Computational Molecular Biology

Computational Biology is a fairly new subject that arose in response to the computational problems posed by the analysis and the processing of biomolecular sequence and structure data. The field was initiated in the late 60's and early 70's largely by pioneers working in the life sciences. Physicists and mathematicians entered the field in the 70's and 80's, while Computer Science became involved with the new biological problems in the late 1980's. Computational problems have gained further importance in molecular biology through the various genome projects which produce enormous amounts of data. For this bibliography we focus on those areas of computational molecular biology that involve discrete algorithms or discrete optimization. We thus neglect several other areas of computational molecular biology, like most of the literature on the protein folding problem, as well as databases for molecular and genetic data, and genetic mapping algorithms. Due to the availability of review papers and a bibliography this bibliography

The analysis of pendolino (peo) mutants reveals differences in the fusigenic potential among Drosophila telomeres

Drosophila telomeres are sequence-independent structures that are maintained by transposition to chromosome ends of three specialized retroelements (HeT-A, TART and TAHRE; collectively designated as HTT) rather than telomerase activity. Fly telomeres are protected by the terminin complex (HOAP-HipHop-Moi-Ver) that localizes and functions exclusively at telomeres and by non-terminin proteins that do not serve telomere-specific functions. Although all Drosophila telomeres terminate with HTT arrays and are capped by terminin, they differ in the type of subtelomeric chromatin; the Y, XR, and 4L HTT are juxtaposed to constitutive heterochromatin, while the XL, 2L, 2R, 3L and 3R HTT are linked to the TAS repetitive sequences; the 4R HTT is associated with a chromatin that has features common to both euchromatin and heterochromatin. Here we show that mutations in pendolino (peo) cause telomeric fusions (TFs). The analysis of several peo mutant combinations showed that these TFs preferentially involve the Y, XR and 4th chromosome telomeres, a TF pattern never observed in the other 10 telomere-capping mutants so far characterized. peo encodes a non-terminin protein homologous to the E2 variant ubiquitin-conjugating enzymes. The Peo protein directly interacts with the terminin components, but peo mutations do not affect telomeric localization of HOAP, Moi, Ver and HP1a, suggesting that the peodependent telomere fusion phenotype is not due to loss of terminin from chromosome ends. peo mutants are also defective in DNA replication and PCNA recruitment. However, our results suggest that general defects in DNA replication are unable to induce TFs in Drosophila cells. We thus hypothesize that DNA replication in Peodepleted cells results in specific fusigenic lesions concentrated in heterochromatinassociated telomeres. Alternatively it is possible that Peo plays a dual function being independently required for DNA replication and telomere capping

Recognition Algorithms for 2-Tree Probe Interval Graphs

This thesis focuses on looking at a particular set of graphs and recognizing if a given graph has certain properties that would make it belong in this family, here called 2-tree Probe Interval Graphs. For these graphs, we create an algorithm to run on a coded script that recursively runs criteria through an input graph from its matrix representation to check the 2-path, and will output either a success that our graph is a 2-tree Probe Interval Graph, or failure if it is not. After the creation of this algorithm, a complexity analysis for the algorithm will be developed, as well as the implementation of di_erent search criteria to hopefully reduce the complexity by some polynomial factor. The recognition for our set of graphs follows to the conceptual idea that triangles are built upon each other in a fashion of adding one vertex and two edges to a previous triangle in the graph. Each new triangle is added to an existing triangle and recursively builds the graph where the new vertex neighbors strictly two vertices with an existing triangle, creating a recursively de_ned 2-path

Chromatin Remodeling in Transgenic Mouse Brain: Implications for the Neurobiology of Depression: A Dissertation

