Erythrocyte pyruvate kinase (R-PK) is a key glycolytic enzyme catalyzing the transphosphorylation of phosphoenolpyruvate (PEP) and ADP to pyruvate and ATP respectively3,4. The substrate PEP and product pyruvate of this reaction are involved in a number of energetic and biosynthetic pathways; hence a tight regulation of R-PK activity is crucial not only for glycolysis, but also for the entire cellular metabolism. Deficiency of R-PK is one of the most common enzymatic defects of RBC, and may be caused by mutations of the PK-LR (pyruvate kinase liver red blood cell) gene31, 32. Clinically, R-PK deficiency manifests itself as a chronic life-long hemolysis ranging from very mild or fully compensated anemia to life-threatening neonatal anemia and pronounced jaundice. Current treatment options are limited to continuous blood transfusions and splenectomy. Thus, there is an urgent need for medications to counter R-PK deficiency without resorting to these complicated procedures. Our aim is to identify novel allosteric modifiers of R-PK using a combination of computational studies and enzyme activity assays. Such compounds could be of medical interest. Human R-PK was expressed in DH-5α cells and was purified by the procedure reported by Wang et al57. However, this method gave a very low yield of R-PIS (5mg/L). In an attempt to increase the yield, we expressed R-PK in Rosetta strain cells. Further, addition of His-tag to the protein's N-terminus simplified purification to a one step Ni-NTA (Nickel- nitrilotriacetic acid) column resulting in a 6-fold increase in the yield. Computational methods were applied to identify small molecules that bind to the allosteric activator fructose 1,6-bisphosphate (FBP) binding site of R-PK to identify compounds that could interact with the protein. The software UNITY, as present in the molecular modeling software Sybyl was used to perform 3D searches against the National Cancer Institute (NCI) chemical database. From these searches we obtained 29 hits that were subjected to further computational analysis. The small molecules were docked into the FBP binding site of R-PK with different docking methods includingFlexX, GOLD and energy minimization. The energy scoring function of HINT was then applied to analyze the interactions between the docked molecules and R-PK. Compounds with highest HINT score were requested fiom NCI and were subjected to further kinetic analysis to identify possible allosteric effectors of R-PK.In the kinetic analysis, we employed a lactate dehydrogenase (LDH) coupled spectrophotometric assay to determine the activity of R-PK in the presence of these compounds. The steady state kinetics of R-PK gave a typical S-shaped curve that fitted a signloidal function indicative of allosterism. All the kinetic parameters of our enzyme were in excellent agreement with native R-PK activity as previously reported5, 57. R-PK activity in the presence of the analyzed compounds revealed both activators and inhibitors of R-PK. X-ray crystallographic analysis of R-PK in the presence of FBP and the identified small molecule effectors are currently in progress. These experiments were initiated to reveal the binding site of the compounds in R-PK, allowing for further optimization of the starting phannacophores and syntheses of new molecular entities for enhanced allosteric activity.In conclusion, we have developed a simple and efficient method for the expression and purification of R-PK. Using computational screening and HINT analysis we have also identified several compounds that interact with R-PK and kinetic analysis revealed both activators and inhibitors of the protein. Crystals of R-PK in the presence of effectors have been obtained and identification of the binding site on R-PK is under investigation. R-PK effectors discovered in this study could prove to be lead compounds for developing medications for the treatment of anemia and other disorders arising from R-PK malfunction.

While it implies aimless movement, the act of wandering is an act of discovery and can become a search for the unexpected. Wandering, as a metaphor for making, creates opportunities to work in ways where the final outcome is unknown. This can be accomplished by the creation of methods and instructions designed for wandering-the act of discovery. In the field of design, these ideas can be incorporated as methods for making that guide the design process in ultimately unknowable ways, resulting in products that bear little trace of preconception. The role of chance and indeterminacy as methods for relinquishing control and generating narrative are explored in three projects relying on video and interactivity.

