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Description
The RNA editing enzyme adenosine deaminase acting on double stranded RNA 2 (ADAR2) converts adenosine into inosine in regions of double stranded RNA. Here, it was discovered that this critical function of ADAR2 was dysfunctional in amyotrophic lateral sclerosis (ALS) mediated by the C9orf72 hexanucleotide repeat expansion, the most common

The RNA editing enzyme adenosine deaminase acting on double stranded RNA 2 (ADAR2) converts adenosine into inosine in regions of double stranded RNA. Here, it was discovered that this critical function of ADAR2 was dysfunctional in amyotrophic lateral sclerosis (ALS) mediated by the C9orf72 hexanucleotide repeat expansion, the most common genetic abnormality associated with ALS. Typically a nuclear protein, ADAR2 was localized in cytoplasmic accumulations in postmortem tissue from C9orf72 ALS patients. The mislocalization of ADAR2 was confirmed using immunostaining in a C9orf72 mouse model and motor neurons differentiated from C9orf72 patient induced pluripotent stem cells. Notably, the cytoplasmic accumulation of ADAR2 coexisted in neurons with cytoplasmic accumulations of TAR DNA binding protein 43 (TDP-43). Interestingly, ADAR2 overexpression in mammalian cell lines induced nuclear depletion and cytoplasmic accumulation of TDP-43, reflective of the pathology observed in ALS patients. The mislocalization of TDP-43 was dependent on the catalytic activity of ADAR2 and the ability of TDP-43 to bind directly to inosine containing RNA. In addition, TDP-43 nuclear export was significantly elevated in cells with increased RNA editing. Together these results describe a novel cellular mechanism by which alterations in RNA editing drive the nuclear export of TDP-43 leading to its cytoplasmic mislocalization. Considering the contribution of cytoplasmic TDP-43 to the pathogenesis of ALS, these findings represent a novel understanding of how the formation of pathogenic cytoplasmic TDP-43 accumulations may be initiated. Further research exploring this mechanism will provide insights into opportunities for novel therapeutic interventions.
ContributorsMoore, Stephen Philip (Author) / Sattler, Rita (Thesis advisor) / Zarnescu, Daniela (Committee member) / Brafman, David (Committee member) / Van Keuren-Jensen, Kendall (Committee member) / Mangone, Marco (Committee member) / Arizona State University (Publisher)
Created2021
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Description
Background: Studies have examined student fruit/vegetable (FV) consumption, selection, and waste related to lunch duration and found that longer duration at lunch was associated with greater consumption, selection, and reduced waste. However, few studies have investigated the relationship between time to eat and FVs. The aim of this research is

