<p>  </p> <p>Shikonin is 1,4-naphthoquinones produced exclusively in Boraginaceae species. The compound and its derivatives are predominantly made in roots where they function in mediating plant-plant (allelopathic) and plant-microbe interactions. Moreover, this compound has been a target for drug development due to its strong anti-cancer properties. Our genome assembly and analysis of <em>Lithospermum erythrorhizon</em> uncovered metabolic innovation events that contributed to the evolution of the shikonin biosynthesis. This metabolic innovation also reveals the evolutionary link between shikonin biosynthesis and ubiquinone biosynthesis, one of the central metabolism functions in aerobic cellular respiration. To explore additional links between these two pathways, we used a transcriptome-based network analysis which uncovered a shikonin gene network model that predicts strong associations between primary metabolic pathway genes and known shikonin biosynthesis genes, as well as links with uncharacterized genes. <em>L. erythrorhizon</em> geranyldiphosphate (GPP) synthase (<em>LeGPPS</em>) is one of the candidates predicted by the network analysis, of which encodes a cytoplasmic enzyme shown in vitro to produce GPP. Knocking down of <em>LeGPPS</em> in <em>L. erythrorhizon </em>hairy roots (<em>LeGPPSi </em>lines) results in reduced shikonin content. This result provides functional evidence that cytoplasmic LeGPPS supplies GPP precursor to the shikonin biosynthesis. <em>LeGPPSi </em>lines also increased ubiquinone content, further supporting our hypothesis on the metabolic and evolutionary connection between shikonin and ubiquinone biosynthesis. Further RNA-seq analysis of the <em>LeGPPSi</em> line showed that downregulating <em>LeGPPS</em> significantly reduces the expression of benzenoid/phenylpropanoid genes, indicating the presence of factors that coordinately regulate the pathways providing the 4-hydroxybenzoic acid and GPP precursors to the shikonin pathway. In addition to <em>LeGPPS</em>, we also found<em> ubiquinone biosynthesis protein COQ4-like </em>gene (<em>LeCOQ4-L</em>) which provided another evolutionary link between shikonin and ubiquinone biosynthesis. The enzymatic activity of canonical COQ4 is unknown. In yeast, the protein is essential for ubiquinone biosynthesis and its metabolon formation. With the existing connections between shikonin and ubiquinone biosynthesis, if LeCOQ4 functions in the same manner as yeast COQ4, it is possible that <em>LeCOQ4-L </em>has an analogous function in shikonin biosynthesis as a structural protein for stabilizing biosynthesis metabolon. This leads us to the characterization of<em> COQ4</em> ortholog in Arabidopsis (<em>AtCOQ4</em>) to gain insight into its functional mechanism. Characterization of <em>atcoq4 </em>T-DNA mutant line showed that reduced <em>AtCOQ4</em> expression resulted in reduced ubiquinone. Further subcellular localization study revealed that AtCOQ4 and <em>LeCOQ4-L</em> localize in mitochondria without conventional transit peptide. We also performed pull-down assay to identify AtCOQ4 interactors which might be the missing enzymes that cannot be identified based on homology. 80 potential AtCOQ4 interactors were found including proteins like AtCHLM, GRIM-19, and AtSSLs. However, further study is needed to verify the protein interactions captured by pull-down assay. Taken all together, our study sheds light on the metabolic innovations that give rise to shikonin biosynthesis from ubiquinone biosynthesis and provide insight into the dynamics of the metabolic networks.</p>

Microvesicle generation is an integral part of the aging process of red blood cells in vivo and in vitro. Extensive vesiculation impairs function and survival of red blood cells after transfusion, and microvesicles contribute to transfusion reactions. The triggers and mechanisms of microvesicle generation are largely unknown. In this study, we combined morphological, immunochemical, proteomic, lipidomic, and metabolomic analyses to obtain an integrated understanding of the mechanisms underlying microvesicle generation during the storage of red blood cell concentrates. Our data indicate that changes in membrane organization, triggered by altered protein conformation, constitute the main mechanism of vesiculation, and precede changes in lipid organization. The resulting selective accumulation of membrane components in microvesicles is accompanied by the recruitment of plasma proteins involved in inflammation and coagulation. Our data may serve as a basis for further dissection of the fundamental mechanisms of red blood cell aging and vesiculation, for identifying the cause-effect relationship between blood bank storage and transfusion complications, and for assessing the role of microvesicles in pathologies affecting red blood cells.