Histone lysine methylation is an important epigenetic mark for regulation of gene expression and chromatin organization. Setdb1 (Set domain, bifurcate 1), one of the histone lysine methyltransferases, specifically methylates histone H3 at lysine 9 (H3K9) and participates in transcriptional repression and heterochromatin formation. The major task of my thesis work was to investigate the epigenetic roles of Setdb1 in regulating brain functions. I started my thesis work by examining Setdb1 expression pattern during mouse brain development. The most robust signal of Setdb1 was detected in the fetal brains at embryonic day 12.5, with a ubiquitous distribution in all the proliferative zones, as well as the cortical plate and other regions comprised of postmitotic neurons. The expression of Setdb1 decreased as the brain developed, and this down-regulation profile was correlated to neuronal maturation as examined in a primary culture model of mouse cortical neurons. I then generated CK-Setdb1 transgenic mice, in which a myc-tagged full length mouse Setdb1 was constantly expressed in postmitotic neurons under the control of the CaMK II alpha promoter (CK). The expression of mycSetdb1 was detected in NeuN positive cells throughout most forebrain regions including cerebral cortex, striatum and hippocampus. A sustained increase of Setdb1 in CK-Setdb1 transgenics was verified at both mRNA and protein levels. Furthermore, an increase of H3K9 trimethylation was detected at major satellite DNA repeats in CK-Setdb1forebrains, which indicated that transgene-expressed mycSetdb1 was functionally active in adult brains. The behavioral phenotype of CK-Setdb1 transgenics was examined by using two separate founder lines. Gross neurological functions including body weight, locomotion activity, motor coordination, and breeding behavior were generally normal in CK-Setdb1 mice. CK-Setdb1 mice were further subjected to behavioral paradigms related to mood and cognitive functions. Intriguingly, as compared to the littermate controls, CK-Setdb1 mice represent a lower level of depression as indicated by decreased total immobility in two different behavioral despair tests. Moreover, CK-Setdb1 mice showed an accelerated extinction in the learned helplessness paradigm after a delayed interval (7 days), indicating a faster recovery from an established status of despair. The potential confounding factors, like memory deficits, were ruled out as CK-Setdb1 mice showed normal or even improved performances in different memory-related paradigms. Anxiety scores and stimulant drug response were normal in CK-Setdb1mice. Taken together, these findings suggested that a specific antidepressant-like phenotype was elicited by the over-expression of Setdb1 in adult mice forebrains. To further study the molecular mechanism underlying Setdb1-associated antidepressant-like behavioral changes, I screened for Setdb1-binding sites in a genome-scale by ChIP-on-chip using a tiling microarray from Affymetrix. Unexpectedly, Setdb1 showed a very restricted binding profile with a high specificity towards ionotropic glutamate receptor genes including the NMDA receptor 2B subunit gene Grin2b, which is a new target for the treatment for major depression. An increase of H3K9 dimethylation at Setdb1-binding site on Grin2b locus was detected in CK-Setdb1 hippocampus, which was correlated to a decrease of Grin2b expression as well as an accelerated desensitization of NMDA receptor. Furthermore, Chromosome Conformation Capture (3C) on Grin2b locus revealed a repressive chromatin loop structure, which tethered the distal Setdb1-binding site (~ 32 Kb downstream of transcriptional start site (TSS)) to a proximal intronic region (~12 Kb downstream of TSS) that is enriched for the binding of KAP1, a well-studied Setdb1-interacting transcriptional corepressor. Taken together, our data indicated that Setdb1 repressed Grin2b expression via H3K9 hypermethylation and higher-order chromatin loop formation, which may contribute to the antidepressant-like phenotype we observed in CK-Setdb1mice. The second part of my thesis work was to investigate the role of Setdb1 in the animal model of a neurodevelopmental disorder - Rett syndrome (RTT). Loss-of-function mutations of the gene encoding methyl-CpG binding protein 2 (MECP2) is the primary cause of RTT. There is an overlap between Setdb1- and Mecp2-associated repressive chromatin machineries, which both include histone deacetylase complex, H3K9 methyltransferase, DNA methyltransferase and heterochromatin protein 1 (HP1). Moreover, in contrast to Setdb1, which is downregulated during the cortical neuronal differentiation, Mecp2 is upregulated and the expression level is positively correlated to neuronal maturation. Therefore, we hypothesized that there is a functional redundancy between Setdb1 and Mecp2, and the up-regulation of Setdb1 in mature neurons will compensate for brain deficiency due to the loss of Mecp2. To test this hypothesis, I crossed CK-Setdb1 transgenic mice with nestincre-Mecp2 conditional knockout mice (Mecp2-/y). The behavior changes of CK-Setdb1/Mecp2-/y mice, including body weight, locomotion, motor coordination, and life span, were then compared to Mecp2-/y mice. No significant improvements in behaviors or survival were observed from CK-Setdb1/Mecp2-/y mice. Because the activation of CK promoter is limited to defined population of postmitotic neurons in forebrain, I tested our hypothesis by generating another strain of Setdb1 overexpression mice – tauSetdb1, in which the expression of mycSetdb1 is under the control of an endogenous pan-neuronal active promoter Tau. However, the introduction of tauSetdb1 also failed to rescue Mecp2 deficiency. The life span of tauSetdb1/ Mecp2-/y was even shorter as compared to Mecp2-/y mice (Kaplan-Meier, p=0.07). In conclusion, up-regulation of Setdb1 in adult brain was not sufficient to rescue Mecp2deficiency in the mouse model of RTT. One of the most challenges to study neuronal dysfunctions in brain diseases is the cellular heterogeneity of central nervous system. Current techniques for chromatin studies, including chromatin immunoprecipitation (ChIP) assays, usually lack of single cell resolution and are unable to examine the neurobiological changes in defined cell populations. In the third part of my thesis work, I developed a modified protocol to isolate neuronal nuclei from brain homogenates via Fluorescence-Activated Cell Sorting (FACS). In general, total nuclei was extracted from frozen brains, neuronal nuclei were then immuno-tagged with NeuN and sorted via FACS. Besides the NeuN labeling-FACS protocol, I also generated CK-H2BeGFP transgenic mice, in which a histone H2B-eGFP (enhanced green fluorescent protein) fusion protein was expressed in the nuclei of postmitotic neurons in mouse forebrain. Nuclei extracted from CKH2BeGFP brain were directly applied for FACS sorting. By using this protocol, we routinely got around 6-8 x106neuronal nuclei from one adult mouse forebrain, which was sufficient for ChIP applications followed by single gene PCR and microarray studies. In conclusion, our protocol permits large-scale studies of chromatin modifications or any other nuclei events in defined cell populations from distinct brain regions. Taken together, my dissertation work will lead to a better understanding of the epigenetic roles of histone H3K9 methyltransferase Setdb1 in brain functions, and may provide new targets for the therapeutic treatment of major depression