By 2007, one half of higher education students are expected to enroll in distributed learning courses. Higher education institutions need to attract students searching the Internet for courses and need to provide students with enough information to select courses. Internet resource discovery tools are readily available, however, users have difficulty selecting relevant resources. In part this is due to the lack of a standard for representation of Internet resources. An emerging solution is metadata. In the educational domain, the IEEE Learning Technology Standards Committee (LTSC) has specified a Learning Object Metadata (LOM) standard. This exploratory study (a) determined criteria students think are important for selecting higher education courses, (b) discovered relationships between these criteria and students' demographic characteristics, educational status, and Internet experience, and (c) evaluated these criteria vis-à-vis the IEEE LTSC LOM standard. Web-based questionnaires (N=209) measured (a) the criteria students think are important in the selection of higher education courses and (b) three factors that might influence students' selections. Respondents were principally female (66%), employed full time (57%), and located in the U.S. (89%). The chi square goodness-of-fit test determined 40 criteria students think are important and exploratory factor analysis determined five common factors among the top 21 criteria, three evaluative factors and two descriptive. Results indicated evaluation criteria are very important in course selection. Spearman correlation coefficients and chi-square tests of independence determined the relationships between the importance of selection criteria and demographic characteristics, educational status, and Internet experience. Four profiles emerged representing groups of students with unique concerns. Side by side analysis determined if the IEEE LTSC LOM standard included the criteria of importance to students. The IEEE LOM by itself is not enough to meet students course selection needs. Recommendations include development of a metadata standard for course evaluation and accommodation of group differences in information retrieval systems.

This thesis examines John Napier of Merchiston’s 1593 commentary on the Book of Revelation within the context of sixteenth century apocalyptic thought in Scotland and England. Napier is usually remembered as a mathematician and this study aims to contribute to a more complete understanding of the man. Its most important contribution to scholarship is its discussion of Napier’s identification of himself as a conduit for divine revelation, chosen by God to expose the mysteries of scripture in the final age of human history. This placed him in the tradition of reformers like Knox but he differed from them in two crucial ways. Firstly, he broke from the texts that had influenced him by controversially predicting the approximate date of the apocalypse. Some of these works, and responses to Napier’s conclusions, are considered. Secondly, he did not regard a call to ministry as a facet of his prophetic status. Instead, he saw his biblical commentary as the expression of an intellectual gift from God. He employed grandiose eschatological themes to appeal to the highest echelons of society in an attempt to affect religious change. His dedicatory epistle to James VI was a direct correspondence that revealed shared knowledge and experiences. Napier’s approaches to the apocalypse and alchemy stemmed from a worldview that presented him as belonging to an intellectual and moral elite, preordained by God to receive and disseminate hidden knowledge at appointed times. The impact of historical events on the content of his work, including the Spanish Armada, Scottish Reformation and resulting sense of unity between Scotland and England, are assessed. The current biographical understanding of Napier is critiqued. The unique aspects of the Plaine Discovery, including the explicit chronology of salvation history that framed its conclusions, are discussed in detail.

Internet-based distance education (IDE) continues to grow in popularity and ubiquity. Acceptance of IDE among Christian higher education institutions has also increased. However, these institutions seek assistance. Such was the case with the nineteen institutions endorsed by the Assemblies of God (AG). The AG's oversight organization (The Alliance for Assemblies of God Higher Education, Alliance) was asked by member institutions for IDE aid, resources, and direction. To understand the current environment of IDE within AG higher education, an organizational discovery case study reviewed the historical IDE trends within AG higher education, surveyed institutional faculty members and administrators as to their IDE beliefs and situations, and analyzed the data collected. From the research findings, the Alliance gained a better understanding of the needs and intentions of its member institutions. It also realized the aid and resources to offer its endorsed institutions, what endorsement requirements were needed for spiritual development in an online distance education setting, and an overall IDE direction that the organization could provide or facilitate. To aid the organizational discovery, a research framework was created that the Alliance could reuse and share with similar organizations for their own internal discovery.

