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End date: 2017 × Start date: 2013 × Item Type: Publication × search.filters.applied.f.resourcesubtype: Undergraduate Thesis ×

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Rotifer Growth Under Astaxanthin Enrichment

2017-12 , Siegfried, Emma

Rotifers and astaxanthin both play an important part in the aquaculture industry. Rotifers are used as a substitute for copepods, the main source of food for larval fish in natural systems, due to the ease with which they can be cultured. Astaxanthin is a carotenoid and antioxidant which brightens the coloring of fish and improves fish health. Rotifers are believed to be a method through which astaxanthin can be bioencapsulated and vectored to larval fish. As a result, it is important to understand the effect of astaxanthin on rotifers themselves. This experiment uses a multitude of different protocols to determine how different astaxanthin compounds effects rotifers on both the individual and population levels. Reproductive tables and fluorescent imaging were used to assess the health of individual rotifers; population density measurements in mass cultures were used to assess rotifer population health. The reproductive ability of rotifers was significantly different from control under multiple astaxanthin treatments. Astaxanthin enrichment also created a higher stable population density in the mass cultures. The fluorescent imaging showed that the rotifers reached peak astaxanthin concentration within the rotifer gut after 3 hours, and but concentration returned to control levels within 24 hours of removal from astaxanthin. These results all point to the fact that astaxanthin helps to increase rotifer health and fitness, and that these rotifers could be used as a vector for astaxanthin to larval fish.

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Gastrointestinal Motility: Kinematic Properties of Segmentation Contraction

2017-05 , Lamarca, Morgan

The kinematic processes of the small intestine play integral roles in overseeing the digestion and transportation of food throughout the gastrointestinal tract. Specifically responsible for governing the flow and digestion of chyme along the gastrointestinal tract are the two fundamental patterns of motility, propulsion and mixing [1]. While peristalsis is the principle muscle contraction for propulsive motility, segmentation contractions are responsible for mixing and chopping the chyme [2]. Previous studies on the contraction kinematics of the small intestine have provided evidence that contraction frequencies alter in response to different types of food. We propose that the pattern of segmental contraction varies in response to the different compositions of intraluminal contents as well. As the composition of chyme alters and it becomes less dense, segmental contraction frequency increases as a response. Our in vivo observational technique conducted on the small intestine of male Sprague-Dawley rats is much less invasive than previous studies, thus allowing us to better quantify the kinematic properties of the small intestine, such as frequency and amplitude of contraction. Understanding and comparing essential patterns of motility across patients can improve medical diagnostics as well as the manufacturing of food supplements and pharmaceutical medications.

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Aggressive Phenotypes in Malawi Cichlids Associated with V1AR Variant

2016-12 , Schappaugh, Nicholas A.

The cichlid model provides a great opportunity to explore diversity in behavioral phenotypes. Different groups of Malawi cichlids exhibit distinct patterns of behavior for a variety of scenarios, including aggressive encounters. These cichlids, characterized by the rocky or sandy habitats they occupy, exhibit strong genetic divergence, possessing large numbers of alternatively fixed variants between them. One such variant exists in the gene avpr1a, also known as V1aR, a major receptor for vasopressin in humans. This gene has been linked to behavioral effects across a variety of animal species, with this specific variant likely to have significant structural implications for the receptor product. Here we investigate the aggressive behaviors of a set of rock and sand hybrid fish for their association with the variant observed in V1aR. While specific metrics of aggression showed similar trends in these hybrids compared to those observed in the parental rock and sand species, ultimately these trends were not significant and were inconclusive. However, these results serve as a preliminary investigation of this gene’s involvement in cichlid aggressive behavior. In future work, further examination of the locus will be conducted utilizing more precise and powerful methods in order to draw stronger conclusions.

