Thesis and Dissertation Announcements

A Prototypical Thermal-Preference Assessment Device for Studying Factors Affecting the Thermal Biology of Small Aquatic Animals

Major Advisor: Dr. David Huffman, Department of Biology, Texas State University

Committee Members: 

Dr. Shannon E. Weigum, Department of Biology, Texas State University

Dr. Alan W. Groeger, Department of Biology, Texas State University

Randy Gibson, Supervisory Biologist, San Marcos Aquatic Resources Center

Zoom link: https://txstate.zoom.us/j/85811727971pwd=M3ozWmFrL3A4ckhxbzBJRWlGVnZvUT09

Passcode 205373

Come see the Thermal Preference Assessment Device (TPAD) I have created for studying the thermal biology of small aquatic organisms such as Melanoides tuberculata. The TPAD was created in response to my review of prior thermal biology studies which revealed a wide range of thermal-gradient device designs and operational theories, many of which were plagued by design flaws that would prevent the formation of a stable, linear gradient. This, coupled with a ubiquitous lack of published performance data and assembly documentation would render replicating these devices difficult at best. The goal of this study was to design and build a proof-of-concept device capable of generating customizable, stable, thermal gradients for use in thermal biology studies, which conformed to the following specifications; 1) quick generation of stable, linear thermal gradients with extremes of 10 °C to 30 °C for at least 24 hours; 2) precise temperature controls, allowing the thermal gradient to be moved back and forth in the trough; and 3) free, unobstructed movement for organisms of varying size classes and modalities within the Testing Arena. The TPAD is driven by a novel counter-current exchange system, powered by computer-controlled thermoelectric Peltier heater/coolers. The Peltier units heat and cool water running counterflow through two pairs of vertically stacked exchange conduits. The exchange conduits are designed so that every segment serves as an individual heater/cooler for the water at that point, with the output temperature increasing incrementally from one end of the Testing Arena to the other. The TPAD was shown to be capable of producing a linear thermal gradient suitable for testing many small organisms of conservation concern from local waters and then maintaining the gradient for 24 hours. The Testing Arena was found to comfortably house organisms including M. tuberculata and Cambarus spp., though there were shaded areas which were favored by cover-seeking organisms. I think, despite not reaching all our original goals, that the TPAD demonstrates the potential of the counter-current exchange model as a refined method of conducting thermal preference tests. Work on the TPAD Design Phase II, a larger, more flexible, and more efficient model based on the success of Phase I, is already underway.

Bio: Matthew Tanner Donelon is from Georgetown, Texas. He graduated with a B.S. in Wildlife Biology from Texas State University in 2019, where he realized his love for innovation in wildlife management strategies. After graduation, Matthew will pursue work as an environmental consultant or engineer.

Optimization of Molecular Buoys for use in the Isolation and Amplification of SARS-CoV-2 RNA from Wastewater

Major Advisor:  Dr. Shannon Weigum

Committee Members:

Dr. Rodney Rohde

Dr. Kelly Woytek

Wastewater based epidemiology (WBE) is an emerging tool used in the surveillance of viruses and viral diseases, such as SARS-CoV-2 and COVID-19. WBE comprises wastewater sampling and processing, isolation of target viruses and purification of target viral nucleic acids, and quantification through molecular means such as RT-qPCR. Isolation and purification are crucial steps in estimating viral loads and positivity rates in a given watershed. Current CDC-recommended methods in viral isolation can be costly, time consuming, or are otherwise inaccessible for widespread use. Our aim is to develop a rapid and accessible bio-separation method using inexpensive materials. In the present work, hollow silica microspheres, or “molecular buoys”, were functionalized with chitosan, a cationic polysaccharide, to capture nucleic acids in diluted stool. Scanning electron microscopy (SEM) was used to examine chitosan surface loading and fluorescently-tagged DNA aptamer (a surrogate oligonucleotide) diluted in stool was used to examine nucleic acid binding capacity. In addition, optimization of nucleic acid capture, reduction of assay time, and limit of detection (LOD) were explored. Under SEM, we observed shell formation on the surface of chitosan-functionalized microspheres that was absent in non-functionalized microspheres. Additionally, optimization of the assay wash protocol reduced assay time, bringing total assay time to around 35 - 40 minutes. Finally, a preliminary LOD was found to be 40 copies/uL in diluted stool. These findings suggest that functionalized hollow silica microspheres may be an effective, low-cost, and rapid method in the isolation of viral nucleic acids for use in WBE.

