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Plants have evolved complex signaling mechanisms to sense and respond to abiotic stresses. These stresses can lead to the excess production of reactive oxygen species (ROS) that damage cellular components and can lead to cellular dysfunction and, ultimately, cell death. Chloroplasts generate large amounts of ROS, including singlet oxygen (1O2), during photosynthesis. This 1O2 can act as a retrograde signal for the selective degradation of damaged chloroplasts and the activation of programmed cell death (PCD). While such processes allow plants to maintain efficient energy production and avoid toxic ROS accumulation, the signaling mechanisms and genes involved are largely unknown. This raises two intriguing questions: How do plants sense chloroplast dysfunction and selectively degrade damaged chloroplasts to sustain healthy chloroplast populations? -and- What mechanisms do plants use to activate PCD when their chloroplasts accumulate too much 1O2 to manage via selective degradation? To answer these questions, my dissertation research has utilized both forward and reverse genetic approaches to identify and characterize genes and pathways involved in 1O2-mediated chloroplast retrograde signaling (CRS), chloroplast quality control (CQC), and PCD. The Arabidopsis plastid ferrochelatase 2 (fc2) mutant has proven to be useful as a system in which to study 1O2-induced CRS, CQC, and PCD. These mutants conditionally accumulate 1O2 under diurnal (cycling) light conditions, leading to enhancements of chloroplast degradation and cell death in stressed fc2 plants. As these fc2 mutants have a clear conditional phenotype that correlates with chloroplast degradation, they are ideal for genetic suppressor screens to identify genes that play a role in 1O2-induced CRS, CQC, and PCD. In such screens, fc2 mutants are further mutagenized to screen for secondary mutations that lead to an fc2 suppressor (fts) phenotype. Such loss-of-function screens have already yielded many suppressor mutan

Translation is a conserved and highly dynamic process that relates all domains of life. This informational processing system is mediated by a core network of translation machinery, which is hypothesized to have been present in the last universal common ancestor of bacteria, archaea, and eukarya and has tightly co-evolved throughout time. Specifically, elongation factor thermal unstable, or EF-Tu, is a universally conserved translational GTPase (trGTPase), highly expressed and essential in translation. The function and efficiency of translation elongation is critical, and therefore the dysregulation has been associated with health and disease in humans, including tumorigenesis and neurodegeneration. In this thesis, I analyze the evolution of EF-Tu, specifically focusing on ways EF-Tu may compensate and adapt in times of cell stress. In Chapter 2, we constructed the phylogeny and shared evolutionary history of essential trGTPases EF-Tu (aEF-1A/eEF-1A) and initiation factor IF2 (aIF5B/eIF5B). Our results show that the GTP-binding domain and tRNA-binding domain of the reconstructed common ancestor (IF2/EF-Tu) is more similar in protein sequence to the modern E. coli IF2, while the domain organization and structure of IF2/EF-Tu’s binding pocket is more comparable to modern EF-Tu. This work indicates that analogous to modern IF2, IF2/EF-Tu may have exhibited limited conformational change between GTP bound/unbound states. Whether the shared IF2/EF-Tu common ancestor was able to conduct both initiation and elongation functions requires further investigation. In Chapter 3, we experimentally evolved an engineered E. coli strain 3000 generations, in which modern EF-Tu was substituted for ancestral EF-Tu (AnEF) and discovered a synonymous mutation in the 5’ region of AnEF (C45T). This synonymous mutation, which appeared after 2,000 generations, is correlated with an increase in AnEF protein and mRNA levels, as well as an increase in polysome abundance during translation. Howeve

The kynurenine pathway is responsible for a majority of tryptophan metabolism. It has been intricately linked to both aging and immune function, yet its exact roles in the two processes has not been fully characterized. The only well-established regulation of the kynurenine pathway is the control of flux via activity of indoleamine 2,3-dioxygenase (IDO). We have chosen C. elegans and M. musculus to establish how some kynurenine pathway components impact aging and immune function and how these components are regulated. We found that accumulation of the kynurenine pathway intermediate metabolite 3-hydroxyanthranilic acid (3HAA) through supplementation or inhibition of the catabolizing enzyme 3-hydroxyanthranilate 3,4-dioxygenase (HAAO) increases lifespan and several aspects of health. We also found that these kynurenine pathway interventions improve pathogen response and decrease infection progression. The kynurenine pathway is normally upregulated during pathogen response in C. elegans but we found that 3HAA may be regulated via tissue separation of synthesis by kynureninase (KYNU) in the hypodermal tissue and degradation by HAAO in the neuronal tissue. The special temporal separation of synthesis and degradation of 3HAA allow for intermediate function of the metabolite not previously characterized. Ultimately, 3HAA’s impact on immune function may be due to antimicrobial activity and localization to the interface between host and pathogen in the gut granules. The special temporal regulation of 3HAA combined with the immune enhancement may, in part, explain the life extending properties of supplementation and HAAO inhibition. This may also provide further insight into the overall role of these kynurenine pathway components in aging and immune function.

