Therapeutic Delivery Technology and its Economic Impact

Therapeutic delivery technology is a current area of high interest in both university and industrial settings. These technologies are being developed in order to deliver therapeutic agents, such as genes, proteins, and drugs, to patients more efficiently. Nanoscale delivery vehicles have proven to be useful for these applications; these vehicles may either be naturally produced or chemically synthesized. The physical properties of these nanomaterials must be characterized correctly using instrumentation that evaluates their size, morphology, and potential for agglomeration. These technologies represent a high-growth economic area that fosters entrepreneurship and innovation. Because of this innovative spirit, research and economic interest will continue to be focused on therapeutic delivery technologies

Quantitative mRNA detection with advanced nonlinear microscopy

Cell-specific information on quantity and localization of key mRNA transcripts in single-cell level are critical to the assessment of cancer risk, therapy efficacy, and effective prevention strategies. While current techniques are not capable to visualize single mRNA transcript beyond the diffraction limit. In this thesis, two nonlinear technologies, second harmonic super-resolution microscopy (SHaSM) and transient absorption microscopy (TAM), are developed to detect and quantify single Human edimer receptor 2 (Her2) mRNA transcripts. The SHaSM is used to detect single mRNA transcript beyond the diffraction limit, while the TAM is employed to detect mRNA without the interference of fluorescence background. The thesis presents the fundamental study on the probes used in SHaSM, the concept and instrumental layout of the two technologies, and the detection as well as quantification of mRNA transcript in cells and tissues by super resolution microscopy and background-free detection microscopy. The first part of my dissertation focuses on the introduction of available mRNA detection methods and nonlinear imaging techniques. In chapter 2, I mainly characterize the SHG emission behavior of individual BTO nanocrystals via time-resolved single molecule spectroscopy, correlation spectroscopy, and confocal microscopy. High-intensity stable emission is collected from individual BTO nanocrystals with a high signal-to-noise ratio; the polar-dependent emission behavior of individual BTO NCs was also investigated theoretically and experimentally; and the dynamics of individual BTO in turbid medium is studied by an improved autocorrelation spectroscopy. The third chapter develops a novel second harmonic super-resolution microscopy (SHaSM), which is capable of detecting individual BTO nanocrystals with the lateral resolution as high as 30 nm. Motivated by the capability of SHaSM to visualize single BTO nanocrystals beyond the diffraction limit, we develop a dimer configuration of BTO nanocrystals for detecting single mRNA transcript beyond the diffraction limit. We validate our SHaSM to resolve single mRNA transcript first in vitro. Preformed BTO dimers are detected and differentiated by the SHaSM and by the SEM as the control. Expression level and localization patterns of Her2 mRNA transcript in single SKBR3, MCF7, and HeLa cell are investigated with the SHaSM. SHaSM can successfully differentiate the Her2 mRNA from the nonspecific BTO monomers, and identify more than one transcript in a diffraction-limited spot for SKBR3 cells. Quantification results agree well with the theoretical estimation and the RNA FISH results, and in addition it shows that the SHaSM has more accurate quantification when detecting over-expressed mRNA transcript. Furthermore we applied the SHG probes and SHaSM to study the heterogeneity of Her2 mRNA transcript in breast cancer tissues. High-specific binding of the SHG probes is observed and high penetration detection can be realized. In addition to the SHaSM, I also develop a background-free method to detect and quantify mRNA transcript. A femto-second transient absorption microscopy (TAM) is developed in the lab. It starts with the theoretical description of the TAM process, and then introduce the fundamental optical properties of the gold nanoparticles in TAM. By chemically treating the gold nanoparticles and conjugating with ODN probes, the gold nanoparticles hybridize to the mRNA molecules and are visualized in the TAM, together with label-free images of cells obtained in the SRS microscopy. mRNA is quantified with single copy sensitivity and is validated by the FISH approach. Super resolution microscopy of Her2 mRNA transcript in single cells will provide more accurate quantification in single cells; what\u27s more, it can be potentially employed to investigate the dynamics of single mRNA transcript beyond the diffraction limit, which is extremely significant in basic biology. TAM microscopy promotes the detection of mRNA transcript at a high speed without fluorescence background, which can be further utilized to investigate the dynamics of RNA regulation. Both these two methods will promote our understandings of the expression level and localization patterns of mRNA transcript in single cells, provide a route to employ mRNA transcript as a marker or indicator for cancer diagnosis and therapy

