Typescript (photocopy). / Digitized by Kansas Correctional Industries

Nanomedicine has emerged in the past decades, and a variety of designs for drug/gene delivery have been reported since the concept of nanomedicine was first demonstrated. However, with the exception of a few notable successes, the clinical translation of nanomedicine has been slow. Specificity and delivery efficiency are the major obstacles; only a few nanomedicine systems can effectively reach and release the therapeutic payload at the target site, thereby limiting the therapeutic efficacy. To tackle these issues, this work aims to design new strategies to improve nanomedicine systems at the gene-, protein- and tissue- levels. We applied CRISPR/Cas9 technology for gene targeting. Delivering CRISPR/Cas9 elements, including Cas9 endonuclease and a corresponding guide RNA, allows for specific gene mutagenesis. A conventional gene delivery carrier often has a highly positive charge density for higher transgene expression, but this may result in unfavorable effects on the Cas9 plasmid transfection. As a large plasmid, strong interaction between the Cas9 plasmid and the polycation with high charge density may hinder the plasmid’s intracellular release. Moreover, high Cas9 expression usually leads to undesirable off-target effects. We addressed these two major obstacles by designing a low-charged density micelle, composed of quaternary ammonium‐terminated poly(propylene oxide) and amphiphilic Pluronic F127. We tested this design on a human papillomavirus (HPV)-induced cervical cancer model to target the HPV oncogene, E7. Our micellar carrier enabled effective Cas9 transfection with a transient Cas9 expression, which offered enhanced Cas9 on-target specificity. This nonviral Cas9‐mediated E7 mutagenesis resulted in significant inhibition of HPV‐induced cancerous activity both in vitro and in vivo. Although CRISPR/Cas9 technology is a powerful toolkit for gene manipulation, gene editing might not be practical for therapeutics in the cancers that develop from endogenous mutations, which may vary among patients and disease stages. Protein-targeting, therefore, may be a more efficient approach. Aptamer and its selection technology, namely SELEX, offer direct evolution to obtain a nucleic acid ligand that specifically recognizes the protein target. Yet, aptamer screening remains unsatisfactory, and the success rate of SELEX is limited. We designed two approaches to improve the aptamer screening. We first employed a microarray platform to deconvolute the aptamer sequence and identified the aptamer functional motif. The resulted protein-targeting motif with an optimal length and showed enhanced structural and functional characteristics compared with its parental sequence. In addition to sequence optimization, conjunction of two distinct aptamers that recognize different epitopes of the protein target is another approach to improve the aptamer’s affinity. In looking for a rapid way to screen this bivalent aptamer pair, we designed a quantum dot (QD)/ Förster resonance energy transfer (FRET) sensor. Using a thrombin aptamer as a model system, we conjugated an anti-thrombin aptamer with QD and stained the other one with the intercalation dye, YOYO-3. If the two aptamers recognized different epitopes of thrombin, the conformational change of the two aptamers would take place when interacting with thrombin, and this would induce YOYO-3 dye’s translocation. YOYO-3 would be transferred from the aptamer to QD surface, resulting in a strong FRET signal. In contrast, if they recognized the same epitope, binding competition between two aptamers would inhibit dye translocation, thereby giving a minimal FRET signal. By measuring the FRET signal, we can verify if the two aptamers may form a bivalent pair. Lastly, we integrated mesenchymal stem cell (MSC) with a nanomedicine system to achieve active tissue-targeting. MSC is known to migrate toward certain types of cancer cells by chasing the chemotaxis release from the cancer cells, but the therapeutic payload that MSC can carry is limited. Forming an MSC spheroid allowed the loading of the nanomedicine system with another type of anti-cancer drug. We therefore designed a hybrid MSC/nanomedicine spheroid, which functioned as an active tumor-targeting platform, enabling effective delivery for both cytotoxic protein and chemotherapeutic drugs. In a heterotopic glioblastoma model, the hybrid spheroid significantly improved the retention of the nanomedicine system at the tumor site, leading to enhanced tumor inhibition in vivo. Collectively, this work demonstrated the effective approaches for gene, protein and tissue targeting by addressing the issues of low specificity and limited delivery efficiency that many current nanomedicine systems face. Particularly, the results may add to the armamentarium of cancer therapeutics, which remains largely challenging and intractable.