Bio:  Alexandra received a Bachelor of Science in Biology from the University of Missouri-Columbia in 2021. She then moved to Austin and worked in a contract lab for a year. She joined the Optical Biosensors and Nanomaterials lab, lead my Dr. Shannon Weigum, in August 2022. After graduation, Alexandra plans to continue her education and pursue a career in virology. 

Optimizing Chitosan-Functionalized Hollow Silica Microspheres for Isolation and Concentration of Nucleic Acids

Major Advisor:  Dr. Shannon Weigum

Committee Members:

Dr. Adeyemi A. Olanrewaju

Dr. Manish Kumar 

This research investigates Wastewater-Based Epidemiology (WBE), a tool utilized for detection and concentration analysis of wastewater samples. WBE is useful for monitoring and management of public health threats like viral outbreaks such as COVID-19. In current research, the development of this assay uses the unique properties of hollow silica microspheres coupled with chitosan as a binding agent, a method effective in the detection of various RNA and antigens in wastewater samples. The utilization of hollow silica microspheres, selected for the high surface area and buoyant properties, aided by chitosan, creates an efficient and selective binding process. This combination can be manipulated for the sensitivity of detection but also supports the capture of a wide range of viral particles and RNA molecules. Capture of oligonucleotides is achieved by chitosan binding to the surface of the hollow silica microspheres. This thesis research served to find the optimal conditions for nucleic acid binding, isolation, and long-term microsphere stability when functionalized with chitosan. The preparation of functionalized microspheres has been reduced. The incubation time of functionalized microspheres with L-Cysteine has been reduced to 10 minutes under end over end rotation.  The optimal binding conditions of oligonucleotides has been found utilizing a surrogate. It was found that the functionalized microspheres can bind nucleic acids at saturation within 1 hour. The incorporation of reduced incubation times has reduced the overall protocol by over 24hrs. A protocol has been developed to stabilize microspheres after lyophilization and storage. The addition of Trehalose, a cryoprotectant, increases stability after lyophilization and storage. SEM or Scanning electron microscopy was used to image the chitosan shell on the hollow silica microspheres. The functionalized microspheres containing various treatments applied was examined for disruptions in the chitosan surface and binding kinetics. The implications of this research are vast and transformative. By increasing efficiency and stability and reducing sample preparation costs, this research increases access to WBE for public healthcare and diagnostic testing. The adoption of methods can aid in early outbreak detection and the assessment of community health, therefore contributing to more informed and effective public health strategies and interventions.

Bio:  Omari began undergraduate studies at Southern Methodist University. He then finished his B.S. in Biology at the University of Houston. In the fall of 2022, he joined Dr. Weigum’s Optical Biosensors and Nanomaterials lab. After Graduation he plans to further his studies and pursue a joint MD/JD program.

Understanding the Lasting Effects of Climate and Wildland Fire on Montane Rattlesnake (Crotalus sp.) Natural History in the American Southwest 

Major Advisor: Dr. Michael Forstner

Committee Members:

