NMR structure of a complex between the VirB9/VirB7 interaction domains of the pKM101 type IV secretion system.

Type IV secretion (T4S) systems translocate DNA and protein effectors through the double membrane of Gram-negative bacteria. The paradigmatic T4S system in Agrobacterium tumefaciens is assembled from 11 VirB subunits and VirD4. Two subunits, VirB9 and VirB7, form an important stabilizing complex in the outer membrane. We describe here the NMR structure of a complex between the C-terminal domain of the VirB9 homolog TraO (TraO(CT)), bound to VirB7-like TraN from plasmid pKM101. TraO(CT) forms a beta-sandwich around which TraN winds. Structure-based mutations in VirB7 and VirB9 of A. tumefaciens show that the heterodimer interface is conserved. Opposite this interface, the TraO structure shows a protruding three-stranded beta-appendage, and here, we supply evidence that the corresponding region of VirB9 of A. tumefaciens inserts in the membrane and protrudes extracellularly. This complex structure elucidates the molecular basis for the interaction between two essential components of a T4S system.

Biological diversity of prokaryotic type IV secretion systems.

Type IV secretion systems (T4SS) translocate DNA and protein substrates across prokaryotic cell envelopes generally by a mechanism requiring direct contact with a target cell. Three types of T4SS have been described: (i) conjugation systems, operationally defined as machines that translocate DNA substrates intercellularly by a contact-dependent process; (ii) effector translocator systems, functioning to deliver proteins or other macromolecules to eukaryotic target cells; and (iii) DNA release/uptake systems, which translocate DNA to or from the extracellular milieu. Studies of a few paradigmatic systems, notably the conjugation systems of plasmids F, R388, RP4, and pKM101 and the Agrobacterium tumefaciens VirB/VirD4 system, have supplied important insights into the structure, function, and mechanism of action of type IV secretion machines. Information on these systems is updated, with emphasis on recent exciting structural advances. An underappreciated feature of T4SS, most notably of the conjugation subfamily, is that they are widely distributed among many species of gram-negative and -positive bacteria, wall-less bacteria, and the Archaea. Conjugation-mediated lateral gene transfer has shaped the genomes of most if not all prokaryotes over evolutionary time and also contributed in the short term to the dissemination of antibiotic resistance and other virulence traits among medically important pathogens. How have these machines adapted to function across envelopes of distantly related microorganisms? A survey of T4SS functioning in phylogenetically diverse species highlights the biological complexity of these translocation systems and identifies common mechanistic themes as well as novel adaptations for specialized purposes relating to the modulation of the donor-target cell interaction.

Biological diversity of prokaryotic type IV secretion systems.

Type IV secretion systems (T4SS) translocate DNA and protein substrates across prokaryotic cell envelopes generally by a mechanism requiring direct contact with a target cell. Three types of T4SS have been described: (i) conjugation systems, operationally defined as machines that translocate DNA substrates intercellularly by a contact-dependent process; (ii) effector translocator systems, functioning to deliver proteins or other macromolecules to eukaryotic target cells; and (iii) DNA release/uptake systems, which translocate DNA to or from the extracellular milieu. Studies of a few paradigmatic systems, notably the conjugation systems of plasmids F, R388, RP4, and pKM101 and the Agrobacterium tumefaciens VirB/VirD4 system, have supplied important insights into the structure, function, and mechanism of action of type IV secretion machines. Information on these systems is updated, with emphasis on recent exciting structural advances. An underappreciated feature of T4SS, most notably of the conjugation subfamily, is that they are widely distributed among many species of gram-negative and -positive bacteria, wall-less bacteria, and the Archaea. Conjugation-mediated lateral gene transfer has shaped the genomes of most if not all prokaryotes over evolutionary time and also contributed in the short term to the dissemination of antibiotic resistance and other virulence traits among medically important pathogens. How have these machines adapted to function across envelopes of distantly related microorganisms? A survey of T4SS functioning in phylogenetically diverse species highlights the biological complexity of these translocation systems and identifies common mechanistic themes as well as novel adaptations for specialized purposes relating to the modulation of the donor-target cell interaction.

Interlinked dual-time feedback loops can enhance robustness to stochasticity and persistence of memory.