Single Chain Reversible Addition-Fragmentation Chain Transfer (RAFT) Poly(N-isopropylacrylamide) for Chemical Sensing

An approach to single chain poly-N-isopropyl acrylamide (polyNIPAM) with a redox tag was polymerized by the reversible addition fragmentation-chain transfer (RAFT) method. Molecularly imprinted polymers (MIPs) technology is the design of an artificial receptor with high selectivity for a specific analyte. The synthesized RAFT polymers were devised to develop conformation-based electrochemical MIP sensors.The material polyNIPAM is attractive as a receptor of a chemical sensor. Due to its thermosensitive properties, polyNIPAM collapses above the lower critical solution temperature (LCST) and returns to its original state when the temperature is reduced below LCST. This reversible aggregation behavior shows that polyNIPAM has a flexible structure vital to generating a conformational change with stimulus and molecular recognition. Beyond the aggregation behaviors, the isopropyl group of these monomers can form hydrophobic interactions, which helps create non-covalent interactions, the same as the use of acidic and basic functional monomers in MIP synthesis. This non-covalent crosslinking can reduce the number of covalent crosslinkers, increasing the binding affinity of the MIPs. Another approach to minimize binding blockage and increase the binding affinity is untangling in THF, which takes approximately one day to reach an equilibrium state. The polyNIPAM size measurements by dynamic light scattering (DLS) were conducted because it confirmed that the reversible aggregation behavior of polyNIPAM was not influenced by an applied voltage. The success of synthesis and characterization of ferrocene contained polyNIPAM illustrates that modifying a redox tag on the RAFT agent is feasible. However, ferrocene was found to not be stable with vinyl-pyridine, which is the basic functional monomer used in the current MIP recipe. Due to this, exploration of other redox tags such as triphenylamine (TPA) and anthraquinone (AQ) were tested. These redox tags were found not to be adequate for our application. As a result, they were declared to no longer be candidates for the project. However, methylene blue (MB), another redox tag option, was studied. It is believed that MB has the potential to make this approach of conformational-based electrochemical MIP sensors work, but it will require more research. For this reason, future work should focus on this redox tag