In Chapter 1, I hypothesize that there is a differential response by agents to changes in sovereign credit or default risk in both quiet (low default risk) and turbulent markets (high default risk). These market conditions create two different states of the market (world) or regimes. Investors and policy makers respond differently in the two regimes but the response in the turbulent market condition is amplified as policy makers attempt to smoothen the fluctuations and uncertainty while investors rebalance their portfolios in an attempt to hedge against downside risk of wealth loss. In the two regimes, the short run and long run dynamic relationships between any two cointegrated assets may change. To capture this phenomenon, this study tests for nonlinearities that may characterize the regimes, how cointegration relationships, short term dynamic interaction and price discovery (speed of adjustment to new information between two assets) may change in alternative regimes. To this end, I employ threshold cointegration, threshold vector error correction model (TVECM) asymmetrical return spillover modeling for sovereign credit default swaps (CDS), bonds and equity markets of seventeen emerging markets from four geographical regions. I find that there is non-linear cointegration and momentum in long-run adjustment process in 43/51 spreads analyzed. All countries analyzed have at least 2/6 possible regime specific asymmetric price discovery process. The study also finds evidence in support of asset substitution hypothesis and news-based hypothesis of financial contagions in sovereign CDS, bond and equity markets. The findings have important implications for asset allocation and portfolio rebalancing decisions by investors, policy intervention in financial markets, risk management and regime specific short and/or long term dynamic interactions among assets held in a portfolio as well as nonlinear speed of adjustment to new information. In chapter 2, I hypothesize that financial intermediaries can be categorized into bank-based institutions (BBIs) and market-based institutions (MBIs). MBIs and BBIs are under different regulatory agencies. Traditionally, only BBIs, regulated by the Fed, are used as conduits of transmitting liquidity and monetary policy into real economy and financial markets yet MBIs also play important role in providing liquidity and stability in financial markets. I use two tools of monetary policy (Federal fund rate and monetary aggregate) under two monetary policy regimes to investigate the impact of monetary policy under each regime on the liquidity of MBIs and BBIs. I investigate whether MBIs be used as conduits of transmitting monetary policy and liquidity in the market and if they should, under what economic and financial conditions (Regimes) should they be used. Moreover, what monetary policy tool is more effective for MBIs relative to BBIs under different regimes? Using Threshold vector auto-regressions and regime specific impulse response functions, I find that liquidity of BBIs and MBIs respond differently to different monetary policy tools under different regimes. Moreover, monetary policies are uncertain and vary over time. The Fed cannot continue to ignore MBIs in formulating and implementing monetary policy. Moreover, monetary aggregate policy is more effective when used on MBIs during contractionary monetary policy intervention (economic downturn) while Federal fund rate is more effective when used on BBIs under expansionary monetary policy.

The gene for methylthioadenosine phosphorylase (MTAP) is absent in almost 30% of cancers, opening a door for selective chemotherapy. One strategy to target the absence of MTAP involves the design of a cytotoxic methylthioadenosine (MTA) analogue. Non-cancerous cells would break down the cytotoxic analogue, since they contain MTAP, but cancerous cells would not, since they do not have MTAP. However, before such a compound can be made, we need to better understand the types of substrates accommodated by MTAP. The purpose of this thesis was therefore to explore a series of MTA analogues, probing the structure-function relationships between MTAP and specific structural modifications of MTA. Nine phenylthioadenosine (PTA) derivatives bearing ortho-, meta-, or para- methyl carboxylate, carboxylate, and hydroxymethyl substituents were synthesized and tested for cytotoxicity and as substrates for MTAP. The biological results of these nine compounds suggested that addition of substituents to the ortho-position was not tolerated by MTAP, and substituents similar to the hydroxymethyl might be accommodated by MTAP. None of the compounds were cytotoxic. This informed the design of ten more PTA derivatives, most of which were synthesized and tested for cytotoxicity and as substrates for MTAP. The range of functionalities included an amine, an acetamide, a urea, an isovaleramide, and an N-nitrosourea group inspired by the known anticancer agent lomustine. The amine derivatives of PTA were the best substrates of all MTA analogues tested (including PTA). The meta-amine derivative and the meta-isovaleramide showed minor cytotoxicity. Finally, the urea derivatives were moderate substrates of MTAP, and this pointed towards the future creation of other nitrosoureas as potential cytotoxic MTAP substrates. / Graduate / 2017-08-25