Multiple interlinked positive feedback loops shape the stimulus responses of various biochemical systems, such as the cell cycle or intracellular Ca2+ release. Recent studies with simplified models have identified two advantages of coupling fast and slow feedback loops. This dual-time structure enables a fast response while enhancing resistances of responses and bistability to stimulus noise. We now find that (1) the dual-time structure similarly confers resistance to internal noise due to molecule number fluctuations, and (2) model variants with altered coupling, which better represent some specific biochemical systems, share all the above advantages. We also develop a similar bistable model with coupling of a fast autoactivation loop to a slow loop. This model's topology was suggested by positive feedback proposed to play a role in long-term synaptic potentiation (LTP). The advantages of fast response and noise resistance are also present in this autoactivation model. Empirically, LTP develops resistance to reversal over approximately 1h . The model suggests this resistance may result from increased amounts of synaptic kinases involved in positive feedback.

The Dissociation of Location and Object Working Memory using fMRI and MEG

Visual working memory (VWM) involves maintaining and processing visual information, often for the purpose of making immediate decisions. Neuroimaging experiments of VWM provide evidence in support of a neural system mainly involving a fronto-parietal neuronal network, but the role of specific brain areas is less clear. A proposal that has recently generated considerable debate suggests that a dissociation of object and location VWM occurs within the prefrontal cortex, in dorsal and ventral regions, respectively. However, re-examination of the relevant literature presents a more robust distribution suggestive of a general caudal-rostral dissociation from occipital and parietal structures, caudally, to prefrontal regions, rostrally, corresponding to location and object memory, respectively. The purpose of the present study was to identify a dissociation of location and object VWM across two imaging methods (magnetoencephalography, MEG, and functional magnetic imaging, fMRI). These two techniques provide complimentary results due the high temporal resolution of MEG and the high spatial resolution of fMRI. The use of identical location and object change detection tasks was employed across techniques and reported for the first time. Moreover, this study is the first to use matched stimulus displays across location and object VWM conditions. The results from these two imaging methods provided convergent evidence of a location and object VWM dissociation favoring a general caudal-rostral rather than the more common prefrontal dorsal-ventral view. Moreover, neural activity across techniques was correlated with behavioral performance for the first time and provided convergent results. This novel approach of combining imaging tools to study memory resulted in robust evidence suggesting a novel interpretation of location and object memory. Accordingly, this study presents a novel context within which to explore the neural substrates of WM across imaging techniques and populations.

Structural studies of cytochrome P4501A1: Identification of substrate and cumene hydroperoxide binding regions in the active site of P4501A1 and characterization of the P4501A1 interaction with cytochrome P450 reductase

Cytochromes P450 are a superfamily of heme-thiolate proteins that function in a concert with another protein, cytochrome P450 reductase, as terminal oxidases of an enzymatic system catalyzing the metabolism of a variety of foreign compounds and endogenous substrates. In order to better understand P450s catalytic mechanism and substrate specificity, information about the structure of the active site is necessary. Given the lack of a crystal structure of mammalian P450, other methods have been used to elucidate the substrate recognition and binding site structure in the active center. In this project I utilized the photoaffinity labeling technique and site-directed mutagenesis approach to gain further structural insight into the active site of mammalian cytochrome P4501AI and examine the role of surface residues in the interaction of P4501A1 with the reductase. ^ Four crosslinked peptides were identified by photoaffinity labeling using diazido benzphetamine as a substrate analog. Alignment of the primary structure of cytochrome P4501A1 with that of bacterial cytochrome P450102 (the crystal structure of which is known) revealed that two of the isolated crosslinked peptides can be placed in the vicinity of heme (in the L helix region and β10-β11 sheet region of cytochrome P450102) and could be involved in substrate binding. The other two peptides were located on the surface of the protein with the label bound specifically to Lys residues that were proposed to be involved in reductase-P450 interaction. ^ Alternatively, it has been shown that some of the organic hydroperoxides can support P450 catalyzed reactions in the absence of NADPH, O2 and reductase. By means of photoaffinity labeling the cumene hydroperoxide binding region was identified. Using azidocumene as the photoaffinity label, the tripeptide T501-L502-K503 was shown to be the site where azidocumene covalently binds to P4501A1. The sequence alignment of cytochrome P4501A1 with cytochrome P450102 predicts that this region might correspond to β-sheet structure localized on the distal side of the heme ring near the I helix and the oxygen binding pocket. The role of Thr501 in the cumene hydroperoxide binding was confirmed by mutations of this residue and kinetic analysis of the effects of the mutations. ^ In addition, the role of two lysine residues, Lys271 and Lys279, in the interaction with reductase was examined by means of site-directed mutagenesis. The lysine residues were substituted with isoleucine and enzymatic activity of the wild type and the mutants were compared in reductase- and cumene hydroperoxide-supported systems. The lysine 279 residue has been shown to play a critical role in the P4501A1-reductase interaction. ^