Localizer:fast, accurate, open-source, and modular software package for superresolution microscopy

We present Localizer, a freely available and open source software package that implements the computational data processing inherent to several types of superresolution fluorescence imaging, such as localization (PALM/STORM/GSDIM) and fluctuation imaging (SOFI/pcSOFI). Localizer delivers high accuracy and performance and comes with a fully featured and easy-to-use graphical user interface but is also designed to be integrated in higher-level analysis environments. Due to its modular design, Localizer can be readily extended with new algorithms as they become available, while maintaining the same interface and performance. We provide front-ends for running Localizer from Igor Pro, Matlab, or as a stand-alone program. We show that Localizer performs favorably when compared with two existing superresolution packages, and to our knowledge is the only freely available implementation of SOFI/pcSOFI microscopy. By dramatically improving the analysis performance and ensuring the easy addition of current and future enhancements, Localizer strongly improves the usability of superresolution imaging in a variety of biomedical studies

DNA-PAINT MINFLUX nanoscopy

MINimal fluorescence photon FLUXes (MINFLUX) nanoscopy, providing photon-efficient fluorophore localizations, has brought about three-dimensional resolution at nanometer scales. However, by using an intrinsic on–off switching process for single fluorophore separation, initial MINFLUX implementations have been limited to two color channels. Here we show that MINFLUX can be effectively combined with sequentially multiplexed DNA-based labeling (DNA-PAINT), expanding MINFLUX nanoscopy to multiple molecular targets. Our method is exemplified with three-color recordings of mitochondria in human cells

New insights into immune mechanisms in idiopathic membranous nephropathy

A Nefropatia Membranosa (NM) é atualmente considerada a causa mais comum de síndrome nefrótica no adulto. A NM idiopática (NMi) comporta-se como uma doença glomerular autoimune, tendo-se identificado até à data três auto-anticorpos circulantes específicos contra antigénicos podócitários, os quais transformaram a abordagem e a monitorização da NMi. O carácter autoimune da NMi foi descrito pela primeira vez em 2009 quando o PLA2R1 foi identificado como o principal antigénio alvo em mais de 70% dos casos de NMI sem causas secundárias aparentes, particularmente em homens. PLA2R1 é geralmente avaliado em casos de NMi através de testes serológicos para anti-PLA2R1, e na biópsia renal através da marcação para PLA2R1, com especificidade superior a 99% e sensibilidade de 78% para o diagnóstico de NMi. THSD7A foi o segundo antigénio a ser descoberto e adicionado ao painel de antigénios da NMi. A recente identificação de NELL-1 pressupõe a revelação de um marcador distinto na histologia renal para diagnóstico de NMi. A avaliação dos níveis de anticorpos revela-se de crucial importância uma vez que estes foram propostos como marcadores da atividade autoimune da NM. Nesta revisão, descrevemos avanços recentes na área de estudo de auto-anticorpos relacionados com a NMi, designadamente o anti-PLA2R1 e o anti-THSD7A, e sua potencial contribuição como biomarcadores da atividade da doença, na monitorização terapêutica, e como preditores de prognóstico.Membranous nephropathy (NM) is recognized as the most common cause of nephrotic syndrome in adults. Idiopathic NM (iMN) behaves as an autoimmune glomerular disease with three circulating autoantibodies specific for native podocyte antigens identified by now, which has transformed the management and monitoring of iMN. The autoimmune character of iMN was described in 2009 when PLA2R1 was identified as a major target antigen in more than 70% of cases of iMN without apparent secondary causes, particularly in men. PLA2R1 is commonly tested in cases of iMN by serologic tests for anti-PLA2R1, and in kidney biopsy by staining for PLA2R1, with more than 99% specificity and 78% sensitivity for the diagnosis of iMN. THSD7A was the second antigen recognized, and added to the panel of iMN antigens. Recently, NELL-1 was identified and is expected to be a distinct biopsy marker for iMN. Measuring antibodies titers is of crucial importance since they have been proposed as biomarkers of MN autoimmune activity. In this review, we describe recent advances of autoantibodies related to iMN, namely the anti-PLA2R1 and the anti-THSD7A, and its potential contribution as disease activity biomarkers, therapeutic monitoring, and outcome predictors