VictorJuiz/thesis huggingface dataset:
A robust platform for high-throughput genomics in microorganisms [electronic resource]
Keywords: Genetics

Accurate, large-scale gene annotation is a major challenge in biology. Traditionally, gene function can be assigned by observing the phenotype of a null mutant under various conditions. This principle has engendered the creation of genome-wide deletion collections in which each gene in a genome is knocked out or disrupted. Phenotype can then be assayed for each gene knockout individually. Parallelization of phenotypic assays via the introduction of molecular barcodes has proved invaluable for interrogating genome-wide collections of mutants in many conditions. This approach has been most visibly successful in the case of the budding yeast Saccharomyces cerevisiae, but it has the potential to bring insight to the genomes of many more microorganisms. The strategy of creating the genome-wide mutant collections that permit parallel phenotypic analysis has not been broadly applied beyond S. cerevisiae due primarily to technical limitations. In this dissertation, we describe a universal approach to rapidly generate comparable tagged, mutant collections. This approach combines DNA tag technology with transposon mutagenesis, and thereby can be generalized to any microorganism amenable to transposon mutagenesis. We created a universal collection of tags whose utility can extend to any application requiring sample tracking or multiplexing. We describe the validation of this tag resource as well as its use in the transposon mutagenesis of two different microorganisms, the bacteria Shewanella oneidensis MR-1 and the yeast Candida albicans. We then describe the expansion of this method to generate a genome-wide tagged transposon mutant collection in C. albicans. We used this collection to identify i) haploinsufficient genes in different nutrient conditions, and ii) mechanisms of drug-induced haploinsufficiency in C. albicans, and illustrated how these data can be used as a resource for genome annotation and hypothesis generation. Moreover, these studies illustrate why direct study of C. albicans is necessary, because relying solely on similarities between its traditional model organism, S. cerevisiae, would result in exclusion of C. albicans-specific genes and processes that are involved in its pathogenesis and may prove to be novel therapeutic targets.
A role for the let-7 primary microRNA in target gene recognition and repression [electronic resource]
Keywords: Microbiology and Immunology.

MicroRNA (miRNA) genes produce three noncoding RNA products: the long primary transcript (pri-miRNA), the ~70 nucleotide pre-miRNA, and the ~22-nt mature miRNA. Only the mature miRNA is considered to be the functional species of a miRNA gene in recognizing cognate target mRNAs and modulating their expression. However, mature miRNAs are processed from the primary transcript through sequential endonucleolytic steps. As a result, the mature miRNA sequence is present in all three RNA products of a miRNA gene. It has thus been intrinsically difficult to determine the contribution of each miRNA gene product to target repression. In fact, direct functional roles for pri- and pre-miRNAs have never been ruled out. Here we show that pri- and pre-miRNAs may not be mere transitory intermediates of mature miRNA biogenesis. We found that ectopic expression of the C. elegans miRNA gene let-7 (cel-let-7) in human culture cells results in the production of truncated pre- and mature miRNAs that lack the first two 5' nucleotides, one of which is the first nucleotide of the miRNA seed region (nucleotide SD1). We found this nucleotide to be required for repression of target reporters by cel-let-7 in these cells, demonstrating that pri-let-7 may have a direct role in target repression. Further, we show that the nucleotide sequence and structure of both the pri-/pre-let-7 loop and stem regions play a key role in miRNA gene function in reporter assays. In vitro and in vivo analyses indicated the significance of these regions may be in the mediation of a physical interaction between pri-let-7 and target RNAs. These observations suggest that regulatory information encoded in the structured pri-miRNAs, but absent from mature miRNAs, could be directly interpreted for target recognition and repression through RNA:RNA interaction. Intriguingly, some mutations in the loop nucleotide sequence also restored processing of the 5' ends of C. elegans pre- and mature let-7 in culture cells, demonstrating that the pri-/pre-miRNA loop region can also regulate the precision of mature miRNA biogenesis. Importantly, in the presence of functional pre- and mature let-7, cel-let-7 activity in target repression consists of both SD1-independent and SD1-dependent components, implying potential contributions by both pri- and mature let-7. Finally, we interrogated the effects of pri-/pre-let-7 loop mutations on their ability to rescue a let-7 loss-of-function mutant phenotype in C. elegans. Our results indicate decreased significance of these parameters in the control of worm vulval development, although context-dependent differences in mature miRNA biogenesis between heterologous culture and live animals may partially explain this discrepancy. Taken together the work presented here reveals a novel layer of regulatory complexity encoded in long primary miRNAs that may have broad implications in understanding the mechanisms by which miRNA genes control target expression.
The role of glycoprotein H in Varicella-zoster virus pathogenesis [electronic resource]
Keywords: Microbiology and Immunology

