Insecticide Susceptibility and Resistance Detection in \u3cem\u3ePhlebotomus argentipes\u3c/em\u3e Sandflies (Diptera: Psychodidae: Phlebotominae)

Leishmaniasis is an understudied disease found predominantly in high heat and humidity areas. The disease is transmitted by sandflies which are blood-feeding, biting insects. There is currently no known vaccine for any form of leishmaniasis and treatment imposes a significant economic impact on already poor and marginalized populations, as well as severe, lingering side effects for afflicted individuals. Annually, there are millions of people around the world afflicted with a form of Leishmaniasis that is transmitted by species of sandflies. The primary sandfly population control method for over half a century has been indoor residual spraying using insecticides. The selective pressure from utilizing insecticides over such a long period has resulted in selection for resistance which requires an assay to monitor and identify these populations. The goal of this thesis is to determine and assess insecticide susceptibility and resistance of the sandfly species, Phelebotomus argentipes. Research started by understanding the response of sandflies to particular insecticides of interest. This was completed by assessing levels of susceptibility of the P. argentipes colony to three insecticides which have been used extensively in India throughout the years. Susceptibility curves were developed to determine lethal concentration (LC) of insecticides capable of reducing populations by 50%, 75%, and 90%. An amplification assay (Polymerase Chain Reaction) was optimized to specifically amplify genetic regions associated with insecticide resistance for P. argentipes sandflies. Whereas, another amplification assay (Loop-mediated Isothermal Amplification) was developed as an effective and rapid procedure for integrated pest management (IPM) professionals to assess the degree of insecticide susceptibility in field collected populations of P. argentipes sandflies. This rapid detection of field collected samples could allow IPM professionals to determine the degree of susceptibility of sandfly populations collected from afflicted areas. Thus, allowing the disease vector to be controlled in a more timely and effective manner

Tales of Teaching: Exploring the Dialectical Tensions of the GTA Experience

In universities across the United States, an increasing number of departments are turning to graduate teaching assistants (GTAs) to teach introductory courses. As GTAs assume a larger percentage of university teaching responsibilities, it becomes even more important to understand the tensions and challenges that GTAs face. The majority of research on GTAs focuses on the perceptions of students and GTA supervisors, and few researchers have talked directly to GTAs. This research fills that gap by studying the GTA experience from the GTA perspective. Using relational dialectics theory, this study identifies three key tensions that emerge from GTAs’ stories about role conflict and identity management: distance-closeness, perfect teacherperfect student, and structure-freedom. Further, it analyzes the strategies GTAs use to manage and negotiate these tensions. After discussing the implications that these tensions have for GTAs and supervisors, the study offers suggestions for coping with tensions constructively. Finally, since these tensions can influence GTAs’ future careers as educators, this study concludes by considering the broader implications of these tensions for students and teachers

Deletion of Gpr27 in vivo reduces insulin mRNA but does not result in diabetes.

Gpr27 is a highly conserved, orphan G protein coupled receptor (GPCR) previously implicated in pancreatic beta cell insulin transcription and glucose-stimulated insulin secretion in vitro. Here, we characterize a whole-body mouse knockout of Gpr27. Gpr27 knockout mice were born at expected Mendelian ratios and exhibited no gross abnormalities. Insulin and Pdx1 mRNA in Gpr27 knockout islets were reduced by 30%, but this did not translate to a reduction in islet insulin content or beta cell mass. Gpr27 knockout mice exhibited slightly worsened glucose tolerance with lower plasma insulin levels while maintaining similar insulin tolerance. Unexpectedly, Gpr27 deletion reduced expression of Eif4e3, a neighboring gene, likely by deleting transcription start sites on the anti-sense strand of the Gpr27 coding exon. Our data confirm that loss of Gpr27 reduces insulin mRNA in vivo but has only minor effects on glucose tolerance

