Cancer cell esterases are often overexpressed and some have chiral specificities different from those of corresponding normal cells. Carboxylesterases in particular are known to be overexpressed in several cancers. Additionally, cancer cells often exhibit high levels of intrinsic oxidative stress that is required for survival and an aggressive phenotype. We hypothesized that these 2 characteristics of cancer cells could be exploited to aid in the detection and treatment of prostate cancer. We have developed a fluorogenic ester probe that is activated by carboxylesterase to help distinguish tumorigenic cells from nontumorigenic prostate cells. Ester prodrugs have the same activation mechanism and have been thought to be a promising approach in cancer therapy. Prodrugs are inactive drugs that can be selectively activated by a specific enzyme. We have developed a chiral ester prodrug strategy using native polyacrylamide gel electrophoresis (n-PAGE) and proteomic methods to compare and identify the esterase profiles of several tumorigenic and nontumorigenic prostate cell lines. Our results showed that cell lysates from LNCaP, DU 145, and PC3 prostate cancer cell lines exhibit differential esterase activity compared with non-tumorigenic RWPE-1 prostate cell lysates when incubated with α- naphthyl acetate or α-naphthyl N-acetyl-alaninate ester substrates and a diazonium salt. We have identified oxidized protein hydrolase (OPH), a serine esterase/protease that catalyzes the removal of N-acylated residues from proteins, to be differentially expressed between some tumorigenic and nontumorigenic prostate cell lines. OPH was found to have high hydrolytic activity towards the S-isomer of α-naphthyl N-acetylalaninate (S-ANAA) chiral ester. LNCaP lysates incubated with N-acetyl-alanyl-p-nitroanilide, a known OPH substrate, had twofold higher OPH activity compared with RWPE-1 lysates. We have also developed and tested novel glutathione depleting prodrugs modeled after S-ANAA that increase oxidative stress and induced apoptosis in tumorigenic prostate cells with little effect on nontumorigenic RWPE-1 cells. These results suggest that ester molecular beacon probes and ester prodrugs may be effective in identifying and treating prostate cancer tumors that overexpress esterases with little effect on normal prostate cells.

Transfer RNAs (tRNAs) are known for their role as adaptors during translation of the genetic information and as regulators for gene expression; uncharged tRNAs regulate global gene expression in response to changes in amino acid pools in the cell. Aminoacylated tRNAs play a role in non-ribosomal peptide bond formation, post-translational protein labeling, modification of phospholipids in the cell membrane, and antibiotic biosynthesis. [1] tRNAs have a highly stable structure that can present a challenge for their detection using conventional techniques. [2] To enable signal amplification and lower detection limits, a split probe - split deoxyribozyme (sDz or BiDz) probe, which uses a double-labeled fluorogenic substrate as a reporter – has been introduced. In this project we developed an assay based on sDz probe to detect yeast tRNAPhe as a proof-of-principle highly structured target. An sDz probe was designed specific to tRNAphe that could efficiently unwind stable secondary and tertiary structure of the target RNA thereby providing an efficient tool for tRNA detection. [3]The efficiency of the developed sDz probe was compared with a currently used state-of-the-art hybridization probe – molecular beacon probe. The results obtained in the project further demonstrate the power of sDz probes for the detection of highly structured RNA analytes. The split probes show signal amplification capabilities in detection of structured analytes, which will benefit diagnostics, fundamental molecular biology research and therapeutic fields.

L'angiogenèse est une étape clef dans le processus de progression tumorale. Elle est caractérisée par une surexpresssion d'un grand nombre de métalloprotéinases matricielles (MMP). Parmis ces MMP, les gélatinases (MMP-2 et MMP-9) sont connues pour jouer un rôle important dans l'angiogenèse tumorale et la croissance de nombreux cancers. Les "Photodynamic Molecular Beacon" (PMB) sont des constructions moléculaires qui peuvent être utilisées dans le traitement de cancers en associant un photosensibilisateur (PS) de type chlorine et un inhibiteur d'états excités, aussi appelé "Quencher", liés par un peptide substrat des gélatinases afin d'inhiber la toxicité du PS dans les cellules non ciblées, et de restaurer sa toxicité uniquement à proximité des gélatinases. Nous avons donc cherché à déterminer le couple PS/quencher permettant la meilleure inhibition de la production d'oxygène singulet, principale source de la toxicité du PS, puis avons synthétisé une famille de PMB ciblant les gélatinases. Différents peptides et bras espaceurs ont été utilisés pour évaluer l'influence de la distance entre le PS et le quencher sur les propriétés photophysiques et l'activation enzymatique du PMB / Angiogenesis is a key step in the tumoral progression process. It is characterized by an over-expression of a number of matrix metalloproteinases (MMP). Among these MMPs, gelatinases (MMP-2 and MMP-9) are known to play a critical role in tumor angiogenesis and the growth of many cancers. Photodynamic Molecular Beacons (PMB) can be designed for cancer treatment by associating a chlorin-like photosensitizer and a black hole quencher linked by a gelatinase substrate peptide with the aim of silencing photosensitizer toxicity in non-targeted cells and restore its toxicity only in surrounding gelatinases. We investigated the PS/quencher pair allowing the best singlet oxygen production inhibition, and then we synthesized a novel family of PMB triggering gélatinases MMP-2 and MMP-9. Different lengths of peptide and spacers were used in order to determinate the influence of the distance between PS and quencher on the PMB photophysical properties and enzymatic activation.