O câncer é a segunda maior causa de mortes no mundo, sendo os tumores de mama os mais prevalentes e letais entre as mulheres. Apesar de vasta quimioterapia disponível, os tratamentos apresentam problemas como alta toxicidade e resistência. Dentre os tumores de mama, o subtipo triplo negativo (TNBC) apresenta o pior prognóstico e a maior limitação de tratamentos. O presente trabalho de doutorado visa o desenvolvimento de novos candidatos para tratamento de tumores de mama triplo-negativos. Fármacos que têm como alvo a proteína tubulina estão entre as terapias anticâncer mais bem-sucedidas e representam a primeira linha de tratamento para tumores do tipo TNBC. Neste contexto, foram desenvolvidas acridinonas que inibem a polimerização da tubulina e são capazes de impedir os principais mecanismos de resistência aos fármacos desta classe. O composto líder não interage com a bomba de efluxo glicoproteína-P, além de ser igualmente potente contra a linhagem celular superexpressando a tubulina βIII, uma isoforma clinicamente relevante. O mecanismo de ação revelou a interação desses compostos com o sítio da colchicina da proteína alvo. Os compostos apresentaram valores de IC50 entre 10 e 12.000 nM contra a linhagem tumoral TNBC MDA-MB-231. Em contrapartida, não foi observada citotoxicidade na linhagem normal de fibroblastos humanos (HFF1). Ensaios de imunofluorescência reforçaram a ação seletiva dos compostos, mostrando que os mesmos perturbaram a rede de microtúbulos nas células MDA-MB-231, mas não nas células HFF1. As substâncias também inibiram a migração celular e a angiogênese in vitro. Os enantiômeros do composto líder foram separados, levando a identificação de um eutômero 10 vezes mais potente contra as células tumorais e 2 vezes mais potente contra a tubulina quando comparado com a mistura racêmica. Um candidato a fármaco, eficaz e seguro, deve apresentar um balanço favorável entre a sua potência e seus parâmetros farmacocinéticos. Dessa forma, o metabolismo e a farmacocinética das acridinonas bioativas foram investigados. Em geral, os compostos foram metabolicamente estáveis, mas requerem otimização da solubilidade e permeabilidade para o desenvolvimento de fármacos administrados por via oral. A avaliação do composto líder revelou propriedades promissoras que justificam a sua consideração em modelos pré-clínicos de prova de conceito. O objetivo é a geração de candidatos a novos fármacos moduladores da tubulina com ação anticâncer. / Cancer is the second most common cause of death globally, being the breast tumors the leading cause of death in women. The number of anticancer medicines grows yearly but still exhibits problems such as high toxicity and resistance. The breast cancer has a number of subtypes and the one presenting the poorest prognosis and biggest therapeutic limitation is called triple negative breast cancer (TNBC). This PhD work aims to develop new small molecules as candidates to the treatment of the triple negative breast cancer. Drugs that target the protein tubulin are among the most successful anticancer therapies and represent the first line treatment to TNBC tumors. In this context, we developed a series of acridinones as tubulin inhibitors that can circumvent common resistance mechanisms to tubulin modulators. The lead compound did not interact with the P-glycoprotein and presented same effectiveness against cell lines overexpressing a clinically relevant tubulin isotype (βIII). In this work, we determined the mechanism of action of these compounds, that bind to the colchicine site in the tubulin. The compounds presented IC50 cytotoxicity values between 10 – 12000 nM against the TNBC cell line MDA-MB-231, with no cytotoxicity against a normal fibroblast cell line (HFF1). Immunofluorescence studies reinforced the compounds selectivity showing they disrupted the microtubules network on MDA-MB-231 cells but not on the HFF1 ones. The substances also inhibited cell migration and angiogenesis in vitro. The enantiomers of the lead compound were purified, and we identified an eutomer 10-fold more potent against the tumor cells and 2-fold more potent against the tubulin when compared to the racemic mixture. The metabolism and pharmacokinetics of the compounds were also studied aiming the development of agents with a favourable balance between potency and pharmacokinetic parameters, essential feature of an effective and safe drug. In general, the compounds were metabolically stable but need an improvement in the solubility and permeability properties to be developed as oral drugs. In summary, the lead compound presents promising properties to be assessed in preclinical proof of concept studies for further development as next-generation antitubulin drugs.

<p><i>Staphylococcus aureus</i> (<i>S. aureus</i>) is a Gram-positive pathogen that causes a wide range of infections in both hospitals and communities, of which the total mortality rate is higher than AIDS, tuberculosis, and viral hepatitis combined. The drug resistant <i>S. aureus </i>is a member of the “ESKAPE” pathogens that require immediate and sustained actions of novel method to combat. However, the current antimicrobial development against <i>S. aureus</i> is in stagnation, which underscores the urgent need for novel antimicrobial scaffolds and targets. <i>S. aureus</i> Ribonuclease P protein (RnpA) is an essential protein that plays important roles in both tRNA maturation and mRNA degradation pathways. The goal of this research was to evaluate RnpA as an antimicrobial target using biophysical methods. The crystal structures of wild-type RnpA in three different constructs were determined, among which the tag-free RnpA construct has a structural model of 2.0 Å resolution and R_crys/R_free= 0.214/0.234, and its crystals are reproducible. This crystal structure of tag-free <i>S. aureus </i>RnpA shows a globular representation with key structural motifs, including the “RNR” Ribonuclease P RNA binding region and a substrate binding central cleft, which shares high similarity to previously solved RnpA structures from other species despite of their low sequence identity. Meanwhile, in a screen of <i>S. aureus </i>RnpA mutants performed by our collaborator, RnpA^P89A was found lacking the mRNA degradation activity while retaining the tRNA maturation function, and causing defects in cell viability. We therefore studied this mutant using differential scanning fluorimetry, crystallography, and circular dichroism. It was shown that RnpA^P89A is thermally less stable than wild-type RnpA by ~2.0 ˚C, but no secondary structural or 3D conformational differences were found between the two proteins. Although the mutant RnpA^P89A requires further characterization, the results of the studies in this thesis have begun to shed light on the relatively new role of <i>S. aureus </i>RnpA in mRNA degradation, and will serve as useful tools in future structure-based drug discovery for multi-drug resistant <i>S. aureus </i>treatment. </p>