Deformation Quantization: Quantum Mechanics Lives and Works in Phase-Space

Wigner's quasi-probability distribution function in phase-space is a special (Weyl) representation of the density matrix. It has been useful in describing quantum transport in quantum optics; nuclear physics; decoherence (eg, quantum computing); quantum chaos; "Welcher Weg" discussions; semiclassical limits. It is also of importance in signal processing. Nevertheless, a remarkable aspect of its internal logic, pioneered by Moyal, has only emerged in the last quarter-century: It furnishes a third, alternative, formulation of Quantum Mechanics, independent of the conventional Hilbert Space, or Path Integral formulations. In this logically complete and self-standing formulation, one need not choose sides--coordinate or momentum space. It works in full phase-space, accommodating the uncertainty principle. This is an introductory overview of the formulation with simple illustrations.Comment: LaTeX, 22 pages, 2 figure

Untersuchungen zur Expression von Uhrengenen in der kortikotrophen AtT-20-Tumorzelllinie der Maus

In den unterschiedlichsten Lebewesen, wie Cyanobakterien, Pilzen, Pflanzen und Tieren können tägliche Rhythmen biologischer Aktivität beobachtet werden, die von einem endogenen zirkadianen Oszillator gesteuert werden. Dieser zirkadiane Oszillator residiert bei Säugern im Nucleus suprachiasmaticus (SCN) des Hypothalamus, unterhält auch unter konstanten Bedingungen einen Rhythmus mit einer Periodenlänge von etwa 24 h und wird unter natürlichen Bedingungen an den täglichen Wechsel der Beleuchtungsverhältnisse über neuronale Signale, die von den Augen kommen, angepasst. Für die Generation dieser endogenen Oszillationen konnte die rhythmische Expression von so genannten Uhrengenen verantwortlich gemacht werden. Nach der heute gültigen Vorstellung bilden diese zusammen mit ihren Proteinprodukten interagierende transkriptionelle-translationale Rückkopplungsschleifen, die für einen vollständigen Durchlauf, bis ein neuer Zyklus beginnt, etwa 24 h brauchen. Dabei aktivieren zwei Transkriptionsfaktoren der bHLH-PAS-Familie, CLOCK und BMAL1, zu Beginn eines zirkadianen Zyklus als Heterodimer über eine hochspezifisches E-Box-Promotorelement die Transkription der Uhrengene Per1-3, Cry1-2 und Rev-Erbα. Im Zytosol bilden die Uhrengenprodukte der CRYs und PERs zusammen mit der Caseinkinase Iε (CKIε) einen heterotrimeren Komplex, der im Kern wiederum die CLOCK-BMAL1-abhängige Transkription blockiert. Überraschend ist, dass nicht nur die Neurone des Schrittmachers im SCN diese Rhythmen endogen produzieren können, sondern auch eine Vielzahl peripherer Zellen, selbst, wenn sie über Jahre in Kultur gehalten wurden. Man nimmt an, dass der Rhythmus peripherer Zellen in vivo sowohl über neuronalen Verbindungen als auch über bisher noch nicht identifizierte humorale Faktoren synchronisiert wird. Es ist bis heute weder geklärt, worin die molekularen Unterschiede peripherer Oszillatoren im Vergleich zum SCN bestehen, noch, wie der Synchronisationsprozess dieser Zellen zu Stande kommt. Auf Grund methodischer Schwierigkeiten bei der Untersuchung des SCN wurde zuletzt vermehrt gefordert, sich diesen Fragen zunächst an Hand eines Modellsystems, wie einer Zellkultur aus immortalisierten Zellen zu nähern. In der vorliegenden Arbeit wurde deshalb untersucht, ob sich die kortikotrophe hypophysäre AtT-20 Tumorzelllinie der Maus prinzipiell für die Erforschung zirkadianer Rhythmen und deren Synchronisation eignet, d.h. ob sie selbst über eine stimulierbare rhythmische Uhrengen-Expression verfügen. Weiterhin sollte eine geeignete Methode gefunden werden, um zirkadiane Rhythmen auf mRNA-Ebene darzustellen. In einem ersten Schritt wurde über RT-PCR Technik erstmals nachgewiesen, dass die essen-tiellen Uhrengene Per1, Per2, Per3, Cry1, Cry2, Bmal1, Clock und CK1ε endogen in AtT-20 Zellen exprimiert werden. Für jedes dieser Gene wurde nun eine Variante der quantitativen Real-Time-PCR (RTQ-PCR), die ΔΔCT-Methode, validiert, die bei hohem Probendurchsatz zuverlässig Expressionsunterschiede wiedergeben kann. Durch Stimulation mit Forskolin, ei-nem Aktivator der Adenylatzyklase, konnte in dieser Arbeit dokumentiert werden, dass kulti-vierte AtT-20 Zellen in der Lage sind, eine rhythmische Expression von Uhrengenen mit einer Periodenlänge von etwa 24 h für mindestens drei Tage zu zeigen. Von allen hier untersuchten Uhrengenen wiesen alle diejenigen eine oszillierende Schwankung des mRNA-Gehaltes auf, die auch im SCN rhythmisch exprimiert werden, namentlich Per1-3, Cry1-2, Bmal1. Im SCN kon-stitutiv exprimierten Uhrengene (Clock, Ck1ε) fluktuieren auch nicht in AtT-20 Zellen. Dar-über hinaus antworteten Zellen auf das hier angewandte Stimulationsprotokoll mit einer initia-len Hochregulierung der Transkription für das Uhrengen Per1, das im SCN eine prominente Rolle bei der Anpassung des endogenen Rhythmus an die exogenen Beleuchtungsverhältnisse spielt und dort als Antwort auf synchronisierende Lichtpulse in ähnlicher Weise induziert wer-den kann. Zeitlich korreliert die Zunahme von Per1-Transkripten – ebenfalls der Situation im SCN entsprechend – mit einer Aktivierung des Transkriptionsfaktors CREB und der Induktion seines