New methods for designing and analyzing microarray experiments for the detection of differential expression

This thesis is divided into three sections all pertaining to microarray experimental design and analysis. Microarrays are a tool used in biological research which enables scientists to measure the relative level of expression many genes within an organism at the same time. Microarrays have also opened new research areas in statistics which are currently being investigated concerning different aspects of data normalization, experimental design, and analysis;The first chapter entails a comparison of two commonly used experimental designs in two-dye microarray experiments. Both designs are applicable only to experiments containing treatments with two levels. One design is shown to be more powerful when constrained by the number of arrays. Also, mixed model analysis is often used for both designs. With small sample sizes, mixed model analysis is shown to give inaccurate results under certain conditions. Due to this problem, an alternative method of analysis is proposed for both experimental designs which eliminates this concern;Two-dye microarray experiments require special consideration in design since they have multiple random effect in the model. This is because arrays are usually viewed as a random factor that should always be contained in a model for the data. Research has been done on comparing two-dye microarray experimental designs by requiring calculation of array differences. This is shown to inhibit the power of the analysis by removing inter-block information. There are also experimental designs that are viable options which can not be compared using this method. An alternative method of analysis is proposed which allows for multiple random effects in the model. Under certain conditions, this method is shown to choose designs that either would not be chosen, or cannot be considered, when using methods based on array differences;The third chapter discusses new methods for analyzing microarray experiments by categories. Most commonly, microarray analysis is performed on a gene-by-gene basis with the goal of finding the genes whose expression differ the greatest between varieties of treatments. However, scientists often would like to know what aspect of cell life is affected most by differences in varieties. There could be cases where a group of genes pertaining to the same task are all have a mild change in expression which would not be found using gene-by-gene analysis. Two different resampling based methods are proposed for solving this problem. Both methods are compared and results are visualized on a directed acyclical graph

Soluble factors from neocortical astrocytes enhance neuronal differentiation of neural progenitor cells from adult rat hippocampus on micropatterned polymer substrates

Rat adult hippocampal progenitor cells (AHPCs) are self-renewing, multipotent neural progenitors that have the ability to differentiate into neurons and glia. Previously, we demonstrated that coculture of AHPCs with postnatal day two, type 1 cortical astrocytes on laminin-coated micropatterned polymer substrates facilitates selective neuronal differentiation of the AHPCs 1. Under this condition, multi-dimensional cell-cell and/or cell-extracellular matrix interactions, as well as possible soluble factors released from astrocytes provided spatial and temporal control selectively enhancing neuronal differentiation and neurite alignment on topographically different regions of the same substrate. To investigate the potential role of astrocyte-derived soluble factors as cues involved in neuronal differentiation, a non-contact co-culture system was used. Under control conditions, approximately 14% of the AHPCs were immunoreactive (IR) for the neuronal marker, class III β-tubulin (TUJ1-IR). When co-cultured in physical contact with astrocytes, neuronal differentiation increased significantly to about 25%, consistent with our previous results. Moreover, under non-contact co-culture conditions using Transwell insert cultures, neuronal differentiation was dramatically increased to approximately 64%. Furthermore, neurite outgrowth from neuronal cell bodies was considerably greater on the patterned substrate, compared to the non-patterned planar substrate under non-contact co-culture conditions. Taken together, our results demonstrate that astrocyte-derived soluble factors provide cues for specific neuronal differentiation of AHPCs cultured on micropatterned substrates. In addition, a suppressive influence on neuronal differentiation appears to be mediated by contact with co-cultured astrocytes. These results provide important insights into mechanisms for controlling neural progenitor/stem cell differentiation and facilitate development of strategies for CNS repair

STARTING VALUES FOR PROC MIXED WITH REPEATED MEASURES DATA

A major advantage of PROC MIXED for repeated measures data is that one could choose from many different correlated error models. However, MIXED uses default starting values that may cause difficulty obtaining REML estimates of the covariance parameters for several of the models available. This can take the form of excessively long run times or even failure to converge. We have written a program to obtain initial covariance parameter estimates that result in greatly improved performance of the REML algorithm. We will use two covariance models frequently of interest in animal health experiments, the first-order ante-dependence model [ANTE(l)] and the Toeplitz model with heterogeneous variances [TOEPH], to illustrate the use of our procedure