Glycoprotein H (gH) plays an essential role in virus binding, entry and fusion of the Herpesviridae. Varicella-zoster virus (VZV) is an important human pathogen that causes varicella (chicken pox) and herpes zoster (shingles). VZV gH function has not be analyzed in depth. gH function was demonstrated to be important for VZV pathogenesis in skin xenografts in vivo by administration of anti-gH mAb 206, a conformation dependent neutralizing antibody. Antibody administration prevented infection in 42% of skin xenografts, and reduced virus replication and lesion formation in the remaining skin xenografts. Antibody binding to gH altered gH localization following endocytosis, preventing gH trafficking to the trans-Golgi network for virus secondary envelopment. Antibody binding to gH within the virus envelope resulted in internalization of virus particles, possibly for targeted degradation. Deletion of ORF 37, which encodes gH, demonstrated that gH was essential for VZV pathogenesis. Mutational analysis demonstrated that the N-terminus of the protein formed a structural epitope required for efficient VZV pathogenesis in vivo. Several neutralizing anti-gH antibodies target this epitope. A region of the C-terminus was required for VZV pathogenesis, and for efficient virus-induced cell-cell fusion. Predicted [alpha]-helices that might act as heptad repeats or fusion peptides were also required for gH function and VZV pathogenesis. Cysteine residues were important for gH maturation and transport, and possibly for correct expression of gH on the cell surface. Altogether, these studies demonstrate the importance of structural and functional domains for gH-dependent fusion and VZV pathogenesis.
The role of processing complexity in word order variation and change [electronic resource]
Keywords: Linguistics.

All normal humans have the same basic cognitive capacity for language. Nevertheless, the world's languages differ in the kind and number of grammatical options they give their speakers to express themselves with. Sometimes, a language's grammatical constructions may differ in how easy they are for comprehenders to process or how readily speakers will choose them. It has been observed that languages which allow more difficult constructions also tend to allow easier ones, and when a language only allows one option, it tends to allow the easiest to process. This correlation is intuitive: languages tend to give their speakers options that they find easy to use. However, the causal process that underlies it is not well understood. How did the world's languages come to have this convenient property? In this dissertation, I discuss a family of evolutionary models of language change in which processing-efficient variants tend to be selected more frequently, and hence over time have the potential to displace less efficient variants, pushing them out of the language. I begin by showing that a psycholinguistic theory, dependency length minimization, accounts for word ordering preferences in data taken from Old and Middle English just as it does in Present Day English. I then discuss computer simulations of a model of language change which implements this bias, predicting observed word order changes in English. Finally, I present experimental studies of online comprehension in Japanese which not only display evidence for the dependency length bias, but also suggest that comprehenders encode it as part of their knowledge about language, using it to help understand the sentences they receive from their peers.
The role of teacher language background in the scoring of science responses from English learner students [electronic resource]
Keywords: Education.

This dissertation explored differences among bilingual and monolingual teacher's scoring and interpretations of English learner (EL) students' responses to open-ended science items. Scores were analyzed using a split plot analysis of variance and interpretations were used to gather qualitative information about what teachers focused on as they read the responses. Analyses of scoring data collected for this study revealed that teacher language background was not significantly related to scores for EL or non-EL student groups. This suggests that teachers, regardless of their language background, interpreted the rubric and student responses similarly when they assigned scores. The presence of language features in certain student responses was, however, associated with scoring. The presence of language features appeared to matter for incorrect responses, particularly for EL students, and not correct responses. In the interpretations, teachers wrote along three characteristics: content focus, use of qualifiers, and noticing student language. Of the three characteristics, noticing student language revealed the most differences among teachers of the different language background groups. These findings, however, warrant further studies since the monolingual population used in this study was geographically unique and their everyday exposure to a second language is not typical for most monolinguals.
Roles for hedgehog signaling in zebrafish development [electronic resource]
Keywords: Biochemistry

The zebrafish has emerged as a powerful model organism for the experimental interrogation of the processes that bring about vertebrate development. In this thesis we study functions of Hedgehog (Hh) signaling, a process that is frequently dysregulated in cancer, that occur during the formation of the zebrafish embryo. First, we investigate a possible link between Hh signaling and the directed migration of germ cells to the presumptive gonad in the early embryo and find that Hh-targeting drugs can perturb germ cell migration, but Hh signaling itself does not regulate this process. Second, we study the formation of motoneurons in the developing ventral spinal cord and find that Hh signaling and retinoic acid signaling work together to induce these cells by the same downstream output of Gli transcription factor activity. Third, we generated transgenic zebrafish that reliably report Hh signaling events at multiple levels of the pathway and we apply these tools in initial studies of zebrafish regeneration. In summary, these studies further our knowledge of the roles for Hh signaling in vertebrate development and will lead insight into the pathology of Hh-related disorders.
The roles of chromatin modifying enzymes in DNA double-strand break repair [electronic resource]
Keywords: Cancer Biology.