This thesis develops an application for interactive experiments with BRDF functions. Arbitrary BRDF function may be specified with an analytic formula, including arbitrary number of parameters. A simple scene is displayed, using specified BRDF. The parameters can be modified which has an immediate impact to the scene's appearance. The application can utilize a programmable GPU for high speed of rendering.

ADAR (Adenosine Deaminases acting on RNA) family proteins are double-strand RNA binding proteins that deaminate specific adenosines into inosines. This A-to-I conversion is called A-to-I RNA editing and is well conserved in the animal kingdom from nematodes to humans. RNA editing is a pre-splicing event on nascent RNA that may affect alternative splicing when the editing occurs in the exon-intron junction or in the intron. Also, editing may change biological function of small RNAs by editing the premicroRNAs or other noncoding RNAs. Editing also alters protein amino acid sequences because inosine in the mRNA base pairs with cytosine and is therefore read as guanosine. In mammals, there are three ADAR family proteins, ADAR1, ADAR2, and ADAR3, encoded by three different genes. So far, no enzymatic activity of ADAR3 is detected. The most frequently edited targets of ADAR1 and ADAR2 are regions covering copies of Alu transposable elements in primates. In addition, loss of some specific editing events leads to profound phenotypes when the editing does not occur correctly. For example, some human neural disorders – such as epilepsy, forebrain ischemia, and Amyotrophic Lateral Sclerosis – are known to be associated with abnormally edited ion channel transcripts. Drosophila has a single ADAR protein (encoded by the Adar gene) that is highly conserved with human ADAR2 (encoded by the ADARB1 gene). To date, 972 editing sites have been identified in 597 transcripts in Drosophila, and approximately 20% of AGO2-associated esiRNAs are edited. Similar to mammals, many ion channel-encoding mRNA transcripts undergo ADAR-mediated A-to-I editing in Drosophila. While Adar1 null mice die at the embryonic stage and Adar2 null mice die shortly after birth due to seizures, Adar null flies are morphologically normal and have normal life span under ideal conditions. However, Adar null flies exhibit severe neurodegeneration and locomotion defects from eclosion, whilst Adar overexpression (OE) is lethal. To better understand the physiological role of RNA editing and ADAR, and to shed light on ADAR-related human disease, I used Drosophila Adar mutant flies as a model organism to investigate phenotypes, and to find chromosomal deletions and specific mutations that rescue the neural-behavioural phenotype of the Adar null mutant flies. Using the publicly available chromosomal deletions collectively covering more than 80% of the euchromatic genome of Chromsome III, I performed a genetic screen to find rescuers of the lethality caused by Adar overexpression. I confirmed that mutation in Rdl (Resistant to dieldrin, the gene encoding GABAA receptor main subunit) rescues. This rescue was not likely caused by effects on Adar expression level or activity. Driven by the hypothesis that the rescue may be due to reduction in GABAergic input to neurons, I recorded spontaneous firing activity of Drosophila larval aCC motor neurons using in vivo extracellular current recording technique. As expected, the neurons overexpressing Adar had much less activities compared with wild type neurons. Also, I found that Adar null fly neurons fired much more and showed epilepsy-like increased excitability. Although feeding PTX (Picrotoxin), a GABAA receptor antagonist, failed to rescue the lethality, reducing the expression of GAD1 to reduce synthesis of GABA was able to rescue the ADAR overexpression lethality. These results suggest that ADAR may finetune neuron activity synergistically with the GABAergic inhibitory signal pathway. I used MARCM (mosaic analysis using a repressible cell marker) to detect cellautonomous phenotypes in Adar null cells in otherwise wild type flies. Although neurodegeneration, observed as enlarged vacuoles formation in neurophils, was detected both in histological staining and EM images, the Adar null neurons marked with GFP from early developmental stages were not lost with age. Nevertheless, swelling in the axons or fragmentation of the axon branches of Adar null neurons was sometimes observed in the midbrain. By comparing the Poly-A RNA sequencing data from Adar null and wild type fly heads, we detected significant upregulation of innate immune genes. I confirmed this by qRT PCR and found that inactive ADAR reduces the innate immune gene transcript levels almost as much as active ADAR does. Further, using the locomotion assay, I confirmed that reintroducing inactive ADAR into Adar null flies can improve the flies’ climbing ability. Based on the Adar null flies having comparatively low viability, I performed a second deficiency screen to find rescuers of Adar null low viability using the same set of deficiencies as in the lethality rescue screen described above. I found seven deletions removing 1 to 37 genes that significantly increased the relative viability of the Adar null flies. However, not all the rescuing deficiencies also improved the Adar null locomotion. One rescuing gene, CG11357 was mapped from one of the rescuing deficiencies, and some mutant alleles of cry, JIL-1 and Gem3 also showed significant effects on the Adar null fly viability. The single gene viability rescuers were also not necessarily locomotion or neurodegeneration rescuers. Although the initial aim was to find neural-behavioural rescuing genes from the viability screen, the viability rescuers found in the screen are more likely to play a role in different aspects of stress response for survival.