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Establishing a Working Protocol for Plasmid Cloning and shRNA Design in Endogenous Brachionus manjavacas Gene TRP7

2016-07-18 , Krishnappan, Sharadha

Current transfection protocol in rotifers only allows for temporary transfection within rotifers and does not allow for the continuous knockdown of endogenous genes, thereby inhibiting the possibility of observing long-term biological effects in response to specific perpetual gene knockdowns. This study aims to address this particular issue by establishing a working protocol for plasmid cloning and shRNA design within an endogenous gene of B. manjavacas with known biological effects, allowing for the exploration into the optimization of a transfection protocol and demonstration of RNAi knockdown of the known gene within the rotifers as subsequent studies. Manipulation of gene expression in rotifers could occur through plasmid vector insertions, which induce silencing of a gene’s expression with short hairpin RNA (shRNA), via RNAi. This would effectively stimulate gene knockdown, allowing for the observation of biological effects such as changes in fecundity and lifespan. With the establishment of a working protocol for plasmid cloning and shRNA design, as a result of this study, the optimization of a transfection protocol for rotifers is explored. With increased efficiency in the transfection of rotifers, populations of rotifers expressing the plasmid can be amassed, allowing for experimental design that examine the varying aging mechanisms and effects that are stimulated due to permanent changes in target gene expression through RNAi. This, in turn, could give rise to the identification of evolutionarily conserved genes that regulate organismal aging, which could lead to further implications in the field of pharmacological intervention in mammalian aging as well as in the field of biogerontology overall.

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Determining how Freshwater Copepods Follow Planar Dextran Trails

2017-05 , Young, Madison R.

In this study two species of freshwater copepods, Hesperodiaptomus shoshone (H. shoshone), and Hesperodiaptomus arcticus (H. arcticus) will be used to determine how some species of freshwater copepods interact with planar dextran trails. When a copepod swims through water, hydrodynamic disturbances with a variety of structures are created; some are vortical, planar, or laminar. Initial studies show that these copepods avidly follow laminar trails in an upstream fashion [Pender Healy]. However, when copepods execute turns or fast swimming, vortices are created. When copepods execute slow turns, planar wakes are formed. The direction of flow in the wakes and the location of the wakes provide information on the location of the copepod that is generating that wake. The intent of this research is to determine if the signals in the wakes can lead the following copepod to the source of the disturbance. Hence, all analyses focused on events where a copepod responded to the signal. Responses include reorientation or angle of entrance, time spent in trail, preference for a particular trail width and edge following behavior [Strouhal number]. The goal is to understand more about their how they sense and respond to changes in their environment and it is hypothesized that both species will interact more with the wide trail and that H. arcticus will spend more time in the trail and enter at a greater angle. It is also hypothesized that both species adhere to ideal Strouhal values. To test this, two dextran (a polysaccharide) trails will be simultaneously dropped into a tank containing the copepods. One of the trails will be 2mm and the other will be 4mm. It is expected that the copepod will wobble or traverse the trail to contact the edges; edge following enables the copepod to stay on track. Alternatively, the copepod may follow the center of the trail where the flow is the fastest and therefore is relying on flow speed rather than the shear found in the edge of the trail. Analyses of the location of the follower relative to the edge versus the center of the trail can assess whether the copepod is sensing flow shear versus flow speed. A MatLab script will be used to find more detailed information (Figures 11-22). Both species prefer to follow the wider trail (Figure 5 and 6), and H. arcticus spend more time in the trail than H. shoshone. Hesperodiaptomus shoshone followed the 4mm trail eighteen times for an average of 2.14 seconds and the 2mm trail five times for an average of 1.36 seconds. The number of encounters was also determined, which confirms that the proportion of encounters resulting in follows is much higher for the 4mm trial than the 2mm trial in both species (Table 3). This data illustrates statistically significant results (p<0.05) that H. shoshone prefer to follow the wider trail and follow for longer periods of time compared to the smaller one. These results were compared to the results for H. arcticus, which followed the 4mm trail twelve times for an average of 5.08 seconds (Figure 6). This comparison between both species on the 4mm trail confirmed that H. arctius spend more time in the trail than H. shoshone (p<0.05). The average angles of entrance were 29.16° for H. shoshone on the 4mm trail and 21.08° on the 2mm trail, and 39.69° for H. arcticus on the 4mm trail (Table 2). When compared, the results demonstrated that H. arcticus enters the trail at a greater angle than H. shoshone on both the 2mm and 4mm trails (p<0.05). There was not enough of the wobbling behavior shown to find Strouhal Values at this time.