molekularen Gegenspielers Icer. Die zeitlich umschriebene Hochregulation der Transkriptionsrate des Repressors Icer während der ersten Stunden nach Applikation des syn-chronisierenden Reizes spricht dafür, dass dieser womöglich in AtT-20, wie auch bereits für Elemente des zirkadianen Systems beschrieben, eine wichtige Rolle bei der Verarbeitung von Synchronisationsreizes im molekularen Uhrwerk spielt. Die genaue Analyse der hier erhobenen Expressions-Rhythmen von Uhrengenen und deren zeitliches Verhältnis zueinander deuteten darauf hin, dass in AtT-20 Zellen ein funktionsfähiges zirkadianes Uhrwerk existiert, das dem des SCN in weiten Teilen gleicht. Die Möglichkeiten der Stimulation und Manipulation (z.B. durch Transfektion) erheben AtT-20 Zellen zu einem Modellsystem für die Erforschung der molekularen Abläufe in der zirkadianen Rhythmusgeneration und –synchronisation. Erkenntnisse aus dieser Forschung können in den unterschiedlichsten klinischen Disziplinen wichtige Anwendungsmöglichkeiten finden.Daily rhythms of biological activity are driven by an endogenous circadian oscillator and can be observed in living organisms as diverse as cyanobacteria, fungi, plants and animals. In mammals, this circadian oscillator resides in the suprachiasmatic nucleus of the hypothalamus and produces robust rhythms of a period length of about 24 hours, even under constant conditions. Under natural conditions, neuronal input from the eyes entrains its activity to the environmental light-dark cycle. Endogenous oscillations are generated by rhythmic expression of so called clock genes and their protein products that form interacting transcriptional-translational feedback loops, covering a cycling period close to 24 hours. The circadian cycle starts when two transcription factors of the bHLH-PAS family, CLOCK and BMAL1, enhance transcription by binding as heterodimers to highly selective E-Box elements of Per1-3, Cry1-2 and Rev-Erbα. In turn, a heterotrimeric complex of the clock gene products of CRYs, PERs and CK1ε translocates into the nucleus and blocks CLOCK-BMAL1 driven transcription. Surprisingly, not only SCN pacemaker neurons are capable to exhibit endogenous oscillations but also a multitude of different peripheral cells, even if they have been cultured for years. One can assume that in vivo the SCN synchronizes rhythms of peripheral cells by means of both neuronal connections and yet unknown humoral factors. To date, it is still not elucidated in which way the molecular setup of pacemaker neurons of the SCN differs from that of peripheral oscillators, nor it is clear, how synchronization of these cells is performed. As the use of the SCN harbours certain methodical difficulties, it has been proposed to clarify these questions for the present by means of culturing immortalized cells. The present work therefore aimed to determine whether the hypophyseal corticotroph AtT-20 cell line of the mouse can serve as a model to investigate generation and synchronization proc-esses of circadian rhythms, i.e. whether they exhibit rhythmic circadian expression of clock genes upon stimulation. Further, a suitable method should be found, to analyse circadian mRNA rhythms. Here it has been shown for the first time by means of the RT-PCR technique that AtT-20 cells express endogenously the essential clock genes Per1, Per2, Per3, Cry1, Cry2, Bmal1, Clock and CK1ε. For each of these genes, a variant of quantitative Real-Time PCR, the ΔΔCT method, has been validated, which offers both high-throughput processing of many samples and a reliable display of differences in expression. This work shows that cultured AtT-20 cells are able to exhibit an almost 24 hour rhythm in clock gene expression for at least 3 days upon stimulation with forskolin, an activator of the adenylate cyclase. From all genes investigated, all those (Per1-3, Cry1-2, Bmal1) showed oscillating changes of mRNA levels that are also known to be rhythmic in the SCN. On the other hand, clock genes that are constitutively expressed in the SCN did also not oscillate in AtT-20 cells (Clock, CK1ε). Further, AtT-20 cells showed an initial up regulation of mRNA of the clock gene Per1 upon stimulation, similar to SCN neurons when synchronized by photic input signals. Similarly, the increase of Per1 transcripts correlates with the activation of the transcription factor CREB and the induction of its molecular opponent ICER. In AtT-20 cells the up regulation of Icer expression is limited to the first hours after stimulation, indicating that this transcriptional repressor plays an important role in the processing of synchronizing stimuli to the molecular clock mechanism, as it has been described in other elements of the circadian system. The analysis of the here presented rhythms of clock gene expression and their phase relation demonstrates that a functional circadian oscillator exists in AtT-20 cells that is very similar in structure and function to that of the SCN. The possibilities to stimulate and manipulate AtT-20 cells mark them to a model system for the exploration of molecular processes, involved in gen-eration and synchronization of circadian rhythms. Findings of this research may furthermore be applied in various clinical disciplines