The mitochondrial genome (kinetoplast or kDNA) of Trypanosoma brucei is highly complex in terms of structure, content and function. It is composed of two types of molecules: 10-50 copies of identical ~23-kb maxicircles and 5,000-10,000 highly heterogeneous 1-kb minicircles. Maxicircles and minicircles form a concatenated network that resembles chainmail. Maxicircles are the equivalent of mitochondrial DNA in other eukaryotes, but 12 out of the 18 protein-coding genes encoded on the maxicircle require post-transcriptional RNA editing by uridylate insertion and removal before a functional mRNA can be generated. The 1-kb minicircles make up the bulk of the kDNA content and facilitate the editing of the maxicircle-encoded mRNAs by encoding short guide RNAs (gRNAs) that are complementary to blocks of edited sequence. It is estimated that there are at least hundred classes of minicircle, each class encoding a different set of gRNAs. At each cycle of cell division the contents of the kDNA genome must be faithfully copied and segregated into the daughter cells. Mathematical modelling of kDNA replication has shown that failure to segregate evenly will eventually result in loss of low copy number minicircle classes from the population. Depending on the type of minicircle that is lost this can result in immediate parasite death or, if the loss occurred in the bloodstream stage, render the cells unable to complete the canonical life-cycle in the tsetse fly vector. In order to investigate minicircle complexity and replication in T. brucei further we i) first established the true complexity of the kDNA genome using a Illumina short read sequencing and a bespoke assembly pipeline, ii) annotated the minicircles to establish the editing capacity of the cells, iii) analysed expression levels of predicted gRNA gene cassettes using small RNA data, and iv) carried out a long term time course to measure how kDNA complexity changes over time and compared this to preliminary model predictions. The structure of this thesis follows these steps. Using these approaches, 365 unique and complete minicircle sequences were assembled and annotated, representing 99% of the minicircle genome of the differentiation competent (i.e. transmission competent) T. brucei strain AnTat90.13. These minicircles encode 593 canonical gRNAs, defined as having a match in the known editing space, and a further 558 non-canonical gRNAs with unknown function. Transcriptome analysis showed that the non-canonical gRNAs, like the canonical set, have 3' U-tails and showed the same length distribution. Canonical and non-canonical sets differ, however, in their sense to anti-sense transcript ratios. In vitro culturing of bloodstream form T. brucei for ~500 generations resulted in loss of ~30 minicircle classes. After incorporating parameters for network size and minicircle diversity determined above, model fitting to longitudinal kDNA complexity data will provide estimations for the fidelity of kDNA segregation. The refined mathematical model for kDNA segregation will permit insight into time constraints for transmissibility during chronic infections due to progressive minicircle loss. It also has the potential to shed light on the selective pressures that may have led to the apparent co-evolution of the concatenated kDNA network structure and parasitism in kinetoplastids.

