CRISPR/Cas9-Mediated Gene Editing of ARG1 in Cell Lines and Primary Cells

Arginase 1 deficiency, a urea cycle disorder resulting from an inability of the body to convert arginine into urea, results in hyperargininemia and sporadic episodes of hyperammonemia. Arginase 1 deficiency can lead to a range of developmental disorders and progressive spastic diplegia in children, and current therapeutic options are limited. Clustered regularly interspaced short palindromic repeat (CRISPR) /CRISPR associated protein (Cas) 9 gene editing systems serve as a novel means of treating genetic disorders such as Arginase 1 (ARG1) deficiency, and must be thoroughly examined to determine their curative capabilities. In these experiments numerous guide RNAs and CRISPR/Cas9 systems targeting the ARG1 gene were designed and observed by heteroduplex assay for their targeting capabilities and cleavage efficiencies in multiple cell lines. The CRISPR/Cas9 system utilized in these experiments, along with a panel of guide RNAs targeting various locations in the arginase 1 gene, successfully produced targeted cleavage in HEK293, MCF7, A549, K562, HeLa, and HepG2 cells; however, targeted cleavage in human dermal fibroblasts, blood outgrowth endothelial cells, and induced pluripotent stem cells was not observed. Additionally, a CRISPR/Cas system involving partially inactivated Cas9 was capable of producing targeted DNA cleavage in intron 1 of ARG1, while a Cas protein termed Cpf1 was incapable of producing targeted cleavage. These results indicate a complex set of variables determining the CRISPR/Cas9 systems’ capabilities in the cell lines and primary cells tested. By examining epigenetic factors and alternative CRISPR/Cas9 gene targeting systems, the CRISPR/Cas9 system can be more thoroughly considered in its ability to act as a means towards editing the genome of arginase 1-deficient individuals.

Design of binary polymeric systems containing ɩ-carrageenan and hydroxypropylcellulose for use in cataract surgery.

This study describes the design and characterisation of the rheological and mechanical properties of binary polymeric systems composed of 2-Hydroxypropylcellulose and ɩ-carrageenan, designed as ophthalmic viscoelastic devices (OVDs). Platforms were characterised using dilute solution, flow and oscillatory rheometry and texture profile analysis. Rheological synergy between the two polymers was observed both in the dilute and gel states. All platforms exhibited pseudoplastic flow. Increasing polymer concentrations significantly decreased the loss tangent and rate index yet increased the storage and loss moduli, consistency, gel hardness, compressibility and adhesiveness, the latter being related to the in-vivo retention properties of the platforms. Binary polymeric platforms exhibited unique physicochemical properties, properties that could not be engineered using mono-polymeric platforms. Using characterisation methods that provide information relevant to their clinical performance, low-cost binary platforms (3% hydroxypropylcellulose and either 1% or 2% ɩ-carrageenan) were identified that exhibited rheological, textural and viscoelastic properties advantageous for use as OVDs.

Stimulating Neoblast-Like Cell Proliferation in Juvenile Fasciola hepatica Supports Growth and Progression towards the Adult Phenotype In Vitro

Fascioliasis (or fasciolosis) is a socioeconomically important parasitic disease caused by liver flukes of the genus Fasciola. Flukicide resistance has exposed the need for new drugs and/or a vaccine for liver fluke control. A rapidly improving 'molecular toolbox' for liver fluke encompasses quality genomic/transcriptomic datasets and an RNA interference platform that facilitates functional genomics approaches to drug/vaccine target validation. The exploitation of these resources is undermined by the absence of effective culture/maintenance systems that would support in vitro studies on juvenile fluke development/biology. Here we report markedly improved in vitro maintenance methods for Fasciola hepatica that achieved 65% survival of juvenile fluke after 6 months in standard cell culture medium supplemented with 50% chicken serum. We discovered that this long-term maintenance was dependent upon fluke growth, which was supported by increased proliferation of cells resembling the "neoblast" stem cells described in other flatworms. Growth led to dramatic morphological changes in juveniles, including the development of the digestive tract, reproductive organs and the tegument, towards more adult-like forms. The inhibition of DNA synthesis prevented neoblast-like cell proliferation and inhibited growth/development. Supporting our assertion that we have triggered the development of juveniles towards adult-like fluke, mass spectrometric analyses showed that growing fluke have an excretory/secretory protein profile that is distinct from that of newly-excysted juveniles and more closely resembles that of ex vivo immature and adult fluke. Further, in vitro maintained fluke displayed a transition in their movement from the probing behaviour associated with migrating stage worms to a slower wave-like motility seen in adults. Our ability to stimulate neoblast-like cell proliferation and growth in F. hepatica underpins the first simple platform for their long-term in vitro study, complementing the recent expansion in liver fluke resources and facilitating in vitro target validation studies of the developmental biology of liver fluke.

