Dissertations and Theses in Biological Sciences

Small Interfering RNA-Mediated Translation Repression Alters Ribosome Sensitivity to Inhibition by Cycloheximide in Chlamydomonas reinhardtii

Xinrong Ma — Mon, 22 Apr 2013 07:30:53 PDT

RNA interference (RNAi) is an evolutionarily conserved gene silencing mechanism in eukaryotes, with regulatory roles in a variety of biological processes, including cell cycle, cell differentiation, physiological and metabolic pathways, and stress responses. RNAi can function by transcriptional silencing, mRNA target cleavage, translation repression and/or DNA elimination. In this study, we used the unicellular green alga Chlamydomonas reinhardtii as a model system to study RNAi-mediated translation repression. We demonstrated that small RNAs (sRNAs) generated from exogenously introduced inverted repeat transgenes, with perfect complementarity to the 3’UTR of a target transcript, can inhibit protein synthesis, without or with only minimal mRNA destabilization. In addition, there are no changes in the polyadenylation status of sRNA-repressed transcripts. Moreover, the translationally repressed mRNAs remain associated with polyribosomes, suggesting that sRNA-mediated silencing occurs at a post-initiation step of translation. Intriguingly, we consistently observed reduced sensitivity of the ribosomes associated with these repressed transcripts to inhibition by antibiotics such as cycloheximide, both in ribosome run-off assays and in in vivo experiments. Our results suggest that sRNA-mediated repression of protein synthesis in Chlamydomonas may involve alterations to the function/structural conformation of translating ribosomes. Additionally, since sRNA-mediated translation inhibition is now known to occur in a number of phylogenetically diverse eukaryotes, this mechanism may have been a feature of an ancestral RNAi machinery.

Advisor: Heriberto Cerutti

Spontaneous male death and monogyny in the dark fishing spider Dolomedes tenebrosus Hentz, 1843 (Araneae, Pisauridae)

Steven K. Schwartz — Fri, 19 Apr 2013 14:51:15 PDT

Male animals typically attempt to mate with multiple females in order to increase their reproductive success. In some species, however, males instead invest in fertilizing the eggs of a single female. Monogyny (male monogamy) is found in a diverse assemblage of taxa, and recent theoretical work reveals that a male-biased sex ratio can favor the evolution of this relatively rare mating system. We integrate this theoretical framework with field observations and laboratory experiments involving the sexually size dimorphic fishing spider, Dolomedes tenebrosus. Results from mating trials revealed a novel form of self-sacrifice behavior where males spontaneously die when they copulate, thus all males are monogynous. Since all males experience obligate death when they mate, we set out to determine if male self-sacrifice in D. tenebrosus is adaptive. Self-sacrifice behavior (complicity in cannibalism or spontaneous death associated with copulation) can be adaptive if it facilitates sexual cannibalism, and if sexual cannibalism results in reproductive benefits for the self-sacrificing male. In a first series of experiments, we examine variation in female cannibalistic behavior and variation in female mating rate. We test the hypothesis that spontaneous male death facilitates postcopulatory sexual cannibalism and that sexual cannibalism reduces the likelihood of female remating. We found that spontaneous male death does indeed facilitate sexual cannibalism, as all females cannibalize males postcopulation. However, sexual cannibalism does not reduce the likelihood of female remating, thus D. tenebrosus males do not appear to receive a paternity advantage from postcopulatory sexual cannibalism. In a second series of experiments, we examine how sexual cannibalism affects female fecundity. We test the hypothesis that the consumption of a male by a female postcopulation results in an increase in the quantity and/or quality of offspring. We found that sexual cannibalism causes females to produce more and higher quality offspring. Specifically, when females were allowed to consume their mating partner, it resulted in a significant increase in the number, the size, and the survivorship of offspring. Our results document a novel case of adaptive self-sacrifice in the first non-araneoid spider, D. tenebrosus, providing evidence that males benefit from sexual cannibalism through paternal effort.

Advisors: Eileen A. Hebets and William E. Wagner Jr.

Telomere-Related Factors and Human Papillomavirus Genome Maintenance

Adam Rogers — Fri, 19 Apr 2013 10:01:48 PDT

Human papillomaviruses (HPVs) are small DNA tumor viruses identified by their characteristic ability to replicate as a nuclear plasmid in mitotically active basal keratinocytes. A key characteristic of the HPV life cycle is the establishment of a stable maintenance phase wherein the virus replicates at low copy number, which likely occurs in cells expressing little to no E1 and E2. It is thought that HPV16 replicates in a once-per-cell-cycle manner during this portion of its life cycle and presumably interacts with host chromosome replication and maintenance factors to facilitate this replication. The adaptive radiation of papillomaviruses in response to changing host factors was well demonstrated in this work with an examination of the evolution of the Papillomaviruses’ E2 proteins and cognate binding sites as the virus has adapted to infect new body tissues. Additionally, the yeast model of HPV replication we utilize in our laboratory showed a varying ability to replicate in S. cerevisiae, again demonstrating that the replication environment plays a significant role in the long-term success of papillomaviruses.