Forebrain-cerebellum interactions revealed by trace eyelid conditioning

While it is commonly assumed that brain systems receive and process information from other brain systems, there are few examples of tractable behaviors that allow such interactions to be studied. With the experiments presented in this dissertation we provide evidence that trace eyelid conditioning, a simple form of associative learning, is mediated by cerebellar learning in response to the output of persistent neural activity in the prefrontal cortex (PFC) and thus may be useful in analyses of PFC-cerebellar interactions. In a series of stimulation and reversible inactivation experiments we provide evidence that trace eyelid conditioning is mediated by cerebellar learning in response to a learned forebrain-driven input. Specifically, we provide evidence that this input is driven by the medial PFC and persists through the stimulus free trace interval of trace eyelid conditioning. In the next set of experiments we show that directly presenting the cerebellum with a pattern of input that mimics the classic persistent activity of PFC neurons reconstitutes trace eyelid conditioning, as assessed by a number of stringent tests. Finally, in set of reversible inactivation experiments, we provide evidence that bidirectional learning during trace eyelid conditioning involves the omission of the persistent, PFC-driven input that the cerebellum learns and responds to during trace eyelid conditioning. Given that persistent activity in PFC is often associated with working memory, these experiments suggest that trace eyelid conditioning may be useful in analyses of working memory mechanisms, cerebellar information processing and their interaction. To facilitate future analyses, we conclude with a working hypothesis of forebrain-cerebellum interactions during trace eyelid conditioning that addresses how persistent activity in PFC is induced and how the cerebellum decodes and uses PFC-driven input. ^

Macromolecular interaction studies on FF1-3 of human CA150 and STAT1-importin alpha5 using biophysical and biochemical methods

Macromolecular interactions, such as protein-protein interactions and protein-DNA interactions, play important roles in executing biological functions in cells. However the complexity of such interactions often makes it very challenging to elucidate the structural details of these subjects. In this thesis, two different research strategies were applied on two different two macromolecular systems: X-ray crystallography on three tandem FF domains of transcription regulator CA150 and electron microscopy on STAT1-importin α5 complex. The results from these studies provide novel insights into the function-structure relationships of transcription coupled RNA splicing mediated by CA150 and the nuclear import process of the JAK-STAT signaling pathway. ^ The first project aimed at the protein-protein interaction module FF domain, which often occurs as tandem repeats. Crystallographic structure of the first three FF domains of human CA150 was determined to 2.7 Å resolution. This is the only crystal structure of an FF domain and the only structure on tandem FF domains to date. It revealed a striking connectivity between an FF domain and the next. Peptide binding assay with the potential binding ligand of FF domains was performed using fluorescence polarization. Furthermore, for the first time, FF domains were found to potentially interact with DNA. DNA binding assays were also performed and the results were supportive to this newly proposed functionality of an FF domain. ^ The second project aimed at understanding the molecular mechanism of the nuclear import process of transcription factor STAT1. The first structural model of pSTAT1-importin α5 complex in solution was built from the images of negative staining electron microscopy. Two STAT1 molecules were observed to interact with one molecule of importin α5 in an asymmetric manner. This seems to imply that STAT1 interacts with importin α5 with a novel mechanism that is different from canonical importin α-cargo interactions. Further in vitro binding assays were performed to obtain more details on the pSTAT1-importin α5 interaction. ^

STUDIES ON CHOLINERGIC FUNCTION AND MEMORY IN MICE (CHOLINOTOXINS, WORKING, REFERENCE MEMORY, RADIAL ARM MAZE)

