The World Health Organization estimates that about 10-30% of pharmaceuticals in the world are either substandard or counterfeit. The number is even higher in the developing countries. From a public health perspective, a key contributor to the development and proliferation drug resistant strains of infections, including tuberculosis (TB), malaria and other infections that are leading killers in resource limited settings is poor quality medicines. Most of the main causes are profit driven corruption in many pharmaceutical companies, the poor manufacture and quality control, and/or the inappropriate storage conditions. Poor quality drugs lead to loss of life, create morbidity, strain the financial structure of the health system and lead to long-term drug resistance that affects us all. The current technology for screening poor quality drugs can be divided into 2 categories: the high end, precise and high cost technologies (such as High Performance Liquid Chromatography) and lower cost and qualitative technologies (such as Thin-Layered Chromatography). The high-end methods can give a precise measurement of active pharmaceutical ingredient (API) concentration and the presence of impurities in the tablets, but require trained personnel, advanced machine and lab set up, not suitable for field testing where most of poor quality pharmaceuticals have been found. The lower cost techniques require little training and simple equipment to operate at a relatively inexpensive price, but only gives qualitative results. In addition, most of current methods do not look at the dissolution profile of the tablets simultaneously with the concentration of API. Therefore, we propose to develop an assay that can quantify the concentrations of multiple APIs simultaneously and measure dissolution rates. In order to address current gaps in knowledge, my research proposal has three main parts in the assay development: 1) Development of an fluorescent/luminescent assay for detection of counterfeit/substandard antimalarial using small-molecules-based methods and field testing in Ghana; 2) Development of a fluorescent assay for detection of water-soluble pharmaceuticals using SELEX; and 3) Design a detection platform using microfluidic chips for real time quantification of multiple active pharmaceutical ingredients. For proof-of-concept, an antimalarial drug (artesunate and amodiaquine) and antibacterial antibiotics (sulfamethoxazole and trimethoprim) are selected to demonstrate the probe development and test the chip performance. Overall, the assay will be rapid, robust, portable, inexpensive, multiplexed, quantitative, specific, and sensitive. At a big picture level, emphasizing drug quality and creating robust mechanisms of drug testing will improve health outcomes and enhance treatment efficacy in resource limited settings.

CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / FAPEMIG - Fundação de Amparo a Pesquisa do Estado de Minas Gerais / INCT - Instituto Nacional de Ciência e Tecnologia / A tuberculose (Tb) é uma infecção heterogênea causada pelo Mycobacterium tuberculosis (Mtb), com altos índices de mortalidade. O principal método diagnóstico consiste em achados bacteriológicos, carecendo de alternativas mais eficazes. Acurácia, agilidade e acessibilidade no diagnóstico são cruciais para a contenção do fluxo infeccioso. Neste trabalho, objetivamos caracterizar o padrão de expressão de 27 citocinas na saliva e no soro de pacientes com Tb em associação com o tempo de tratamento, avaliar a aplicação de peptídeos sintéticos, TBC10 e TB2C10, em ensaios imunoenzimáticos e biossensor para o diagnóstico da Tb pela saliva e selecionar ácidos nucleicos ligantes de proteínas específicas. 27 citocinas, em soro e saliva de 86 indivíduos, foram dosadas simultaneamente por kit multiplex. Os indivíduos foram agrupados em TB (doentes sem tratamento), TBT (doentes em curso de tratamento), TBTDO (doentes ao término do tratamento), PPD+ (saudáveis reativos ao teste tuberculínico PPD) e C- (saudáveis não reativos ao PPD). Nove citocinas apresentaram maior expressão em TB que em C-. A expressão IL-2, IL-15 e IL-4 na saliva foi significativamente diferente entre doentes e PPD+. Todos os grupos positivos para Tb apresentaram maior expressão de IL-2, IL15, IL-8, IL-9 na saliva que indivíduos saudáveis. Os valores obtidos para IP-10 no soro permitiu distinguir os grupos positivos entre si e negativo, apresentando potencial pra monitoramento do tratamento de Tb. Seis citocinas, IL-13, IL-10, IL-6, IL-2, IL-15 e IL-8, foram significativamente mais expressas no soro dos grupos positivos que de saudáveis. O score associando as quatro citocinas com melhor representatividade na saliva e as seis no soro, permitiu predizer a infecção por Mtb com assertividade de 77% na saliva e 96% no soro. A quantificação de citocinas pode auxiliar no diagnóstico de Tb. Para validação dos peptídeos sintéticos, TBC10 e TB2C10 foram testados em imunoensaios pra detecção de IgA específica de tuberculose presente em saliva. Ambos foram capazes de discriminar doentes de saudáveis com sensibilidade de 94,12% e especificidades 76,92% e 84,62%, com valores de p<0,0001. Foi realizado um biopanning de anticorpo para caracterização molecular dos ligantes de IgA específica de Tb, a partir de um fago C10. Obteve-se um scFv (fragmento variável de cadeia única) capaz de se ligar a TBC10, TB2C10 e a proteínas de Mtb, o que permitiu delinear os prováveis epítopos de proteínas de Mtb reconhecidos pela IgA. SELEX foi utilizado para selecionar moléculas de DNA ligante de TBC10 e TB2C10. Quatro aptâmeros apresentaram capacidade de reconhecer os biomarcadores e também proteínas de Mtb. Tanto o scFv quanto os aptâmeros de DNA selecionados a partir dos biomarcadores puderam reconhecer proteínas de Mtb, confirmando a fidelidade dos epítopos das proteínas e dos biomarcadores. Os biomarcadores TBC10 e TB2C10 apresentaram alto potencial de aplicação para diagnóstico de Tb através da saliva, os aptâmeros, úteis para confirmação da identidade dos epítopos ligantes a IgA, apresentaram potencial para fins diagnósticos e terapêuticos, assim como os scores utilizados a partir da expressão de citocinas em soro e saliva permitem predizer a infecção ativa de tuberculose. / Tuberculosis (TB) is a heterogeneous infection caused by Mycobacterium tuberculosis (Mtb), with high mortality rates. The main diagnostic method consists of bacteriological findings, lacking more effective alternatives. Accuracy, agility and accessibility in diagnosis are crucial for the containment of infectious flow. In this work, we aimed to characterize the expression pattern of 27 cytokines in the saliva and serum of patients with Tb in association with the time of treatment, to evaluate the application of synthetic peptides, TBC10 and TB2C10, in immunoenzymatic and biosensor assays for the diagnosis of Tb by saliva and select specific protein binding nucleic acids. 27 cytokines, in serum and saliva of 86 individuals, were simultaneously dosed by multiplex kit. The individuals were grouped into TB (untreated patients), TBT (patients undergoing treatment), TBTDO (patients at the end of treatment), PPD + (healthy individuals reactive tuberculin test PPD) and C- (healthy non-reactive to PPD). Nine cytokines presented greater expression in TB than in C-. The expression IL-2, IL-15 and IL-4 in saliva was significantly different between patients and PPD +. All Tb-positive groups had higher expression of IL-2, IL-15, IL-8, IL-9 in saliva than healthy individuals. The values obtained for IP-10 in the serum allowed to distinguish the positive groups from each other and negative, presenting potential for monitoring the treatment of Tb. Six cytokines, IL-13, IL-10, IL-6, IL-2, IL-15 and IL-8 were significantly more expressed in serum than the positive groups. The score associated with the four cytokines with better representativeness in the saliva and the six in the serum allowed to predict Mtb infection with assertiveness of 77% in saliva and 96% in serum. Quantification of cytokines may aid in the diagnosis of Tb. For the validation of the synthetic peptides, TBC10 and TB2C10 were tested in immunoassays for the detection of specific IgA of tuberculosis present in saliva. Both were able to discriminate healthy patients with sensitivity of 94.12% and specificities 76.92% and 84.62%, with values of p <0.0001. An antibody biopanning was performed for molecular characterization of Tb-specific IgA ligands from a C10 phage. A scFv (single chain variable fragment) was obtained capable of binding to TBC10, TB2C10 and Mtb proteins, which allowed to delineate the likely epitopes of Mtb proteins recognized by IgA. SELEX was used to select TBC10 and TB2C10 binding DNA molecules. Four aptamers were able to recognize the biomarkers and also Mtb proteins. Both the scFv and the DNA aptamers selected from the biomarkers were able to recognize Mtb proteins, confirming the fidelity of protein epitopes and biomarkers. The biomarkers TBC10 and TB2C10 presented high potential of application for diagnosis of Tb through saliva, the aptamers, useful for confirming the identity of IgA binding epitopes, presented potential for diagnostic and therapeutic purposes, as well as the scores used from the expression of cytokines in serum and saliva allow prediction of active tuberculosis infection. / Tese (Doutorado)

<div> <div> <div> <p>The cosmos of noncoding RNAs (ncRNAs) has been thriving in recent years; so much so that researchers are discovering them much faster than they can uncover their functions. The subset of these RNAs that have been characterized have been noted to perform and regulate a plethora of remarkably diverse and essential biological functions. This diversity in function is accompanied by a large array of dynamic and elegantly folded 3-dimensional structures. In this collection of work, we will journey through the discovery of the first catalytic noncoding RNAs (ribozymes), explore a new method for identifying uniquely structured ribozymes, and detail the design of a technique to select for highly structured RNAs with a high affinity for an RNA binding partner. Although these topics vary widely within the field of RNA, this work strives to showcase the integral relationship between intricate macromolecular structures with their chemical and cellular functions. </p> </div> </div> </div>