To further investigate these cellular factors, we investigated the role telomeric maintenance factors may play in these processes. We have performed ChIP assays that have shown that components of the telomere maintenance complex (shelterin) can bind to at least four sites in the HPV genome, each of which contain nine-base telomere-repeat sequences (TTAGGGTTA). We have shown that mutating these sites has a detrimental effect on the virus’s ability to replicate under certain conditions. The shelterin complex interacts with a number of important chromosome replication and maintenance proteins with such diverse functions as DNA replication, chromosome segregation, and DNA repair, making it an ideal target for coercion by a DNA virus utilizing a stable low-copy replication strategy. Interaction between Telomer Repeat Binding Factor 2 (TRF2) and Epstein Barr Virus Nuclear Antigen 1 (EBNA1) protein (a structural and functional homologue of E2) is required for replication of plasmids containing the Epstein Barr Virus latent origin of replication. Kaposi’s Sarcoma Herpesvirus (KSHV) Latency Associated Nuclear Antigen (LANA) protein (another homologue of E2) also interacts with TRF2. Results from our Far-Western and co-immunoprecipitation assays show that E2 interacts with TRF2 and other shelterin components. In summary, these results suggest that TRF2, TRF1, Rap1, Pot1, and Tin2, plus certain DNA repair proteins, may regulate the maintenance phase of the HPV lifecycle. E2 appears to be capable of mediating these interactions.

Advisor: Peter C. Angeletti

EVOLUTION OF HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 CLADE C ENVELOPE V1-V5 REGION DURING DISEASE PROGRESSION IN NON-HUMAN PRIMATE MODEL

For Yue Tso — Tue, 16 Apr 2013 08:25:36 PDT

Human immunodeficiency virus type 1 (HIV-1) clade C strain is the fastest spreading HIV-1 strain globally, especially in Africa. It has been decades since the acquired immune deficiency syndrome (AIDS) pandemic first started. However, an effective anti-HIV-1 vaccine is not yet available, which is partly due to the highly variable nature of HIV-1 envelope gene and the absence of a suitable animal model. Strengthening of the understanding of envelope evolution during disease progression will contribute significantly towards future anti-HIV-1 treatment and preventions.
Non-human primates have been an essential animal model for many biomedical research areas. Using simian-human immunodeficiency virus (SHIV) expressing the HIV-1 clade C V1-V5 envelope regions from Zambia and infecting rhesus macaque, we demonstrated close genetic evolution of the HIV-1 envelope in both non-human primates and patient during disease progression. Utilizing this animal system, we further examined the evolution of the biological properties of HIV-1 envelope and its association with disease progression. In addition, we applied next generation sequencing technology to analyze the genetic evolution of HIV-1 envelope in a different non-human primate, pigtailed macaque, which was infected with a closely related SHIV strain. Our analysis showed that HIV-1 envelope underwent similar genetic evolution through the disease course in three different primate species; the human host and two non-human primate species.
In summary, our studies have validated the relevance of SHIV infected non-human primates as the ideal animal model for HIV-1 research. In addition, we have also chronicled the changes in biological characteristics of the HIV-1 envelope through disease progression. Thus, our results underscore the dynamic evolutionary relationships between the intrinsic properties of the HIV-1 envelopes, their evolution and host immune response. Findings from this study may contribute to the eventual resolution of the AIDS pandemic and our knowledge of the roles that HIV-1 envelope plays during disease progression.

Advisor: Charles Wood

COMPARATIVE STUDIES OF DIFFERENTIAL GENE CALLING METHODS FOR RNA-SEQ DATA

Ximeng Zheng — Thu, 06 Dec 2012 12:40:48 PST

RNA-Seq is a recently developed technology that can reveal RNA expression profile by taking advantage of deep sequencing. This new technology has many advantages over microarray technologies. Although RNA-Seq is expected to overtake microarray experiments due to their massive amounts of data produced, it presents many challenges to bioinformatics research regarding efficient data processing and storage, and accuracy in data interpretation. One of the challenges and also an important aspect of expression profiling is to detect differentially expressed genes between different experimental conditions. Several statistical methods have been developed over the past few years. In this study, we chose two representative methods: one parametric method, DESeq, and one nonparametric method, NOISeq. We compared the performance of these two methods using simulated and real datasets. We showed that both DESeq and NOISeq identified over-expressed genes more correctly than under-expressed ones. While DESeq was more likely to call longer genes as differentially expressed, NOISeq did not show such bias. When the underlying variation increased, both methods showed higher falsepositive rates at the same threshold. When replicates were not available, both methods showed lower true-positive and higher false-positive rates. Finally, we explored a strategy to combine the results from DESeq and NOISeq when replicates are available. We showed that it is possible to improve differential gene-calling results by combining the results obtained from the two methods. NOISeq is recommended when no replicate is available.