Sustainability Enhancement of a Turbine Vane Manufacturing Cell through Digital Simulation based Design

Modern manufacturing systems should satisfy emerging needs related to sustainable development. The design of sustainable manufacturing systems can be valuably supported by simulation, traditionally employed mainly for time and cost reduction. In this paper, a multi-purpose digital simulation approach is proposed to deal with sustainable manufacturing systems design through Discrete Event Simulation (DES) and 3D digital human modelling. DES models integrated with data on power consumption of the manufacturing equipment are utilized to simulate different scenarios with the aim to improve productivity as well as energy efficiency, avoiding resource and energy waste. 3D simulation based on digital human modelling is employed to assess human factors issues related to ergonomics and safety of manufacturing systems. The approach is implemented for the sustainability enhancement of a real manufacturing cell of the aerospace industry, automated by robotic deburring. Alternative scenarios are proposed and simulated, obtaining a significant improvement in terms of energy efficiency (−87%) for the new deburring cell, and a reduction of energy consumption around −69% for the coordinate measuring machine, with high potential annual energy cost savings and increased energy efficiency. Moreover, the simulation-based ergonomic assessment of human operator postures allows 25% improvement of the workcell ergonomic index.

Stochastic Modeling of Reversible Biochemical Reaction-Diffusion Systems and High-Resolution Shock-Capturing Methods for Fluid Interfaces

Thesis (Ph.D.)--University of Washington, 2016-08

Synthetic Polymer and Microbubble Systems for Gene Delivery to the Brain

Thesis (Ph.D.)--University of Washington, 2016-08

Nonlinear Dynamics Near the Cell Membrane of Chara Corallina

The phenomenon of patterned distribution of pH near the cell membrane of the algae Chara corallina upon illumination is well-known. In this paper, we develop a mathematical model, based on the detailed kinetic analysis of proton fluxes across the cell membrane, to explain this phenomenon. The model yields two coupled nonlinear partial differential equations which describe the spatial dynamics of proton concentration changes and transmembrane potential generation. The experimental observation of pH pattern formation, its period and amplitude of oscillation, and also its hysteresis in response to changing illumination, are all reproduced by our model. A comparison of experimental results and predictions of our theory is made. Finally, a mechanism for pattern formation in Chara corallina is proposed.

Development of a whole cell biochip for toxicant detection.

In the development of biosensors for ecotoxicity testing it is desirable to produce a small, portable system that can be used in the field. Toxicity testing using bioluminescence is widely used in the laboratory utilising natural and genetically modified (lux/ luc-marked) bacteria and other microorganisms. It is currently not possible to use genetically manipulated microorganisms in field testing and a biosensor, therefore, that incorporates naturally luminescent organisms may be preferred. In the development of a biosensor it is aimed to use the naturally luminescent bacterium Vibrio fischeri as a toxicity detection system on a chip. The bacterium will be immobilised in a polymeric matrix. Current work deals with the optimisation of light output and light preservation within the bacterium prior to immobilisation in polyvinyl alcohol. An examination of a range of physicochemical conditions within the polymer will be made, including cell density, thickness of polymer film, growth and light induction environment, and, preservation conditions, in order to develop a testing system giving consistent results over the lifetime of the biosensor. Data will be presented on light production using different culture media for the growth of V. fischeri and retention of light under immobilised conditions. .

Sampling strategies for characterization of neuropeptides using mass spectrometry and functional implications into cell-cell signaling

