dopamine in social motivation and social play, experiments were conducted in mice with reduced dopamine transporter (DAT) expression. On this basis it was predicted that reduced DAT expression increases dopamine actions and should thereby increase social motivation and social play. Separate experiments were conducted to measure social preference and social behavior in same-sex mouse pairs. During these tests ultrasonic vocalizations (USV) were also measured. USV is the primary means of communication in mice. Social play was examined in isolated and socially housed heterozygous DAT KO mice (DAT +/- mice) and compared to Wildtype (DAT +/+) mice. Methods: All studies examine DAT +/+ and DAT +/- adolescent (28-35 day old) mice. Social interaction is examined in same-sex pairs of mice in a novel environment; one is isolated for 24 hrs and one is not. Behavior and USV are measured during the 10 min test. 40-60 min after interaction test, both subjects are euthanatized and perfused prior to preparation of brain tissue slices for examination of c-fos immunohistochemistry (a marker for cellular activity). Social preference is studied using a three chambered apparatus where the test subject is given a choice between engaging with another mouse (contained in a small cage in one side of the apparatus) or just the cage in the opposite chamber. The measure is the preference of the target mouse for the social stimulus. There are 3 phases: I. Exploration for 10 min; II. Social choice for the target mouse; III. Social choice for novel target mouse. Results: Initial results in the social interaction test show that DAT genotype has different effects in male and female mice on both behavior and USV. The pattern of USV was affected by both sex and genotype. Moreover, differences in USV are associated with specific social behaviors. Results for the preference task are still preliminary but indicate that DAT genotype also has effects on both behavior and USV that are sex dependent. Male DAT +/- mice have increased USV compared to DAT +/+ mice, but this is not seen in female mice.

Conclusions: Changes in DAT genotype affects social behavior and social communication, which warrants further study. Additionally, Isolated mouse have a preference for a novel cage mate, but the lack of physical interaction reduces USV calls.

Quantifying the concentration of pesticides in the human plasma using solid phase micro extraction (SPME) and LC/-MS/MS
Faculty Mentor: Emanuela Gionfriddo

Cannabis product has received increasingly more public attention in the last decades, with more states has or are leaning toward legalizing marijuana product for both medicinal and recreational purposes. However, there are currently very minimal regulation on the cultivation and production of these cannabis product, in which the amount of pesticide residues on the final product is particularly concerning as many studies have demonstrated the effects of pesticides on human health when exposed regularly and/or with an higher than acceptable amount as dementia and cancer 1-3. Solid phase micro extraction (SPME), a novel non-exhaustive extraction technique that provide a high through-put, sustainable, less laborious and more selective sample processing method that has a wide range of application from clinical to environmental testing 4, 5. To provide a better understanding of the current level of cannabis pesticide residue accumulated in human, the goal of this project is to optimized a procedure to accurately quantify the concentration of 95 cannabis pesticides in human plasma using SPME and compare it to the frequently used extraction method QuEChERS. Post-processed samples were analyzed via UHPLC-MS/MS. The study showed that Hydrophilic-Lipophilic Balance fiber and desorption solution composed of Acetonile:Methanol:Water (2:2:1) provide the optimal extraction and desorption condition for the pesticide analytes available in the provided mixture.

Mapping the autoinhibitory sequence that controls targeting of the apical scaffold ANKS4B during enterocyte brush border assembly
Faculty Mentor: Scott W. Crawley