Background: Studies have examined student fruit/vegetable (FV) consumption, selection, and waste related to lunch duration and found that longer duration at lunch was associated with greater consumption, selection, and reduced waste. However, few studies have investigated the relationship between time to eat and FVs. The aim of this research is to analyze the relationship between objective time to students took to eat (“time to eat”) as it relates to their fruit and vegetable consumption, selection, and plate waste.in elementary, middle, and high schools. Methods: A secondary analysis of cross-sectional study of 37 Arizona schools to discover the differences in the selection, consumption, and waste of FVs from students (Full N = 2226, Elementary N = 630, Middle School N = 699, High School N = 897) using objective time to eat measures. Zero-inflated negative binomial regressions examined differences in FV grams selected, consumed, and wasted adjusted for sociodemographics including race, ethnicity, eligibility for free or reduced lunch, academic year, and sex and clustering for students within schools. Results are presented across school level (elementary, middle, and high school). Results: The average time taken to eat ranged from 10-12 minutes for all students. The association of time to eat and lunch duration were not closely related (r=0.03, p = 0.172). In the count model for every additional minute spent, there was a 0.5% greater likelihood of selecting FVs for elementary kids among those who took any FVs. In the zero-inflated model, it was found that there was a statistically significant relationship between time spent eating and the selection of fruits and vegetables. For the total sample and high schoolers, a minute more of eating time was associated with a 4.3% and 8.8% greater odds of selecting FV. This means that longer eating time increased the likelihood of choosing fruits and vegetables. The results indicated that the longer students took to eat, the higher the likelihood of consuming more of FVs. Each 10 more minutes spent eating (i.e., time to eat) is associated with a 5% increase in grams of FV selected relative to mean (for those that chose FV) over 1 week this equates to 32 g increase of FV selected. However, for middle schoolers, the time to eat was not found to be significant in relation to the grams of fruits and vegetables consumed. There was some significance in the sociodemographic factors such as gender (all) and other (middle school). There was a relationship between time taken to eat and waste as a proportion for fruits and vegetables. For example, among those among the students who wasted something (as a proportion of selection), each additional 10 minutes of eating time was associated with a .6% decrease in waste relative to the mean (for those who chose fruits and vegetables) over a week, resulting in a decrease in waste percentage of 16.5%. Among high schoolers, males had a slightly higher odds of wasting a proportion of fruits and vegetables. Conclusions: This study aimed to examine the association between the time students take to eat during lunch and their fruit and vegetable (FV) consumption, selection, and plate waste. The findings revealed that the time to eat was related to FV consumption, depending on the school level. However, it was not significantly associated with FV selection or waste. The study emphasized the need for further research on time to eat, distinguishing it from the duration of lunch. Longer lunch periods and adequate time could influence better food choices, increased FV consumption, and reduced waste. The study highlighted the importance of interventions and school policies promoting healthier food choices and providing sufficient time for students to eat. Future research should validate these findings and explore the impact of socialization opportunities on promoting healthier eating habits. Understanding the relationship between lunch duration, time to eat, and students' dietary behaviors can contribute to improved health outcomes and inform effective strategies in school settings.
ContributorsDandridge, Christina Marie (Author) / Adams, Marc (Thesis advisor) / Whisner, Corrie (Committee member) / Bruening, Meg (Committee member) / Arizona State University (Publisher)
Created2023
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Description
MicroRNAs (miRNAs) are 17-22 nucleotide non-coding RNAs that regulate gene expression by targeting non-complementary elements in the 3’ untranslated regions (3’UTRs) of mRNAs. miRNAs, which form complex networks of interaction that differ by tissue and developmental stage, display conservation in their function across metazoan species. Yet much remains unknown regarding

MicroRNAs (miRNAs) are 17-22 nucleotide non-coding RNAs that regulate gene expression by targeting non-complementary elements in the 3’ untranslated regions (3’UTRs) of mRNAs. miRNAs, which form complex networks of interaction that differ by tissue and developmental stage, display conservation in their function across metazoan species. Yet much remains unknown regarding their biogenesis, localization, strand selection, and their absolute abundance due to the difficulty of detecting and amplifying such small molecules. Here, I used an updated HT qPCR-based methodology to follow miRNA expression of 5p and 3p strands for all 190 C. elegans miRNAs described in miRBase throughout all six developmental stages in triplicates (total of 9,708 experiments), and studied their expression levels, tissue localization, and the rules underlying miRNA strand selection. My study validated previous findings and identified novel, conserved patterns of miRNA strand expression throughout C. elegans development, which at times correlate with previously observed developmental phenotypes. Additionally, my results highlighted novel structural principles underlying strand selection, which can be applied to higher metazoans. Though optimized for use in C. elegans, this method can be easily adapted to other eukaryotic systems, allowing for more scalable quantitative investigation of miRNA biology and/or miRNA diagnostics.
ContributorsMeadows, Dalton Alexander (Author) / Mangone, Marco (Thesis advisor) / LaBaer, Joshua (Committee member) / Murugan, Vel (Committee member) / Wilson-Rawls, Jeanne (Committee member) / Arizona State University (Publisher)
Created2023
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Description
Precise modulation of gene expression is essential for proper tissue and cell-specific differentiation and function. Multiple distinct post-transcriptional regulatory mechanisms, such as miRNA (microRNA)-based regulation and alternative polyadenylation (APA), are an intrinsic part of this modulation and orchestrate intricate pathways to achieve and maintain balanced gene expression.MiRNA-based regulation and APA