Dr. Sarah Fritts

 Dr. Jenifer Jensen

 Dr. Weston Nowlin 

Dr. Andrew Holycross 

Dr. Ivana Mali

Climate change, and its’ effects on wildland fire has recently been one of the most dynamic subjects in the ecological study of western United States. Understanding the effects of fire is essential for future management of Southwestern ecosystems and the conservation of at-risk species. In July 2019, the Miller Fire burned ~2,331 hectares of forest and grassland in the Peloncillo Mountains of New Mexico and Arizona, and subsequently the Foster Fire burnt an additional 3,074ha in 2022. These fires have resulted in a unique opportunity to study the effects of fire in conjunction with climatic trends on the ecology of one of the region’s most ubiquitous taxa, montane Rattlesnakes (Crotalus). I propose a large scale, multi-year project assessing many of the effects that fire and climate patterns may have had on the ecology of rattlesnakes, primarily Crotalus lepidus klauberi, C. molossus, and C. willardi obscurus. This study will use analysis of individual health, phenotypic variation, behavior, and ecology to gauge effects that these fires and past fire history may have on the species that inhabit this unique region of the US. Additionally, this project will seek to inform future fire management practices to be better stewards of these species as we return a healthy fire regime to this region.

Bio:  Saunders Drukker is from Central Texas and graduated with a BS in Ecology and Biodiversity from Sewanee: The University of the South in Sewanee Tennessee. He then worked in the Fire Science Lab at Tall Timbers Research Station in north Florida, studying fire behavior and forestry before joining Dr. Forstner’s herpetology lab at Texas State in 2019. His research interests focus on the ecology and conservation of the Madrean Sky Islands in New Mexico and Arizona particularly in relation to fire and anthropogenic climate change. He is a sitting board member of the Central Texas Herpetological Society, as well as the Rattlesnake Conservancy’s Montane Rattlesnake Working Group. 

Drawing as a Generative Learning Activity: An Exploratory, Qualitative Investigation on a Nature Journaling Seminar Course

Major Advisor: Dr. Kristy L. Daniel

Committee Members:

Dr. Carrie Jo. Bucklin

Dr. Paula S. Williams

Drawing encourages critical thinking skills by pushing learners to use discernment and prior knowledge of a subject matter to create an external representation of their mental understanding. While visual representations play a key role in science education, it is less common to prompt students to draw their own visual representations to communicate their uniquely constructed ideas. This exploratory, qualitative study investigated drawing as a generative learning activity in a nature journaling seminar course by examining students’ use of drawing techniques taught in class, as well as dissecting their reported reflections on how drawing impacted their journaling experience. The students (n=11) were required to complete a nature journal using prompts developed to encourage reflection, drawing technique practice, and scientific thinking. We coded and analyzed student responses for trends in drawing technique usage, such as hatching and use of color. Student drawings reflected a steady increase of technique usage as more techniques were introduced in class, with a decline once drawing instruction ceased. Students reported that the practice of drawing nature observations increased their mindfulness and attention to detail in their written entries. Our findings provide insight to the benefit of drawing practices in nature journaling, as well as the effects of drawing instruction on student perceptions. Teaching drawing techniques with instructional guidance can provide students with the opportunity to take an active role in their education and deepen their understanding of, and connection with, the natural world. 

Bio: Hannah Baratang graduated with two A.S. degrees in Chemistry and Organismal Biology from Central Texas College at the age of 17. She graduated with a B.S. in Biology from Texas A&M - Central Texas at the age of 19. She joined the Daniel Biology Education Research Group at Texas State University in the Spring of 2023 to pursue a M.S. in Biology under Dr. Daniel's supervision. Since joining the research group, she has been an active member of Research Rangers, developed and illustrated the Good Gall-y! outdoor science activity, and independently organized a series of live music events in the San Marcos DIY scene.