Enterocytes interact with the luminal environment of the gut to mediate nutrient absorption using actin-based membrane protrusions known as microvilli.   These microvilli are assembled on the apical surface of enterocytes during their development to form a highly organized densely packed array called the brush border (BB). BB assembly is controlled by a protocadherin-based adhesion complex known as the intermicrovillar adhesion complex (IMAC). This complex mediates adhesion between neighboring microvilli to promote proper BB assembly. ANKS4B is a critical scaffold found as a component of the IMAC. ANKS4B localizes to the tips of brush border microvilli, interacting with another IMAC scaffold known as USHIC and IMAC myosin motor protein Myo7B in a stable, tripartite complex. ANKS4B is comprised of an ankyrin-repeat domain (ANKRmin), a linker region (LK), a central unstructured region (CEN), a C-terminal sterile α-motif (SAM) that ends with a canonical PDZ-binding motif (PBM). ANKS4B was previously shown to target to the BB using its LK segment.  The LK segment of ANKS4B exhibits autonomous targeting to the BB through direct binding to apical membrane.  This autonomous targeting ability allows ANKS4B to act as the primordial scaffold that assembles the IMAC in the apical domain during the initial stages of BB assembly. The BB-targeting of ANKS4B is strictly regulated,  however, prior to BB assembly, ANKS4B lies dormant in the cytoplasm in which the membrane-binding ability of the LK segment is masked.  Upon interaction with the other IMAC scaffold, USH1C, ANKS4B is thought to become activated and target to the BB where it assembles the IMAC.  We used domain truncation analysis to map the sequence found in ANKS4B that regulates the apical targeting ability of the LK segment. Our approach involved creating a series of C-terminal truncations of ANKS4B to test their ability to target to the BB as EGFP-fusion proteins in an enterocyte cell culture model.  We observed that deletion of the C-terminal PBM of ANKS4B (which blocks its interaction with USH1C) locked ANKS4B in an auto-inhibited state that could not be activated.  While further truncation of the SAM domain did not relieve this inhibition, progressive truncation into the N-terminal region of the CEN domain allowed ANKS4B to regain its robust BB-targeting ability.  More specifically, we were able to map the auto-inhibitory domain of ANKS4B to a 23 amino acid sequence located between the LK segment and the CEN domain.  Together, these results shed light on the molecular determinants that control ANKS4B targeting during BB assembly.

Fission Yeast Polycystin Pkd2p Promotes Cell Growth  Synergistically with the MOR Pathway
Faculty Mentor: Dr. Qian Chen

Mutations of human Polycystins leads to a common genetic disorder, Autosomal Dominant Polycystic Kidney Disease marked by growth of numerous kidney cysts. However, the cellular role of Polycystins in regulating cystic growth remains poorly understood. In our lab, we recently discovered that the Fission Yeast Polycystins homologue Pkd2p, is a novel regulator of cell growth. The temperature sensitive mutant of pkd2—pkd2-B42 showed severely reduced growth rate when exposed to restrictive temperatures. Morphological studies revealed that Pkd2p mutant cells failed tip growth and volume expansion. To understand Pkd2p’s role in cell growth, we examined whether it has any relationship with the MOR signaling pathway. MOR or morphogenesis Orb6 related network, is a conserved Hippo like Kinase Pathway essential for polarized cell growth in fission yeast. The double mutant of Pkd2-B42 and the MOR mutants showed reduced viability compared to either of the single mutant, revealing a negative genetic interaction. This implies that MOR and Pkd2p might be acting in the same or complimentary pathways to control cell growth. MOR mutants lack cell growth through tip expansion however, they can continue depolarized lateral growth therefore, attaining a spherical shape. Morphological analysis of the double mutant cells showed that even the lateral growth was prevented. Furthermore, another morphological characteristic is a high percentage of the cells which appeared darkened as if they had shrunk. In conclusion, we can say that Pkd2p helps in cell expansion and synergistically acts with the MOR pathway in promoting cell growth.