Participation in laboratories during an undergraduate’s science education provides unique opportunities to engage in constructing knowledge using the very tools, techniques, and practices scientist use while investigating phenomenon. Learning outcomes from participating in laboratory experiences that position students as generators and critiquers can develop students' understanding of the nature of science and shape how they view themselves in science. Science-practice based laboratory curricula such as inquiry-based laboratory courses or course-based undergraduate research experiences have shown positive outcomes in students’ affect and even influencing student trajectories in STEM. One such affective learning outcome is the development of a student’s science identity. We focused on investigating mechanisms leading to a student’s change in their science identity as they participated in an authentic inquiry laboratory course. We used a microgenetic case study and operationalized a community of science practice framework as a lens to investigate how a science identity formed in a student as a result of their participation in a novel introductory biology laboratory course. Our results detail the impact the learning community played in our case study participants experience and the importance of curricular decisions to grant students epistemic agency and authority to construct knowledge. We also investigated differences in student’s affect that resulted from students participating in two different introductory biology curricula. In this comparison study, we had the unique opportunity to investigate how two different curricula support learning outcomes differentially. We used a quantitative approach to measure change in students perceived science self-efficacy, science identity, and science values using a validated Likert scale survey in a pre-post method. We also measured the impact of curricula on student’s science self-efficacy, science identity, and science values f

Tremendous progress has been made over the last century characterizing the structure, function, and evolutionary dynamics of protein-coding genes. Over the last three decades, biologists have sequenced the genomes of a variety of organisms, and it quickly became apparent that a relatively small portion of most genomes encode protein-coding genes. We now know that the non-protein-coding space contains repetitive sequences along with non-coding RNAs. Many of these non-coding RNAs encode housekeeping transcripts such as: ribosomal RNAs, transfer RNAs, small nucleolar RNAs, and small nuclear RNAs. However, the remaining transcripts include regulatory RNAs that have become the focus of intense investigation over the past two decades. These include the microRNAs and the small interfering RNAs which both act to knock down gene expression. More recently, a class of transcripts called long non-coding RNAs (lncRNAs) have been interrogated. One well described, functional lncRNA is the telomerase RNA which is necessary for genome stability via the maintenance of chromosome ends following DNA replication (Dew-Budd et al. 2020). While lncRNAs have been known for more than two decades, their functions have remained enigmatic; there are few functionally characterized lncRNAs. However, lncRNAs that have been characterized, especially in mammalian systems, appear to fine tune a plethora of developmental and regulatory processes, along with responses to environmental cues. LncRNAs are frequently expressed at low levels and in specific tissues; they are often poorly conserved among closely related species (Palos et al. 2021). As high-throughput RNA-sequencing becomes more widely available and used, researchers have employed the resulting data to identify lncRNAs en masse. For mammals, a variety of high-quality resources have been used to generate informative annotations to allow for improved hypothesis generation. These resources include GENCODE (Frankish et al. 2021), coupled with lnc

Prions modulate cellular phenotypes by adopting alternative folds called amyloid that change their function and act as templates to convert natively folded protein to the amyloid state. The initial appearance of an amyloid is rare but enhanced by the presence of other aggregated proteins. Previous work has suggested this enhancement arises from the ability of pre-existing aggregates to template amyloid assembly but fails to explain all experimental observations. Our studies in S. cerevisiae suggest an alternative role for pre-existing aggregates: the titration of chaperones, which normally act to disassemble nascent amyloid nuclei. Using the [PSI+]/[PIN+] dual prion system, we demonstrate that the presence of [PIN+] reduces the binding of the molecular chaperone Hsp104 to Sup35 amyloid, the determinant of the [PSI+] prion. In turn, this change reduces the fragmentation of Sup35 amyloid, promoting appearance of the [PSI+] prion. Notably, the combination of [PIN+] and reduced levels of Hsp104 can inhibit prion appearance and propagation for some conformational variants of Sup35 amyloid, providing further evidence of titration. Using conformational variants of [PIN+] known to promote different levels of [PSI+] formation, we demonstrate that differential chaperone titration explains differences in [PIN+] variant promotion of [PSI+] induction. Thus, [PIN+] acts as a decoy for chaperones and thereby promotes persistence of nascent aggregates.

