Multistable Decision Switches for Flexible Control of Epigenetic Differentiation

It is now recognized that molecular circuits with positive feedback can induce two different gene expression states (bistability) under the very same cellular conditions. Whether, and how, cells make use of the coexistence of a larger number of stable states (multistability) is however largely unknown. Here, we first examine how autoregulation, a common attribute of genetic master regulators, facilitates multistability in two-component circuits. A systematic exploration of these modules' parameter space reveals two classes of molecular switches, involving transitions in bistable (progression switches) or multistable (decision switches) regimes. We demonstrate the potential of decision switches for multifaceted stimulus processing, including strength, duration, and flexible discrimination. These tasks enhance response specificity, help to store short-term memories of recent signaling events, stabilize transient gene expression, and enable stochastic fate commitment. The relevance of these circuits is further supported by biological data, because we find them in numerous developmental scenarios. Indeed, many of the presented information-processing features of decision switches could ultimately demonstrate a more flexible control of epigenetic differentiation

Selective Interaction of Syntaxin 1A with KCNQ2: Possible Implications for Specific Modulation of Presynaptic Activity

KCNQ2/KCNQ3 channels are the molecular correlates of the neuronal M-channels, which play a major role in the control of neuronal excitability. Notably, they differ from homomeric KCNQ2 channels in their distribution pattern within neurons, with unique expression of KCNQ2 in axons and nerve terminals. Here, combined reciprocal coimmunoprecipitation and two-electrode voltage clamp analyses in Xenopus oocytes revealed a strong association of syntaxin 1A, a major component of the exocytotic SNARE complex, with KCNQ2 homomeric channels resulting in a ∼2-fold reduction in macroscopic conductance and ∼2-fold slower activation kinetics. Remarkably, the interaction of KCNQ2/Q3 heteromeric channels with syntaxin 1A was significantly weaker and KCNQ3 homomeric channels were practically resistant to syntaxin 1A. Analysis of different KCNQ2 and KCNQ3 chimeras and deletion mutants combined with in-vitro binding analysis pinpointed a crucial C-terminal syntaxin 1A-association domain in KCNQ2. Pull-down and coimmunoprecipitation analyses in hippocampal and cortical synaptosomes demonstrated a physical interaction of brain KCNQ2 with syntaxin 1A, and confocal immunofluorescence microscopy showed high colocalization of KCNQ2 and syntaxin 1A at presynaptic varicosities. The selective interaction of syntaxin 1A with KCNQ2, combined with a numerical simulation of syntaxin 1A's impact in a firing-neuron model, suggest that syntaxin 1A's interaction is targeted at regulating KCNQ2 channels to fine-tune presynaptic transmitter release, without interfering with the function of KCNQ2/3 channels in neuronal firing frequency adaptation

A solid-state nanopore-based platform for molecular diagnostics

Thesis (Ph.D.)--Boston UniversityPLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at [email protected]. Thank you.The ability to identify and characterize infectious pathogens is essential for proper diagnosis, treatment and disease management. Many pathogens display similar pathophysiological traits, but respond to different treatment regimes and can develop resistance to prescribed treatments. This creates a need for development of novel methods to provide accurate, rapid, and cost-effective genomic characterization of infecting pathogens. While diagnostic methods that utilize a pathogen's genomic information do exist today, currently their use is severely hindered by high costs, methodological complexity, and lengthy turnaround. One relatively new approach that could address these issues utilizes the known genomic sequence variations of pathogen strains to create a barcode of detectable genomic 'tags', specific to each strain. Unfortunately, current optical methods to detect such 'tags' lack accuracy and resolution. This project explores the feasibility of using solid-state nanopores to circumvent these apparent roadblocks, laying the foundation for a purely electronic pathogen barcoding method. The first aim of this project is to evaluate the platform's ability to detect minute fluctuations in the DNA's helical structure through analysis of transient changes to the ion current as the DNA passes through a nanopore. The second aim of this project is to detect local points of DNA structure variation. In this aim we utilize highly sequence-specific synthetic peptide nucleic acid (PNA) probes to locally tag predesigned short sequences along a DNA molecule. The sequence specificity of the tags and the nanopore's ability to discriminate between tagged and untagged regions along the DNA allow identification of specific sequences in a long DNA molecule. The final aim of this project is to develop the ability to localize the PNA probes along the DNA molecule. This enables us to conduct a proof-of-concept demonstration of the combined PNAjnanopore method through discrimination between two variants of a gene from two nearly identical human immunodeficiency virus (HIV) subtypes. This body of work forms the foundation for a highly capable nanopore-based molecular diagnostics platform.2031-01-0

Maximum directed cuts in acyclic digraphs

It is easily shown that every digraph with m edges has a directed cut of size at least m/4, and that 1/4 cannot be replaced by any larger constant. We investigate the size of a largest directed cut in acyclic digraphs, and prove a number of related results concerning cuts in digraphs and acyclic digraphs.