Une des limitations de la thérapie photodynamique est la faible sélectivité des photosensibilisateurs (PS) pour les tissus tumoraux. La recherche de nouveaux PS plus sélectifs s’est orientée vers la synthèse de PS couplés à des motifs afins pour des récepteurs membranaires surexprimés dans certains cancers. Le récepteur à l’acide folique est surexprimé dans les carcinomes ovariens et des PS ont été conjugués à de l’acide folique. Des PS ont aussi été couplés à un peptide spécifique de neuropiline 1 surexprimé au niveau des cellules tumorales de médulloblastome. Une autre stratégie pour augmenter la sélectivité du traitement vise à produire les espèces réactives de l’oxygène spécifiquement au niveau du site tumoral. L’activité de clivage enzymatique de marqueurs biologiques surexprimés dans les zones tumorales est utilisée. Les gélatinases (MMP-2 et -9) et leur activateur MMP-14 sont connues pour jouer un rôle primordial dans l'angiogenèse tumorale et la croissance du glioblastome multiforme. Différents photodynamic molecular beacons (PMB), associant un PS et un quencher lié par un peptide substrat des gélatinases et MMP-14, ont été développés / One limitation of photodynamic therapy is the low selectivity of photosensitizers (PS) to tumour tissue. The search of new PS more selective began to focus on the synthesis of PS coupled with substrate specific of the membrane receptors overexpressed in certain cancers. The acid folic receptor is overexpressed in ovarian carcinomas and PS were conjugated with folic acid. PS were also coupled with a specific peptide of neuropilin 1 overexpressed in tumoral cells of medulloblastoma. Another strategy for increasing the selectivity of the treatment is to produce reactive oxygen species specifically at the tumor site. The activity of enzymatic cleavage of biomarkers overexpressed in tumour areas is used. The gelatinases (MMP-2 and MMP-9) and their activator MMP-14 are known to play a key role in tumour angiogenesis and the growth of glioblastoma multiform. Different photodynamic molecular beacons (PMB), composed of a photosensitizer and a quencher linked together by a peptide substrate of gelatinases or MMP-14, were designed.

Global threats to treatment of bacterial infections due to antibiotic resistance (AR) have been on the rise in recent years. Current diagnostic tests identify bacteria by using blood culture, which takes more than 24 hours. This study focuses on the fluorescent labeling of DNA derived from bacterial AR genes (KPC & VIM) and other model DNAs using oligreen dye (OG) and molecular beacons (MB). A NanoDrop 3300 fluorospectrometer was used to take fluorescence measurements. Linear dynamic range and labeling efficiency were dependent on the following optimized conditions: dilution factor of OG (200 fold), buffer (20 mM Tris HCl, pH 8), and heat treatment of 95 °C for 15 min.Fluorescence analysis of a target DNA with a designed MB showed signal-to-background of 10 with our buffer only and 20 with our buffer and 25% ethanol. I also demonstrated a simple microfluidic device capable of detecting AR genes using model DNAs, magnetic beads, and designed MBs for assays of µ50 L volume. This study provides a first step towards detecting MB-DNA complexes by a simple, low cost, and fast non-amplified method, which may be used to detect AR genes in clinical samples in the future.