Precise modulation of gene expression is essential for proper tissue and cell-specific differentiation and function. Multiple distinct post-transcriptional regulatory mechanisms, such as miRNA (microRNA)-based regulation and alternative polyadenylation (APA), are an intrinsic part of this modulation and orchestrate intricate pathways to achieve and maintain balanced gene expression.MiRNA-based regulation and APA function through sequence motifs located in the 3’ Untranslated Region (3’UTR) of mRNA transcripts. MiRNAs are short (~22 nt) non-coding RNA molecules that bind target sequences within the 3’UTR of an mRNA transcript, inhibiting its translation or promoting its degradation. APA occurs during RNA transcription termination and leads to the preparation of mature mRNAs with different 3’UTR lengths, allowing shorter 3’UTRs to bypass miRNA regulation. In addition to these two post-transcriptional forms of regulation, co-transcriptional mechanisms such as alternative RNA splicing, which produces distinct gene products from a precursor mRNA, are also important in controlling gene expression. While miRNA-based regulation, APA, and alternative RNA splicing are important regulatory mechanisms, there is a lack of comprehensive understanding of how they interact and communicate with each other. This thesis studies these three forms of gene regulation in the nematode C. elegans, with the goal of extracting rules and mechanisms used by each of them in development to establish and maintain somatic tissue identity. After isolating miRNA targets in multiple C. elegans somatic tissues, it was found that miRNAs can modulate the abundance of hnRNPs and SR proteins, which are known to control alternative RNA splicing in a dosage-dependent manner.To identify tissue-specific miRNAs, a nuclear fluorescent cell sorting (FACS)-based methodology named Nuc-Seq, was developed to isolate and sequence tissue-specific miRNAs from body muscle tissue. Nuc-Seq identified 2,848 muscle-specific protein-coding genes and 16 body muscle-specific miRNAs. This data was used to develop a high-quality body muscle-specific miRNA-APA Interactome which allows studies in regulatory processes in detail. Taken together, this work highlights some of the complexity of pre- and post-transcriptional gene regulation and sheds light on how miRNA-based regulation, APA, and alternative RNA splicing are interconnected and are responsible for the establishment and maintenance of tissue identity.
ContributorsSchorr, Anna L (Author) / Mangone, Marco (Thesis advisor) / Harris, Robin (Committee member) / Sharma, Shalini (Committee member) / Varsani, Arvind (Committee member) / Arizona State University (Publisher)
Created2023
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Description
Tools designed to help match people with behaviors they identify as likely to lead to a successful behavioral outcome remain under-researched. This study assessed the effect of a participant-driven behavior-matching intervention on 1) the adoption of a new behavior related to fruit and vegetable (F&V) consumption, 2) study attrition, and

Tools designed to help match people with behaviors they identify as likely to lead to a successful behavioral outcome remain under-researched. This study assessed the effect of a participant-driven behavior-matching intervention on 1) the adoption of a new behavior related to fruit and vegetable (F&V) consumption, 2) study attrition, and 3) changes in F&V consumption. In this two-arm randomized controlled trial, 64 adults who did not meet standard F&V recommendations were allocated to an intervention (n=33) or control group (n=31). Participants in the intervention group ranked 20 F&V-related behaviors according to their perceived likelihood of engagement in the behavior and their perception of the behavior’s efficacy in increasing F&V consumption. Participants in the intervention group were subsequently shown the list of 20 behaviors in order of their provided rankings, with the highest-ranked behaviors at the top, and were asked to choose a behavior they would like to perform daily for 4 weeks. The control group chose from a random-order list of the same 20 behaviors to adopt daily for 4 weeks. During the study period, text messages were sent to all participants 90 minutes before their reported bedtime to collect Yes/No data reflecting successful behavior engagement each day. The binary repeated-measures data collected from the text messages was analyzed using mixed-effects logistic regression, differences in attrition were assessed using log-rank analysis, and change scores in F&V consumption were compared between the two groups using the Man-Whitney U test. P<0.05 indicated significance. The rate of successful behavior adoption did not differ significantly between the two groups (b=0.09, 95%CI= -0.81, 0.98, p=0.85). The log rank test results indicated that there was no significant difference in attrition between the two groups (χ2=2.68, df=1, p=0.10). F&V consumption increased significantly over the 4 weeks in the total sample (Z=-5.86, p<0.001), but no differences in F&V change scores were identified between the control and intervention groups (Z=-0.21, p=0.84). The behavior-matching tool assessed in this study did not significantly improve behavior adoption, study attrition, or F&V intake over 4 weeks.
ContributorsCosgrove, Kelly Sarah (Author) / Wharton, Christopher (Thesis advisor) / Adams, Marc (Committee member) / DesRoches, Tyler (Committee member) / Grebitus, Carola (Committee member) / Johnston, Carol (Committee member) / Arizona State University (Publisher)
Created2023
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Description
Objective: It’s not well understood how youth perceive existing fruit and vegetable (FV) marketing materials available in schools. This ancillary study sought to assess the acceptability of FV marketing materials freely available to schools among adolescents in grades 6-12.

