Microfluidics and Nanofluidics Handbook

The Microfluidics and Nanofluidics Handbook: Two-Volume Set comprehensively captures the cross-disciplinary breadth of the fields of micro- and nanofluidics, which encompass the biological sciences, chemistry, physics and engineering applications. To fill the knowledge gap between engineering and the basic sciences, the editors pulled together key individuals, well known in their respective areas, to author chapters that help graduate students, scientists, and practicing engineers understand the overall area of microfluidics and nanofluidics. Topics covered include Finite Volume Method for Numerical Simulation Lattice Boltzmann Method and Its Applications in Microfluidics Microparticle and Nanoparticle Manipulation Methane Solubility Enhancement in Water Confined to Nanoscale Pores Volume Two: Fabrication, Implementation, and Applications focuses on topics related to experimental and numerical methods. It also covers fabrication and applications in a variety of areas, from aerospace to biological systems. Reflecting the inherent nature of microfluidics and nanofluidics, the book includes as much interdisciplinary knowledge as possible. It provides the fundamental science background for newcomers and advanced techniques and concepts for experienced researchers and professionals

DEFINITION OF BIOLOGICAL RESPONSES THROUGH THE ANALYSIS OF GENE EXPRESSION PROFILES

The aim of this PhD project was the development of a pipeline for the analysis of expression data and a set of of different strategies to extract biological informations from micrarray experiments. The computational pipeline for processing raw microarray data (images) to define gene expression levels, to provide experiment quality assessment and significativity statistical tests, was implemented in R, using mostly Bioconductor packages. The first fase had as purpose the determination of the gene function combining experiments of silecing with the gene expression analysis. Caspase-2 is a member of a cystein-protease family that carry out important roles in the apoptosis and in the inflammation. Altough it is highly conserved from the evolutionary point of view, in the literature several contradictory results are found. Being expressed at high level during the neurological development and with a strong involvement in the apoptotic processes in the adult central nervous system, we decided to proceed with the silecing of the gene that codifies for this enzyme using glioblastoma cells, a very aggressive cerebral tumor. The comparative analysis of expression profiles of silenced cells respect to the control ones, highlighted the relation between CASP2 and genes involved in the cholesterol metabolism. Previuos studies have suggested for this enzime a role in the control of intracellular level of this metabolite. Therefor, we decided to use data stored in public databases in order to to extend the investigation, including all the other caspases and all the genes in same way connected to cholesterol. After we had obtained the data related to several different experiments, we went ahead with the computation of the correlation between expression levels and, then, based of these values, with the clustring analysis in order to see which among the caspases has the same corralational profile. After that, the analysis was expanded to normal brain and liver tissues, in order to know whether the situation observed in the patological condition is unique or if it can be overlayed to that present in normal tissues. In the second phase, I performed an analysis of expression data with a completely different purpose. The aim of this project was the definition of the signaling pathways and of the resistence mechanisms induced by the treatment of cancer cells obtained from patients affected by cronic lymphocytic leukemia and treated with a new category of ubiquitin proteasome system (UPS) inhibitors. Through the comparison of trascriptional profiles before and after the treatment, many genes connected with the drug action at cellular level, whose expression was altered by the UPS inhibitor, were identified. Furthermore, considering the difference in terms of responsiveness of the analized patients, we could determine some genes responsible of the different efficacy of the farmacological treatment

Chemical Approaches for ‘Undruggable’ Targets: The Discovery of Ligands for Small Heat Shock Proteins.