During the past few years, gold nanoparticles (AuNPs) have received considerable attention in many fields due to their optical properties, photothermal effect and biocompatibility. AuNPs, particularly AuNCs and AuNRs, exhibit great potential in diagnostics and imaging. In the present study, AuNCs were used to selectively image and quantify intracellular antioxidants. It was reported by Chen et al. that the strong fluorescence of AuNCs is quenched by highly reactive oxygen species (hROS). Most of applications depend on fluorescence quenching, however, for our project we designed turn-on fluorescent sensors using AuNCs that sense antioxidants. In the presence of antioxidants, AuNCs fluorescence switch on, while in the absence of antioxidants their fluorescence immediately turn off due to hROS effect. AuNRs were also used for cellular imaging in which AuNRs were conjugated to Cy3-labelled molecular beacon (MB) DNA. Next, the previous complex was loaded in two different strains of magnetotactic bacteria (MTB). MTB were used as a targeted delivery vehicle in which magnetosomes direct the movement of bacteria. The DNA sequence was specific to a certain sequence in mitochondria. The exposure of MTB to an alternating magnetic field (AMF) leads to the increase of temperature inside the bacteria, which destruct the cell wall, and hence, bacterial payload is released. When MD-DNA hybrid with the target sequence, AuNR and Cy3 separate from each other, the fluorescence of the Cy3 is restored.

The hybridization between two complementary strands of nucleic acid is the basis for a number of applications in DNA and RNA analysis, including in vivo RNA monitoring, microarrays, SNPs detection, and so on. The short oligonucleotide probes form Watson/Crick base pairs (A-T and G-C) with the analyzed nucleic acid. Molecular beacon (MB) probe is one of the most advantageous tools for nucleic acid analysis in real-time. A traditional MB probe consists of a DNA strand folded in hairpin motif with a fluorophore attached to the 5'end and a quencher attached to the 3' end. The loop segment is complementary to the analytes. Upon hybridization to a complementary single-stranded nucleic acid, MB probe switches to the elongated conformation, which separates the fluorophore from the quencher, resulting in high fluorescence signal. However, DNA or RNA folded in hairpin motifs are difficult to analyze by a conventional MB probes. Inefficient formation of the duplex between the secondary analyte and the MB probe results in low or undetectable fluorescent signal. In this project, we developed a tripartite probe consisting of one MB probe and two adaptor strands to genotype single nucleotide polymorphism (SNPs) in DNA hairpin motifs in real-time fluorescent assays. Each adaptor strand contains a fragment complementary to the analyte and a fragment complementary to an MB probe. One adaptor strand hybridizes to the analyte and unwinds its secondary structure, and the other strand forms stable complex only with the fully complementary analyte sequence. The tri-component probe promises to simplify nucleic acid analysis at ambient temperatures in such application as in vivo RNA monitoring and isothermal detection of specific DNA/RNA targets.

No description available.

<p>Among the great challenges in biology are the precise quantification of specific sets of proteins and analyses of their patterns of interaction on a much larger scale than is possible today. </p><p>This thesis presents a novel protein detection technique - proximity ligation - and reports the development and application of a nucleic acid amplification technique, RCA. Proximity ligation converts information about the presence or co-localization of specific proteins to unique sets of nucleic acid sequences. For detection of target proteins or protein complexes the coincident binding by pairs or triplets of specific protein-binding reagents are required. Oligonucleotide-extensions attached to those binding reagents are joined by a DNA ligase and subsequently analyzed by standard molecular genetic techniques. The technique is shown to sensitively detect an assortment of proteins using different types of binders converted to proximity probes, including SELEX aptamers and mono- and polyclonal antibodies. I discuss factors important for using the technique to analyze many proteins simultaneously.</p><p>Quantification of target molecules requires precise amplification and detection. I show how rolling circle amplification, RCA, can be used for precise quantification of circular templates using modified molecular beacons with real-time detection. The combination of proximity-probe templated circularization and RCA results in a sensitive method with high selectivity, capable of visualizing individual immobilized proteins. This technique is used for localized detection of a set of individual proteins and protein complexes at sub-cellular resolution.</p>

Among the great challenges in biology are the precise quantification of specific sets of proteins and analyses of their patterns of interaction on a much larger scale than is possible today. This thesis presents a novel protein detection technique - proximity ligation - and reports the development and application of a nucleic acid amplification technique, RCA. Proximity ligation converts information about the presence or co-localization of specific proteins to unique sets of nucleic acid sequences. For detection of target proteins or protein complexes the coincident binding by pairs or triplets of specific protein-binding reagents are required. Oligonucleotide-extensions attached to those binding reagents are joined by a DNA ligase and subsequently analyzed by standard molecular genetic techniques. The technique is shown to sensitively detect an assortment of proteins using different types of binders converted to proximity probes, including SELEX aptamers and mono- and polyclonal antibodies. I discuss factors important for using the technique to analyze many proteins simultaneously. Quantification of target molecules requires precise amplification and detection. I show how rolling circle amplification, RCA, can be used for precise quantification of circular templates using modified molecular beacons with real-time detection. The combination of proximity-probe templated circularization and RCA results in a sensitive method with high selectivity, capable of visualizing individual immobilized proteins. This technique is used for localized detection of a set of individual proteins and protein complexes at sub-cellular resolution.