Self assembled monolayers (SAMs) are widely used for surface modification. Alkylsilane monolayers are one of the most widely deposited and studied SAMs. My work focuses on the preparation, patterning, and application of alkysilane monolayers. 3-aminopropyltriethoxysilane (APTES) is one of the most popular silanes used to make active surfaces for surface modification. To possibly improve the surface physical properties and increase options for processing this material, I prepared and studied a series of amino silane surfaces on silicon/silicon dioxide from APTES and two other related silanes by chemical vapor deposition (CVD). I also explored CVD of 3-mercaptopropyltrimethoxysilane on silicon and quartz. Several deposition conditions were investigated. Results show that properties of silane monolayers are quite consistent under different conditions. For monolayer patterning, I developed a new and extremely rapid technique, which we termed laser activation modification of semiconductor surfaces or LAMSS. This method consists of wetting a semiconductor surface with a reactive compound and then firing a highly focused nanosecond pulse of laser light through the transparent liquid onto the surface. The high peak power of the pulse at the surface activates the surface so that it reacts with the liquid with which it is in contact. I also developed a new application for monolayer patterning. I built a technologically viable platform for producing protein arrays on silicon that appears to meet all requirements for industrial application including automation, low cost, and high throughput. This method used microlens array (MA) patterning with a laser to pattern the surface, which was followed by protein deposition. Stencil lithography is a good patterning technique compatible with monolayer modification. Here, I added a new patterning method and accordingly present a simple, straightforward procedure for patterning silicon based on plasma oxidation through a stencil mask. We termed this method subsurface oxidation for micropatterning silicon (SOMS).

No description available.

Protein-Arrays sind das Mittel der Wahl, um eine Vielzahl von Proteinen parallel zu untersuchen. Ziele dieser Untersuchungen sind meistens Proteininteraktionsnetzwerke zu entdecken oder besser verstehen zu können. Bisher wurden die benötigten Proteine fast ausschließlich mit biologischen Methoden gewonnen. Diese bieten allerdings keinen generellen Zugang zu posttranslational-modi-fizierten (PTM)-Proteinen. Somit war es bisher nicht möglich den Einfluss von PTMs auf Protein-Protein-Interaktionen (PPIs) im Arrayformat zu untersuchen. Die chemische Synthese kann dagegen Proteine mit ortsspezifischen PTMs liefern. Daher ist es verwunderlich, dass bislang noch keine Berichte über chemisch hergestellte PTM-Protein-Arrays existieren, besonders da PTMs meist entscheidend für proteomische Interaktionsnetzwerke sind. In der vorliegenden Arbeit wird eine Methodik beschrieben, die es ermöglicht PTM-modifizierte Protein-Domänen-Arrays auf der Oberfläche zu synthetisieren und zu analysieren. Mit der Methodik wurden 20 SH3-Domänen synthetisiert und 64 PPIs gemessen. Neben vier Hefe-SH3-Domänen wurden je acht humane (Phospho)SH3-Domänen der Abl- und Arg(Abl2)-Tyrosinkinase synthetisiert und funktionell untersucht. Es wurde gefunden, dass die Ligandenspezifität von Abl-SH3-Domänen durch Phosphorylierung feinreguliert wird. Je nach Phosphorylierungsmustern wurde die Affinität für spezifische Liganden erhöht oder erniedrigt. Der Ursprung dieser Phosphoregulierung wurde für die Abl-SH3-Domäne mit Hilfe der NMR-Spektroskopie und durch Zellexperimente versucht zu entschlüsseln und weiter validiert. / Protein-arrays are the method of choice to investigate a variety of proteins in a parallel fashion. Objectives of these studies are mostly to discover or to investigate protein interaction networks. So far, the necessary proteins were almost exclusively gained by biological methods. Unfortunately, generic access to proteins bearing post-translational modifications (PTM) is not provided by these techniques. Therefore, it was not possible to investigate the impact of PTMs on protein-protein-interactions (PPIs) on arrays so far. Chemical synthesis in contrast offers proteins with site-specific PTM incorporation. In this context, it is surprising, that chemical methods of PTM-protein array synthesis remained virtually unexplored, especially since these modifications are usually crucial for proteomic interaction networks. In this thesis, a methodology is described, that allows to synthesize and functional analyse post-translationally modified protein domain arrays on the surface. By using this methodology, 20 SH3 domains were synthesized and 64 protein-pep-tide interactions were measured. In addition to 4 yeast SH3 domains, 8 human (phospho) SH3 domains of the Abl and Arg(Abl2) tyrosine kinase were synthesized and functionally investigated. The experiments revealed that phosphorylation might serve as a means to fine tune the ligand recognition. Depending on the phosphorylation pattern the affinity to specific interaction partners were enhanced or reduced. The origin of this phosphoregulation was further investigated for the Abl SH3 domain by means of NMR spectroscopy and cellular experiments.

