Molecular genetic tools for manipulation of the oleaginous yeast Rhodotorula toruloides

Rhodotorula (Rhodosporidium) toruloides is an oleaginous basidiomycete yeast with great biotechnological potential. Capable of accumulating lipid up to 76 % of its dry biomass and well suited to the metabolism of lignocellulosic hydrolysate, it is a good candidate for production of advanced biofuels as well as a host of other potential roles in industry. However, molecular genetic tools for manipulation of this yeast are lacking and its high genomic GC content can make routine cloning difficult. Agrobacterium tumefaciens-mediated transformation of R. toruloides CBS 14 was demonstrated, and plasmid vectors were developed for transformation of R. toruloides, including elements for Saccharomyces cerevisiae in-yeast assembly. In-yeast assembly is robust to the manipulation of GC-rich DNA and of large plasmids. Using these vectors and an EGFP reporter, a screen to identify inducible promoters was performed, and promoters from the genes NAR1, ICL1, CTR3, and MET16 identified. These promoters have independent induction/repression conditions and different levels and rates of induction. Minimal inducible promoters were determined, which are as small as 200 bp. As well as showing tight regulation of the EGFP marker, the NAR1 promoter was able to drive conditional rescue of a leu2 mutant strain. In parallel, as a proof of principle for production of advanced biofuels, hydrocarbon biosynthesis pathways were expressed in R. toruloides and analysed by GC-MS. After co-expression of Synechococcus elongatus fatty acyl-ACP reductase and fatty aldehyde decarbonylase, and E. coli ferredoxin and ferredoxin reductase, production of the alkane heptadecane was observed. To increase the availability of free fatty acids (FFA) for production of hydrocarbons by other pathways, Thermomyces lanuginosus lipase 2 was expressed, resulting in a 1.3-fold increase in the concentration of FFAs.BBSR

Sop Acute Kidney Injury (SAKI): Predictive models in the management of acute kidney injury