Advisor: Etsuko Moriyama

Transcriptional analysis of cervical epithelial cell responses to HIV-1

Andrew A. Block — Thu, 06 Dec 2012 11:30:42 PST

Human Immunodeficiency Virus type 1 (HIV-1) infection causes a growing pandemic throughout the world, of which women comprise 51% of people who live with HIV-1, more than 60% in sub-Saharan Africa. HIV-1 infections of women are mainly acquired through female reproductive tract where cervical and vaginal epithelial cells are the first line of defense. Although HIV-1 does not directly infect epithelial cells, HIV-1 obligatorily interacts with and crosses over epithelial layer to infect susceptible target cells, mainly CD4+ T cells, in the lamina propria to initiate an infection. However, the mechanism and ramification of the interaction of HIV-1 and epithelial cells in vaginal transmission of HIV-1 remain to be elucidated. We hypothesized that cervical epithelial cells are not a passive barrier, but actively respond to HIV-1 to modulate the mucosal milieu and facilitate HIV-1 transmission. We tested this hypothesis by studying the responses of cervical epithelial cells to HIV-1 through profiling genome-wide transcription, analyzing of cytokine and chemokine proteins, and confirming some differentially expressed key genes in rhesus macaques model of SIV infection. We found: 1) cervical epithelial cells actively respond to HIV-1. Five hundred forty-three transcripts/genes in cervical epithelial cells were significantly altered in expression at four hours post exposure to HIV-1, of which many relate to important signaling pathways, such as innate immune responses, pattern recognition receptors, apoptosis, biosynthesis, and energy production, 2) HIV-1 increases the expression of CXC Chemokines (IL-8, CXCL1 and CXCL3) in cervical epithelial cells. IL-8 and CXCL1 are potent chemotactic for multinuclear neutrophils (MNP), monocytes and a minority of lymphocytes, and CXCL3 is predominant chemotactic for monocytes, 3) HIV-1 increases the expression of key inflammatory enzymesCOX-1 and COX-2. COX-1 is responsible for the production of prostaglandins that are important for homeostasisi, and COX-2 is a key enzyme to convert arachidonic acid to prostaglandins, key inflammatory mediators, and 4) the increased expression of IL-8 and COX-2 revealed using microarraywas mapped to the endocervical epithelial cells of the macaques intravaginally inoculated with SIV in vivo. Our date lead to a role model of epithelial cells in HIV-1 vaginal transmission, that is the axis of HIV-1, epithelial cells, proinflammatory molecules (IL-8, CXCL1, CXCL3, COX-1 and COX-2), cell recruitment (MNP, monocytes and T cells), and inflammation. This model implies that moderating epithelial proinflammatory response to HIV-1 may be a utilityto prevent of HIV-1 vaginal transmission.

Advisor: Qingsheng Li

Characterization of Binding and Fusion Efficiencies Mediated by the V1-V5 Env Derived from Transmitted and Non-transmitted Viruses Isolated from a Perinatal Transmission Cohort from Zambia

Mackenzie Waltke — Thu, 11 Oct 2012 13:25:34 PDT

Human Immunodeficiency Virus Type 1 (HIV-1) is the etiological agent of acquired immunodeficiency syndrome (AIDS), which affects over 34 million people worldwide. In sub-Saharan Africa where access to antiretroviral therapies (ART) is limited, mother-to-child transmission (MTCT) rates remain high and represent a major concern in the global HIV/AIDS epidemic. Little is known about the biological properties of viruses that are transmitted perinatally, including how the biological functions of envelope (Env) influence transmissibility. Previously, transmitted viruses were found to have an advantage in replicative fitness mediated by Env V1-V5. In this study viruses derived from transmitting mother infant pairs (MIPs) were used to determine if the binding and fusion activities are influenced by Env V1-V5 and if any differences correlate to replicative fitness and transmission. Fusion and binding assays were used to measure the biological functions of Env from individual clones of five MIP. RESULTS: Neither binding nor fusion is predictive of transmission. However, clonal variation in both functions is observed, demonstrating V1-V5 is capable of influencing fusion and binding. Fusion correlates with infectivity, but does not correlate with binding. The results of this study have provided new insights to better understand the functional properties of Env and its role in MTCT.

Advisor: Charles Wood

THE PSEUDOMONAS SYRINGAE TYPE III SECRETION SYSTEM: THE TRANSLOCATOR PROTEINS, THEIR SECRETION, AND THE RESTRICTION OF TRANSLOCATION BY THE PLANT IMMUNE SYSTEM

