Methods and technologies for high-throughput and high-content small animal screening

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2012.Cataloged from PDF version of thesis.Includes bibliographical references (p. 157-170).High-throughput and high-content screening (HTS and HCS) of whole animals requires their immobilization for high-resolution imaging and manipulation. Here we present methods to enable HTS and HCS of the nematode Caenorhabditis elegans (C. elegans). First we present microfluidic technologies to rapidly isolate, immobilize, image and manipulate individual animals. These technologies include 1. a high-speed microfluidic sorter that can isolate and immobilize C. elegans in a well defined geometry for screening phenotypic features in physiologically active animals, 2. an integrated chip containing individually addressable screening-chamber devices for incubation and exposure of individual animals to biochemical compounds and high-resolution time-lapse imaging of multiple animals and 3. a design for delivery of compound libraries in standard multiwell plates to microfluidic devices and also for rapid dispensing of screened animals into multiwell plates. We then present an improved immobilization method that restrains animals with sufficient stability to perform femtosecond laser microsurgery and multiphoton imaging, without any apparent effects on animal health. We subsequently screen the contents of a small-molecule library for factors affecting neural regeneration following femtosecond laser microsurgery of C. elegans using these technologies. This screen identifies the kinase inhibitor staurosporine as a strong inhibitor of neural regeneration, and does so in a concentration and neuronal cell type-specific manner. Finally, we present a simple device for immobilizing C. elegans inside standard microtiter plates that is compatible with existing HTS systems. The device consists of an array of metal pins connected to individually-controlled thermoelectric coolers. 'We use this to perform femtosecond laser microsurgery on C. elegans in microtiter plates and to analyze the regeneration dynamics over time. This analysis shows that neurons tend regenerate in single short bursts that occur stochastically within the first two days post-surgery.by Christopher B. Rohde.Ph.D

Роль текстурированного инструмента в повышении эффективности процесса обработки резанием

Текстурирование поверхности является одним из перспективных методов, который влияет на улучшение трибологических свойств инструментов. Текстуры поверхности имеют разный геометрический размер, форму и, как правило, создаются либо на боковой поверхности, либо на передней поверхности режущего инструмента. Качество текстуры в значительной степени зависит от производственных процессов, которые были использованы

A Multi-Method Study of a Chalcolithic Kiln in the Bora Plain (Iraqi Kurdistan): The Evidence From Excavation, Micromorphological and Pyrotechnological Analyses

Pyrotechnology has always been a core topic in the archaeological debate concerning phases of deep cultural transformations, such as the Chalcolithic period in the Near East (c. 6000–3500 BC). However, previous studies on pyrotechnological installations, such as pottery kilns, pertaining to this period, have often been mainly descriptive, with a limited use of archaeometric investigations. This work presents a multi-method investigation of a Chalcolithic kiln recently discovered in the Bora Plain (part of the larger Peshdar Plain, in Iraqi Kurdistan), which combines stratigraphic analysis, pyrotechnological, micromorphological, and micro-remains analyses. Since this kiln represents the first Chalcolithic architectural feature excavated in the Bora Plain, this work offers precious insights into the pyrotechnology of the period, which is still relatively poorly understood, through the reconstruction of the kiln’s use and processes. The analytical outputs can be used to compare with other Near East kilns from the Chalcolithic and later periods

Gain of Function Mutations in CgPDR1 of Candida glabrata Not Only Mediate Antifungal Resistance but Also Enhance Virulence

CgPdr1p is a Candida glabrata Zn(2)-Cys(6) transcription factor involved in the regulation of the ABC-transporter genes CgCDR1, CgCDR2, and CgSNQ2, which are mediators of azole resistance. Single-point mutations in CgPDR1 are known to increase the expression of at least CgCDR1 and CgCDR2 and thus to contribute to azole resistance of clinical isolates. In this study, we investigated the incidence of CgPDR1 mutations in a large collection of clinical isolates and tested their relevance, not only to azole resistance in vitro and in vivo, but also to virulence. The comparison of CgPDR1 alleles from azole-susceptible and azole-resistant matched isolates enabled the identification of 57 amino acid substitutions, each positioned in distinct CgPDR1 alleles. These substitutions, which could be grouped into three different “hot spots,” were gain of function (GOF) mutations since they conferred hyperactivity to CgPdr1p revealed by constitutive high expression of ABC-transporter genes. Interestingly, the major transporters involved in azole resistance (CgCDR1, CgCDR2, and CgSNQ2) were not always coordinately expressed in presence of specific CgPDR1 GOF mutations, thus suggesting that these are rather trans-acting elements (GOF in CgPDR1) than cis-acting elements (promoters) that lead to azole resistance by upregulating specific combinations of ABC-transporter genes. Moreover, C. glabrata isolates complemented with CgPDR1 hyperactive alleles were not only more virulent in mice than those with wild type alleles, but they also gained fitness in the same animal model. The presence of CgPDR1 hyperactive alleles also contributed to fluconazole treatment failure in the mouse model. In conclusion, this study shows for the first time that CgPDR1 mutations are not only responsible for in vitro/in vivo azole resistance but that they can also confer a selective advantage under host conditions

"On the one hand" as a cue in the comprehension of discourse structure

Given evidence of anticipation within sentences (for upcoming sounds, words, and syntacti

Recovering discourse relations: Varying influence of discourse adverbials

Discourse relations are a bridge between sentence-level semantics and discourse-level semantics. They can be signalled explicitly with discourse connectives or conveyed implicitly, to be inferred by a comprehender. The same discourse units can be related in more than one way, sig-nalled by multiple connectives. But mul-tiple connectives aren’t necessary: Multi-ple relations can be conveyed even when only one connective is explicit. This paper describes the initial phase in a larger ex-perimental study aimed at answering two questions: (1) Given an explicit discourse adverbial, what discourse relation(s) do naive subjects take to be operative, and (2) Can this be predicted on the basis of the explicit adverbial alone, or does it depend instead on other factors?

An international guideline with six personalised titration schedules for preventing myocarditis and pneumonia associated with clozapine

White blood cell (WBC) monitoring has reduced clozapine-treated patient deaths associated with agranulocytosis to a rarity. However, clozapine protocols and package inserts worldwide provide no instructions for preventing myocarditis or pneumonia during clozapine titrations. Prescribers worldwide are largely unaware of that. Meanwhile, as they worry about agranulocytosis, their clozapine-treated patients are at risk of dying from pneumonia or myocarditis. Consequently, an international guideline with 104 authors from 50 countries/regions was recently published to provide personalised clozapine titration schedules for adult inpatients. This forum article reviews pneumonia and myocarditis occurring during clozapine titration, as well as the three most innovative aspects of this new guideline: (1) personalised titration, (2) C reactive protein (CRP) measures, and (3) dose predictions based on blood levels. Clozapine metabolism is influenced by 3 levels of complexity: (1) ancestry groups, (2) sex-smoking subgroups, and (3) presence/absence of poor metabolizer status. These 3 groups of variables should determine the maintenance dose and speed of clozapine titration; they are summarised in a table in the full-text. The international clozapine titration guideline recommends measuring CRP levels simultaneously with WBC, at baseline and weekly at least for the first 4 weeks of titration, the highest risk period for clozapine-induced myocarditis

Оценка экономической эффективности при подборе оптимальной программы уплотняющего бурения

В представленной работе приводится сравнение и обоснование экономической эффективности бурения горизонтальных стволов с многостадийным гидравлическим разрывом пласта в качестве уплотняющего бурения. Рассматриваются экономические показатели бурения горизонтальных и наклонно-направленных скважин