Under constant volume muscle contraction myofilament overlap increases and muscle expands radially. While myofilament overlap is considered a major factor in force generation, considerably less is known about the role of myofilament expansion accompanying shortening during contraction. Recent theoretical evidence suggests that the spacing between filaments will indeed be an important determinant of force generation at any level of filament overlap, however, there are scant data that show the extent to which filament overlap changes in intact muscles. To address this issue we performed work loops on intact Manduca sexta flight muscles while simultaneously measuring myofilament spacing, force, and length using time resolved small angle x-ray diffraction imaging. Muscles were stimulated at 25 Hz and held at a temperature of 35C while simultaneously measuring length and force. Five x-ray images were taken during each wingbeat cycle for 100 contractions. These images were averaged over all cycles, and lattice spacing was measured for each phase. During work loops with a 6% strain cycle, lattice expansion changes by 2.77 ± 0.839%. Thus we do not reject the constant volume assumption, which would predict a 2.4% change in radial dimension for a 6% longitudinal strain. Taken together these data suggest that lattice expansion during muscle shortening is a critical component of muscle force generation.

Flight control in dynamic environments is a complicated task that many animals accomplish with ease. It requires rapid integration of sensory input as well as balancing long-term planning with reflexive behaviors. The giant hawk moth Manduca sexta is a useful model organism for studying flight control because its physiology is well understood and it is easy to rear in captivity and work with experimentally. In order to make flight control adjustments, Manduca constantly integrates information from slow-sensing visual sensors and fast-sensing mechanosensory organs. Both visual motion and mechanical rotation elicit strong abdominal reflexes in Manduca during tethered flight, however the exact role of the abdomen in flight is unclear. Previous work has suggested that abdominal reflexes help maintain flight stability, but it has not been shown that the abdomen is actively used for control. The goal of my research is to determine if Manduca can actively use their abdomen to stabilize the visual field. To answer this question, I have created a virtual flight arena composed of a projection system, magnetic tether, and IR sensor. The visual stimulus is projected onto an acrylic dome within which the moth is suspended by the tether. The experiment consists of a system where the moth controls the velocity of a horizontal bar displayed on the dome with its abdominal position. The bar has an initial velocity that the moth counteracts by moving its abdomen in order to stabilize the visual field. The precision of the moth’s abdominal control capabilities can then be tested by differing the initial velocities of the bar and varying the way that abdominal position controls the bar’s velocity. In addition to answering my research question, this project provides a framework to study the general multi-input multi-output problem in neural control.

Cooperation is a fascinating paradox of evolution. Why would an organism incur a cost to itself in order to benefit another individual? The Achilles’ heel of cooperation is that it can be exploited by cheaters. Cheaters reap the benefits of cooperation, but do not incur the same costs as the cooperators. Cheating is therefore considered a major problem in the evolution of cooperation. One possible way of reducing the vulnerability of cooperating organisms to cheating is through communication. Communication allows individuals to cooperate only when the conditions are favorable. My research involves a form of bacterial communication called quorum sensing, which allows bacteria to accomplish cooperative tasks that require a collective action. Pseudomonas aeruginosa, an opportunistic pathogen that causes severe infections in immuno-compromised individuals and patients with cystic fibrosis, uses quorum sensing to regulate the production of toxins and enzymes. We used a wild-type cooperating strain of P. aeruginosa and several non-cooperating cheater strains to investigate the relationship between cooperators and cheaters. We predicted that the cheater strains would benefit when grown together with cooperators in a well-mixed environment, but suffer in a structured environment. We tested this prediction through competition assays in both a shaken liquid environment (unstructured) and a soft-agar environment (structured). Combined with further research, our results may suggest that communication helps a cooperating population avoid collapse when faced with cheaters.

Cooperative interactions are observed at all levels of biological organization. Cooperation facilitates major transitions in evolution such as from unicellularity to multicellularity. How a cooperative system evolves from its incipient state is unknown. I have used a synthetic cooperative system termed CoSMO (Cooperation that is Synthetic and Mutually Obligatory) as a model for incipient cooperation. CoSMO is an engineered yeast system consisting of two non-mating strains, differentially labeled with fluorescence. It relies on direct exchange of distinct metabolites between the two cooperators. Evolved CoSMO cocultures exhibit an improvement in viability, defined as an increased ability to survive reductions in population density. I have determined that this viability improvement is heritable, by measuring the viability of reconstituted evolved CoSMO pregrown in rich media which allow cells to grow in the absence of cooperation. Furthermore, by mixing an evolved strain with its ancestral partner, I determined that a single evolved cooperator was responsible for community-level viability improvement. I am characterizing phenotypic changes in evolved cooperators that contribute to increased viability. The phenotypic changes can potentially be categorized as either “self-helping”, such as through increased starvation tolerance and higher affinity for metabolites, or “partner-helping”, such as through increased metabolite release. In all cases, I will identify the molecular basis for these adaptations through genome-wide re-sequencing. Furthermore, genomic analysis on entire evolved CoSMO cocultures will be performed to reveal the diversity and frequency of relevant mutations in the population over time. Lastly, I will perform similar analyses on replicate populations. If similar adaptive changes that result in improvement in system viability are observed, a deterministic process to improve viability is implied. Otherwise, diverse mechanisms can achieve improved fitness. In both cases, we will gain insights into how cooperative systems evolve from inception.