<p dir="ltr">Bacteriophages are viruses that infect, replicate in, and kill bacteria. In industries that utilize microbes for production, like <i>E.coli</i> in the production of insulin or <i>A. globiformis</i> in the production of cheese, bacteriophages can pose a huge threat to manufacturing. However, bacteriophages aren’t entirely detrimental: we can use the destructive nature of bacteriophages to kill bacterial infections in the human body. This process is known as phage therapy, and while it isn’t a new concept, it is being seen as an increasingly necessary alternative to traditional antibiotics due to the increasing rise of antimicrobial resistance. Because bacteriophages have an entirely different mechanism of destroying bacteria, they can be used in tandem with traditional antibiotic regimens to help wipe out infections. Also, phages have a highly specific host range, meaning that an injection of a certain type of phage will only infect the bacteria it is targeting, sparing important gut microbes.</p><p dir="ltr">The search for new phages to treat infections has resulted in the discovery of over 25,000 actinobacteriophages, with about 4898 of them being sequenced. This is extremely important and necessary, but 49% of these sequenced phages are all mycobacteriophages. This bias towards mycobacteriophages is likely because they infect the genus mycobacterium, where the deadly <i>M. tuberculosis</i> resides. The discovery of new phages using less studied hosts results in novel phages that exhibit rarely seen morphologies, phenotypes, and genotypes. This leads to a better overall understanding of the phage proteome and can lead to new breakthroughs in phage therapy.</p><p dir="ltr">The purpose of this research is to study the differences between different types of phages and try to determine the impact it may have on phage therapy. This thesis is divided into three chapters. In the first chapter, novel phages from different hosts, including <i>M. smegmatis</i> and <i>A. globiformis</i>, were discovered and annotated, and the differences between them were characterized. The discovery of arthrobacteriophages immediately resulted in rare and previously unseen phage characteristics. In the second chapter, proteomic mass spectrometry data of various diverse mycobacteriophages was analyzed to determine differences. Despite being from multiple clusters and lifecycles, the expression data had more similarities than differences. In the third chapter, an alternative method of extracting DNA from phages is explored to determine the result of discrepancies in gel quality from <i>M. smegmatis</i> and <i>A. globiformis.</i><i> </i>Although a large amount of nucleic material was derived, it was not stable DNA and was unsuitable for use. The reason for poor gel quality is still unknown.</p>

<p>Building a robust deep neural network (DNN) framework turns out to be a very difficult task as adaptive attacks are developed that break a robust DNN strategy. In this work we first study the bootstrap distribution of DNN weights and biases. We bootstrap three DNN models: a simple three layer convolutional neural network (CNN), VGG16 with 13 convolutional layers and 3 fully connected layers, and Inception v3 with 42 layers. Both VGG16 and Inception v3 are trained on CIFAR10 in order for bootstrapping networks to converge. We then compare the bootstrap NN parameter distributions with those from training DNN with different random initial seeds. We discover that the bootstrap DNN parameter distributions change as the DNN model size increases. And the bootstrap DNN parameter distributions are very close to those obtained from training with different random initial seeds. The bootstrap DNN parameter distributions are used to create a graybox defense strategy. We randomize a certain percentage of the weights of the first convolutional layers of a DNN model, and create a random ensemble of DNNs. Based on one trained DNN, we have infinitely many random DNN ensembles. The adaptive attacks lose the target. A random DNN ensemble is resilient to the adversarial attacks and maintains performance on clean data.</p>

<p dir="ltr">Photosynthesis is the basis of primary productivity on Earth. Oxygenic photosynthesis utilizes the nearly inexhaustible energy of radiant solar light to fix atmospheric carbon dioxide into usable forms of chemical energy and produces dioxygen as a product. Central to this process are several large hetero-oligomeric protein complexes that comprise the photosynthetic electron transport chain. Photosystem II (PSII) initiates electron transport through the light-driven oxidation of water, in-turn relinquishing protons and oxygen. Through this reaction, electrons are used to form the reductant NADPH, while protons form a proton-motive gradient that is used to drive synthesis of ATP. As a result of this highly energetic reaction, PSII is often subject to oxidative photodamage due to the production of reactive oxygen species. Inevitably, accumulation of oxidative photodamage disrupts the catalytic activity of PSII, resulting in a loss of photosynthetic activity. To deal with the nearly constant incurred photodamage to PSII, oxygenic photoautotrophs undergo a disassembly and repair cycle that results in the complete turnover of the damaged D1 subunit of PSII. Due to its high tendency for damage, the D1 subunit has a half-life of under one hour in high light intensity. Despite our current understanding of photoinhibition and PSII repair, it is still unclear how D1 is replaced so rapidly in response to damaging conditions. Previous research has indicated a role for phosphorylation of PSII in D1 turnover, however the mechanism has not been totally resolved. In the first chapter of this thesis, our current understanding of PSII phosphorylation and oxidative damage is reviewed in the context of PSII repair. In the second chapter, the role of protein phosphorylation in the PSII repair cycle is investigated in the model organism <i>Arabidopsis</i>. Using several PSII phosphorylation mutants, we demonstrate that phosphorylation seems to mediate disassembly of large PSII supercomplexes and dimers into smaller subcomplexes. In the third chapter, the role of oxidative photodamage is investigated in mediating PSII disassembly. Here, we use several <i>in vitro</i> assays to demonstrate that photodamage is sufficient to induce the disassembly of smaller PSII subcomplexes. In the fourth chapter, a technique for determining the stoichiometry of photosynthetic complexes is examined, with implications for understanding PSII repair. Finally, in the fifth chapter, several conclusions and unanswered questions from this thesis are discussed.</p>