Emerson Crabill — Tue, 24 Jul 2012 14:07:02 PDT

Pseudomonas syringae is a Gram-negative plant pathogen whose virulence is dependent upon its type III secretion system (T3SS), a nanosyringe that facilitates translocation, or injection, of type III effector (T3E) proteins into eukaryotic cells. The primary function of P. syringae T3E proteins is suppression of plant immunity. Bacterial proteins called translocators form a translocon that forms a pore in the host plasma membrane which is traversed by T3Es. HrpK1, a putative P. syringae translocator, is a type III-secreted protein important for virulence and T3E injection, but not secretion of T3Es. Harpins are a group of proteins specific to plant pathogens that are also important for T3E translocation. P. syringae pv. tomato DC3000 has 4 harpins – HrpZ1, HrpW1, HopAK1, and HopP1. Here, HrpK1 is confirmed to be a translocator. HrpK1 had a greater impact on T3E translocation than the harpins. HrpK1 and HrpZ1 disrupted liposomes. Both proteins interacted with phosphatidic acid which interfered with T3E translocation. HrpJ, a type III-secreted protein required for HrpZ1 secretion, was also required for secretion of HrpK1, HrpW1, and HopAK1. A hrpJ mutant secreted elevated levels of the Hrp pilus protein HrpA1. HrpJ appears to control transition from Hrp pilus secretion to translocator secretion. Secretion was complemented by secretion incompetent HrpJ derivatives indicating that HrpJ controls secretion from inside the bacteria. The hrpJ mutant expressing secretion incompetent HrpJ was reduced in virulence but was complemented by HrpJ expressed inside plant cells. Additionally, transgenic Arabidopsis plants expressing HrpJ were reduced in their immune responses indicating that HrpJ can suppress plant immunity. Plants pretreated with an inducer of pathogen-associated molecular pattern-triggered immunity are unable to produce an HR. Plants, as an immune response, have evolved the ability to block T3E translocation when plant immunity has been induced prior to bacterial inoculation. This is especially true in non-host interactions whereas virulent bacteria appear to be able to attenuate injection restriction in host plants via T3E activity.

Adviser: James R. Alfano

Foraging Challenges: Unsuitable Prey and Limited Information

Travis M. Hinkelman — Wed, 18 Jul 2012 06:21:58 PDT

Food acquisition is a complicated task. The profitability of potential food items depends on numerous factors, including the spatial distribution, probability of detection and capture, and suitability of the food. Animals faced with such challenges can use relatively simple mechanisms to maximize foraging efficiency. However, mechanisms that maximize foraging efficiency under some ecological conditions (e.g., prey scarcity) may produce ostensibly suboptimal behavior under different ecological conditions (e.g., prey abundance). In the work presented here, we explore two facets of foraging: (1) consuming unsuitable prey, and (2) searching for resources with limited information about resource location. To explore the consequences of consuming unsuitable prey on predator behavior, we first measured the suitability of two aphid species, black bean aphids and pea aphids, for a native predatory insect, the convergent ladybird beetle. Ladybird larvae had lower larval survival, longer developmental times, and lower adult weights on a diet of bean than pea aphids. We found that ladybird larvae killed bean aphids even if pea aphids were abundant, presumably because bean aphids were easier to capture than the pea aphids. Consumption of even a single bean aphid had pronounced short-term (< 1 day) effects on predator behavior. Ladybird larvae had longer handling times, longer bouts of inactivity, shorter bouts of intensive search, and lower patch-leaving tendencies after eating a bean aphid than after eating a pea aphid. The general lethargy from eating bean aphids may reduce the foraging efficiency of ladybird larvae. We built a simulation model to explore the performance of composite search strategies on landscapes where resource distributions ranged from dispersed to clumped. The search strategies involved switching between intensive and extensive modes based on either resource encounters or sensory cues. We found that the search strategy based on sensory cues outperformed the search strategy based on resource encounters across all resource distributions and was more robust to changes in the resource distribution.

Adviser: Brigitte Tenhumberg

Sociality and Reconciliation in Monk Parakeets (Myiopsitta monachus)

Lynnsey Lee Morrison — Thu, 26 Apr 2012 08:37:04 PDT

For animals living within socially complex groups, it is beneficial for all individuals to maintain group cohesion. Conflicts often arise in groups, which could potentially have high costs both to the subordinate and dominant members, and could lead to group instability. It has been shown numerous times that highly social group members participate in affiliative relationships with other group members discriminately. They also perform related behavior patterns such as reconciliation at a high rate, which could strengthen group stability. Reconciliation occurs when an affiliative interaction takes place between two individuals following an agonistic encounter. The rate of affiliation is higher after the conflict than had no conflict occurred. In several species, reconciliation appears to be shown more often between individuals with “mutually valuable relationships.” I tested the occurrence of reconciliation in a group of six individual monk parakeets. Monk parakeets likely live in complex societies and thus are good candidates for tests of reconciliation. First I developed an ethogram and quantified social behaviors. Multidimensional scaling of a hypergeometric similarity matrix allowed me to functionally define behaviors as affiliative or agonistic. A weighted score was assigned to each behavior based on these analyses. Each dyadic interaction between pairs of birds was then analyzed to see if they were significantly affiliative or agonistic. Among the 15 dyads, I found that six dyads showed behavior patterns that were primarily affiliative and four showed patterns that were primarily agonistic. In the reconciliation experiment, seven out of 13 dyads reconciled (54%), including five of the six dyads that were primarily affiliative. The results are consistent with the hypothesis that reconciliation occurs mainly in “mutually valuable relationships.” This is the first study to demonstrate reconciliation in an avian species.