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Exploring Cichlid fish tooth regeneration turnover rates

2017-05 , Tome, Maya

Polyphyodonts are organisms who can continuously replace their teeth throughout their lives, yet this mechanism is extremely complex. There are many factors that can affect tooth density and turnover rate, including genetic predisposition and environmental stimuli. In this study, we use pulse chase experiments to investigate the tooth turnover rates of polyphyodont cichlid fish species with differing tooth densities and morphologies. The experiments were carried out with 15-20 day old cichlids from the species Cynotilapia afra and Metriaclima zebra, which are unicuspid and bicuspid respectively. Alizarin-red and Calcein green fluorescent dye were applied in succession with varied timing, and dye incorporation patterns were then analyzed to distinguish recently replaced teeth from older teeth. C. afra have a lower density unicuspid teeth and M. zebra have a higher density of bicuspid teeth. Preliminary results suggest that the C. afra had slower tooth regeneration when compared to the M. zebra jaws, as Alizarin dye incorporation was much more prevalent in the unicuspid jaws; however, the M. zebra jaws featured many more half red/half green teeth and a larger number of Calcein-only replacement teeth that began to form a distinct, second row. Thus, after compiling the preliminary results, the data suggests that the unicuspid species C. afra has a slower tooth turnover rate than the bicuspid species M. zebra, but a larger sample size is needed to confirm these assertions.

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Generalizing Disease Associations to Non-Studied Populations

2016-12 , Patel, Kane

This study determines whether risk allele frequencies (RAFs) for common diseases can be generalized in genome wide association surveys (GWAS) that are done in different populations other than the original study population. To test this, the study compares RAFs gathered from the NHRI-EBI GWAS Catalog and 1000 Genomes Project by study population and checks if there is bias towards the study population. If the trend is present, the study looks to answer the question of whether or not this is due to an inherent bias from the study population, or a pre-ascertained bias in the genotyping single nucleotide polymorphism (SNP) chip array. To test bias in the technology, the study compares allele frequencies for disease SNPs and non-disease SNPs on Illumina1M and Affymetrix 6.0 genotyping arrays. If the bias still persists, then there is an inherent bias due to the study population alone. At this point, the study will examine the role of other contributing factors to differences in disease allele frequencies across populations. These include: type of disease, number of participants in the GWAS, whether alleles have a large effect, etc. This study potentially contributes the overall field of population genetics and personalized medicine. Essentially, the goal is to ensure that the information attained can be used to create models that could correct potential bias in GWAS studies.

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Determining how Freshwater Copepods Follow Planar Dextrain Trails

2017-05 , Young, Madison Ruth

In this study two species of freshwater copepods, Hesperodiaptomus shoshone (H. shoshone), and Hesperodiaptomus arcticus (H. arcticus) will be used to determine how some species of freshwater copepods interact with planar dextran trails. When a copepod swims through water, hydrodynamic disturbances with a variety of structures are created; some are vortical, planar, or laminar. Initial studies show that these copepods avidly follow laminar trails in an upstream fashion [Pender Healy]. However, when copepods execute turns or fast swimming, vortices are created. When copepods execute slow turns, planar wakes are formed. The direction of flow in the wakes and the location of the wakes provide information on the location of the copepod that is generating that wake. The intent of this research is to determine if the signals in the wakes can lead the following copepod to the source of the disturbance. Hence, all analyses focused on events where a copepod responded to the signal. Responses include reorientation or angle of entrance, time spent in trail, preference for a particular trail width and edge following behavior [Strouhal number]. The goal is to understand more about their how they sense and respond to changes in their environment and it is hypothesized that both species will interact more with the wide trail and that H. arcticus will spend more time in the trail and enter at a greater angle. It is also hypothesized that both species adhere to ideal Strouhal values. To test this, two dextran (a polysaccharide) trails will be simultaneously dropped into a tank containing the copepods. One of the trails will be 2mm and the other will be 4mm. It is expected that the copepod will wobble or traverse the trail to contact the edges; edge following enables the copepod to stay on track. Alternatively, the copepod may follow the center of the trail where the flow is the fastest and therefore is relying on flow speed rather than the shear found in the edge of the trail. Analyses of the location of the follower relative to the edge versus the center of the trail can assess whether the copepod is sensing flow shear versus flow speed. A MatLab script will be used to find more detailed information (Figures 11-22). Both species prefer to follow the wider trail (Figure 5 and 6), and H. arcticus spend more time in the trail than H. shoshone. Hesperodiaptomus shoshone followed the 4mm trail eighteen times for an average of 2.14 seconds and the 2mm trail five times for an average of 1.36 seconds. The number of encounters was also determined, which confirms that the proportion of encounters resulting in follows is much higher for the 4mm trial than the 2mm trial in both species (Table 3). These data illustrates statistically significant results (p<0.05) that H. shoshone prefer to follow the wider trail and follow for longer periods of time compared to the smaller one. These results were compared to the results for H. arcticus, which followed the 4mm trail twelve times for an average of 5.08 seconds (Figure 6). This comparison between both species on the 4mm trail confirmed that H. arctius spend more time in the trail than H. shoshone (p<0.05). The average angles of entrance were 29.16° for H. shoshone on the 4mm trail and 21.08° on the 2mm trail, and 39.69° for H. arcticus on the 4mm trail (Table 2). When compared, the results demonstrated that H. arcticus enters the trail at a greater angle than H. shoshone on both the 2mm and 4mm trails (p<0.05). There was not enough of the wobbling behavior shown to find Strouhal Values at this time.