In this dissertation, there are developed different analytical strategies to discover and characterize mammalian brain peptides using small amount of tissues. The magnocellular neurons of rat supraoptic nucleus in tissue and cell culture served as the main model to study neuropeptides, in addition to hippocampal neurons and mouse embryonic pituitaries. The neuropeptidomcis studies described here use different extraction methods on tissue or cell culture combined with mass spectrometry (MS) techniques, matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI). These strategies lead to the identification of multiple peptides from the rat/mouse brain in tissue and cell cultures, including novel compounds One of the goals in this dissertation was to optimize sample preparations on samples isolated from well-defined brain regions for mass spectrometric analysis. Here, the neuropeptidomics study of the SON resulted in the identification of 85 peptides, including 20 unique peptides from known prohormones. This study includes mass spectrometric analysis even from individually isolated magnocellular neuroendocrine cells, where vasopressin and several other peptides are detected. At the same time, it was shown that the same approach could be applied to analyze peptides isolated from a similar hypothalamic region, the suprachiasmatic nucleus (SCN). Although there were some overlaps regarding the detection of the peptides in the two brain nuclei, different peptides were detected specific to each nucleus. Among other peptides, provasopressin fragments were specifically detected in the SON while angiotensin I, somatostatin-14, neurokinin B, galanin, and vasoactive-intestinal peptide (VIP) were detected in the SCN only. Lists of peptides were generated from both brain regions for comparison of the peptidome of SON and SCN nuclei. Moving from analysis of magnocellular neurons in tissue to cell culture, the direct peptidomics of the magnocellular and hippocampal neurons led to the detection of 10 peaks that were assigned to previously characterized peptides and 17 peaks that remain unassigned. Peptides from the vasopressin prohormone and secretogranin-2 are attributed to magnocellular neurons, whereas neurokinin A, peptide J, and neurokinin B are attributed to cultured hippocampal neurons. This approach enabled the elucidation of cell-specific prohormone processing and the discovery of cell-cell signaling peptides. The peptides with roles in the development of the pituitary were analyzed using transgenic mice. Hes1 KO is a genetically modified mouse that lives only e18.5 (embryonic days). Anterior pituitaries of Hes1 null mice exhibit hypoplasia due to increased cell death and reduced proliferation and in the intermediate lobe, the cells differentiate abnormally into somatotropes instead of melanotropes. These previous findings demonstrate that Hes1 has multiple roles in pituitary development, cell differentiation, and cell fate. AVP was detected in all samples. Interestingly, somatostatin [92-100] and provasopressin [151-168] were detected in the mutant but not in the wild type or heterozygous pituitaries while somatostatin-14 was detected only in the heterozygous pituitary. In addition, the putative peptide corresponding to m/z 1330.2 and POMC [205-222] are detected in the mutant and heterozygous pituitaries, but not in the wild type. These results indicate that Hes1 influences the processing of different prohormones having possible roles during development and opens new directions for further developmental studies. This research demonstrates the robust capabilities of MS, which ensures the unbiased direct analysis of peptides extracted from complex biological systems and allows addressing important questions to understand cell-cell signaling in the brain.

'Cooperative Automated worm Response and Detection ImmuNe ALgorithm (CARDINAL) inspired by T-cell Immunity and Tolerance'

The role of T-cells within the immune system is to confirm and assess anomalous situations and then either respond to or tolerate the source of the effect. To illustrate how these mechanisms can be harnessed to solve real-world problems, we present the blueprint of a T-cell inspired algorithm for computer security worm detection. We show how the three central T-cell processes, namely T-cell maturation, differentiation and proliferation, naturally map into this domain and further illustrate how such an algorithm fits into a complete immune inspired computer security system and framework.

Oil Retention and Pressure Drop in Horizontal and Vertical Suction Lines with R410A / POE ISO 32

In refrigeration systems a small amount of compressor lubricant is entrained in the refrigerant and circulated through the system, where some is retained in each component. The suction line to the compressor has the largest potential for oil retention. This paper presents results from an experimental apparatus that has been constructed to circulate POE (polyolester) oil and R410A at a controlled mass flux, OCR (oil in circulation ratio), and apparent superheat, and to directly measure the pressure drop and mass of oil retained in horizontal and vertical suction lines. The bulk vapor velocity and overall void fraction are determined from direct mass and temperature measurements. The oil retention, pressure drop, and flow regimes near the minimum ASHRAE recommended mass flux condition are explored. It was found that oil retention begins to increase sharply even above the minimum recommended flux, so conditions near the minimum should be avoided. Two relationships were developed to predict the oil retention in the vertical and horizontal suction lines. The average error from the predictions method was 10.9% for the vertical tube, and 7.9% for the horizontal tube.

Technology strategies in the process of student retention and efficiency of academic management

This article discusses the application of Information and Communication Technologies and strategies for best practices in order to capture and maintain faculty students' attention. It is based on a case study of ten years, using a complete information system. This system, in addition to be considered an ERP, to support the activities of academic management, also has a strong component of SRM that provides support to academic and administrative activities. It describes the extent to which the presented system facilitates the interaction and communication between members of the academic community, using the Internet, with services available on the Web complementing them with email, SMS and CTI. Through a perception, backed by empirical analysis and results of investigations, it demonstrates how this type of practice may raise the level of satisfaction of the community. In particular, it is possible to combat failure at school, avoid that students leave their course before its completion and also that they recommend them to potential students. In addition, such a strategy also allows strong economies in the management of the institution, increasing its value. As future work, we present the new phase of the project towards implementation of Business Intelligence to optimize the management process, making it proactive. The technological vision that guides new developments to a construction based on Web services and procedural languages is also presented.