Herbivory affects patterns of plant reproductive effort and seed production

Natalie M. West — Mon, 23 Apr 2012 13:41:21 PDT

Insect herbivory can have a major influence on plant reproduction, and potentially drive selection for strategies that reduce or resist herbivore effects. I used a combination of field experiments and ecological modeling to examine how modifications in the patterns and timing of reproductive investment might ameliorate the consequences of herbivore damage for plant reproduction. I performed experiments to examine how changes in reproductive effort after apical damage and reduction of insect herbivory affected seed production in two thistles native to Nebraska. I then used field data to parameterize a life history model predicting the resource allocation among buds and size and timing of flowering that would optimize fitness under a continual risk of herbivory.

In monocarpic Cirsium canescens, insect herbivores had a severe impact on plant seed production. Plants did modify reproductive effort in response to apical damage. High seed production from a large apical head, as well as increased flowering and seed production with apical damage from later flower heads, played a role in improving seed production. However, changes in flowering and investment patterns were insufficient to compensate for high insect damage; plants had lower seed set under ambient herbivory. We found similar effects in the iterocarpic Cirsium undulatum, although plant responses were not consistent between years. The combination of these two experiments allowed us to quantify the influence of plant reproductive response on the consequences of insect damage, and how it varies between plants with different life history strategies.

To better understand how the risk of insect herbivory might shape optimal plant allocation patterns, I constructed a stochastic dynamic programming model (SDP) to examine the optimal allocation between flower heads through time, and the size and time at which buds should flower to maximize fitness. The model predicts optimal allocation patterns should vary with survival risk, and plants should favor strategies that reduce the duration of risk. Both the model and experiments demonstrate the pressure insect herbivores can exert over plant reproductive strategies, and broaden our understanding of how ecological interactions can affect influence basic life history decisions.

Advisors: Svata Louda & Brigitte Tenhumberg

ASSESSING PATTERNS OF HYBRIDIZATION AND MULTIPLE MECHANISMS OF REPRODUCTIVE ISOLATION BETWEEN ERYTHRONIUM ALBIDUM AND ITS CONGENER E. MESOCHOREUM

Kathy Roccaforte — Wed, 18 Apr 2012 13:04:07 PDT

Although there are approximately 250,000 extant angiosperm species, we still have much to learn about the speciation process, including the ways in which species boundaries are maintained among closely related taxa. Species are formed when populations become reproductively isolated from each other via genetic and morphological barriers that act before hybrid formation (prezygotic) or after hybrids are formed (postzygotic).

Erythronium albidum Nutt. and Erythronium mesochoreum Knerr (Liliaceae) differ in ploidy, are likely sister species, and have been reported to hybridize, making them well-suited for assessing the strength of multiple reproductive barriers. First, I assessed the frequency of hybrid occurrence at four contact zones throughout the U.S. Midwest. Hybrids were identified based on genome size using flow cytometry. I found that hybrids occurred infrequently, indicating that reproductive isolation between the study species is strong.

Next, I assessed the contributions of numerous pre- and postzygotic barriers to species boundary maintenance between E. albidum and E. mesochoreum. Using herbarium records from Midwestern states and study plots in eastern Nebraska, I found that flowering phenology for each species differs significantly on a broad geographic scale but can overlap substantially on local scales. This indicates that flowering asynchrony is not a consistently strong reproductive barrier. Further, by capturing insects visiting E. albidum and E. mesochoreum flowers, I found that the plants’ pollinator assemblages are significantly non-overlapping, which may serve as a strong reproductive barrier by severely limiting interspecific pollen transfer. Finally, in a hand-pollination experiment, I found that hybrid seed set was significantly lower than conspecific seed set.

Overall, these studies show that multiple reproductive barriers contribute to the maintenance of species boundaries between E. albidum and E. mesochoreum. However, these barriers varied in strength. Though many previous studies have emphasized the role of individual reproductive barriers for species formation and perpetuation, my results highlight the importance of considering the role of multiple barriers in species boundary maintenance among plants.

Advisors: Sabrina E. Russo and Diana Pilson

Gene Duplication and the Evolution of Hemoglobin Isoform Differentiation in Birds