Thesis advisor: Marc A.T. Muskavitch / Malaria is a life-threatening infectious disease caused by inoculation of the apicomplexan Plasmodium parasite into vertebrate hosts. Transmission of the parasite is mediated by the Anopheles mosquito, which has the capacity to efficiently transmit the parasite from host to host, as the disease vector. There are many factors that make anopheline mosquitoes competent vectors for disease transmission. The hematophagous (blood-feeding) behavior of the female mosquito is one of most fundamental factors in physical transmission of parasites, because the ingestion of blood from an infected host allows parasite entry into the mosquito and the completion of parasite sexual reproduction. In addition to this blood-feeding behavior, there are a host of biological (i.e., parasite replication) and behavioral factors (i.e., mosquito chemosensation, host preference) that contribute to the high vectorial capacity of these vector species. There are over four hundred Anopheles species worldwide, approximately forty of which are considered epidemiologically critical human malaria vectors. Anopheles gambiae, the primary vector in malaria-endemic sub-Saharan Africa, is responsible for the largest number of malaria cases in the world and is therefore one of the most important vectors to study and target with control measures. Currently, vector-targeted control strategies remain our most effective tools for reduction of malaria transmission and incidence. Although control efforts based on the deployment of insecticides have proven successful in the past and are still widely used, the threat and continuing increases of insecticide resistance motivate the discovery of novel insecticides. In this thesis, I provide evidence that G protein-coupled receptors (GPCRs) may serve as “druggable” targets for the development of new insecticides, through the modulation of developmental and sensory processes. In Chapter II, “A critical role for the Drosophila dopamine 1-like receptor Dop1R2 at the onset of metamorphosis,” I provide evidence supporting an essential role for this receptor in Drosophila melanogaster metamorphosis via transgenic RNA interference and pharmacological methods. In An. gambiae, we find that the receptor encoded by the mosquito ortholog GPRDOP2 can be inhibited in vitro using pharmacological antagonists, and that in vivo inhibition with such antagonists produces pre-adult lethality. These findings support the inference that this An. gambiae dopamine receptor may serve as a novel target for the development of vector-targeted larvicides. In Chapter III, “RNAi trigger delivery into Anopheles gambiae pupae,” I describe the development of a method for injection directly into the hemolymph of double strand RNA (dsRNA) during the pupal stage, and I demonstrate that knockdown of the translational product of the SRPN2 gene occurs efficiently, based on reductions in the levels of SRPN2 protein and formation of melanized pseudo-tumors, in SRPN2 knockdown mosquitoes. This method was developed for rapid knockdown of target genes, using a dye-labeled injection technique that allows for easy visualization of injection quality. This technique is further utilized in Chapter IV, “Uncovering the Role of an Anopheles gambiae G Protein-Coupled Receptor, GPRGR2, in the Detection of Noxious Compounds,” where the role for GPRGR2 in the detection of multiple noxious compounds is elucidated. We find that pupal stage knockdown of this receptor decreases the ability of adult Anopheles gambiae to identify multiple noxious compounds. While these findings provide a strong link between GPRGR2 and a very interesting mosquito behavior, they may also provide opportunities to develop better field-based strategies (i.e., insecticides baited traps) for vector control. The goal of this thesis is to understand the functional roles of selected mosquito GPCRs that may serve as targets for the development of new vector-targeted control strategies. Exploiting these GPCRs genetically and pharmacologically may provide insights into novel vector control targets that can be manipulated so as to decrease the vectorial capacity of An. gambiae and other malaria vectors in the field, and thereby decrease the burden of human malaria. / Thesis (PhD) — Boston College, 2015. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.