Studies of Single-Molecule Dynamics in Microorganisms

Fluorescence microscopy is one of the most extensively used techniques in the life sciences. Considering the non-invasive sample preparation, enabling live-cell compliant imaging, and the specific fluorescence labeling, allowing for a specific visualization of virtually any cellular compound, it is possible to localize even a single molecule in living cells. This makes modern fluorescence microscopy a powerful toolbox. In the recent decades, the development of new, "super-resolution" fluorescence microscopy techniques, which surpass the diffraction limit, revolutionized the field. Single-Molecule Localization Microscopy (SMLM) is a class of super-resolution microscopy methods and it enables resolution of down to tens of nanometers. SMLM methods like Photoactivated Localization Microscopy (PALM), (direct) Stochastic Optical Reconstruction Microscopy ((d)STORM), Ground-State Depletion followed by Individual Molecule Return (GSDIM) and Point Accumulation for Imaging in Nanoscale Topography (PAINT) have allowed to investigate both, the intracellular spatial organization of proteins and to observe their real-time dynamics at the single-molecule level in live cells. The focus of this thesis was the development of novel tools and strategies for live-cell SingleParticle Tracking PALM (sptPALM) imaging and implementing them for biological research. In the first part of this thesis, I describe the development of new Photoconvertible Fluorescent Proteins (pcFPs) which are optimized for sptPALM lowering the phototoxic damage caused by the imaging procedure. Furthermore, we show that we can utilize them together with Photoactivatable Fluorescent Proteins (paFPs) to enable multi-target labeling and read-out in a single color channel, which significantly simplifies the sample preparation and imaging routines as well as data analysis of multi-color PALM imaging of live cells. In parallel to developing new fluorescent proteins, I developed a high throughput data analysis pipeline. I have implemented this pipeline in my second project, described in the second part of this thesis, where I have investigated the protein organization and dynamics of the CRISPR-Cas antiviral defense mechanism of bacteria in vivo at a high spatiotemporal level with the sptPALM approach. I was successful to show the differences in the target search dynamics of the CRISPR effector complexes as well as of single Cas proteins for different target complementarities. I have also first data describing longer-lasting bound-times between effector complex and their potential targets in vivo, for which only in vitro data has been available till today. In summary, this thesis is a significant contribution for both, the advances of current sptPALM imaging methods, as well as for the understanding of the native behavior of CRISPR-Cas systems in vivo

Reconstruction and measurement of (100) MeV energy electromagnetic activity from π0 arrow γγ decays in the MicroBooNE LArTPC

We present results on the reconstruction of electromagnetic (EM) activity from photons produced in charged current νμ interactions with final state π0s. We employ a fully-automated reconstruction chain capable of identifying EM showers of (100) MeV energy, relying on a combination of traditional reconstruction techniques together with novel machine-learning approaches. These studies demonstrate good energy resolution, and good agreement between data and simulation, relying on the reconstructed invariant π0 mass and other photon distributions for validation. The reconstruction techniques developed are applied to a selection of νμ + Ar → μ + π0 + X candidate events to demonstrate the potential for calorimetric separation of photons from electrons and reconstruction of π0 kinematics

Trafficking of Hepatitis C Virus Core Protein during Virus Particle Assembly

Hepatitis C virus (HCV) core protein is directed to the surface of lipid droplets (LD), a step that is essential for infectious virus production. However, the process by which core is recruited from LD into nascent virus particles is not well understood. To investigate the kinetics of core trafficking, we developed methods to image functional core protein in live, virus-producing cells. During the peak of virus assembly, core formed polarized caps on large, immotile LDs, adjacent to putative sites of assembly. In addition, LD-independent, motile puncta of core were found to traffic along microtubules. Importantly, core was recruited from LDs into these puncta, and interaction between the viral NS2 and NS3-4A proteins was essential for this recruitment process. These data reveal new aspects of core trafficking and identify a novel role for viral nonstructural proteins in virus particle assembly