Considerable evidence suggests that central cholinergic neurons participate in either acquisition, storage or retrieval of information. Experiments were designed to evaluate information processing in mice following either reversible or irreversible impairment in central cholinergic activity. The cholinergic receptor antagonists, atropine and methylatropine were used to reversibly inhibit cholinergic transmission. Irreversible impairment in central cholinergic function was achieved by central administration of the cholinergic-specific neurotoxins, N-ethyl-choline aziridinium (ECA) and N-ethyl-acetylcholine aziridinium (EACA).^ ECA and EACA appear to act by irreversible inhibition of high affinity choline uptake (proposed rate-limiting step in acetylcholine synthesis). Intraventricular administration of ECA or EACA produced persistent reduction in hippocampal choline acetyltransferase activity. Other neuronal systems and brain regions showed no evidence of toxicity.^ Mice treated with either ECA or EACA showed behavioral deficits associated with cholinergic dysfunction. Passive avoidance behavior was significantly impaired by cholinotoxin treatment. Radial arm maze performance was also significantly impaired in cholinotoxin-treated animals. Deficits in radial arm maze performance were transient, however, such that rapid and apparent complete behavioral recovery was seen during retention testing. The centrally active cholinergic receptor antagonist atropine also caused significant impairment in radial arm maze behavior, while equivalent doses of methylatropine were without effect.^ The relative effects of cholinotoxin and receptor antagonist treatment on short-term (working) memory and long-term (reference) memory in radial arm maze behavior were examined. Maze rotation studies indicated that there were at least two different response strategies which could result in accurate maze performance. One strategy involved the use of response algorithms and was considered to be a function of reference memory. Another strategy appeared to be primarily dependent on spatial working memory. However, all behavioral paradigms with multiple trails have reference memory requirements (i.e. information useful over all trials). Performance was similarly affected following either cholinotoxin or anticholinergic treatment, regardless of the response strategy utilized. In addition, rates of behavioral recovery following cholinotoxin treatment were similar between response groups. It was concluded that both cholinotoxin and anticholinergic treatment primarily resulted in impaired reference memory processes. ^

Design, Calibration, and Evaluation of Depth-of-Interaction-Capable PET Detector Modules

High-resolution, small-bore PET systems suffer from a tradeoff between system sensitivity, and image quality degradation. In these systems long crystals allow mispositioning of the line of response due to parallax error and this mispositioning causes resolution blurring, but long crystals are necessary for high system sensitivity. One means to allow long crystals without introducing parallax errors is to determine the depth of interaction (DOI) of the gamma ray interaction within the detector module. While DOI has been investigated previously, newly available solid state photomultipliers (SSPMs) well-suited to PET applications and allow new modules for investigation. Depth of interaction in full modules is a relatively new field, and so even if high performance DOI capable modules were available, the appropriate means to characterize and calibrate the modules are not. This work presents an investigation of DOI capable arrays and techniques for characterizing and calibrating those modules. The methods introduced here accurately and reliably characterize and calibrate energy, timing, and event interaction positioning. Additionally presented is a characterization of the spatial resolution of DOI capable modules and a measurement of DOI effects for different angles between detector modules. These arrays have been built into a prototype PET system that delivers better than 2.0 mm resolution with a single-sided-stopping-power in excess of 95% for 511 keV g's. The noise properties of SSPMs scale with the active area of the detector face, and so the best signal-to-noise ratio is possible with parallel readout of each SSPM photodetector pixel rather than multiplexing signals together. This work additionally investigates several algorithms for improving timing performance using timing information from multiple SSPM pixels when light is distributed among several photodetectors.

A comparative analysis of hospital -based physician monitoring systems

The purpose of the multiple case-study was to determine how hospital subsystems (such as physician monitoring and credentialing; quality assurance; risk management; and peer review) were supporting the monitoring of physicians? Three large metropolitan hospitals in Texas were studied and designated as hospitals #1, #2, and #3. Realizing that hospital subsystems are a unique entity and part of a larger system, conclusions were made on the premises of a quality control system, in relation to the tools of government (particularly the Health Care Quality Improvement Act (HCQIA)), and in relation to itself as a tool of a hospital.^ Three major analytical assessments were performed. First, the subsystems were analyzed as to their "completeness"; secondly, the subsystems were analyzed for "performance"; and thirdly, the subsystems were analyzed in reference to the interaction of completeness and performance.^ The physician credentialing and monitoring and the peer review subsystems as quality control systems were most complete, efficient, and effective in hospitals #1 and #3. The HCQIA did not seem to be an influencing factor in the completeness of the subsystem in hospital #1. The quality assurance and risk management subsystem in hospital #2 was not representative of completeness and performance and the HCQIA was not an influencing factor in the completeness of the Q.A. or R.M. systems in any hospital. The efficiency (computerization) of the physician credentialing, quality assurance and peer review subsystems in hospitals #1 and #3 seemed to contribute to their effectiveness (system-wide effect).^ The results indicated that the more complete, effective, and efficient subsystems were characterized by (1) all defined activities being met, (2) the HCQIA being an influencing factor, (3) a decentralized administrative structure, (4) computerization an important element, and (5) staff was sophisticated in subsystem operations. However, other variables were identified which deserve further research as to their effect on completeness and performance of subsystems. They include (1) medical staff affiliations, (2) system funding levels, (3) the system's administrative structure, and (4) the physician staff "cultural" characteristics. Perhaps by understanding other influencing factors, health care administrators may plan subsystems that will be compatible with legislative requirements and administrative objectives. ^