Efficient DNA double-strand break (DSB) repair is essential for maintaining the stability of the genome. A single long-lived DSB can cause cell lethality (Bennett et al., 1996). Humans with defects in DSB repair are sensitive to DNA damaging agents and are predisposed to developing cancers, as exemplified by such diseases as Nijmegan breakage syndrome, Ataxia telangiectasia, and breast cancer susceptibility in women with mutations in the BRCA1 and BRCA2 genes (Duker, 2002). Repair of DNA damage requires activation of cell cycle checkpoint controls, recruitment of repair proteins to DNA lesions, and transcriptional activation of relevant genes. As shown by our data, deletion of particular histone modification genes produces sensitivity to ionizing radiation in Saccharomyces cerevisiae, suggesting a role for chromatin modification enzymes in the repair process as well. Because damage recognition and repair of lesions are both influenced by chromatin structure, we began by studying the role of histone acetylation in the DNA damage response. Acetylation of the N-terminal tails of histone H4 opens the chromatin to allow repair enzymes to access broken DNA. Through a screen of the yeast deletion pool, we found that deletion of BRE1 and DOT1 genes causes sensitivity to ionizing radiation. Ubiquitination of H2B on lysine 123 (H2B-K123) by ubiquitin ligase Bre1 is necessary for methylation of H3 on lysine 79 (H3-K79) by Dot1. Through the histone modifications they catalyze, these proteins are involved in many aspects of the DNA repair response, as outlined in Sections C, D, and E. Our studies focused on homologous recombination repair defects, genome-wide expression patterns in BRE1 and DOT1 deletion mutants, an analysis of the data regarding proteins purported to bind to methylated H3-K79, and optimization of a protein purification strategy to find Dot1 binding partners. Our yeast deletion pool screen also predicted a role for N-terminal acetyltransferase complex NatB in DNA double-strand break repair. In Section F, we build a case for acetylation of DNA end-binding protein Mre11.
Rounding by sampling [electronic resource]
Keywords: Management Science and Engineering

In this thesis we introduce a new technique for designing approximation algorithms for a class of discrete optimization problems. The idea, that we call "Rounding by Sampling'', is basically a technique for rounding the optimal fractional solution of the problem (obtained from the relaxation of the original problem into a continuous domain) back to the integral domain. The technique is based on sampling from a maximum entropy distribution over the combinatorial structures hidden in the feasible solutions. The main implication of our technique is that it keeps the combinatorial structures intact, while aiming to preserve the solutions quantitatively. We present our technique on combinatorial structures such as spanning trees and matchings. Our results suggest a new approach to attack discrete optimization problems: the path goes through the well-known idea of relaxation the problem into the continuous domain and then finding a (fractional) optimal solution in the relaxed domain using the known techniques of optimization literature, e.g. interior point method, ellipsoid method, etc. After that, our technique comes to help: it enables us to transform the fractional solution back to an integral feasible solution of the original problem, while ensuring that we do not lose much, quantitatively. Using this approach, we show how to design approximation algorithms for two of the most well-known problems in discrete optimization: the traveling salesman problem and the fair allocation of indivisible goods.
Routing to mobile users by exploiting mobility prediction in wireless mesh networks [electronic resource]
Keywords: Electrical Engineering