abstract: Biomarkers find a wide variety of applications in oncology from risk assessment to diagnosis and predicting and monitoring recurrence and response to therapy. Developing clinically useful biomarkers for cancer is faced with several challenges, including cancer heterogeneity and factors related to assay development and biomarker performance. Circulating biomarkers offer a rapid, cost-effective, and minimally-invasive window to disease and are ideal for population-based screening. Circulating immune biomarkers are stable, measurable, and can betray the underlying antigen when present below detection levels or even no longer present. This dissertation aims to investigate potential circulating immune biomarkers with applications in cancer detection and novel therapies. Over 600,000 cancers each year are attributed to the human papillomavirus (HPV), including cervical, anogenital and oropharyngeal cancers. A key challenge in understanding HPV immunobiology and developing immune biomarkers is the diversity of HPV types and the need for multiplexed display of HPV antigens. In Project 1, nucleic acid programmable protein arrays displaying the proteomes of 12 HPV types were developed and used for serum immunoprofiling of women with cervical lesions or invasive cervical cancer. These arrays provide a valuable high-throughput tool for measuring the breadth, specificity, heterogeneity, and cross-reactivity of the serologic response to HPV. Project 2 investigates potential biomarkers of immunity to the bacterial CRISPR/Cas9 system that is currently in clinical trials for cancer. Pre-existing B cell and T cell immune responses to Cas9 were detected in humans and Cas9 was modified to eliminate immunodominant epitopes while preserving its function and specificity. This dissertation broadens our understanding of the immunobiology of cervical cancer and provides insights into the immune profiles that could serve as biomarkers of various applications in cancer. / Dissertation/Thesis / Doctoral Dissertation Molecular and Cellular Biology 2018

Indiana University-Purdue University Indianapolis (IUPUI) / Monomers like triethylene glycol dimethacrylate (TEGDMA) leach from dental composites and adhesives due to incomplete polymerization or polymer degradation. The release of these monomers causes a variety of reactions that can lead to cell death. This death can be either necrotic, which is characterized mainly by inflammation and injury to the surrounding tissues, or apoptotic, which elicits little inflammatory responses, if any at all. TEGDMA-induced apoptosis in human pulp has been reported recently. However, the molecular mechanisms and the apoptotic (pro and anti) proteins involved in this process remain unclear. The objective of this study was to determine the apoptotic proteins expressed or suppressed during TEGDMA-induced apoptosis. Human pulp fibroblasts (HPFs) were incubated for 24 hours with different TEGDMA concentrations (0.125-1.0 mM). Cytotoxicity was determined using the cytotoxicity Detection KitPLUS (Roche Applied Science, Mannheim, Germany). TEGDMA was shown to cause cell cytotoxicity at concentrations of 0.50 mM and up. The highest concentration with no significant cytotoxicity was used. Cells were incubated with or without 0.25 mM TEGDMA for 6 h and 24 h. Cell lysates were then prepared and the protein concentrations determined using the Bradford protein assay. A Human Apoptosis Array kit (Bio-Rad Hercules, CA ) was utilized to detect the relative levels of 43 apoptotic proteins. The results of this study showed statistically significant increases of multiple examined pro-apoptotic proteins. The anti-apoptotic proteins were also altered. Pro-apoptotic proteins involved in the intrinsic and extrinsic apoptotic pathways were increased significantly. The results indicated that TEGDMA has effects on both the extrinsic and intrinsic apoptotic pathways.