Synthesis of Small Molecule Derivatives of CK-666 as Potential Inhibitors of the Arp2/3 Complex

The Actin-Related Protein (Arp) 2/3 Complex is an actin nucleating factor intrinsically involved in cellular regulation of actin networks during life-essential processes such as motility. Overexpression of the Arp2/3 complex has indicated as a factor allowing the proliferation of certain metastatic cancers. This work describes the synthesis and in vitro biochemical testing of several molecules predicted by computational docking to be inhibitors of Arp2/3 Complex, and therefore of potential interest in clinical applications. The molecules are designed based off of the structure of known Arp2/3 inhibitor CK-666, which was discovered via high-throughput screening. Details of the synthesis of the tryptamine derivatives are discussed, and the bulk actin polymerization assay used to determine potency of the new compounds is discussed

X-56A Structural Dynamics Ground Testing Overview and Lessons Learned

The X-56A Multi-Utility Technology Testbed (MUTT) is a subscale, fixed-wing aircraft designed for high-risk aeroelastic flight demonstration and research. Structural dynamics ground testing for model validation was especially important for this vehicle because the structural model was directly used in the development of a flight control system with active flutter suppression capabilities. Structural dynamics ground tests of the X-56A MUTT with coupled rigid-body and structural modes provided a unique set of challenges. An overview of the ground vibration test (GVT) and moment of inertia (MOI) test setup and execution is presented. The series of GVTs included the wing by itself attached to a strongback and complete vehicle at two mass conditions: empty and full fuel. Two boundary conditions for the complete-vehicle test were studied: on landing gear and suspended free-free. Pitch MOI tests were performed using a compound pendulum method and repeated with two different pendulum lengths for independent verification. The original soft-support test configuration for the GVT used multiple bungees, resulting in unforeseen coupling interactions between the soft-support bungees and the vehicle structural modes. To resolve this problem, the soft-support test setup underwent multiple iterations. The various GVT configurations and boundary-condition modifications are highlighted and explained. Lessons learned are captured for future consideration when performing structural dynamics testing with similar vehicles

Hsp90-Mediated Maturation of Kinases and Nuclear Steroid Hormone Receptors: A Dissertation