Methods: Middle and high school adolescents (n=40; 50% female; 52.5% Hispanic) in

Objective: It’s not well understood how youth perceive existing fruit and vegetable (FV) marketing materials available in schools. This ancillary study sought to assess the acceptability of FV marketing materials freely available to schools among adolescents in grades 6-12.

Methods: Middle and high school adolescents (n=40; 50% female; 52.5% Hispanic) in the Phoenix, AZ area were asked to rank marketing materials (n=35) from favorite to least favorite in four categories: table tents, medium posters, large posters and announcements. Favorites were determined by showing participants two items at a time and having them choose which they preferred; items were displayed to each adolescent in a random order. Adolescents participated in a 20-30 minute interview on their favorite items in each category based on acceptance/attractiveness, comprehension, relevance, motivation and uniqueness of the materials. A content analysis was performed on top rated marketing materials. Top rated marketing materials were determined by the number of times the advertisement was ranked first in its category.

Results: An analysis of the design features of the items indicated that most participants (84%) preferred marketing materials with more than 4 color groups. Participant preference of advertisement length and word count was varied. A total of 5 themes and 20 subthemes emerged when participants discussed their favorite FV advertisements. Themes included: likes (e.g., colors, length, FV shown), dislikes (e.g., length, FV shown), health information (e.g., vitamin shown), comprehension (e.g., doesn’t recognize FV), and social aspects (e.g., peer opinion). Peer opinion often influenced participant opinion on marketing materials. Participants often said peers wouldn’t like the advertisements shown: “…kids my age think that vegetables are not good, and they like food more than vegetables.” Fruits and vegetable pictured as well as the information in the marketing materials also influenced adolescent preference.

Conclusion: Students preferred advertisements with more color and strong visual aspects. Word count had minimal influence on their opinions of the marketing materials, while information mentioned and peer opinion did have a positive effect. Further research needs to be done to determine if there is a link between adolescent preferences on FV marketing materials and FV consumption habits.
ContributorsPisano, Sydney Alexis (Author) / Bruening, Meg (Thesis advisor) / Adams, Marc (Committee member) / Grgich, Traci (Committee member) / Arizona State University (Publisher)
Created2019
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Description
The fundamental building blocks for constructing complex synthetic gene networks are effective biological parts with wide dynamic range, low crosstalk, and modularity. RNA-based components are promising sources of such parts since they can provide regulation at the level of transcription and translation and their predictable base pairing properties enable large

The fundamental building blocks for constructing complex synthetic gene networks are effective biological parts with wide dynamic range, low crosstalk, and modularity. RNA-based components are promising sources of such parts since they can provide regulation at the level of transcription and translation and their predictable base pairing properties enable large libraries to be generated through in silico design. This dissertation studies two different approaches for initiating interactions between RNA molecules to implement RNA-based components that achieve translational regulation. First, single-stranded domains known as toeholds were employed for detection of the highly prevalent foodborne pathogen norovirus. Toehold switch riboregulators activated by trigger RNAs from the norovirus RNA genome are designed, validated, and coupled with paper-based cell-free transcription-translation systems. Integration of paper-based reactions with synbody enrichment and isothermal RNA amplification enables as few as 160 copies/mL of norovirus from clinical samples to be detected in reactions that do not require sophisticated equipment and can be read directly by eye. Second, a new type of riboregulator that initiates RNA-RNA interactions through the loop portions of RNA stem-loop structures was developed. These loop-initiated RNA activators (LIRAs) provide multiple advantages compared to toehold-based riboregulators, exhibiting ultralow signal leakage in vivo, lacking any trigger RNA sequence constraints, and appending no additional residues to the output protein. Harnessing LIRAs as modular parts, logic gates that exploit loop-mediated control of mRNA folding state to implement AND and OR operations with up to three sequence-independent input RNAs were constructed. LIRA circuits can also be ported to paper-based cell-free reactions to implement portable systems with molecular computing and sensing capabilities. LIRAs can detect RNAs from a variety of different pathogens, such as HIV, Zika, dengue, yellow fever, and norovirus, and after coupling to isothermal amplification reactions, provide visible test results down to concentrations of 20 aM (12 RNA copies/µL). And the logic functionality of LIRA circuits can be used to specifically identify different HIV strains and influenza A subtypes. These findings demonstrate that toehold- and loop-mediated RNA-RNA interactions are both powerful strategies for implementing RNA-based computing systems for intracellular and diagnostic applications.
ContributorsMA, DUO (Author) / Green, Alexander (Thesis advisor) / Mangone, Marco (Committee member) / Liu, Yan (Committee member) / Arizona State University (Publisher)
Created2019
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Description
Multicellular organisms use precise gene regulation, executed throughout development, to build and sustain various cell and tissue types. Post-transcriptional gene regulation is essential for metazoan development and acts on mRNA to determine its localization, stability, and translation. MicroRNAs (miRNAs) and RNA binding proteins (RBPs) are the principal effectors of post-transcriptional