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by the loss of upper and lower motor neurons. Despite one hundred and fifty years since the first characterization of the disease, an effective treatment for patients has remained elusive. ALS patients often present with systemic metabolic defects that correlate with disease progression. Exactly how metabolic alterations contribute to disease pathogenesis is poorly understood. Our laboratory has developed a Drosophila model of ALS based on the genetic overexpression of TAR DNA binding protein 43 (TDP-43), a protein that is found in pathological aggregates in over 97% of all ALS patients. Using this model, we find metabolic alterations in long chain fatty acids and glycolysis end-products - suggesting altered mitochondrial function and altered glucose metabolism. Dietary supplementation with either medium chained fatty acids, ketone precursors, or glucose improves the locomotor function of TDP-43 expressing flies. Indeed, increased glucose availability through genetic expression of human glucose transporter 3 in motor neurons improves TDP-43 induced locomotor defects, improves synaptic vesicle function, and increases the number of boutons at the neuromuscular junction. Moreover, Pfk mRNA, an indicator of glycolytic flux, is upregulated in flies, patient derived motor neurons differentiated from induced pluripotent stem cells and ALS spinal cords. Interestingly, Pfk overexpression rescues TDP-43 induced locomotor defects – suggesting that increasing the rate of glycolysis in motor neurons is neuroprotective. Together, these findings show that there are clear metabolic alterations in the central nervous system of a Drosophila model of ALS that can be mitigated by either increasing the availability of medium-chained fatty acids, or increasing the rate of glycolysis.

Gene evolution is one of the most significant contributors to the extensive number and diversity of organisms that have arisen on planet Earth. Gene duplication is a major driving force in gene evolution by allowing the development of novel functions and the creation of new pathways. Every gene within an organism’s genome has been retained through numerous duplication events and has a unique evolutionary history that dictates structure and function. As organisms retain increasing numbers of related genes, more complex pathways evolve. Whole genome duplications have occurred numerous times within the land plant lineage and are a contributing factor in the complexity of plant genomes. The plant-specific epigenetic pathway of RNA-directed DNA Methylation (RdDM) arose through evolution of specialized genes following gene duplications in the ancestor of plants. RdDM is responsible for de novo methylation associated with transcriptional gene silencing of transposons and other repetitive sequences. Evolution of RdDM components is dynamic and ongoing with many lineage specific paralogs, leading to further complexity of the pathway. Understanding the evolutionary history and relationship among gene families can drive the formation of hypotheses regarding the molecular necessity of related genes. RdDM requires the cooperative activity of plant-specific DNA-dependent RNA polymerases (Pol) IV and V, which evolved from RNA Pol II subunits. Although many subunits are shared among the polymerases, the largest subunit of Pol IV and V are unique and harbor distinct carboxyl terminal domains. In addition to separate largest subunits, Pol IV and V possess other specialized subunits and interacting partners that influence their functions. The evolutionary origin of these subunit and interacting proteins is unclear and requires further investigation. Determining the origin and evolutionary history of these proteins will be valuable for translating research from model organisms to other

Experts in the field of molecular and cellular biology (MCB) use domain-specific reasoning strategies to navigate the unique complexities of the phenomena they study and creatively explore problems in their fields. One primary goal of instruction in undergraduate MCB is to foster the development of these domain-specific reasoning strategies among students. However, decades of evidence-based research and many national calls for undergraduate instructional reform have demonstrated that teaching and learning complex fields like MCB is difficult for instructors and learners alike. Therefore, how do students develop rich understandings of biological mechanisms? It is the aim of this dissertation work to explore features of expertise and knowledge building in undergraduate MCB by investigating knowledge organization and problem-solving strategies. Semi-structured clinical think-aloud interviews were conducted with introductory and upper-division students in MCB. Results suggest that students must sort ideas about molecular mechanism into appropriate mental categories, create connections using function-driven and mechanistic rather than associative reasoning, and create nested and overlapping ideas in order to build a nuanced network of biological ideas. Additionally, I characterize the observable components of generative multi-level mechanistic reasoning among undergraduate MCB students constructing explanations about in two novel problem-solving contexts. Results indicate that like MCB experts, students are functionally subdividing the overarching mechanism into functional modules, hypothesizing and instantiating plausible schema, and even flexibly consider the impact of mutations across ontological and biophysical levels. However "filling in" these more abstract schema with molecular mechanisms remains problematic for many students, with students instead employing a range of developing mechanistic strategies. Through this investigation of expertise and knowledge buildin