Studying the Subcellular Localization of TM4SF3 Mutants at Predicted Palmitoylation Sites
Faculty Mentor: Dr. Lirim Shemshedini

Prostate cancer persists as the second-leading cause of cancer-related death. As such, the purpose of the following study was to analyze the subcellular localization of TM4SF3 in prostate cancer cells through the mutations of putative palmitoylation sites. It is known that tetraspanins are palmitoylated at cysteine sites, but there is nothing known about the palmitoylation of TM4SF3 proteins, which are transmembrane proteins that stabilize the Androgen Receptor (AR). AR plays a key role in the progression of prostate cancer. Results from this study were gathered through fluorescence microscopy and supported the argument that when palmitoylation sites were mutated, TM4SF3 proteins (tagged with mCherry) shifted localization into the cytoplasm. In prostate cancer cells, AR, accompanied by the presence of androgen, can either be cytosolic or nuclear. The membrane localization of TM4SF3 prevents its interaction with AR, which explains why the internalization of TM4SF3 is necessary. While it is not clear how the internalization of TM4SF3 occurs or how the non-membrane bound fraction of TM4SF3 is maintained, a possible mechanism that explains the localization of TM4SF3 is through palmitoylation. It has been previously shown in our lab that when 2 Bromopalmitate (2-BP) which causes depalmitoylation is applied, it leads to decreased TM4SF3 and AR levels and decreases prostate cancer proliferation as a whole. 

Building an Integrated Database Management System for the Comprehensive PTSD Clinical Study
Faculty Mentor: Hong Xie

An integrated database management study was created for the PTSD clinical study conducted at the University of Toledo. SAS Software was used to build the database structure and to conduct statistical analysis of the data. Database validation was done by comparing the analysis results from SAS and Excel. This database integrates thousands of mental health self-report surveys, clinical interview records, and brain MRI imaging data. The most advanced feature of this database system is that it can easily be updated in SAS when new data is inputted into the raw data file in Excel. Although this database is based on the PTSD study, it can be used as a template for other clinical research studies. Overall, the integrated database management system is an efficient tool for clinical research studies with large amounts of data.

 

Detection of Cyanobacteria in FFPE Tissue
Faculty Mentor: Dr. David Kennedy and Dr. Steven Haller

This project researches the detection of cyanobacteria in formalin-fixed/paraffin-embedded (FFPE) tissue. Cyanobacteria are microscopic organisms found within harmful algal blooms (HABs) capable of producing many different toxins. A protocol using the GeneJET FFPE DNA extraction/purification kit was optimized in order to obtain high quality DNA for analysis to examine the effects of cyanobacteria. The results of the kit for a small sample of health and carcinogenic tissue were analyzed. The 260/280 absorption ratio to determine sample purity was within the acceptable range of 1.7-1.9. Additionally, the DNA concentration was above 100 ng/uL for 3 sets of duplicate samples with the last set showing values in the 70 range. Over this past semester, many additional samples from a variety of disease states and target organs have been collected and will be processed using this protocol.

Palladium-Catalyzed γ,γ’-Diarylation of Free Alkenyl Amines
Faculty Mentor: Dr. Michael Young

The regioselective dicarbofunctionalization of amines is an important area of research to obtain complex organic molecules. We have reported a palladium-catalyzed γ,γ’-diarylation of free alkenyl amines through interrupted chain walking for Z-selective alkenyl amines. Through a Mizoroki-Heck pathway, a β-hydride elimination followed by chain walking to generate the terminal olefin and subsequent alkenyl amine, a wide scope of 1,3-dicarbofunctionalized amines can be manufactured. Mechanistic studies have been performed to obtain what we propose to be the catalytic cycle of the reaction.

Exploring New Methods for Synthesis of Complex Amines
Faculty Mentor: Dr. Michael Young

This presentation will address my work in two separate areas. The first will focus on the use of more sustainable transition metals to achieve ortho-arylation of benzylamines, including nickel, copper, and ruthenium. The second portion involves the selective arylation of allylamines, first targeting more earth abundant metals but ultimately settling on palladium as a catalyst. Using this system, several new cinnamylamine products were produced.