Produção de carboidratos a partir do efluente de laticínios tratado por cianobactéria

Nesta tese foi demonstrado o potencial de produção de carboidratos por Aphanothece microscopica Nägeli cultivada no efluente oriundo de uma indústria de laticínios. Para tanto, o trabalho é composto de quatro artigos que objetivaram avaliar a produção de carboidratos em função da temperatura, inóculo e razões C/N e N/P do elfluente, bem como a possibilidade de reúso da água residuária. Foram utilizadas temperaturas de (10, 20 e 30ºC) e inóculo (100, 200 e 300 mg.L-1). A melhor condição indicada foi quando utilizou-se a temperatura de 30°C e 200 mg.L-1 de inóculo. Na sequência, considerando a temperatura e a concentração celular selecionada, foi estudada a influência das razões C/N e N/P na produção de carboidratos. Para tal, C/N (20, 40 e 60) e N/P (5, 10 e 15) na produção de carboidratos extracelulares foram avaliadas em cultivos a 30°C, tendo como inóculo 200 mg.L-1. Os melhores resultados obtidos, foram quando foi utilizado C/N 60 e N/P 10. Uma vez definidas as melhores condições de produção de carboidratos, foi estudado o processo de separação de biomassa do meio de cultivo, a partir dos coagulantes FeCl3, Al2(SO4)3 e tanino. O efeito dos coagulantes na separação da biomassa foram estudados, quanto ao pH (6,0, 7,0 e 8,0) e concentração de coagulantes (50, 300 e 550 mg.L-1), utilizando como parâmetro de medida, a eficiência de remoção de DQO, turbidez e sólidos suspensos (SS). Os resultados demonstraram que as concentrações de coagulantes influenciaram significativamente ao nível de significância de 5 %, na separação da biomassa, com eficiência significativa na remoção da DQO, turbidez e SS. A melhor condição avaliada foi a que utilizou tanino na concentração de 300 mg.L-1 e pH 7,0, o que resultou em uma água residuária com remoção média de 96 % da turbidez, com potencial de ser reutilizada. Por fim, foi realizada a identificação de carboidratos gerados por Aphanothece microscopica Nägeli. Os resultados evidenciaram uma biomassa com até 33,5 % de carboidratos totais, perfazendo uma fração de carboidratos extracelulares, na fase estacionária de crescimento celular, de aproximadamente 25 % e 8 % os carboidratos da parede celular. Ficou demonstrado ainda que a composição dos carboidratos extracelulares do microorganismo em estudo é constituído por mono e dissacarídeos perfazendo concentrações na ordem de 12,88 % de glicose, 3,54 % de rafinose, 3,43 % sacarose, 2,13 % de frutose e 2,45 % de ribose. Ficou demonstrado o potencial de produção de carboidratos por Aphanothece microscopica Nägeli quando cultivada no efluente da indústria de laticínios.

Cell-cell communication in three dimensional microenvironments

Cellular behavior is dependent on a variety of extracellular cues required for normal tissue function, wound healing, and activation of the immune system. Removed from their in vivo microenvironment and cultured in vitro, cells lose many environmental cues and that may result in abberant behavior, making it difficult to study cellular processes. In order to mimic native tissue environments, optical tweezer and microfluidic technologies were used to place cells within defined areas of the culture environment. To provide three dimensional supports found in natural tissues, hydrogel scaffolds of poly (ethylene glycol) diacrylate and the basement membrane matrix Matrigel were used. Optical tweezer technology allowed precision placement and formation of homotypic and heterotypic arrays of human U937, HEK 293, and porcine mesenchymal stem cells. Alternatively, two microfluidic devices were designed to pattern Matrigel scaffolds. The first microfluidic device utilized laminar flow to spatially pattern multiple cell types within the device. Gradients of soluble molecules were then be formed and manipulated across the Matrigel scaffolds. Patterning Matrigel using laminar flow techniques require microfluidic expertise and do not produce consistent patterning conditions, limiting their use difficult in most cell culture laboratories. Thus, a buried Matrigel polydimethylsiloxane (PDMS) device was developed for spatial patterning of biological scaffolds. Matrigel is injected into micron sized channels of PDMS fabricated by soft lithography and allowed to thermally cure. Following curing, a second PDMS device was placed on top of the buried Matrigel channels to support media flow. In order to validate these systems, a cell-cell communication model system was developed utilizing LPS and TNFα signaling with fluorescent reporter systems to monitor communication in real time. We demonstrated the utility of microfluidic devices to support the cell-cell communication model system by co culturing three cell types within Matrigel scaffolds and monitoring signaling activity via fluorescent reporters.

The Application of a Dendric Cell Algorithm to a Robotic Classifier

The dendritic cell algorithm is an immune-inspired technique for processing time-dependant data. Here we propose it as a possible solution for a robotic classification problem. The dendritic cell algorithm is implemented on a real robot and an investigation is performed into the effects of varying the migration threshold median for the cell population. The algorithm performs well on a classification task with very little tuning. Ways of extending the implementation to allow it to be used as a classifier within the field of robotic security are suggested.