Major advisor: Dr. Dana M. García

Committee members:

Dr. Gar Yee Koh

Dr. Robert J. C. McLean

Join Zoom Meeting: https://txstate.zoom.us/j/88507100096?pwd=NHNIWU5DVzhZR3dCWGpoNUk3Z2pwUT09

Meeting ID: 885 0710 0096 
Passcode: Passcode115682

Vitamin D is an essential vitamin, and worldwide many people experience vitamin D deficiency, leading many individuals around the globe to suffer from different illnesses such as cardiovascular disease, tuberculosis, diabetes, and cystic fibrosis. Vitamin D is well known for its main function in maintaining bone health; however, in recent years vitamin D has been recognized for its function in autoimmune health, integrity of the gut barrier, and gut microbiome composition. In this study, I hypothesized that vitamin D supplementation through diet would have an impact on gut microbiome composition and gut integrity, in the latter case by influencing expression of mRNA encoding tight junction proteins ZO-1, occludin, and claudin. Using C57BL/6J mice as a model, I assigned mice to 4 diet groups as follows: 1) standard diet (AIG-93G purified diet; CTR), 2) CTR + antibiotics (AB), 3) CTR + 5000 international units (IU) of vitamin D3 (VD), and 4) VD + antibiotics (VD + AB). Antibiotics consisting of 0.5 g/L vancomycin, 1 g/L neomycin sulfate, 1 g/L metronidazole, and 1 g/L ampicillin were given in water throughout the experimental period. The microbiome was assessed using DNA extraction, 16s PCR sequencing, and analysis using microbiome analyst. The results indicated that the supplementation of VD via diet had a positive impact on gut microbiome composition, leading to an increase in representation of Firmicutes phyla in the VD group in comparison to the CTR, while also showing an increase in the Dubosiella newyorkensis species in VD. Furthermore, qRT-PCR suggested that vitamin D supplementation improved gut integrity, since ZO-1 and occludin genes having higher rates of mRNA expression in mice consuming a VD diet in comparison mice on the control diet. This study indicates that vitamin D supplementation can potentially be used as a treatment for certain illnesses by treating underlying causes such as gut dysbiosis and permeability of the gastrointestinal tract.

Bio: Ethan Constantine is from the Dallas TX area and graduated from Texas A&M University Corpus Christi in 2020 with a B.S. in Biomedical Sciences. In his free time, he enjoys working out, running, and trains in kick boxing and muay tai. After graduating from Texas State University, he plans to take a gap year before continuing his education.

Nuclear tRNA-derived RNA fragments (tRFs) trigger immunity in Arabidopsis and potentially function as a mobile signal in systemic acquired resistance

Major Advisor:  Dr. Hong-Gu Kang

Committee Members:

Dr. Nihal Dharmasiri (Texas State University)

Dr. Sunethra Dharmasiri (Texas State University)

Dr. Sibum Sung (The University of Texas at Austin)

Dr. Joe Louis (University of Nebraska-Lincoln)

Zoom Link: https://txstate.zoom.us/j/89437707227

Plants respond to stress through rapid and extensive changes in gene expression, orchestrated by intricate modifications in chromatin structure. They also possess a unique long-distance immunity known as Systemic Acquired Resistance (SAR), which offers protection against future attacks, indicating the presence of delicate signaling molecules in these immune responses. However, the identity and mechanisms of these signaling molecules remain poorly understood.

Our research on effector-triggered immunity (ETI) to Pseudomonas syringae pv tomato (Pst) in Arabidopsis revealed the significant role of DCL1 (Dicer-Like 1) in modulating ETI, emphasizing small RNAs' (sRNAs) importance. Deep-sequencing of nuclear sRNAs preceding defense gene induction uncovered the rapid accumulation of a 31-nt tRNA fragment (tRF^31Asp2) in a DCL1-dependent manner. Remarkably, infiltration of Arabidopsis with tRF^31Asp2 alone induced over 500 defense genes and immunized the plants against Pst and aphids. Additionally, we discovered that tRF31^Asp2 exhibited sequence-specific binding to genomes, including those within defense genes, indicating that the interaction between the tRF and the genome likely triggers transcriptional activation. Furthermore, our investigation revealed that tRF^31Asp2 was mobile, with its mobility enhanced by avirulent Pst, and translocated to the nucleus of distal tissue. To further explore this intriguing observation, nuclear sRNAs were deep-sequenced in leaves neighboring those infected. As a control, DIR1, a lipid transport protein known to be involved in SAR and itself capable of movement within plants, was used. Surprisingly, 16-nt-long tRFs, particularly those generated from Asp-tRNA (tRF^16Asp), were induced by avirulent Pst in a DIR1-dependent manner, suggesting that these tRFs might be a SAR mobile signal.