Small heat shock proteins (sHSPs) are molecular chaperones that protect against protein aggregation in response to stress. These chaperones have been linked to a number of protein misfolding diseases, including neurodegenerative disorders and cataracts. However, sHSPs are considered “undruggable” because they lack enzymatic activity or obvious ligand-binding sites. Thus, sHSPs are emblematic of potential drug targets emerging from large-scale genetic efforts. They are clearly linked to disease, yet it is not clear how to develop drug-small molecules to act on this knowledge. In this thesis work, I employed a number of high throughput biophysical approaches to discover cryptic binding sites on two sHSPs: Hsp27 and alphaB-crystallin (cryAB). Using a multifaceted approach involving computational approaches, differential scanning fluorimetry, fragment-based NMR screening and rational design, three unanticipated binding sites were discovered. Medicinal chemistry and structural efforts yielded four classes of small molecules, with representatives that bind each of the three cryptic pockets. One of these chemical series was developed as a potential new therapeutic for the topical treatment of cataract. These molecules bind the native dimer of cryAB and promote its chaperone functions, reversing cataracts in mouse and human lens models. Another series binds to a region of Hsp27 that is important for its interactions with the Hsp70 class of chaperones, showing promise as a chemical probe for understanding how chaperones regulate protein homeostasis. This thesis work has significantly advanced our knowledge of sHSP ‘druggability’ and revealed at least three binding sites for further development. Moreover, these efforts represent a detailed, head-to-head comparison of modern HTS methods to discover ligands for cryptic binding sites. The strengths and weaknesses of these approaches are important in designing screening campaigns for other ‘undruggable’ targets emerging from genetic studies.PHDMedicinal ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/109032/1/makleyln_1.pd

Development of a "genome-proxy" microarray for profiling marine microbial communities, and its application to a time series in Monterey Bay, California

Thesis (Ph. D.)--Joint Program in Biological Oceanography (Massachusetts Institute of Technology, Dept. of Biology; and the Woods Hole Oceanographic Institution), 2008.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Includes bibliographical references (p. 155-181).This thesis describes the development and application of a new tool for profiling marine microbial communities. Chapter 1 places the tool in the context of the range of methods used currently. Chapter 2 describes the development and validation of the "genome proxy" microarray, which targeted marine microbial genomes and genome fragments using sets of 70-mer oligonucleotide probes. In a natural community background, array signal was highly linearly correlated to target cell abundance (R² of 1.0), with a dynamic range from 10²-10⁶ cells/ml. Genotypes with >/=~80% average nucleotide identity to those targeted crosshybridized to target probesets but produced distinct, diagnostic patterns of hybridization. Chapter 3 describes the development an expanded array, targeting 268 microbial genotypes, and its use in profiling 57 samples from Monterey Bay. Comparison of array and pyrosequence data for three samples showed a strong linear correlation between target abundance using the two methods (R²=0.85- 0.91). Array profiles clustered into shallow versus deep, and the majority of targets showed depth-specific distributions consistent with previous observations. Although no correlation was observed to oceanographic season, bloom signatures were evident. Array-based insights into population structure suggested the existence of ecotypes among uncultured clades. Chapter 4 summarizes the work and discusses future directions.by Virginia Rich.Ph.D

Multi-modal optical microscopy image analysis and matching techniques for spatially encoded bead-based microarrays