The overarching aim of this PhD thesis is to develop methods, which will ultimately improve the management of patients with acute kidney injury (AKI). Over the years one inherent problem in both diagnosing AKI clinically and reviewing and comparing studies published in the literature has been the numerous definitions used to define AKI. 87 With now accepted definitions of AKI, the first question raised was to determine the true impact of AKI, in terms of incidence and outcomes, for both the patient (morbidity and mortality) and the healthcare economy. A retrospective observational database study was performed from secondary care in East Kent (adult catchment population of 582,300). All adult patients (18 years or over) admitted between 1st February 2009 and 31st July 2009, were included. Patients receiving chronic renal replacement therapy (RRT), maternity and day case admissions were excluded. AKI was defined by the acute kidney injury network (AKIN) criteria. A time dependent risk analysis with logistic regression and Cox regression was used for the analysis of in-hospital mortality and survival. The incidence of AKI in the 6 month period was 15,325 pmp/yr (adults) (69% AKIN1, 18% AKIN2 and 13% AKIN3). In-hospital mortality, length of stay and ITU utilisation all increased with severity of AKI. Patients with AKI had an increase in care on discharge and an increase in hospital readmission within 30 days.  In comparison with patients with no AKI those with AKI stage 1 had a 52% longer length of stay (LOS) in hospital, a 2.8-fold increased risk of admission to the intensive therapy unit (ITU), a 39% longer ITU stay (in those who went to ITU), and a 2.4-fold greater in-hospital mortality. Furthermore, patients with AKI stage 1 had twice the long-term risk of death, a 33% higher likelihood of an increase in care, and a 42% higher risk of re-admission within 30 days. In those patients with AKI stage 3 (the subject of the NCEPOD report) 100 hospital LOS doubled, there was a 22 times higher risk of admission to ITU and ITU LOS was also doubled, consistent with national data from the Intensive Care National Audit and Research Centre. A further study using this data in collaboration with Marion Kerr (health economist) at the Department of Health, suggested the annual number of excess inpatient deaths, with AKI in England may be greater than 40,000, 106 and the annual cost of AKI-related inpatient care in England is estimated at £1.02 billion. 106 With the problem now evident and clearly defined, the first stage in improving management was to alert clinicians to the presence of AKI as soon as possible to allow early recognition and intervention. Here the development of a static AKI alert report delivered to the critical care outreach team and specialist renal team is documented. A qualitative analysis was then used to explore the effect of professional interactions, information sharing, and personal and professional characteristics on the use of electronic clinical information and clinical decision support. Key areas highlighted in the qualitative analysis included real-time delivery of AKI alerts, clear responsibility of care to be with the clinical teams with advice from the critical care outreach nurses and renal consultants as required, and improved communication with the clinical teams looking after the patients. This work informed a development partnership with a commercial company (Careflow Connect Limited) to deliver real-time alerting of acute kidney injury to clinicians at the point of care and allow collaboration within the clinical team and also with the specialist renal and critical care outreach teams. However, in any disease process, while we can optimise our measures in place (as above) to alert to the presence of a disease (in this case acute kidney injury (AKI)) and manage it effectively and efficiently at recognition, the ultimate form of treatment is the prevention of the disease occurring in the first place. Hence, in order to achieve this we need to determine the patient at risk. Firstly, potential risk factors were explored. Three time points were also defined where significant clinical decision making takes place and at which points the use of risk models would have greatest impact on clinical care and patient management. These were the point of admission to hospital to guide renal function testing and inform admission planning, and secondly, at 24 hours after admission, often on the post-take ward round to highlight patients who are likely to develop new or worsening AKI if already present, in the first 72 hours of hospital admission so that appropriate management decisions can be made on the ward round. The study population included hospital admissions to the three acute hospitals of East Kent Hospitals University NHS Foundation Trust (EKHUFT) in 2011, excluding maternity and elective admissions. For validation in a second population the study included hospital admissions to Medway NHS Foundation Trust. The study developed and assessed traditional methods to provide risk models for the prediction of new or worsening AKI in patients presenting to hospital and in their management within the first 24 hours of admission. Ordinal logistic regression with uni-variable analyses were used to inform the development of multi-variable analyses. Backward selection was used to retain only statistically significant variables in the final models. The models were validated using actual and predicted probabilities, Area Under the Receiver Operating Characteristic (AUROC) curve analysis and the Hosmer Lemeshow test. The analysis identified key variables which predict AKI both at admission and 72 hours post admission. Validation demonstrated area under ROC of 0.75 and 0.68 respectively. Predicting worsening AKI during admission was unsuccessful. These models were also re-defined with use of the NHS England algorithm to define AKI which produced similar results with area under ROC of 0.73 and 0.67 respectively. The work reported here has demonstrated the significant morbidity and mortality both long and short term of patients who experience acute kidney injury managed in hospital and has developed methods of alerting the presence of AKI to the point of care in real-time to ensure efficient intervention with an aim to improve these outcomes. Qualitative work has also highlighted the complexity regarding the implementation and delivery of alerting systems to the clinical front line. The work reported in this thesis has also demonstrated that routinely available data can be used to highlight patients at risk of acute kidney injury both at the point of admission to hospital and following admission

Tiled microprocessors

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2007.Includes bibliographical references (p. 251-258).Current-day microprocessors have reached the point of diminishing returns due to inherent scalability limitations. This thesis examines the tiled microprocessor, a class of microprocessor which is physically scalable but inherits many of the desirable properties of conventional microprocessors. Tiled microprocessors are composed of an array of replicated tiles connected by a special class of network, the Scalar Operand Network (SON), which is optimized for low-latency, low-occupancy communication between remote ALUs on different tiles. Tiled microprocessors can be constructed to scale to 100's or 1000's of functional units. This thesis identifies seven key criteria for achieving physical scalability in tiled microprocessors. It employs an archetypal tiled microprocessor to examine the challenges in achieving these criteria and to explore the properties of Scalar Operand Networks. The thesis develops the field of SONs in three major ways: it introduces the 5-tuple performance metric, it describes a complete, high-frequency SON implementation, and it proposes a taxonomy, called AsTrO, for categorizing them.(cont.) To develop these ideas, the thesis details the design, implementation and analysis of a tiled microprocessor prototype, the Raw Microprocessor, which was implemented at MIT in 180 nm technology. Overall, compared to Raw, recent commercial processors with half the transistors required 30x as many lines of code, occupied 100x as many designers, contained 50x as many pre-tapeout bugs, and resulted in 33x as many post-tapeout bugs. At the same time, the Raw microprocessor proves to be more versatile in exploiting ILP, stream, and server-farm workloads with modest to large amounts of parallelism.by Michael Bedford Taylor.Ph.D