Multicellular organisms use precise gene regulation, executed throughout development, to build and sustain various cell and tissue types. Post-transcriptional gene regulation is essential for metazoan development and acts on mRNA to determine its localization, stability, and translation. MicroRNAs (miRNAs) and RNA binding proteins (RBPs) are the principal effectors of post-transcriptional gene regulation and act by targeting the 3'untranslated regions (3'UTRs) of mRNA. MiRNAs are small non-coding RNAs that have the potential to regulate hundreds to thousands of genes and are dysregulated in many prevalent human diseases such as diabetes, Alzheimer's disease, Duchenne muscular dystrophy, and cancer. However, the precise contribution of miRNAs to the pathology of these diseases is not known.

MiRNA-based gene regulation occurs in a tissue-specific manner and is implemented by an interplay of poorly understood and complex mechanisms, which control both the presence of the miRNAs and their targets. As a consequence, the precise contributions of miRNAs to gene regulation are not well known. The research presented in this thesis systematically explores the targets and effects of miRNA-based gene regulation in cell lines and tissues.

I hypothesize that miRNAs have distinct tissue-specific roles that contribute to the gene expression differences seen across tissues. To address this hypothesis and expand our understanding of miRNA-based gene regulation, 1) I developed the human 3'UTRome v1, a resource for studying post-transcriptional gene regulation. Using this resource, I explored the targets of two cancer-associated miRNAs miR-221 and let-7c. I identified novel targets of both these miRNAs, which present potential mechanisms by which they contribute to cancer. 2) Identified in vivo, tissue-specific targets in the intestine and body muscle of the model organism Caenorhabditis elegans. The results from this study revealed that miRNAs regulate tissue homeostasis, and that alternative polyadenylation and miRNA expression patterns modulate miRNA targeting at the tissue-specific level. 3) Explored the functional relevance of miRNA targeting to tissue-specific gene expression, where I found that miRNAs contribute to the biogenesis of mRNAs, through alternative splicing, by regulating tissue-specific expression of splicing factors. These results expand our understanding of the mechanisms that guide miRNA targeting and its effects on tissue-specific gene expression.
ContributorsKotagama, Kasuen Indrajith Bandara (Author) / Mangone, Marco (Thesis advisor) / LaBaer, Joshua (Committee member) / Newbern, Jason (Committee member) / Rawls, Alan (Committee member) / Arizona State University (Publisher)
Created2019
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Description
The RASopathies are a collection of developmental diseases caused by germline mutations in components of the RAS/MAPK signaling pathway and is one of the world’s most common set of genetic diseases. A majority of these mutations result in an upregulation of RAS/MAPK signaling and cause a variety of both physical