Bead encoding is a key problem central to all multiplexed bead-based microarrays. Most existing bead-based microarrays require complicated and costly bead fabrication and/or sophisticated bead decoding hardware to achieve high multiplexing. Recently, the development of spatial bead encoding techniques have opened up the possibility of using pattern matching and image processing to develop highly multiplexed, high-throughput bead-based arrays. In this thesis, we improve the existing spatial bead encoding scheme, and develop a pipeline of computational methods, which allows for automated spatial bead encoding. Six novel computational methods, that automate the proposed bead encoding scheme, are developed in this thesis. The proposed scheme improves the previously reported spatial encoding schemes by making them better suited for use in real world scenarios. In the spatial bead encoding scheme, a sequential bead deposition method is used to capture the identities of the beads in bright or dark field images of the array. While, a second fluorescence image of the array is used to quantify the target analyte concentrations associated with the beads. By aligning the two images using the patterns formed by the beads, both the bead identities (i.e. the target analyte associate with the bead) and the analyte concentrations are decoded. The six computational methods developed in this thesis, are grouped into three categories: bright and dark field image processing, fluorescence image processing, and bead pattern matching. These methods are developed such that they require minimal parameter tuning, and are able to deal with noisy images acquired in uncontrolled environments As part of the bright and dark field image processing we have developed two novel methods: a fully automatic method for detecting the underlying micro-well grid structure in the images, and an unsupervised learning based method to classify the micro-wells as either empty or containing a bead. We show the ability of the proposed methods to deal with extreme amounts of noise and distortions using multiple datasets. While, microarray image gridding is a well-studied problem in the context of fluorescence images of planar microarrays, to the best of our knowledge no methods have yet been developed for processing either bright or dark field images of bead-based microarrays. Unlike in bright field images, where all the beads are visible, in fluorescence images only those beads that are expressed in the sample are visible. Therefore, in fluorescence image analysis the first step is to detect the beads in the image. To this end, we have developed an unsupervised, simultaneous bead detection and segmentation method which uses the coherence in shape and size of the beads to circumvent the need for parameter optimization. The main challenge in this segmentation is the large dynamic range across which the beads are expressed. We have also developed a probabilistic non-linear gridding method that attempts to establish the micro-well grid structure from the detected beads. While similar gridding techniques have been developed in the past, we make two contributions in this regard: first, we put the gridding method in a probabilistic framework, and second, we show that in certain cases it is impossible to ascertain if the correct grid has been detected without using additional information about the physical properties of the array. The final step in the proposed bead encoding scheme is to establishing one-to-one correspondences between the beads detected in the bright field and fluorescence images. We have developed two methods for this matching. When the micro-well grid structure in the fluorescence image is successfully established, the grid information can be used to reduce the matching problem to a binary grid alignment problem. A grid matching method that is very fast and can handle a large number of beads (>50,000) has been developed for this purpose. Finally, to perform matching when the fluorescence image grid is not available, a novel point pattern matching method that is affine invariant and robust to small non-linear distortions has been developed. The point pattern matching method addresses the problem of matching large point sets in the presence of large amounts of outliers and occlusion. The proposed method is evaluated using several real and simulated datasets.Doctor of Philosophy (SCE

Genetic and Molecular Investigation of the Schnitzler Syndrome

The Schnitzler Syndrome (SchS) is a rare, autoinflammatory condition with an unknown pathological mechanism, but treatment with IL-1 inhibition provides remarkable efficacy. Exhibiting two main defining features: (1) an urticarial rash and (2) an IgM gammopathy, this IL-1 mediated disease phenotypically bears stark resemblance to NLRP3-associated inflammatory disease. The latter monogenic entity is known to show gain-of-function and pathological mutations in the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome. Furthermore, 20% of SchS patients go on to develop overt lymphoproliferative diseases, namely Waldenström's Macroglobulinaemia (WM). This condition presents with a specific mutation in the Myeloid Differentiation Primary Response (MYD88) gene in over 90% of patients. Against the backdrop of these imperative findings, the work presented in this thesis therefore examines the role of these immunological constituents in SchS, via the assessment of mutations in NLRP3 and MYD88 alongside a panel of genes frequently mutated in haematological malignancies. Identification of a causative gene would not only improve molecular diagnosis, but allows for potential unearthing of genotype-phenotype correlations. Since the identification of this condition in 1972, the features and consequences of the IgM gammopathy has remained elusive. In a bid to delineate the latter, examination of the heavy chain of the immunoglobulin repertoire would therefore indicate aspects of the adaptive immune response integral to formation of the monoclonal component. A biased repertoire would therefore indicate the existence of a clonal B-cell population. Additionally, isolation of SchS-IgM and interrogation of a protein array comprising of over 15,700 human proteins further indicates whether this element causes pathological effects. Exploration of the genetic and molecular components did not expose a common mechanism through which SchS manifests, ruling out the notion that NLRP3, MYD88 and other associated genes are universally causative of this enigmatic disease. Assessment of the IgH repertoire indicated that SchS patients show evidence of expanded B-cell populations, and together with protein array analysis demonstrating the preference of IgM binding to nuclear antigens, this study supports the theory that SchS is a clonal disorder. The breadth and depth of these findings broadens the currently limited scientific knowledge pertaining to SchS, forming the basis upon which further investigations can commence