 

Lower Extremity Assessment Protocol: Efficacy of Single Leg Hop Test in Determining Back to Sport Fitness
Faculty Mentor: Dr. Grant Norte

Anterior Cruciate Ligament (ACL) injuries are one of the most common sports injuries with an incidence rate of 68.6 per 100,000 people in the US. After ACL reconstruction (ACLR) a cascade of neuromuscular and physiological changes occurs including persistent quadriceps muscle weakness and altered biomechanics. Despite these changes a vast majority of these individuals attain passing scores in the single leg hop test (SLH), a prominent return to sport diagnostic test. Purpose: The aim of this study was to compare lower extremity kinematic strategies of individuals with ACLR and healthy controls during the propulsion phase of the SLH test. Methods: 16 participants with history of unilateral ACLR (7 males aged 20.14± 1.77 years, 36.27±23.96 months from surgery) and 17 healthy matched controls with no history of lower extremity surgery (10 males aged 21.10±1.52 years) participated in this cross-sectional study. Participants completed three SLH tests on both limbs. Distance was recorded and normalized for height of participants. Participants were fitted with 50 retroreflective markers so that 3D motion analysis could be utilized to measure joint angles and joint range of motion (ROM) during the SLH task. Dependent and independent t-tests were used to compare outcomes between limbs and between groups. Cohen’s d effect sizes were calculated with 95% confidence intervals. Results: ACLR involved limbs vs contralateral limb showed less frontal plane ROM (8.2 ± 2.7 vs. 10.4 ± 4.2°, p = .052), less peak knee flexion (55.9 ± 5.4 vs. 59.5 ± 5.5°, p = .018), and less peak ankle dorsiflexion (33.1 ± 3.2 vs. 35.2 ± 4.5°, p = .030). ACLR contralateral limbs vs healthy controls the ACLR contralateral limb showed greater frontal plane ROM (10.4 ± 4.2 vs. 7.7 ± 2.9°, p = .032). No significant differences were identified in SLH distance between the involved and uninvolved limb (Involved: 0.73 ± 0.12 vs. Uninvolved: 0.80 ± 0.16, p = .071), or between individuals with ACLR and healthy controls (ACLR: 0.73 ± 0.12 vs. con: 0.72 ± 0.14, p = .917). Conclusions: Participants with ACLR showed significant differences between limbs and the control group in terms of joint flexion angles and ROM, however showed no difference in SLH test distance between limbs and groups. Individuals with ACLR are able to accomplish similar SLH distances through the use of altered biomechanics which indicates the use of compensatory movement strategies. This shows that the SLH test is measures the quantity of a movement and not the quality of the movement.

Reliability and Validity of Pose Estimation in Two-Dimensional Kinematic Analysis
Faculty Mentor: Dr. David Bazett-Jones

Introduction: Novel motion analysis technology that stems from the rise of artificial intelligence may be beneficial for those who rely on traditional 2D motion analysis. Pose estimation models, which are a form of artificial intelligence, can identify landmarks on the human body from any 2D video or image. 2D pose estimation increases post-processing efficiency and allows for evaluation of an entire biomechanical profile. The aim of our study was to investigate the reliability and validity of 2D pose estimation to measure sagittal and frontal plane joint angles during a single-leg squatting task in adolescents.
Methods: 22 adolescents participated in this study. In a single visit, participants were instrumented with reflective markers and completed a series of seven single-leg squats on their right leg. 3D kinematic data were recorded using a 12-camera system and 2D sagittal and frontal plane videos were simultaneously recorded using an iPod Touch from each plane, respectively. 3D kinematic data were processed in Visual3D and exported to a custom MATLAB code. 2D video data were processed with the OpenPose BODY_25 model and a custom Python code. For each squat repetition, peak sagittal and frontal plane angles were extracted at the instance of peak knee flexion. Joint angles were averaged across all squat repetitions for analysis. To assess reliability, 2D videos were re-processed with the 2D pose estimation model and ICCs compared results to the initial measurements. To assess validity, Pearson’s correlation coefficients and paired t-tests were used to compare the relationship between 3D and 2D measured joint angles.
Results: 2D pose-estimation demonstrated perfect repeatability with an ICC of 1.0 for all joint angles. Moderate to strong positive relationships (r=.63-.95) were found for sagittal plane joint angles. In the frontal plane, poor relationships (r=-.51-.17) were found. When comparing the mean values of 2D and 3D joint angles, clinically non-significant differences were observed for trunk forward lean, knee flexion, pelvic drop, and knee abduction while clinically meaningful differences were observed for hip flexion, ankle dorsiflexion, and hip adduction.
Discussion: 2D pose estimation was found to be a reliable method for measuring sagittal and frontal plane joint angles during a single-leg squat. 2D pose estimation was found to be valid in evaluating sagittal plane joint angles. However, 2D pose estimation does not appear to correct the frontal plane limitations that are inherent with 2D motion analysis. Future research is warranted to compare 2D pose estimation to commonly used traditional 2D motion analysis software. Overall, 2D pose estimation is a reliable and valid measure of sagittal plane angles during a single-leg squatting task in adolescents.