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Screening for the Functionality of RNA Templated Repair of Double-Strand Breaks in Saccharomyces Cerevisae

2017-05 , Gordon, Katherine

Double-strand breaks (DSBs) in DNA are detrimental, as they can cause mutations and genomic rearrangements, which in turn leads to cancer and other diseases. Recent research reveals that DSBs can be repaired by RNA-templated homologous recombination. However, RNA-templated repair of DSBs is not well understood. In order to better understand the mechanism of RNA-templated repair of DSBs, the current research aims to identify the proteins that facilitate the repair. The research utilizes a system wherein RNA-templated repair of DSBs is known to occur. A yeast overexpression plasmid library was produced in order to test the ability of fragments of the yeast genome to facilitate RNA-templated repair of a DSB when these are highly expressed in the yeast cells. In order to test the ability of added gene fragments to facilitate RNA-templated repair of DSBs, the experimental candidates were exposed to galactose in order to induce a DSB, to activate the transcription of the overexpressed gene fragment, and to initiate the transcription of anti-sense RNA used to repair the break. The candidates were then moved to medium without histidine in order to assess the frequency of repair. Once a large number of colonies (~50,000) are screened, we expect to identify several proteins that facilitate RNA-templated repair of DSBs. Identifying the specific genes that facilitate this repair mechanism will assist in characterizing the functionality of the RNA-templated DNA repair mechanism. Identifying these genes will also allow for better predictions for how this same phenomenon may occur in human cells.

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Catalytic abilities of the Schistosoma mansoni hammerhead ribozyme with mutated substrates in ice

2016-12 , Calvird, Audrey

The synthesis of biomolecules in an environment similar to a pre-biotic Earth within the field of evolutionary chemistry has applications in understanding abiogenesis and the evolution of early biological systems on Earth. The RNA world is a prebiotic environment proposed and subsequently heavily studied in effort to better understand how biochemical reactions started on Earth. This study proposes to assess the catalytic ability of a Hammerhead Ribozyme (HHRz) isolated from the protozoa Schistosoma mansoni (Schist HHRz). It has been previously shown that the Schist HHRz can catalyze the synthesis of extended sequences of ribonucleic acid (RNA) by ligating two RNA substrates in ice, as well as cleave the same substrate back to the original separate substrates in the presence of Mg2+ (Lie et al. 2016). The same study showed that the Schist HHRz is able to catalyze the ligation with mutated substrates (Lie et al. 2016). This study aims to evaluate Schist HHRz’s ability to ligate a mutated substrate in comparison to the wild type substrate in ice, as well as compare the kinetics of the Schist HHRz to cleave the mutated and wild type substrates in the presence of Mg2+. This study will utilize the techniques specified by Lie et al. (2016) to further investigate the extent of the ligation of a mutated substrate catalyzed by the Schist HHRz compared to that of the wild type substrate. This study also assays the kinetics of the cleavage reaction catalyzed by the same Schist HHRz of both the wild type substrate and mutated substrate. This study hopes to improve the understanding of pre-biotic biochemistry in efforts to identify possible mechanisms or models for RNA polymerization by ribozymes on early Earth.

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