Glypican-3 (GPC3), the cellular membrane proteoglycan, has been established as a tumor biomarker for early diagnosis of hepatocellular carcinoma (HCC). GPC3 is highly expressed in more than 70% HCC tissues detected by antibody-based histopathological systems. Recently, aptamers, a short single-strand DNA or RNA generated from systematic evolution of ligands by exponential enrichment (SELEX), were reported as potential alternatives in tumor-targeted imaging and diagnosis. In this study, a total of 19 GPC3-bound aptamers were successfully screened by capillary electrophoresis (CE)-SELEX technology. After truncated, AP613-1 was confirmed to specifically target GPC3 with a dissociation constant (KD) of 59.85 nM. When modified with a phosphorothioate linkage, APS613-1 targeted GPC3 with a KD of 15.48 nM and could be used as a specific probe in living Huh7 and PLC/PRF/5 imaging, GPC3-positive cell lines, but not in L02 or A549, two GPC3-negative cell lines. More importantly, Alexa Fluor 750-conjugated APS613-1 could be used as a fluorescent probe to subcutaneous HCC imaging in xenograft nude mice. Our results indicated that modified AP613-1, especially APS613-1, was a potential agent in GPC3-positive tumor imaging for HCC early diagnosis.

No description available.

<p>Adaptation of nucleic acid <em>in vitro</em> selection for whole cell targets has been demonstrated to be an effective means of isolating useful sequences with applications in biomarker detection and therapeutics. The problem of efficient delivery of materials across cell membranes is common to a variety of research and medical fields. Existing aptamers isolated in surfacing binding selections have been successfully adapted for cell targeted therapies through complex modifications. However, better aptamers may be derived from a selection optimized to isolate internalized sequences directly. A cell selection experiment with the goal of identifying circular random-sequence DNA aptamers with the ability to facilitate their own internalization into MCF7 cells was conducted. Several classes of sequences isolated from this selection were shown to target cell nuclei at a rate significantly greater than control sequences as determined by qPCR relative recovery assays supported by <em>in situ</em> RCA fluorescence microscopy data. The localization of functional DNA sequences at the subcellular and intercellular levels suggests a receptor mediated mechanism. Techniques for the selection, purification and fluorescent detection of small circular DNAs were also developed for this study. Further work to characterize and identify targets should be pursued to better understand the mechanism of internalization and judge the suitability of G18d sequences as a delivery platform.</p> / Master of Science (MSc)

<p>The process of in vitro selection has led to the isolation of many catalytic DNA molecules, called deoxyribozymes, which can catalyze a range of biologically-relevant reactions. Despite these advances, questions still remain as to why DNA, which seems more suited to information storage than catalysis can efficiently catalyze chemical reactions. In this thesis, a group of deoxyribozymes that can catalyze their own phosphorylation using NTP substrates are used a model system to study how DNA is able to fold into complex structures necessary for catalysis. Using a variety of structural probing techniques, these studies elucidated a common secondary structure shared by three deoxyribozymes, which do not appear to share a common ancestor sequence. This suggests that this motif may be most efficient motif to catalyze self-phosphorylation by DNA. It also more generally demonstrates that DNA can undergo convergent evolution to reach the same complex folding arrangement. A fourth deoxyribozyme was found to fold into a complex tertiary structure containing a novel quadruplex-helix pseudoknot motif. The finding of this pseudoknot and comparison with other quadruplexes found in other functional nucleic acids led us to investigate whether these stable motifs could be incorporated into nucleic acid libraries to improve the process of in vitro selection and give researchers a better chance of isolating functional nucleic acids. Design and characterization of structured libraries revealed that DNA libraries could be made in which the majority of sequences are folded into quadruplex arrangements. The incorporation of this quadruplex scaffold into DNA sequence libraries may ease the isolation of functional nucleic acids that contain this useful structural motif. In the final part of this thesis, a self-phosphorylating deoxyribozyme was converted from a tool for study of DNA structure to a sensor for GTP and Mn²⁺, demonstrating that deoxyribozyme substrates can be converted into targets for biosensors.</p> / Doctor of Philosophy (PhD)