Among heat shock proteins, Hsp90 is unusual because it is not required for the proper folding of most cellular proteins but rather is disproportionally linked to the activation of signal transduction proteins including over forty kinases and many steroid hormone receptors. Mutated forms of many Hsp90 clients are causative agents in cancer, making Hsp90 a promising pharmacological target. Many small molecular inhibitors have been identified that competitively bind to the ATP binding site of Hsp90, some of which are in clinical trials as anticancer agents. Although the activation of kinase and hormone receptor clients by Hsp90 and its co-chaperones has been extensively studied, the molecular mechanism of client protein activation is poorly understood. Hsp90 is a dimeric chaperone containing three domains: the N-terminal (N) and middle (M) domains contribute directly to ATP binding and hydrolysis and the C-terminal (C) domain mediates dimerization. At physiological concentration, Hsp90 predominantly forms dimers, but the possibility that full-length monomers might also function in cells has not been tested. In Chapter 3, we used a single-chain strategy to design a full-length Hsp90 monomer (NMCC). The resulting construct was predominantly monomeric at physiological concentration and did not function to support yeast viability as the sole Hsp90. NMCC Hsp90 was also defective at ATP hydrolysis and the activation of kinase and steroid hormone receptor clients in yeast cells. The ability to support yeast growth was rescued by the addition of a coiled-coil dimerization domain, indicating that the parental single-chain construct is functionally defective because it is monomeric. After finding that a full-length Hsp90 monomer containing only one ATPase site was unable to support yeast viability or activate Hsp90 clients, we set out to further explore the role of ATPase activity in client protein activation. Approximately 10 % of the yeast proteome binds to Hsp90 making it important to study Hsp90 function in the cellular environment where all binding partners are present. In Chapter 4, we observed that co-expression of different Hsp90 subunits in Saccharomyces cerevisiae caused unpredictable synthetic growth defects due to cross-dimerization. We engineered super-stabilized Hsp90 dimers that resisted cross-dimerization with endogenous Hsp90 and alleviated the synthetic growth defect. We utilized these super-stabilized dimers to analyze the ability of ATPase mutant homodimers to activate known Hsp90 client proteins in yeast cells. We found that ATP binding and hydrolysis by Hsp90 are both required for the efficient maturation of the glucocorticoid hormone receptor (GR) and v-src confirming the critical role of ATP hydrolysis in the maturation of steroid hormone receptors and kinases in vivo. In addition to its role in the activation of signal transduction client proteins, Hsp90 has been shown to suppress the in vitro aggregation of numerous hard-to-fold proteins. In Chapter 5, we examine the role of charge in Hsp90 anti-aggregation activity. The charge on Hsp90 is largely concentrated in two highly acidic regions. We found that deletion of both charge-rich regions dramatically impaired Hsp90 anti-aggregation activity. Addition of an acid-rich region with a distinct amino acid sequence to our double-deleted Hsp90 construct rescued the anti-aggregation activity of Hsp90 indicating that the net charge contributes to its anti-aggregation activity. The in vitro anti-aggregation activity of Hsp90 studied in Chapter 5 occurs in the absence of ATP. However, all of the biologically important functions of Hsp90 in cells identified to date, including the maturation of kinases and nuclear steroid hormone receptors, clearly require ATP hydrolysis. Why does Hsp90 robustly hinder the aggregation of hard-to-fold proteins without ATP in vitro, but in vivo uses ATP hydrolysis for all of its essential functions? By utilizing separation of function Hsp90 variants (that specifically lack in vitro anti-aggregation activity) we have begun to address this question. We find that anti-aggregation deficient Hsp90 is unable to support yeast growth under stressful conditions, potentially due to reduced cellular expression. Interestingly, the ATP-independent anti-aggregation activity of Hsp90 has no measureable impact on cellular function. Thus, hindering the aggregation of most hard-to- fold proteins by Hsp90 (independent of ATP hydrolysis) does not appear to be important for cell function. These results suggest a cellular model where the Hsp40/60/70 machinery is responsible for hindering the aggregation of most hard-to-fold proteins while Hsp90 assists in the maturation of a select set of clients in an ATP-dependent fashion, potentially aided by its inherent anti-aggregation properties

Stroke Severity Affects Timing: Time From Stroke Code Activation to Initial Imaging is Longer in Patients With Milder Strokes.

Optimizing the time it takes to get a potential stroke patient to imaging is essential in a rapid stroke response. At our hospital, door-to-imaging time is comprised of 2 time periods: the time before a stroke is recognized, followed by the period after the stroke code is called during which the stroke team assesses and brings the patient to the computed tomography scanner. To control for delays due to triage, we isolated the time period after a potential stroke has been recognized, as few studies have examined the biases of stroke code responders. This code-to-imaging time (CIT) encompassed the time from stroke code activation to initial imaging, and we hypothesized that perception of stroke severity would affect how quickly stroke code responders act. In consecutively admitted ischemic stroke patients at The Mount Sinai Hospital emergency department, we tested associations between National Institutes of Health Stroke Scale scores (NIHSS), continuously and at different cutoffs, and CIT using spline regression, t tests for univariate analysis, and multivariable linear regression adjusting for age, sex, and race/ethnicity. In our study population, mean CIT was 26 minutes, and mean presentation NIHSS was 8. In univariate and multivariate analyses comparing CIT between mild and severe strokes, stroke scale scores4

A Needs Assessment Study for an Ongoing Professional Development Program Called Some Saint for Subs for Substitute Teachers in Florida Public School Districts

The purpose of this study was to determine if there was a need for an ongoing professional development program for substitute teachers in Florida public school districts. For some time now, many public school districts in Florida have faced huge teacher and substitute teacher shortages which has a substantial academic impact on the students who attend these schools. A survey was administered to principals, assistant principals, certified teachers, and substitute teachers who work in a public Florida school district to determine if there was a need for talented substitute teachers in their area. Some questions on the survey were modeled after Marzano evaluation questions for certified teachers to see how a talented substitute teacher could be identified as effective while other questions focus on the demographics and substitute teaching experience of those who currently work for a public school district in Florida. Findings include that Florida school districts needed and would benefit from an ongoing professional development program for substitute teachers. The researcher also found that the ongoing professional development program created for substitute teachers, Some Saint for Subs, could increase student achievement as well as solve the substitute inadequacy in Florida public school districts. Recommendations include time (a full school year) and money to thoroughly review the effectiveness of Some Saint for Subs