My study identifies a potent candidate molecule that modulates transcriptional reprogramming in response to infection and signals from neighboring leaves. This groundbreaking discovery unveils a mechanism in which sRNAs carry precise targeting information, guiding essential transcriptome changes. This paves the way for a promising new technological approach, enabling the precise engineering of stress responses.

Bio: Dinesh was born in the far-western part of Nepal. He earned his B.S. Biotechnology degree from Purbanchal University, Nepal in 2011 and M.S. Biotechnology from the Bangalore University, India in 2016. He then moved to San Marcos for pursuing his Ph. D in plant molecular immunology lab of Dr. Hong-Gu Kang in Fall, 2017. After graduation, Dinesh plans to continue his passion for research. Apart from academics, he is passionate about sports. 

Neuromodulation of Olfactory Immune Responses in Rainbow Trout (Oncorhynchus Mykiss)

Major Advisor: Dr. Mar Huertas  

Committee Members:

Dr. Dana Garcia, Texas State University
Dr. Lauren Fuess, Texas State University
Dr. Yuan Lu, Xiphophorus Genetic Stock Center, Texas State University
DR. Erika Calvo-Ochoa, Hope College 
 
Zoom Link: https://txstate.zoom.us/j/84687267464?pwd=eFlKSE5jNEpUTlk3b0hDQ1JCQkxjdz09

Meeting ID: 846 8726 7464    Passcode: 338248

Trout can smell viruses and the resulting olfactory signals can activate the fish brain immune system. Preliminary results in our laboratory showed that trout can also smell bacterial odorants with high sensitivity and its detection elicits an avoidance behavioral response, thus the trout olfactory immune function is likely tunned for a variety of pathogenic odors. However, the brain integration process for these olfactory immune responses is unknown. The integration of environmental signals within the vertebrate brain is mediated by different neuromodulators which subsequently trigger behavioral responses and immune system activation. I hypothesize that the integration of olfactory bacterial signals is mediated by brain neurosteroid neuromodulation, which leads to the activation of the neuroimmune system and results in the avoidance behavior in rainbow trout. To test our hypothesis, we will first measure neurosterodoigenic brain responses in trout after nasal exposure to inactivated Yersinia ruckeri by measuring mRNA expression of different neurosteroidogenic enzymes and neurosteroid production by ELISA. Second, we will do a detailed mapping of the olfactory-immune pathway by pinpointing the specific locations of neurosteroidogenic enzymes and activated immune factors in the rainbow trout brain using multiple “in situ” hybridization and spatial transcriptomics techniques. Preliminary results showed a time (15 mins, 4 hours, 24 hours, and 7 days after bacteria odor exposure) and location-dependent (nose, olfactory bulb, telencephalon, diencephalon, optic tectum, and cerebellum) expression of several neurosteroidogenic enzymes, which were parallel to localized production of neurosteroids. Our findings indicate the involvement of neurosteroids in the rapid bacterial odor identification via the olfactory system in rainbow trout. Investigating the olfactory-immune brain pathway for bacterial odor detection in rainbow trout will help to understand the role of neurosteroids in olfactory processing. Moreover, it can help to identify specific targets of action that can contribute to designing a more effective nasal vaccine to treat enteric red mouth diseases caused by Y. ruckeri in rainbow trout.

Bio: Maruf is from Bangladesh and graduated with his bachelor’s from Bangabandhu Sheikh Mujibur Rahman Agricultural University in Bangladesh from the faculty of Fisheries before getting his Master’s degree in Marine Biology from the University of Texas Rio Grande Valley, Brownsville, TX. His love for biology and a desire to work on fish physiology led him to focus on fish physiology and olfaction in his Ph.D. degree. He is supported by his lab, friends, and family on his academic journey.