Browsing by Subject "Capillary Electrophoresis"
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Item ADVACING ORGANELLE ANALYSIS: DEVELOPMENT OF TECHNIQUES FOR THE ENRICHMENT OF ENDOCYTIC ORGANELLES AND TO DETERMINE AUTOPHAGOSOME PROPERTIES(2013-07) Satori, Chad PatrickLysosomes and endocytic organelles are intracellular bodies present in eukaryotic cells responsible for the degradation of endocytosed extracellular targets. Autophagosomes traffic proteins, organelles, and other intracellular components to lysosomes to facilitate degradation during the degradative process of autophagy. Multiple disorders have been connected to malfunctions in lysosomes (Nieman pick, galactosialidosis, Danon disease) and autophagosomes (Alzheimers, Parkinson's, Huntington's).Methods are needed to enrich organelles in order to study their properties without contamination from unwanted organelles. Current methods to enrich endocytic organelles do not result in highly enriched organelles (differential centrifugation), are time consuming and tedious (density gradient centrifugation), and can damage membranes. Methods are also needed to determine endocytic and autophagy organelle properties such as organelle molecular composition, organelle-specific biotransformation of anti-cancer drugs, individual organelle surface properties and marker protein levels, and pH. The work described in this thesis develops new techniques to improve our ability to enrich endocytic organelles and determine their properties. This work includes: (1) the magnetic enrichment of endocytic organelles and determination of pH by capillary cytometry, (2) the determination of the biotransformation of N-L-leucyldoxorubicin to doxorubicin, (3) the development of a workflow to determine preliminary identifications of enriched autophagosome samples and (4) determine temporal changes in individual autophagy organelle numbers, surface charge, and LC3-II levels from basal and rapamycin enhanced autophagy levels. These methods will improve our understanding of how lysosomes and autophagosomes contribute to disease, leading to better therapeutic strategies that may improve and lengthen people's lives. Endocytic organelle enrichment was done by trafficking dextran coated magnetic iron oxide nanoparticles to lysosomes and endocytic organelles prior to magnetic separation. No detectable enzymatic activity from mitochondria and peroxisomes were observed in the enriched endocytic organelle fractions suggesting that the enriched lysosomes were in high enrichment. A majority of enriched, individual endocytic organelles had an acidic pH as determined by capillary cytometry suggesting the enriched endocytic organelle fraction had intact membranes. Enriched endocytic organelle fractions were then used to determine the biotransformation of N-L-leucyldoxorubicin to doxorubicin. Previous reports had suggested endocytic organelles may be important for intracellular biotransformation. About 45% of the biotransformation from uterine sarcoma cell post nuclear fraction occurred in the enriched endocytic organelle fraction suggesting intracellular biotransformation may be more critical to prodrug activation than previously believed.Ultra high performance liquid chromatography coupled to near-simultaneous low- and high-collision energy mass spectrometry was used to determine preliminary identifications of compounds enriched or unique to enriched autophagosome fractions. A workflow was developed to detect and confirm features (unidentified compounds with a characteristic chromatographic tR and m/z value) in the enriched sample as well as making and confirming identifications from online databases. Multiple high-relevancy preliminary identifications were made that are relevant to autophagy as supported by literature searches. Following validation, these preliminary identifications could prove to be important to maintain autophagosome function and autophagy. Capillary electrophoresis coupled to laser induced fluorescence detection (CE-LIF) was used to determine temporal changes in the detected number of individual autophagy organelle events (phagophores, autophagosomes, amphisomes, and autolysosomes), of GFP-LC3-II levels, and of surface charge by CE-LIF. Pharmacological treatment with vinblastine was used to accumulate autophagosomes and phagophores from basal and rapamycin enhanced autophagy do detect temporal changes in autophagy organelles characteristic of the autophagy level and its autophagy flux. The dramatic contrast between time dependent changes in individual organelle properties between basal and rapamycin enhanced autophagy conditions demonstrates an anticipated complexity of autophagy flux which likely plays critical role in response to drug treatments, aging, and disease.Item Aptamer selection using capillary electrophoresis-SELEX(2008-10) Mosing, Renee KarenSELEX is a method used for the combinatorial selection of aptamers, or single stranded nucleic acid sequences that bind with high affinity and specificity to target molecules. Although successful, SELEX is a very time consuming, laborious process. We introduced a modification to this process called capillary electrophoresis-SELEX. This protocol proved to be