For controversial subjects like evolution, language used by experts can actually reinforce naïve conceptions held by students. For example, students interpret the word “purpose” to mean that traits evolve to serve an immediate function, but experts mean that a trait increases reproductive fitness. Students also often incorrectly assume that evolution follows a predetermined goal, that individuals choose to evolve in a certain direction, and that fit individuals are stronger, bigger, or faster. In this study, I tested the hypothesis that an upper division biology textbook includes some naïve conceptions and unintentionally reinforces others. I analyzed nine chapters of Biology of Microorganisms (13th Ed) by Brock et al., a standard text for teaching microbiology. In my preliminary analysis of these chapters, I found 15 statements that seemed to imply or promote misconceptions. For example, Brock et al. stated that “[e]volution selected for improvements and diversification of these early cells…” (34) which could be misconstrued by students to mean that cells evolve towards a predefined, yet unspecified, improvement. Students may interpret this quote as supporting the inaccurate idea that evolution has a goal in mind and that individual organisms can choose to adapt. Only four chapters discussed evolution as a process, and the others talked about evolutionary patterns. Understanding the patterns is an essential component of evolutionary thinking, for example, interpreting how different lineages are related to each other. However, the text did not explicitly describe the process through which different species diverged from their most recent common ancestor. This result confirms what has been found in other studies, that even though evolution is a central theme in biology, discussion of evolutionary process is often restricted to only a few chapters in textbooks. By being aware of potentially negative impacts behind ambiguous words, educators can address common misconceptions and clarify existing naïve conceptions.

Retrotransposons exist as genetic elements within chromosomal DNA that can replicate themselves and move to new locations within a genome, using a process involving an RNA intermediate. They are a widespread class of transposable elements, abundant in plants, fungi, mammals and yeast. One type of retrotransposon is a Long Interspersed Nuclear Element (LINE). Unlike most retrotransposons, LINEs have no long terminal repeats (LTR) and they possess a poly A tail which defines the 3’ terminus of the element. They are several kilobases long and contain two open reading frames (ORFs) encoding a gag protein (ORF1) and endonuclease and reverse transcriptase domains (ORF 2) conferring the element’s ability for retrotransposition. Due to incomplete insertion attempts, many 5' truncated copies of LINE elements are present in the Rhododendron genome. A family of LINE elements called RLINE and several related 5’ truncation fragments have been discovered in Rhododendron williamsianum. I used PCR primers to amplify the regions of the genome in R. williamsianum where truncated RLINE insertions were found. The amplified DNA was then sequenced by Sanger sequencing. I am analyzing ten 5’ truncation fragments ranging from 692- 2918 base pairs. By phylogenetic analysis of aligned sequences common to the various full-length and deleted RLINES, I will endeavor to determine the evolutionary relationships of the truncated fragments to the full length RLINE and to one another. So far, I have sequenced and compared 8 different RLINE fragments of Rhododendron williamsianum, and will continue characterizing RLINE insertions.

Presently, the United States uses 19.6 million barrels of oil per day. This amount represents 25% of the world’s total consumption, making the development of an alternative biofuel one of the world’s top energy and environmental priorities. Rose Ann Cattolico’s laboratory is working to create that alternative fuel through the use of algae lipids. A number of different tests are being conducted on physiology of selected algal strains with the goal of increasing fatty acid production. A most promising algal strain has been identified. This organism (referred to as P3) was recovered from a lake in Colorado. P3 was put through reiterative flow cytometry selection, creating what is now called P4. The P4 cultures were then subjected to antibiotic selection, creating P5.5 and P5.6. My study is to: (a) determine the taxonomic identity of this organism using molecular techniques. I hypothesize that this alga is a new species in the taxon Chrysochromulina and (b) document whether P3 has mutated while in culture. To achieve these goals I will do the following experiments. (a) The closest taxonomic relatives to Chrysochromulina sp (eg. C. parva) will be compared by sequencing the 18S ribosomal RNA, psbA , and nad5 genes. The results of these experiments will help determine the relationship of Chrysochromulina sp. and other members of this taxon. (b) The divergence of “derived strains” from P3 will be determined, which all iterations of P3 – P5.6 will be examined, using the same gene set presented above. Gene sequencing will also be used to check for other iterations of P3 that have been maintained in different media. My studies will reveal whether P3 has mutated as a result of directed evolution in the laboratory.

Deuterostome adult body plans greatly differ between echinoderms, chordates and hemichordates. Genetic analysis has helped to understand the evolution of deuterostomes and the relationships between species. In this project, we have sequenced the Hox genes in Ptychodera flava, an indirect developer of the hemichordates, which has larvae that are morphologically similar to echinoderms. This information, along with gene expression data, gives insight to the similarities in developmental patterns between phyla as well as insight to their evolution. Once the genomic organization of Ptychodera flava Hox genes and Hox gene expression is complete, we can begin to deduce the developmental pattern of the common ancestor of hemichordates and echinoderms. Since the Hemichordate phyla contains both indirect and direct developers that are equally related to echinoderms, which are indirect developing, a deep comparison between gene expression and genomic organization will help us understand their common ancestor. We approached this by sequencing Bacterial Artificial Chromosomes (BACs) that contain the P. flava Hox complex and by examining gene expression via in situ hybridization. We are specifically interested in whether the Hox complex is rearranged, as in echinoderms, or co-linear, as in chordates and where the genes are expressed during different stages of development. Results show that P. flava Hox 5 through Hox 11/13c are collinear on the chromosome, but the arrangement of Hox 1 through Hox 4 is still unknown.