Michael T. Grispo — Thu, 12 Apr 2012 17:11:57 PDT

The majority of bird species co-express two functionally distinct hemoglobin (Hb) isoforms in definitive erythrocytes: HbA (the major adult Hb isoform, with α-chain subunits encoded by the α^A-globin gene) and HbD (the minor adult Hb isoform, with α-chain subunits encoded by the α^D-globin gene). The α^D-globin gene originated via tandem duplication of an embryonic α-like globin gene in the stem lineage of tetrapod vertebrates, which suggests the possibility that functional differentiation between the HbA and HbD isoforms may be attributable to a retained ancestral character state in HbD that harkens back to a primordial, embryonic function. To investigate this possibility and to examine other aspects of the evolution of the avian α-like globin genes, in collaborative effort with the Roy E. Weber lab, Joana Projecto-Garcia, Chandrasekhar Nataraja, and Hideaki Moriyama, we conducted a combined analysis of protein biochemistry and sequence evolution to characterize the structural and functional basis of Hb isoform differentiation in birds. The main objectives were: (1) to characterize the O₂-binding properties of HbA and HbD in species that are representative of several major avian lineages; (2) to gain insight into the possible structural basis of the observed functional differentiation between the HbA and HbD isoforms; and (3) to determine whether functional differentiation between the HbA and HbD isoforms is primarily attributable to post-duplication substitutions that occurred in the α^A- and α^D-globin gene lineages, or whether the differentiation is attributable to substitutions that occurred in the single-copy, pre-duplication ancestor of the α^D- and α^E-globin genes, in which case the distinctive properties of HbD may represent a retained ancestral character state that is shared with embryonic Hb. Functional experiments involving purified HbA and HbD isofoms from 12 different bird species confirmed that HbD is characterized by a consistently higher O₂-affinity in the presence of allosteric effectors such as organic phosphates and Cl^- ions. In the case of both HbA and HbD, analyses of oxygenation properties under the two-state Monod-Wyman-Changeux allosteric model revealed that the pH-dependence of Hb-O₂ affinity stems from changes in the O₂ association constant of deoxy (T-state) Hb. Ancestral sequence reconstructions indicated that the replacement substitutions that distinguish the avian α^A- and α^D-globin genes occurred exclusively on post-duplication branches of the gene family phylogeny, suggesting that the observed functional differences between the HbA and HbD isoforms are not attributable to the retention of an ancestral (pre-duplication) character state in the α^D-globin gene.

Adviser: Jay F. Storz

MSH1 INFLUENCE ON PLANT MITOCHONDRIAL GENOME RECOMBINATION AND PHENOTYPE IN TOBACCO

Peibei Sun — Fri, 24 Feb 2012 13:52:34 PST

Plant mitochondrial genomes are composed of unusually complex structures, due to active recombination at numerous repeated sequences in the genome. The maintenance of mitochondrial genome stability is under the control of identifiable nuclear genes. In plants, three nuclear genes (MSH1, RECA3 and OSB1) have been shown to participate in recombination surveillance and the suppression of illegitimate recombination in mitochondria. Disruption of these loci in Arabidopsis results in reproducible mitochondrial genome rearrangements. We demonstrated that repeat-mediated de novo recombination was also enhanced in both Arabidopsis and tobacco during passage through in vitro culture. Furthermore, in vitro conditions led to suppression of MSH1 and RECA3 expression. Subsequent regeneration processes restored normal MSH1 transcript levels and mitochondrial DNA configuration in tobacco. Our results show the utility of in vitro culture as an effective means to study the dynamic features of plant mitochondrial genomes and to facilitate more complete mitochondrial sequence assembly in plants. Disruption of the nuclear gene MSH1, which functions in maintaining mitochondrial and chloroplast genome stability, produces an array of unusual plant growth phenotypes and imparts stress tolerance in Arabidopsis. Similarly, transgenic suppression of MSH1 by RNA interference in crop plants (tobacco, tomato and soybean) produces mitochondrial genome alterations and the associated phenotype of cytoplasmic male sterility. We have observed other phenotypes in MSH1-RNAi tobacco, including dwarfism, enhanced branching, altered leaf morphology, and delayed flowering. The dwarfed growth phenotype was partially reversed by application of gibberellic acid (GA). We have characterized these novel phenotypes, and shown them to be heritable in lines lacking the RNAi transgene. We have also investigated their behavior in crossing.

Life history tradeoffs and phenotypic plasticity: The tale of a flight polyphenic field cricket

Chandreyee Mitra — Mon, 05 Dec 2011 08:32:36 PST

Most organisms are resource limited. Such limitations can result in tradeoffs between life history traits -- any traits that affect survival or reproduction. Flight polyphenic field crickets are thought to be a classic example of such a life history tradeoff, in which individuals tradeoff investment in flight capability and investment in reproduction. This polyphenism results from the interaction of two morphological traits: wing morphology (short or long) and flight muscle morphology (functional pink or non-functional white), and is affected by both genes and the environment. I examined life history traits of a flight polyphenic field cricket, Gryllus lineaticeps. First, I investigated whether females and males of flight capable and flightless morphs express alternative reproductive tactics congruent with their alternative life histories. I hypothesized that individuals in poor environments invest in flight capability, making it easier to locate mates, at the cost of early reproduction, while individuals in good environments invest in early reproduction, at the cost of flight capability. My results supported these hypotheses in both females and males. Next, I investigated costs, benefits, and constraints on the environment specific expression of life history traits. Having found that individuals with developed flight muscles pay a reproductive cost, I asked whether they gain flight capability. I found that only individuals with both long wings and developed flight muscles can fly. In addition, I found that flight muscle development and breakdown have correlated effects on other traits such as jumping ability, a trait used to escape predators and therefore likely to have survival consequences, leading to the conclusion that, how resources are allocated between flight capability and reproduction may be constrained from tracking environmental shifts due to selection for/against correlated traits. Lastly, I examined this life history tradeoff in the field. I found that different morphs of field caught individuals tradeoff flight capability and reproduction, by varying investment in reproductive tissues and lipids used for energy storage and egg production. This series of experiments provides a comprehensive look at life history evolution in a phenotypically plastic species.