<p>The food-borne pathogen, <em>Listeria monocytogenes</em>, is a global health concern as it has been responsible for multiple food contamination outbreaks over the past century. Current detection methods like the enzyme-linked immunoassays (ELISA), and polymerase chain reaction (PCR) take over 24 h to attain results, are costly, require specialized equipment and trained personnel. In this study we investigated the use of functional nucleic acid (FNA) to develop a rapid and cost-effective detection method for <em>L. monocytogenes</em>. We carried out in<em> vitro</em> selection in order to isolate a fluorescently labeled DNA-RNA hybrid strand that can be bound and cleaved by specific endoribonucleases (RNase) from <em>L. monocytogenes</em>. We termed these DNA-RNA hybrid strands RNase-cleaved fluorescent substrate (RFS). Since no past studies have isolated RNases from <em>L. monocytogenes</em>, we first identified the genes based on sequence similarities with well characterized RNases. We purified and characterized RNase HII, RNase III and RNase G. Since this study focused primarily on developing RFS for RNase HII, we performed an in depth <em>in vitro</em> biochemical analysis to characterize this enzyme. We found that RNase HII from <em>L. monocytogenes</em> plays an important role in DNA replication and repair. Furthermore, we obtained six sequence classes by <em>in vitro</em> selection which could interact with RNase HII. The key nucleotide regions involved with RNase HII interactions were identified. In the final study, we showed the sequences isolated by <em>in vitro</em> selection could also be used as a tool to study ribonuclease function and identify new interaction between enzyme and substrate.</p> / Master of Science (MSc)

Clostridium difficile, the causative agent of C. difficile infection (CDI), causes hundreds of thousands of hospital-acquired infections in the United States and Canada annually. Furthermore, the prevalence and severity of CDI has been on the rise in developed countries, especially with the appearance of “hypervirulent” strains. Detection of CDI is thus of great importance. Traditional detection methods can be time consuming or lack the desired sensitivity. On the other hand, aptamers pose great prospects as diagnostic and therapeutic agents. Aptamers are nucleic acid ligands with molecular recognition capabilities rivaling those of antibodies. They are obtained by a process of in vitro selection known as systematic evolution of ligands by exponential enrichment (SELEX). However, the current approach may result in aptamers that experience non-specific binding in complex or biological samples. Here, we propose a multiple domain selection (MDS) method for aptamer isolation. This method utilizes independent selections on separate components of a single target in order to obtain uniquely specific aptamers. The aptamers can then be unified into a heterobivalent construct able to recognize two sites on one target. We hypothesize the combined aptamer would result in greater affinity and specificity for the target, resulting in greatly increased aptamer utility in current and future applications. In the current study, we have cloned and purified full length C. difficile DnaK as well as the N-terminal domain (NTD) and C-terminal domain (CTD) of the protein. MDS was performed on each target and the resulting aptamers were combined into a heterobivalent construct. The construct resulted in an approximately 100-fold affinity increase relative to the single aptamer for DnaK, and could detect much smaller quantities of target. Although it experienced low level recognition of high concentrations of purified E. coli DnaK, there was no detectable non-specific binding in several biological samples. / Thesis / Master of Science (MSc)

Asthma is a chronic disease affecting over 300 million people worldwide. Airway inflammation is a central feature of asthma. Quantitative sputum cytometry is the most validated method to assess this and to adjust anti-inflammatory therapy, yet it is underutilized due to rigorous processing that requires expensive specialized technicians. To address these limitations, this thesis focuses on the development of several point of need biosensors that rapidly quantify respiratory biomarkers as alternatives to traditional laboratory tests. The project began by developing a paper based sensor for detection of myeloperoxidase (MPO), a neutrophil biomarker. A test was developed using commercially available antibodies, showing direct correlation between the test line colour intensity and total neutrophils. This work was expanded to include a second protein target, eosinophil peroxidase (EPX), for identification of eosinophils. Although the test performed well using neat samples, it failed to identify EPX in clinical sputum samples. Analyzing pre-treatment methods identified that a quick immunoprecipitation technique using protein A/G beads followed by syringe filtration allowed for the device to successfully quantify EPX, eliminating the need for a centrifuge. However, the limited supply of commercial anti-EPX antibodies combined with the need for sample pre-treatment prompted investigation into alternative detection avenues. Nucleic acid aptamers were explored, with aptamer selection for EPX producing several aptamer candidates. Binding affinity and specificity tests were performed, with the T1-5 aptamer emerging. T1-5 was capable of selectively binding EPX over MPO with high affinity. This aptamer was integrated into a simple pull-down assay, capable of detecting EPX with an order of magnitude lower limit of detection than the antibody test. Overall this work has developed multiple sensors with the potential to overcome the limitations of accessibility to sputum cytometry, rapidly identify the presence and type of airway inflammation, and deliver personalized treatment strategies that not only reduce the global healthcare burden, but also greatly improve a patient’s quality of life. / Thesis / Doctor of Philosophy (PhD)