The ascomycete fungus Magnaporthe oryzae, causative agent of rice blast disease, poses a threat to global food security, destroying enough rice to feed 60 million people each year. Characterization of the host-pathogen interaction between rice and M. oryzae is critical, as better understanding of the system may lead to better disease control strategies. The sequenced genome and repertoire of molecular tools available have made M. oryzae an ideal model system for understanding general plant-pathogen interactions as well. The objective of this dissertation was to characterize the M. oryzae homologs of Histoplasma capsulatum RYP (Required for Yeast Phase) genes that are required for transition to the parasitic phase. H. capsulatum is a human pathogen that undergoes a dimorphic switch from filamentous to yeast cell growth at 37°C, the host body temperature. Four H. capsulatum RYP genes were identified in a forward genetic screen to identify genes required for entry into the yeast phase. RYP1 is a member of the Gti1_Pac2 family, which contains previously characterized regulators of dimorphic switching. RYP2 and RYP3 are homologs of vosA and velB, members of the Velvet family, best characterized in Aspergillus nidulans, where they coordinate morphological differentiation with secondary metabolism. RYP4 is a zinc binuclear cluster protein, a main class in the zinc finger transcription factor family. Deletion of the M. oryzae RYP1 homolog, RIG1 (Required for Infectious Growth), resulted in a non-pathogenic mutant on susceptible rice cultivars, even upon removal of the host penetration barrier. Δrig1 was blocked in the transition to infectious hyphal growth, similar to H. capsulatum ryp1, which could not transition to the yeast phase. Deletion mutants of M. oryzae RYP2, RYP3, and RYP4 homologs were similar to the wild type in somatic growth and pathogenicity indicating that although RIG1 is a pathogenicity factor conserved in plant and animal pathogens, such conservation doe

Transition metals such as copper, zinc and nickel are required in many enzymatic processes that require redox changes. When transition metal concentration exceeds a certain threshold, their redox and metal binding properties make these elements extremely toxic. Bacteria regulate the cellular concentration of these important, yet toxic, elements using elaborate homeostatic systems. One such mechanism is the chemiosmotic extrusion of copper by the Cus system in the Gram-negative bacterium Escherichia coli. This work studies the regulation of the Cus system in response to copper and silver ions. Copper is an essential cofactor required in many enzymatic processes. But its redox properties can lead to toxicity. Silver is chemically similar to copper, but is not bioactive and its presence in cells can lead to extreme cytotoxicity. Transcription from cusCFBA genes is controlled by the CusR/CusS TCS in response to elevated levels of copper or silver in the periplasmic space of E. coli. Extracellular signals are transduced into the cell through phosphotransfer reactions between the prototypical histidine kinase CusS and the response regulator CusR. Copper sensing by the periplasmic domain of CusS is proposed to initiate signal transduction in the Cus system. Despite the frequency with which bacteria employ histidine kinases to sense their environment, signal recognition and incorporation by the protein is not well understood. The goal of this research is to investigate the role of CusS in regulating metal homeostasis in E. coli and characterize the periplasmic domain of the protein to determine its metal binding properties. The experiments described in this work reveal that the CusS is essential for copper and silver resistance and regulates expression from the cusCFBA promoter region. Signal recognition occurs by direct metal binding by the periplasmic domain of CusS. Metal binding causes a change in the secondary structure of the domain and its tendency to dimerize is enh

The Arabidopsis HSP101 protein belongs to the Hsp100/ClpB family of AAA+ proteins (ATPases Associated with various cellular Activities). This family of proteins, in collaboration with the Hsp70 chaperone system, has the remarkable ability to solubilize protein aggregates and refold proteins back to their native forms. Thus, their chaperone activity is necessary for acquired thermotolerance in organisms as diverse as bacteria and plants. My dissertation project focused on understanding the mechanism of HSP101 action using Arabidopsis thaliana as a model system. The first approach used genetics to screen for suppressors of a specific missense mutant allele of HSP101, hot1-4, in order to find interacting cofactor proteins or key substrates of HSP101, or other processes involved in thermotolerance. Four extragenic suppressors that can overcome the heat-hypersensitive phenotype caused by the hot1-4 mutation were isolated and one of them (shot1) was identified as a mutation in a mitochondrion-targeted protein. Although shot1 mutations do not directly interfere with HSP101 function, they reveal independent mechanisms required for thermotolerance, which involve reduced oxidative stress. The second approach used to investigate HSP101 function was to affinity-purify HSP101 and identify associated proteins. For this purpose, transgenic Arabidopsis plants were generated expressing affinity-tagged wild-type and mutant variants of HSP101. As predicted, cytosolic Hsp70s were identified as an interacting partner of HSP101. Surprisingly, 26S proteasome regulatory subunits were also identified, suggesting a possible link between the protein degradation and reactivation pathways. Further experiments were also undertaken to define the importance of different domains of HSP101, as well as the localization of HSP101. Transgenic Arabidopsis plants expressing N- or C-terminally truncated HSP101 indicate that the N-terminal domain of HSP101 is required for full activity in protecting plants