Advisers: William E. Wagner Jr. and Anthony J. Zera

ANALYSIS OF TURNIP CRINKLE VIRUS EFFECTS ON THE INNATE AND ADAPTIVE IMMUNITY IN ARABIDOPSIS THALIANA

Teresa J. Donze — Wed, 30 Nov 2011 10:13:48 PST

The ability to understand the interactions between plants and the variety of pathogens they encounter on a daily basis is an important area of research. In the following work presented in this dissertation, I sought to better understand the mechanisms that Turnip crinkle virus (TCV) employs to elude the defense responses of the host plant Arabidopsis thaliana. It was previously determined that TCV coat protein (CP) interacts with a transcription factor, TIP, within the about 10 amino acid region near the N-terminus of the CP called the R-domain. When this interaction was disrupted by making single amino acid substitutions through the R-domain region, I observed a marked variation in symptom severity and alterations in both basal and resistance gene mediated responses. To further explore the effect of the TCV CP-TIP interaction on virus invasiveness and the plant defense systems, I analyzed virus accumulation and defense gene expression from the susceptible (Col-0) and resistant (Di-17) lines throughout a time course of infection. I discovered that the wildtype TCV (wtTCV) had a transient replicative advantage over CP mutants that were not able to bind TIP. This effect occurred within the first 4 to 6 days of infection. Research reported here demonstrates that the ability of wtTCV to bind TIP causes a suppression of the basal defense response that facilitates viral invasion of the systemic leaves in the susceptible ecotype Col-0.

Further experiments confirmed that TIP-CP binding also had an effect on the R-gene meditated defense conditioned by the HRT (Hypersensitive Response to TCV) gene in the resistant line Dijon-17. This was demonstrated by monitoring virus accumulation and symptom development between wtTCV and several CP mutants with altered TIP binding ability. I demonstrate that expression of the TIP gene in the presence or absence of the R-gene HRT altered development of disease symptoms and systemic spread of the virus.

A primary outcome of the research reported in this dissertation is the demonstration that the interaction of TCV CP with the TIP transcription factor modulates both major defense layers of the plant immune system. These are the basal defense layers referred to as Pathogen Triggered Immunity (PTI) defense and the development of systemic acquired resistance modulated by R-gene mediated defense referred to as the Effector Triggered Immunity (ETI) defense.

Advisor: T. Jack Morris

EFFECT OF DIET QUANITITY AND QUALITY ON FEMALE SAMPLING BEHAVIOUR AND MATING PREFERENCES IN A FIELD CRICKET

Heidi L. Bulfer — Tue, 29 Nov 2011 07:43:23 PST

Understanding the adaptive significance of variation in female mating behaviour is important because variation may often be favored by selection instead of a change in mean mating behaviour, particularly in variable environments. Females are known to adjust their mating behaviour to a variety of intrinsic and extrinsic factors including nutrition. There are multiple reasons why female behaviours might vary with nutrition; we tested two of these hypotheses: the search cost hypothesis and the direct benefits hypothesis. These hypotheses are not mutually exclusive, but they make contrasting predictions under some conditions. Low nutrition females may have less available energy to support the costs of searching for and sampling males. If sampling males is energetically costly, the search cost hypothesis predicts that low nutrition females will sample fewer males, and because they invest less in sampling, will show less biased mate choices. Alternatively, in species with male provided direct benefits, low nutrition females often benefit more from mating with preferred males. When this is the case, the direct benefits hypothesis predicts that low nutrition females will show stronger preferences for traits correlated with benefit quality and thus show more biased mate choices. The direct benefits hypothesis does not make an obvious prediction about female sampling behaviour, although greater sampling might be required to identify high benefit males. We tested these hypotheses using the variable field cricket, Gryllus lineaticeps. In this species, females receive fecundity benefits from mating with high chirp rate males but only in low nutrition females. We manipulated either diet quantity or quality (protein to carbohydrate ratio) and measured female mate preference. In the diet quality experiment, we also measured sampling behaviour. Diet quality influenced female sampling behaviour: females provided a high quality diet sampled more extensively. However, neither diet quantity nor diet quality influenced female preference. These results partially support the search cost hypothesis. Possible explanations for why diet treatments did not influence female chirp rate preference are discussed.

Advisor: William E. Wagner, Jr.

The Role of Human Ubc9 During the Human Immunodeficiency Virus Replication Cycle

Christopher R. Bohl — Tue, 29 Nov 2011 07:43:20 PST

Human immunodeficiency virus type 1 (HIV-1) is a retrovirus and the causative agent of the acquired immune deficiency syndrome (AIDS) pandemic. The retrovirus replication cycle is divided into early infectious events, which involve the infection and integration of the viral DNA into target cell chromosomes; and late events, which involve the expression of viral genes and assembly of infectious virions. To complete the replication cycle, HIV-1 utilizes various cellular pathways.