significantly more efficient, which greatly decreased the time requirement of the process from weeks to days. This improvement was observed despite the lower loading capacity and resolution limited injections on CE which introduced approximately 1013 sequences to the separation instead of the 1015 sequences introduced in more traditional selections protocols. In fact, ssDNA aptamers with picomolar affinity for HIV-1 RT were identified in 4 rounds. Further sequence/structure characterization of these sequences demonstrated no homology, indicating that several sequences can bind with high affinity to the target. Interestingly, the aptamers were 10 fold more selective for the original target (HIV-1 RT) than other reverse transcriptases. Despite this result, the aptamers did not demonstrate inhibition of reverse transcriptase activity. The success of these collections prompted investigation of more challenging targets such as mitochondria and bacteria. These targets are difficult to purify and have surface chemistries that are constantly changing. Aptamers for these targets must identify a feature on the surface that is consistent in order to be conserved throughout the process. Our experiments indicated that the aptamers may have bound to features on the surface that are not very abundant, making affinity characterization cumbersome. Future experiments aim to determine if the aptamers are becoming more refined for specific features rather than just focusing on increase in affinity. Finally, initial experiments were performed for a model that will drive selections toward a specific binding site on the surface of the HIV assembly protein, capsid. Further experiments are proposed to allow aptamer binding to specific sites, and for aptamer characterization.Item Beyond the cycle: investigating the sequencing, binding affinity, and utility of aptamers selected with CE-SELEX.(2023-02) Brinza, NicholasAptamers are single stranded DNA or RNA sequences that bind with high affinity to a target molecule. The discovery of sequences that can bind to a target of interest starts with a mixture of random sequences which are refined through repeated incubation with the target followed by amplification of binding sequences in a process known as systematic evolution of ligands by exponential enrichment (SELEX). Aptamers produced by multiple SELEX rounds can assist bioanalytical scientific research through detecting molecules of interest in biosensors and have the potential to improve drug delivery to specific cells in humans. However, developing aptamers capable of useful application is difficult due to multiple unique challenges. Quantifying the ability for a pool of aptamers produced by SELEX to bind to a target has no single perfect method. In the field of aptamer research, scientists employ a variety of binding affinity tests which contain specific advantages and disadvantages. Reported values for binding affinity vary between different methods. When a pool does show evidence of most sequences binding, identifying the best sequences in a mixture of more than thousands of unique base pair arrangements provides a separate challenge. Finally, even after finding a sequence with evidence of strong binding to a desired target, there remains uncertainty if the aptamer will bind to the target as well in a real-world application as it does in a carefully controlled lab environment. This study examines aptamers at three distinct stages of development for separate capillary electrophoresis SELEX (CE-SELEX) experiments. For an aptamer already selected against transferrin receptor 1 (TfR1) with high affinity, a combination of flow cytometry and confocal microscopy provided evidence the aptamer did not significantly internalize into cancerous liver cells used to model human drug delivery. A separate aptamer pool, previously developed over 4 rounds of selection against low-density lipoprotein receptor (LDLR), is analyzed with preexisting bioinformatic tools as well as custom Python code on next generation sequencing (NGS) data, revealing a primer dimer sequence from polymerase chain reaction (PCR) which impacted the selection of aptamer length candidates but remained at a lower frequency than one possible aptamer candidate. After performing 4 rounds of CE-SELEX against mouse leptin, testing the binding affinity of rounds 1-3 with CE showed weak evidence of binding in early rounds which was lost in later rounds after correcting for an earlier false positive result. Documenting CE problems and optimization, even for an unsuccessful aptamer selection, may help improve future CE research, while detailing the experimental set up, discovery, and correction of the false positive result is important to help prevent future misleading conclusions.Item Comprehensive Multidimensional Separations of Biological Samples using Capillary Electrophoresis coupled with Micro Free Flow Electrophoresis(2017-12) Johnson, AlexanderMicro free-flow electrophoresis (μFFE) is a continuous separation technique in which analytes are streamed through a perpendicularly applied electric field in a planar separation channel. Analyte streams are deflected laterally based on their electrophoretic mobilities as they flow through