Comment on ``Conduction states in oxide perovskites: Three manifestations of Ti3+^{3 +} Jahn-Teller polarons in barium titanate''

In this comment to [S. Lenjer, O. F. Schirmer, H. Hesse, and Th. W. Kool, Phys. Rev. B {\bf 66}, 165106 (2002)] we discuss the electronic structure of oxygen vacancies in perovskites. First principles computations are in favour of rather deep levels in these vacancies, and Lenjer et al suggest that the electrons' interaction energy is negative, but data on electroconductivity are against.Comment: 2 pages, no figure

A Whole-Genome RNA Interference Screen Reveals a Role for Spry2 in Insulin Transcription and the Unfolded Protein Response.

Insulin production by the pancreatic β-cell is required for normal glucose homeostasis. While key transcription factors that bind to the insulin promoter are known, relatively little is known about the upstream regulators of insulin transcription. Using a whole-genome RNA interference screen, we uncovered 26 novel regulators of insulin transcription that regulate diverse processes including oxidative phosphorylation, vesicle traffic, and the unfolded protein response (UPR). We focused on Spry2-a gene implicated in human type 2 diabetes by genome-wide association studies but without a clear connection to glucose homeostasis. We showed that Spry2 is a novel UPR target and its upregulation is dependent on PERK. Knockdown of Spry2 resulted in reduced expression of Serca2, reduced endoplasmic reticulum calcium levels, and induction of the UPR. Spry2 deletion in the adult mouse β-cell caused hyperglycemia and hypoinsulinemia. Our study greatly expands the compendium of insulin promoter regulators and demonstrates a novel β-cell link between Spry2 and human diabetes