Uridylyl insertion/deletion mRNA editing is essential for mitochondrial gene expression in Trypanosoma brucei and governed by multi-protein complexes called editosomes. The final step in each cycle of this post-transcriptional process is that of re-ligating the edited mRNA fragments. The ~20S RNA editing core complex contains two RNA editing ligases, REL1 and REL2, located, respectively, in a deletion and an insertion subcomplex. While REL1 is clearly essential for RNA editing, REL2 knockdown by RNAi has not resulted in a detectable phenotype. To explain these findings, alternative scenarios have been suggested: (a) REL2 is not functional in vivo; (b) REL1 can function in both insertion and deletion editing, whereas REL2 can only function in insertion editing; (c) REL1 has an additional role in repairing erroneously cleaved mRNAs. To further investigate respective functions of the two RELs this study used three complimentary approaches: (i) genetic complementation with chimeric ligase enzymes, (ii) deep sequencing of RNA editing intermediates after ligase inactivation, and (iii) evolutionary analysis. In vivo expression of two chimeric ligases, providing a REL2 catalytic domain at REL1’s position in the deletion subcomplex and a REL1 catalytic domain at REL2’s position in the insertion subcomplex, did not rescue the growth defect caused by REL1 ablation. Although the results were not fully conclusive they suggest that it is the specific catalytic properties of REL1 rather than its position within the deletion subcomplex that makes it essential. In order to identify in vivo substrates of REL1, specific editing intermediates that accumulated after genetic knockdown of REL1 expression were captured by 5’ linker and deep sequenced using Ion Torrent and Illumina technology. Analyses of such unligated editing intermediates with bespoke bioinformatics tools suggest that REL1 functions in deletion editing as expected, but also in the repair of miscleaved mRNAs, implying a novel role for this ligase. Neither role can be fulfilled by REL2, at least not with sufficient efficiency. Sequencing data also suggest that either REL1 is not involved in ligation of addition editing substrates, or that REL2 in this case can fully compensate for loss of REL1. REL1, REL2 and KREPA3 sequences were subjected to analysis using MEGA5 and the HyPhy package available on the Datamonkey adaptive evolution server. Results indicated that all three editosome genes are under much stronger purifying than diversifying selective forces. In general this selection pressure to conserve protein sequence increased from KREPA3 to REL2 to REL1, suggesting a requirement to maintain catalytic function for both ligases. Taken together, these experiments reveal a novel function for REL1 during RNA editing, providing a rationale for its essentiality. Deductively, the results also suggest REL2, which was previously thought to be non-essential, may still be required by the cell at its position in the addition subcomplex. Evolutionary analysis suggests that the RELs and KREPA3 are under the same evolutionary forces to maintain their respective functions in RNA editing.

Clustered regularly interspaced short palindromic repeats (CRISPR/CRISPR-associated (Cas) protein system, CRISPR/Cas9, uses single-guide RNA to guide Cas9 to the target site for genome editing. In this study, the CRISPR/Cas9 system was used to knockout KED in tomato (Solanum lycopersicum). KED was first identified while screening the wounded tobacco (Nicotiana tabacum) leaves. We found that alignment of the protein sequence of SlKED (Solanum lycopersicum KED) and NtKED (Nicotiana tabacum KED) showed 55.1% identity. To investigate, we generated SlKED knockout tomato plants with a single base pair deletion, a five base pair deletion and a three base pair deletion with a single base pair insertion. We performed wounding assays and analyzed gene expression and found that the wounded SlKED knockout plant showed no gene induction. Furthermore, the biological assay results revealed that the tobacco hornworm (Manduca sexta) gained more mass when fed on the SlKED knockout plant. Our studies show that the KED gene plays a role in wound-induced mechanism and suggested it may involve in the plant defense system against biological stress and insect feeding. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2019. / FAU Electronic Theses and Dissertations Collection