With the advent of ubiquitous wireless networks, supporting user mobility has become crucial. Routing algorithms face challenges in supporting mobility while remaining energy efficient. Mobility of human or vehicle makes wireless links in these mobile networks much more volatile than the links in wireless networks of stationary nodes, and the wireless connectivity varies frequently due to moving speeds. Hence reliable data delivery to mobile users is often hard to achieve. In this thesis, we propose a new routing algorithm for wireless mesh networks with mobile users based on the following components: (i) predicting likely next association node of mobile user (short-term mobility prediction), and (ii) predicting a sequence of the future association nodes of mobile user (long-term mobility prediction). Our approach is to understand and characterize the mobility of the mobile users by looking at connectivity patterns over stationary mesh nodes using past history of connectivity information. Our main contribution is the design of techniques that can be used by routing algorithms to leverage the predictive knowledge of user mobility to efficiently deliver data to those users. This work enables a long-term routing plan through a network optimization process, called "data stashing". The data stashing scheme enables reliable data delivery from stationary mesh nodes to mobile users. In this scheme, each mesh node selects a set of possible association nodes on which to stash its data, to be picked up whenever the mobile user passes the stashing node. We show that data stashing significantly decreases routing cost for delivering data from stationary mesh nodes to multiple mobile users compared to immediate routing protocols where mesh nodes immediately deliver data to the last known association nodes of mobile users. We also show that the scheme provides better load balancing, avoiding collisions and consuming energy resources evenly throughout the network, leading to longer overall network lifetime over the immediate routing protocols. More importantly, we demonstrate that given even limited information about the future node associations of mobile users, optimization of routing paths leads to significant improvements in routing performance.
Rule-based response assessment framework [electronic resource]
Keywords: Biomedical Informatics.

The Rule-Based Response Assessment Framework is a goal-driven, context-driven, and knowledge-based temporal interpretation framework that automates response assessment over clinical data. The goal of this work is to enable consistent application of standardized response criteria for clinical trial data analysis, and to enable research and development of response criteria. The Rule-Based Response Assessment Framework defines the dimensions of response assessment including the knowledge and tasks required to apply response criteria to patient data. The Framework consists of several knowledge models and several sub-methods that make up the Response Assessment Method. The framework is implemented using ontologies and rules with the semantic web formalisms of OWL and SWRL respectively. The framework is applied to two response criteria in the oncology domain and one response criterion for rheumatoid arthritis. I demonstrate that the implementation of the Rule-Based Response Assessment Framework is extensible, reusable and enables consistent application of response criteria to patient data and comparison of response criteria within a clinical domain. Future research directions will incorporate the Rule-Based Response Assessment Framework into a larger informatics infrastructure for application and development of oncology response criteria.
Ruthenium-catalyzed redox- and cycloisomerizations [electronic resource] : atom economic syntheses of carbo- and heterocycles
Keywords: Chemistry

A facile and 100% regioselective cycloisomerization--6-pi-cyclization method for obtaining pyridines is described. The unsaturated ketones and aldehydes derived from the cycloisomerization of primary and secondary propargyl diynols in the presence of CpRu(MeCN)3PF6 are converted to 1-azatrienes which in turn undergo a subsequent electrocyclization--dehydration to provide pyridines with excellent regiocontrol. The ruthenium-catalyzed cycloisomerization is a mild, atom-economical method for obtaining the requisite dienone and dienal substrates. The cycloisomerization--6-pi-cyclization sequence may be carried out in one pot or in two independent steps. Additionally, the azatriene cyclization may be carried out in 1-3 hours under microwave irradiation or, for more sensitive substrates, at lower temperatures for longer periods of time (6--24 hours at 90 °C). An atom-economical method for the convenient synthesis of tetrahydropyrans and tetrahydrofurans is also described. Enones and enals derived from the [IndRu(PPh3)2Cl]-catalyzed redox-isomerization of primary and secondary propargyl alcohols undergo a subsequent intramolecular conjugate addition to provide cyclic ethers in excellent yields. Lastly, we have developed complementary methods for the transition metal-catalyzed enyne cycloisomerizations of cyclic olefins. By using distinct ruthenium and palladium catalysts, decalins and 7,6-bicycles can be obtained with dichotomous stereochemical outcomes. The change in mechanism that accompanies the change in metal affords trans-fused 1,4-dienes with ruthenium and their cis-fused diastereomers under palladium catalysis. In the reactions under ruthenium catalysis, a coordinating group is required, and acts to direct the metal to the same side of the carbocycle, resulting in the observed trans diastereoselectivity. Subtle changes in the carbocyclic substrate led to the discovery of a heretofore-unobserved mechanistic pathway, providing bicyclic cycloisomerization products under palladium catalysis, tricyclic products under ruthenium catalysis in DMA, and a 1:1 mixture of the two under ruthenium catalysis in acetone. The different coordination requirements of the two paths allow for the reaction to be shuttled through the metallacycle pathway (generating tricyclic products) when DMA is used as a solvent. This method of obtaining these cyclobutene products complements the existing methods for catalyzing the [2+2] cycloaddition of alkynes. While other methods have been demonstrated for larger ring sizes with palladium, and for specific alkyne termini with platinum and gold, this method is unique in its substrate scope. To the best of our knowledge, this is the first example of a [2+2] cycloaddition catalyzed by CpRu(MeCN)3PF6.