Elliptic Genera and 3d Gravity

We describe general constraints on the elliptic genus of a 2d supersymmetric conformal field theory which has a gravity dual with large radius in Planck units. We give examples of theories which do and do not satisfy the bounds we derive, by describing the elliptic genera of symmetric product orbifolds of $K3$, product manifolds, certain simple families of Calabi-Yau hypersurfaces, and symmetric products of the "Monster CFT." We discuss the distinction between theories with supergravity duals and those whose duals have strings at the scale set by the AdS curvature. Under natural assumptions we attempt to quantify the fraction of (2,2) supersymmetric conformal theories which admit a weakly curved gravity description, at large central charge.Comment: 50 pages, 9 figures, v2: minor corrections to section

Complications Dawn for Kinetochore Regulation by Aurora

Organisms must faithfully segregate their chromosomes during cell division; mistakes in this process can be costly and even fatal to the organism (1, 2). During mitosis, replicated chromosomes attach to the spindle, a dynamic system of microtubules organized around two poles. Chromosomes attach to the spindle via kinetochores, structures that form on centromeres and bind the ends of microtubules. For accurate segregation, kinetochores on sister chromosomes must attach to microtubules from opposite poles; incorrect attachments lead to missegregation (3). In PNAS, Umbreit et al. (4) expand our understanding of how kinetochore–microtubule interactions can be regulated to correct improper attachments. The authors use in vitro studies to demonstrate that a component of the kinetochore, the Ndc80 complex, can directly influence the dynamics of the microtubules it is bound to and how the complex can be regulated to correct errors in chromosome attachment. Kinetochores are complicated machines. They can stay attached to microtubule ends as they grow and shrink, regulate the dynamics of microtubules, regulate their own activity, and signal to the remainder of the cell. The outer layer of the kinetochore contains the dumbbell-shaped Ndc80 complex (5): One globular domain [the N-terminal domains of Hec1 (Ndc80 in budding yeast) and Nuf2] binds microtubules (6) and is connected by a long coiled coil to the other globular domain (composed of the C-terminal domains of Spc24/Spc25), which connects to other kinetochore components (7) (Fig. 1A). Hec1 contains a conserved calponin homology domain and an unstructured N-terminal tail: Both regions can bind to microtubules independently, but they must act together to produce high-affinity binding (5⇓⇓–8). When sister kinetochores attach...(see full text).Molecular and Cellular Biolog

Timing of vessel imaging for suspected large vessel occlusions does not affect groin puncture time in transfer patients with stroke.

BACKGROUND: Access to endovascular therapy (ET) in cases of acute ischemic stroke may be limited, and rapid transfer of eligible patients to hospitals with endovascular capability is needed. OBJECTIVE: To determine the optimal timing of diagnostic CT angiography to confirm large vessel occlusion (LVO). METHODS: Of 57 emergency department transfers to Mount Sinai Hospital (MSH) for possible ET from January 2015 through March 2016, 39 (68%) underwent ET, among whom 22 (56%) had CT angiography before transfer and 17 (44%) had CT angiography on arrival. We compared mean outside hospital arrival to groin puncture (OTG) time between the two groups using t-tests and Wilcoxon rank sum tests. OTG was defined as the difference between groin puncture and outside hospital arrival time minus ambulance travel time. RESULTS: Average age was 73±13 years and average National Institute of Health Stroke Scale score was 19±5. There was no difference in average OTG time between the two groups (191 min for CT angiography at outside hospital vs 190 min for CT angiography at MSH (p=0.99 for t-test and 0.69 for rank sum test)). Among the 18 patients who were transferred but did not receive ET, 10 had no LVO, 5 had large established infarcts on arrival and 3 had post-tissue plasminogen activator hemorrhage. In 9/10 patients without LVO, CT angiography was not performed before transfer. CONCLUSIONS: CT angiography timing in the transfer process does not affect OTG time, but 90% of patients without LVO had not had CT angiography before transfer. Hence, it might be beneficial to obtain a CT angiogram at the outside hospital, if it can be acquired and read rapidly, to avoid the cost and potential clinical deterioration associated with unnecessary transfers