The RASopathies are a collection of developmental diseases caused by germline mutations in components of the RAS/MAPK signaling pathway and is one of the world’s most common set of genetic diseases. A majority of these mutations result in an upregulation of RAS/MAPK signaling and cause a variety of both physical and neurological symptoms. Neurodevelopmental symptoms of the RASopathies include cognitive and motor delays, learning and intellectual disabilities, and various behavioral problems. Recent noninvasive imaging studies have detected widespread abnormalities within white matter tracts in the brains of RASopathy patients. These abnormalities are believed to be indicative of underlying connectivity deficits and a possible source of the behavioral and cognitive deficits. To evaluate these long-range connectivity and behavioral issues in a cell-autonomous manner, MEK1 loss- and gain-of-function (LoF and GoF) mutations were induced solely in the cortical glutamatergic neurons using a Nex:Cre mouse model. Layer autonomous effects of the cortex were also tested in the GoF mouse using a layer 5 specific Rbp4:Cre mouse. Immunohistochemical analysis showed that activated ERK1/2 (P-ERK1/2) was expressed in high levels in the axonal compartments and reduced levels in the soma when compared to control mice. Axonal tract tracing using a lipophilic dye and an adeno-associated viral (AAV) tract tracing vector, identified significant corticospinal tract (CST) elongation deficits in the LoF and GoF Nex:Cre mouse and in the GoF Rbp4:Cre mouse. AAV tract tracing was further used to identify significant deficits in axonal innervation of the contralateral cortex, the dorsal striatum, and the hind brain of the Nex:Cre GoF mouse and the contralateral cortex and dorsal striatum of the Rbp4:Cre mouse. Behavioral testing of the Nex:Cre GoF mouse indicated deficits in motor learning acquisition while the Rbp4:Cre GoF mouse showed no failure to acquire motor skills as tested. Analysis of the expression levels of the immediate early gene ARC in Nex:Cre and Rbp4:Cre mice showed a specific reduction in a cell- and layer-autonomous manner. These findings suggest that hyperactivation of the RAS/MAPK pathway in cortical glutamatergic neurons, induces changes to the expression patterns of P-ERK1/2, disrupts axonal elongation and innervation patterns, and disrupts motor learning abilities.
ContributorsBjorklund, George Reed (Author) / Newbern, Jason M (Thesis advisor) / Neisewander, Janet (Committee member) / Smith, Brian (Committee member) / Orchinik, Miles (Committee member) / Mangone, Marco (Committee member) / Arizona State University (Publisher)
Created2018
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Description
College students are a niche of young adults, characterized by abnormal sleeping habits and inactive lifestyles. Many students entering college are as young as 18 years old and graduate by 22 years old, a window of time in which their bones are still accruing mineral. The purpose of this cross-sectional

College students are a niche of young adults, characterized by abnormal sleeping habits and inactive lifestyles. Many students entering college are as young as 18 years old and graduate by 22 years old, a window of time in which their bones are still accruing mineral. The purpose of this cross-sectional study was to determine whether sleep patterns and physical activity observed in college students (N= 52) 18-25 years old at Arizona State University influenced bone biomarkers, osteocalcin (OC) and N-terminal telopeptide of type 1 collagen (NTX-1) concentrations. Students completed various dietary and health history questionnaires including the International Physical Activity Questionnaire short form. Students wore an actigraphy watch for 7 consecutive nights to record sleep events including total sleep time, sleep onset latency and wake after sleep onset. Total sleep time had a significant, negative correlation with OC (r = -0.298, p-value =0.036) while sleep onset latency had a significant, positive correlation with NTX-1 serum concentration (r = 0.293, p-value = 0.037). Despite correlational findings, only sleep percent was found to be significant (beta coefficient = 0.271 p-value = 0.788) among all the sleep components assessed, after adjusting for gender, race, BMI and calcium intake in multivariate regression models. Physical activity alone was not associated with either bone biomarker. Physical activity*sleep onset latency interactions were significantly correlated with osteocalcin (r = 0.308, p-value =0.006) and NTX-1 (r = 0.286, p-value = 0.042) serum concentrations. Sleep percent*physical activity interactions were significantly correlated with osteocalcin (r = 0.280, p-value = 0.049) but not with NTX-1 serum concentrations. Interaction effects were no longer significant after adjusting for covariates in the regression models. While sleep percent was a significant component in the regression model for NTX-1, it was not clinically significant. Overall, sleep patterns and physical activity did not explain OC and NTX-1 serum concentrations in college students 18-25 years old. Future studies may need to consider objective physical activity devices including accelerometers to measure activity levels. At this time, college students should review sleep and physical activity recommendations to ensure optimal healthy habits are practiced.
ContributorsMahmood, Tara Nabil (Author) / Whisner, Corrie (Thesis advisor) / Dickinson, Jared (Committee member) / Petrov, Megan (Committee member) / Adams, Marc (Committee member) / Arizona State University (Publisher)
Created2019