Design decision in the implementation of a raw architecture workstation

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 1999.Includes bibliographical references (p. 53).by Michael Bedford Taylor.S.M

Symmetries and reversing symmetries of area-preserving polynomial mappings in generalised standard form

We determine the symmetries and reversing symmetries within G, the group of real planar polynomial automorphisms, of area-preserving nonlinear polynomial maps L in generalised standard form, L: x'=x+p(y), y'=y+q(x'), where p and q are polynomials. We do this by using the amalgamated free product structure of G. Our results lead to normal forms for polynomial maps in generalised standard form and to a classification of the group structures of the reversing symmetry groups for such maps.Comment: 22 page

Detection of chromosomal inversions using non-repetitive nucleic acid probes

A method for the identification of chromosomal inversions is described. Single-stranded sister chromatids are generated, for example by CO-FISH. A plurality of non-repetitive, labeled probes of relatively small size are hybridized to portions of only one of a pair of single-stranded sister chromatids. If no inversion exists, all of the probes will hybridize to a first chromatid. If an inversion has occurred, these marker probes will be detected on the sister chromatid at the same location as the inversion on the first chromatid

Do acute elevations of serum creatinine in primary care engender an increased mortality risk?

Background: The significant impact Acute Kidney Injury (AKI) has on patient morbidity and mortality emphasizes the need for early recognition and effective treatment. AKI presenting to or occurring during hospitalisation has been widely studied but little is known about the incidence and outcomes of patients experiencing acute elevations in serum creatinine in the primary care setting where people are not subsequently admitted to hospital. The aim of this study was to define this incidence and explore its impact on mortality. Methods: The study cohort was identified by using hospital data bases over a six month period. Inclusion criteria: People with a serum creatinine request during the study period, 18 or over and not on renal replacement therapy. The patients were stratified by a rise in serum creatinine corresponding to the Acute Kidney Injury Network (AKIN) criteria for comparison purposes. Descriptive and survival data were then analysed. Ethical approval was granted from National Research Ethics Service (NRES) Committee South East Coast and from the National Information Governance Board. Results: The total study population was 61,432. 57,300 subjects with ‘no AKI’, mean age 64.The number (mean age) of acute serum creatinine rises overall were, ‘AKI 1’ 3,798 (72), ‘AKI 2’ 232 (73), and ‘AKI 3’ 102 (68) which equates to an overall incidence of 14,192 pmp/year (adult). Unadjusted 30 day survival was 99.9% in subjects with ‘no AKI’, compared to 98.6%, 90.1% and 82.3% in those with ‘AKI 1’, ‘AKI 2’ and ‘AKI 3’ respectively. After multivariable analysis adjusting for age, gender, baseline kidney function and co-morbidity the odds ratio of 30 day mortality was 5.3 (95% CI 3.6, 7.7), 36.8 (95% CI 21.6, 62.7) and 123 (95% CI 64.8, 235) respectively, compared to those without acute serum creatinine rises as defined. Conclusions: People who develop acute elevations of serum creatinine in primary care without being admitted to hospital have significantly worse outcomes than those with stable kidney function

Spatial Guilds in the Serengeti Food Web Revealed by a Bayesian Group Model

Food webs, networks of feeding relationships in an ecosystem, provide fundamental insights into mechanisms that determine ecosystem stability and persistence. A standard approach in food-web analysis, and network analysis in general, has been to identify compartments, or modules, defined by many links within compartments and few links between them. This approach can identify large habitat boundaries in the network but may fail to identify other important structures. Empirical analyses of food webs have been further limited by low-resolution data for primary producers. In this paper, we present a Bayesian computational method for identifying group structure using a flexible definition that can describe both functional trophic roles and standard compartments. We apply this method to a newly compiled plant-mammal food web from the Serengeti ecosystem that includes high taxonomic resolution at the plant level, allowing a simultaneous examination of the signature of both habitat and trophic roles in network structure. We find that groups at the plant level reflect habitat structure, coupled at higher trophic levels by groups of herbivores, which are in turn coupled by carnivore groups. Thus the group structure of the Serengeti web represents a mixture of trophic guild structure and spatial pattern, in contrast to the standard compartments typically identified. The network topology supports recent ideas on spatial coupling and energy channels in ecosystems that have been proposed as important for persistence. Furthermore, our Bayesian approach provides a powerful, flexible framework for the study of network structure, and we believe it will prove instrumental in a variety of biological contexts.</p