Retinal tissue engineering using mouse retinal progenitor cells and a novel biodegradable, thin-film poly(e-caprolactone) nanowire scaffold

Retinal progenitor cells (RPCs) can be combined with nanostructured polymer scaffolds to generate composite grafts in culture. One strategy for repair of diseased retinal tissue involves implantation of composite grafts of this type in the subretinal space. In the present study, mouse retinal progenitor cells (RPCs) were cultured on laminin-coated novel nanowire poly(e-caprolactone)(PCL) scaffolds, and the survival, differentiation, and migration of these cells into the retina of C57bl/6 and rhodospsin −/− mouse retinal explants and transplant recipients were analyzed. RPCs were cultured on smooth PCL and both short (2.5 μm) and long (27 μm) nanowire PCL scaffolds. Scaffolds with adherent mRPCs were then either co-cultured with, or transplanted to, wild-type and rhodopsin −/− mouse retina. Robust RPC proliferation on each type of PCL scaffold was observed. Immunohistochemistry revealed that RPCs cultured on nanowire scaffolds increased expression of mature bipolar and photoreceptor markers. Reverse transcription polymerase chain reaction revealed down-regulation of several early progenitor markers. PCL-delivered RPCs migrated into the retina of both wild-type and rhodopsin knockout mice. The results provide evidence that RPCs proliferate and express mature retinal proteins in response to interactions with nanowire scaffolds. These composite grafts allow for the migration and differentiation of new cells into normal and degenerated retina

Control of Neural Stem Cell Survival by Electroactive Polymer Substrates

Stem cell function is regulated by intrinsic as well as microenvironmental factors, including chemical and mechanical signals. Conducting polymer-based cell culture substrates provide a powerful tool to control both chemical and physical stimuli sensed by stem cells. Here we show that polypyrrole (PPy), a commonly used conducting polymer, can be tailored to modulate survival and maintenance of rat fetal neural stem cells (NSCs). NSCs cultured on PPy substrates containing different counter ions, dodecylbenzenesulfonate (DBS), tosylate (TsO), perchlorate (ClO4) and chloride (Cl), showed a distinct correlation between PPy counter ion and cell viability. Specifically, NSC viability was high on PPy(DBS) but low on PPy containing TsO, ClO4 and Cl. On PPy(DBS), NSC proliferation and differentiation was comparable to standard NSC culture on tissue culture polystyrene. Electrical reduction of PPy(DBS) created a switch for neural stem cell viability, with widespread cell death upon polymer reduction. Coating the PPy(DBS) films with a gel layer composed of a basement membrane matrix efficiently prevented loss of cell viability upon polymer reduction. Here we have defined conditions for the biocompatibility of PPy substrates with NSC culture, critical for the development of devices based on conducting polymers interfacing with NSCs

Biophysical mechanisms of single-cell interactions with microtopographical cues

Biophysical cues encoded in the extracellular matrix (ECM) are increasingly being explored to control cell behavior in tissue engineering applications. Recently, we showed that cell adhesion to microtopographical structures (“micropegs”) can suppress proliferation in a manner that may be blunted by inhibiting cellular contractility, suggesting that this effect is related to altered cell-scaffold mechanotransduction. We now directly investigate this possibility at the microscale through a combination of live-cell imaging, single-cell mechanics methods, and analysis of gene expression. Using time-lapse imaging, we show that when cells break adhesive contacts with micropegs, they form F-actin-filled tethers that extend and then rupture at a maximum, critical length that is greater than trailing-edge tethers observed on topographically flat substrates. This critical tether length depends on myosin activation, with inhibition of Rho-associated kinase abolishing topography-dependent differences in tether length. Using cellular de-adhesion and atomic force microscopy indentation measurements, we show that the micropegs enhance cell-scaffold adhesive interactions without changing whole-cell elasticity. Moreover, micropeg adhesion increases expression of specific mechanotransductive genes, including RhoA GTPase and myosin heavy chain II, and, in myoblasts, the functional marker connexin 43. Together, our data support a model in which microtopographical cues alter the local mechanical microenvironment of cells by modulating adhesion and adhesion-dependent mechanotransductive signaling