 

The Influence of Sex and Maturation on Biomechanics of Adolescent Running
Faculty Mentor: Dr. David Bazett-Jones

Introduction: Differences in running biomechanics have been reported between male and female adult long-distance runners but it is unknown when these sex differences develop. Therefore, the purpose of this study was to compare running biomechanics in male and female adolescent long-distance runners of different stages of physical maturation.
Methods: Uninjured adolescent long-distance runners (n=53 [female=29, male=24] age=15.3±2.0 years) underwent a three-dimensional running analysis and were stratified as mid, or post-pubertal. Participants ran a 20-meter runway embedded with force plates at a self- selected, comfortable speed. Temporal-spatial parameters, stance phase peak joint angles, and ground reaction forces were compared among groups using two-way ANCOVAs (sex, maturation; covariate= running velocity) with post-hoc Bonferroni pairwise comparisons (p≤.025).
Results: A significant interaction between sex and maturation was found for cadence (p<.01). Post-hoc analysis revealed post-pubertal males ran with a significantly lower cadence than mid-pubertal females (p<.01), post-pubertal females (p<.001), and mid-pubertal males (p<.01). No significant interactions or main effects were found for stride length, stance duration, peak joint angles, or ground reaction forces (p=.12-.99).
Discussion: High school cross-country runners with low step rates were previously found to be at a higher risk of sustaining a running-related injury. The lower step rate demonstrated by post-pubertal males may put them at a greater risk of sustaining an injury. Overall, running biomechanics appear to be similar between mid- and post-pubertal male and female long-distance runners. Coaches and clinicians should treat adolescent long-distance runners individually, regardless of sex or stage of maturation.

Undergraduate Research Project: Writing a Composition
Faculty Mentor: Lee Heritage

This is my presentation for my Spring 2021 undergraduate research project. The project's goal was to create an original piece of music designed for live performance. The piece was initially intended to be performed by the University of Toledo Symphony Orchestra, with a backup plan being a presentation of the work at the Spring New Music Festival. Both of these were unfortunately cancelled due to the pandemic, but the piece was still completed regardless.

This project involved a cycle of me working on material privately and then presenting it to my mentor in a weekly meeting. During these meetings, we would discuss various aspects of the orchestral composition. Everything from writing and theory to layout and presentation was discussed. After each meeting, I would spend the following week incorporating the advice into the piece, all while continuing to make progress on it. This cycle continued until the piece was complete, at which point it was edited, and the audio and sheet music was made renderable.

Despite the music not being able to be performed at this time, I still feel that I have grown as a composer, and that the project was greatly beneficial to my academic career. It provided me with experience in writing music for an extracurricular purpose which will be of great importance to me as I pursue one of my career goals of composition.

The global pandemic has certainly affected everyone's research in some way, and I am proud to say that I was still able to see the project to completion despite these setbacks, and was able to develop and learn along the way.