These days, fact-checking is a fashionable term in the worlds of both politics and the media. On broadcast news, tickers run below the speeches of politicians, with claims annotated in real-time and occasionally labeled as false. In newspapers like the Washington Post and online information hubs like Politifact.com, writers invoke the term to flag reporting that aims to correct or clarify the public record. At times, "fact-checking" efforts are themselves called out for partisan bias or personal gain. The term is now practically mainstream, used in everyday conversation to indicate disbelief. ("I'm going to have to fact-check you," CNN anchor Jake Tapper said to former Baltimore mayor Stephanie Rawlings-Blake in August 2016, expressing surprise that she was the mother of a 12-year-old.) Given the proliferating parties of interest that now claim to be engaged in some sort of fact-checking endeavor--from policy think tanks to Facebook--it's no wonder that a term originally reserved for the pursuit of journalistic accuracy now suffers from muddied public understanding. This study focuses on fact-checking in the context of print magazines: the media genre that innovated a formal version of the practice nearly a century ago. Magazine fact-checking, unlike the "fact-checking" tickers of broadcast news and newspaper postmortems, focuses not on setting the record straight after the fact, but rather on getting the story right before it goes to print. If a magazine fact-checker does her work well, she'll remain invisible to the reader. And that's because the published story, after her fact-checking, will afford the reader an experience uninterrupted by questionable logic, unreliable sources, or suspect data. Magazine fact-checkers aim for this level of perfection by employing a rigorous process that goes far beyond the verification of names, dates, and numerical figures. To illustrate this process, and explain my personal investment in this craft, I share my own experience working as the head of a city magazine's fact-checking department. To gain perspective on magazine fact-checking as practiced elsewhere in the nation, I interview other fact-checkers, writers, and academics. I also draw on case studies, media history, and personal anecdotes to examine some of the fundamental questions that inform the practice. (Among them: what is a fact? When does information become true? And what are the limits of a fact-checker's pursuit of truth?) In the world of fact-checking, there are best practices in the craft, and nuances to consider. Fact-checking also wades into deeper waters: those of philosophy, ethics, and social bias. But at its core, fact-checking is quite simply an application of critical thinking skills: skills that can be honed, and used for good. At a time when the media has lost the faith of many Americans, the magazine fact-checker can play a critical role in building that trust, one scrupulously vetted story at a time.

No description available.

Software systems are getting larger and more complex, while the requirements are becoming vaguer and more rapidly changing. These trends make current software development more and more likely a team work. To integrate multiple developers into a coherent structured management process and make team software development a positive-sum game for both higher productivity and better quality, many team soft ware development methodologies have been proposed and practised. An emerging methodology is collaborative programming, which allows a group of programmers to work together on the same source code for design, implementation of individual components, and integration of individual components. Compared with other team software methodologies that only address needs in some phases or situations, collaborative programming is more adaptive to the variety of different phases or situations in a team software development process. A core technical component in collaborative programming is collaborative editing, which allows a group of programmers to view and edit the same source code. To support different phases or situations in an Internet-based collaborative programming process, collaborative editing must meet the requirements of supporting unconstrained, responsive, real-time collaborative editing; unconstrained, syncretic, non-real-time collaborative editing; and smooth, flexible switching between real-time and non-real-time collaborative editing. This thesis research contributes several novel techniques to address these requirements, and an Internet-based collaborative programming environment to integrate those techniques. These research contributions have advanced state-of-the-art technologies on col laborative editing for supporting Internet-based collaborative programming. First, we contribute a collaborative highlighting gestural communication technique for unconstrained, responsive, real-time collaborative editing. This technique is particularly effective in improving the quality of real-time interaction on text-based source code documents. The contribution to the operational transformation technology is the extension of the technology to support group awareness. It includes a package of operational transformation functions and transformation control algorithms for consistency maintenance in collaborative highlighting, and a flexible undo solution that has the capability of undoing any highlighting operation at any time. Second, we contribute a flexible operation-based merging technique for unconstrained, syncretic, non-real-time collaborative editing, which is efficient and has the capability of textually integrating all changes, and automatically detecting and resolving syntactic conflicts according to application-dependent user-specified policies. The contribution to the operational transformation technology is the extension of the technology to support unconstrained, syncretic, non-real-time collaborative editing. Its includes a log compression algorithm, a textual merging algorithm, and a syntactic merging algorithm. Moreover, we contribute a flexible notification technique to support flexible collaborative editing: unconstrained, responsive, real-time collaborative editing; uncon strained, syncretic, non-real-time collaborative editing; and smooth, flexible switching between them. The contribution to the operational transformation technology is the extension of the technology to support flexible collaborative editing. It includes a new transformation control algorithm that has a linear time complexity, two notification algorithms that support propagation and acceptance of any notifications at any time, and a notification propagation protocol that is efficient for both real-time and non-real-time collaborative editing.