the separation channel. The continuous nature of µFFE separations makes it uniquely suitable as the second dimension for multidimensional separations. The focus of this work is the development of coupling capillary electrophoresis (CE) to µFFE as a high speed two-dimensional (2D) separation platform, followed by an investigation of orthogonality of the two techniques, and finally a novel label-free detection method for µFFE separations. A new µFFE device was fabricated and coupled to CE via capillary inserted directly into the µFFE separation channel. High peak capacity separations of trypsin digested BSA and small molecule bioamines demonstrated the power of CE × µFFE. Since both methods rely on electrophoretic mobility to separate, an investigation on the orthogonality of the two techniques was carried out. µFFE can operate in many different separation modes to increase the orthogonality CE × µFFE. Lastly, fluorescent labeling of the analytes can cause the sample to lose its dimensionality affecting 2D separation peak capacity and coverage. A novel absorption detector was studied to demonstrate the first ever label free absorption detection on a µFFE device. A separation was performed on visible dyes and their detection limits quantified.Item Development and Applications of In Vitro-Microdialysis: A Sampling Platform for Fast Analysis of Non-Electroactive Analytes(2016-04) Stading, AmyWhen considering the measurement of release events from cells, it can be done at levels as small as the single cell and performed in systems increasingly larger and more complex up to in vivo studies. Though in vitro systems lack the physiological relevance of in vivo, their simple and controlled environment is highly advantageous in preliminary mechanistic studies. In spite of this, there exists a serious gap in our ability to perform in vitro measurements on a wide array of analytes within a meaningful time frame. While electrochemical techniques are unparalleled in their ability to temporally resolve minute signals in biological systems, there is only a small class of targets which are suitable for this type of analysis. When analyzing non-electroactive analytes, measurements are often plagued by slow temporal responses (5+ minutes). Fluorescent imaging offers opportunities to monitor faster dynamics of non-electroactive analytes, but the target analyte must be either natively fluorescent or labeled, which can result in nonspecific binding and cytotoxicity. In both of these cases fast dynamics can be observed, but the array of analytes is small and only a few can be monitored simultaneously. In this work, a novel in vitro sampling platform is described which is capable of simultaneously monitoring approximately 15 non-electroactive analytes with 20 second temporal resolution. Cells were cultured on the surface of a microdialysis probe coupled with an analytical system for analysis. Small molecules released from the cells upon stimulation diffuse across the porous membrane because of the close proximity. A high-speed CE, built in house, enabled analysis of the collected dialysate. The ability of our platform to detect basal and stimulated release of amines was confirmed by transferring the probe between artificial cerebrospinal fluid (aCSF) and a potassium-spiked (100 mM K+-aCSF) stimulant solution. A variety of cell models were tested for compatibility with the in vitro-microdialysis platform, both single cell type and co-cultures were initiated. Adherence of viable cells was confirmed by labeling cells with either fluorescein diacetate (FDA) or specific antibody labelling, followed by imaging under a microscope. As a step towards continuously monitoring the change of non-electroactive analytes released from cultured cells, microdialysis was coupled directly to micro free flow electrophoresis (µFFE) device instead of the high-speed CE instrument.Item Development of a high-speed capillary electrophoresis assay for the analysis of branched chain amino acids released from 3T3-L1 adipocytes(2016-08) Harstad, RachelWe have developed a high-throughput assay for monitoring branched chain amino acid (BCAA) uptake and release dynamics in 3T3-L1 cells using microdialysis coupled to high speed capillary electrophoresis (CE). Isoleucine, leucine, and valine are important indicators of lipogenesis, as are alanine, glutamate, and glutamine, which are by-products produced through the catabolism of BCAAs. The major focus of this work will be on the development and optimization of a high-speed (high-throughput) CE assay for the separation of fluorescently labeled amino acids; particularly the BCAAs and their downstream metabolites. Development of this assay allows for us to ask important biological questions related to lipogenesis using an in vitro cell model. In combination with bulk culture, release and uptake rates of BCAAs were monitored and assessed as a function of various biological stimuli, including several concentrations of glucose, circulating concentrations of BCAAs, as well as insulin and artificial sweeteners. Changes in release profiles were also monitored as cells progressed through the differentiation cycle in order to see if development stage affects lipogenesis.