We identified the Ubc9 E2 SUMO conjugating enzyme as a HIV-1 Gag interaction partner. When this interaction was disrupted in HIV-1 producer cells by Ubc9 siRNA, the virus that was released from the cells was approximately 10-fold less infectious than virus released from control cells. The decreased virion infectivity was found to be due to decreased levels of intracellular mature Env, which in turn decreased the amount of Env packaged into assembling virions. Surprisingly, the defect in mature Env levels in Ubc9 knockdown cell was dependent upon Gag expression. The mechanism of decreased mature Env in Ubc9 knockdown cells was examined and we found that gp120/gp41 appeared to be preferentially degraded at a post gp160 cleavage step, but before transport/association with the plasma membrane and lipid rafts. The intracellular gp120 levels were restored when cells were treated with lysosome inhibitors, which suggested that the decreased intracellular Env stability is due to increased lysosomal degradation; however, inhibiting Env degradation did not restore Env packaging into assembled virions. Since lysosomal degradation is linked with autophagy, we examined autophagy in Ubc9 knockdown cells and found that autophagy was increased approximately 3-fold compared to control cells. Autophagy inhibitors were unable to block autophagy, and we were unable to determine if increased lysosomal degradation of gp120 was occurring through an autophagy dependent or independent mechanism.

We have also examined the potential role of Ubc9 during the HIV-1 early infectious events. Decreased LTR driven gene expression was detected following the infection of Ubc9 knockdown cells with VSV-G pseudotyped HIV-1 virions. These results suggested that Ubc9 plays a functional role during the early events of the HIV-1 replication cycle at a post-entry step.

Adviser: Charles Wood

THE EFFECTS OF SOCIAL CONTEXT & CACHE SURVIVAL ON PINYON JAY CACHING BEHAVIOR

Christine L. Keefe — Fri, 18 Nov 2011 12:23:22 PST

I examined how pinyon jays (Gymnorhinus cyanocephalus) assess and determine the risk of pilferage to their caches. Jays were allowed to cache in an open room while alone or while being observed by a conspecific. In a counterbalanced design, jays cached in the opposite treatment once they had finished recovering their caches. I compared birds’ behaviors between treatments in order to determine whether jays consider the presence of an observer in measuring the local competitive environment. Once all jays had completed alone- and observed- treatments, I ran the experiment once more to determine if individuals were consistent in their cache protection strategies. Results from this experiment reveal that pinyon jays did not respond to audience effects. Birds showed distinct patterns of behavior, but individuals were consistent in their behavior across treatments and replications. Jays serving as observers were tested for their ability to recover caches they had watched being made. These birds were able to accurately recover observed caches, though not as reliably as cachers.

I then used base levels of cache protection activity as assessed from the first experiment to divide birds into two groups so each had similar mean levels of cache protection behaviors. To evaluate whether pinyon jays directly assess pilferage through cache-loss, I removed 50% of the seeds cached for birds receiving the cache-removal treatment, while birds receiving the non-removal treatment were allowed to recover all the seeds they cached. Birds that participated in the non-removal treatment later participated in a cache-removal treatment so I could compare the responses to cache-removal between the two groups. To assess whether experience pilfering might influence caching decisions, I compared responses between previous observers and previous cachers. Jays that had their caches removed ate fewer seeds and cached fewer seeds when compared to the non-removal group. Of the seeds they cached, more of those seeds were cached behind shielding landmarks. These behavioral patterns were not evident in the group that participated in both treatments. Birds with experience as pilferers were more exploratory, ate more food, and expressed higher levels of cache protection behaviors during the removal treatment.

Adviser: Alan C. Kamil

The complexities of wolf spider communication: Exploring courtship signal function in Rabidosa rabida

Dustin J. Wilgers — Fri, 14 Oct 2011 11:40:51 PDT

Evidence of signal complexity is seemingly pervasive across animal communication systems. Exploring signal function may provide insight into how these displays evolved and are maintained. This dissertation examines the courtship signal function in a grassland wolf spider. Rabidosa rabida lives in an extremely complex environment, and males use complex displays incorporating both visual and seismic modalities. Using several approaches I provide insight into the content and efficacy of the various signal components, as well as how variation in these displays influence female mating decisions in isolation and combined.

First, I manipulated male and female body condition using diet quantity manipulations and performed mate choice trials using females of each diet across two different age classes. Female mate choice decisions varied with diet and age. Overall, younger females were choosy, mating more often with good condition males, while older females mated indiscriminately. Next, to determine which signal components may be useful in female mate assessment, I explored the condition-dependence of the signal components and tested their efficacy by performing mate choice trials in environments that differed in modality transmission. Both visual and seismic components are condition-dependent, and are sufficient to maintain copulation success when detected in isolation. Thus, each signal component may serve as both a content- and efficacy-backup when facing variable sensory environments. Lastly, I manipulated both foreleg ornamentation and the seismic display, and presented them to females both in isolation and combined, to determine if and how variation in each component influences female mating decisions. Females were choosy based on the seismic display alone, and only discriminated males based on foreleg ornamentation when detected along with a seismic signal, suggesting an inter-signal interaction.

Together, these experiments suggest that the sources of selection acting on male R. rabida are just as complex as the courtship displays used during mating interactions. The courtship signal components making up the display appear to function by maintaining both copulation success and mate assessment across a variety of environments encountered.