5,000 research outputs found
Disruption of the developmental programme of Trypanosoma brucei by genetic ablation of TbZFP1, a differentiation-enriched CCCH protein
No full textThe regulation of differentiation is particularly important in microbial eukaryotes that inhabit multiple environments. The parasite Trypanosoma brucei is an extreme example of this, requiring exquisite gene regulation during transmission from mammals to the tsetse fly vector. Unusually, trypanosomes rely almost exclusively on post-transcriptional mechanisms for regulated gene expression. Hence, RNA binding proteins are potentially of great significance in controlling stage-regulated processes. We have previously identified TbZFP1 as a trypanosome molecule transiently enriched during differentiation to tsetse midgut procyclic forms. This small protein (101 amino acids) contains the unusual CCCH zinc finger, an RNA binding motif. Here, we show that genetic ablation of TbZFP1 compromises repositioning of the mitochondrial genome, a specific event in the strictly regulated differentiation programme. Despite this, other events that occur both before and after this remain intact. Significantly, this phenotype correlates with the TbZFP1 expression profile during differentiation. This is the first genetic disruption of a developmental regulator in T. brucei. It demonstrates that programmed events in parasite development can be uncoupled at the molecular level. It also further supports the importance of CCCH proteins in key aspects of trypanosome cell function
The unbalanced Ururguay Round outcome : the new areas in future WTO negotiations
No full textThe Uruguay Round involved a grand North-South bargain: The North reduced import barriers, particularly in textiles and agriculture. The South adopted new domestic regulations in such areas as services and intellectual property-changes that would lead to increased purchases from the North. In mercantilist economics, apples for apples-imports for imports. In real economics, apples for oranges. The authors argue that while the North's reduction of import barriers benefits both the North and the South, the new domestic regulations adopted by countries of the South could prove costly to those countries. To begin with, the regulations will be expensive to implement. And while the cost side of their impact is secured by a legal obligation (in the case of intellectual property rights, for example, the cost is higher prices for patented goods), the benefits side is not so secured.Environmental Economics&Policies,Economic Theory&Research,Payment Systems&Infrastructure,Decentralization,Rules of Origin,Economic Theory&Research,Environmental Economics&Policies,TF054105-DONOR FUNDED OPERATION ADMINISTRATION FEE INCOME AND EXPENSE ACCOUNT,Trade and Regional Integration,World Trade Organization
Mobility of the SecA 2-helix-finger is not essential for polypeptide translocation via the SecYEG complex
No full textThe bacterial ATPase SecA and protein channel complex SecYEG form the core of an essential protein translocation machinery. The nature of the conformational changes induced by each stage of the hydrolytic cycle of ATP and how they are coupled to protein translocation are not well understood. The structure of the SecA-SecYEG complex revealed a 2-helix-finger (2HF) of SecA in an ideal position to contact the substrate protein and push it through the membrane. Surprisingly, immobilization of this finger at the edge of the protein channel had no effect on translocation, whereas its imposition inside the channel blocked transport. This analysis resolves the stoichiometry of the active complex, demonstrating that after the initiation process translocation requires only one copy each of SecA and SecYEG. The results also have important implications on the mechanism of energy transduction and the power stroke driving transport. Evidently, the 2HF is not a highly mobile transducing element of polypeptide translocation
The measurement of vascular and neurological function in workers exposed to hand-transmitted vibration
No full textFour methods for measuring disorders of vascular function and neurological function associated with occupational exposure to hand-transmitted vibration have been defined by reference to the available literature. For measuring vascular function the methods are: i) measures of the finger systolic blood pressure (FSBP) response to local cooling and ii) measures of the finger skin temperature (FST) response to local cooling. For measuring neurological function the methods are: i) measures of vibrotactile thresholds at the fingertips and ii) measures of thermal thresholds at the fingertips.Measures of the FSBP and the FST response to cold provocation were appraised in 109 dockyard workers. The FST test did not differentiate between 82 healthy subjects and 27 subjects with vibration-induced white finger (VWF) whilst the FSBP test was found to be sensitive, specific and responsive to VWF. Vibrotactile and thermal thresholds were found to be sensitive, specific and responsive to symptoms of numbness in another study of 104 dockyard workers, of whom 67 reported neurological disorders. It was concluded that whilst the above tests could be useful for monitoring the vascular and neurological disorders, a number of improvements to the measurement methods could be worthwhile. Further experiments were carried out to investigate these improvements.The simultaneous measurement of FSBPs on multiple test fingers was developed to improve the practicality of this test when measuring FSBPs on more than one test finger. Increased central sympathetic activity was hypothesised to result from increasing the stimulus by cooling more fingers. In two experiments on 12 healthy subjects, it was found that FSBPs measured simultaneously on four test fingers gave similar results to FSBP measurements on one test finger. Simultaneous FSBP measurements on four test fingers had comparable repeatability to measurements on one test finger. It was concluded that measuring FSBPs on multiple test fingers is a useful improvement to this test.When measuring FSBPs, changing the order of presentation and the period of recovery between thermal stimuli was hypothesised to influence the results by altering central sympathetic activity. In 12 healthy subjects it was found that the order of presentation of thermal stimuli was not important but that inter-subject variability increased when recovery was allowed between thermal stimuli. It was concluded that minimising the time interval between successive applications of thermal provocation reduces undesirable inter-subject variability. Another study on 12 healthy subjects showed that different reference measurement locations give different results. It was concluded that the thumb is suitable location for making reference measurements.The FSBP test and the FST test both involve application of cold provocation. The two tests are sometimes performed in succession but multiple thermal provocations may have cumulative effects on central sympathetic activity. When the two vascular tests were performed in succession on 36 subjects, including 12 subjects with VWF, any effects of the order of test presentation were small although a test performed first tended to be more repeatable. It was concluded that if both tests are performed consecutively, greater emphasis should be placed on the test performed first. The data for the FST test were reanalysed and showed that the sensitivity and specificity to VWF of this test is improved by changing the method of interpreting the results. Three methods of interpreting the FST response to cold provocation that represent an improvement to the test are suggested.The two vascular tests have been shown in the literature to be repeatable for healthy subjects but not for subjects with VWF. The repeatability of the vascular tests was assessed in 36 subjects (12 manual workers, 12 office workers and 12 subjects with VWF). The repeatability of both tests was found to be low amongst workers with VWF; some of these subjects showed a negative test result on one occasion and a positive test result on another occasion. It was concluded that a repeat test may be required when a false negative result is obtained.For the vibrotactile threshold test, the skin-stimulus contact force is usually controlled. Controlling the skin indentation would simplify measurement equipment. An experiment on ten healthy subjects investigated the relationship between skin-stimulus contact force, skin indentation and vibrotactile thresholds. It was concluded that the vibrotactile threshold test could be improved by implementing control of skin-indentation. Skin indentations giving comparable vibrotactile thresholds to those obtained using controlled contact forces were identified.It is concluded that a test battery comprising the four test methods identified from the literature and subsequently developed during the course of this research can be used to monitor disorders of both vascular and neurological function associated with occupational exposure to hand-transmitted vibration. A number of recommendations are made for further improvements that might be achievable as a result of further work
The development of an endoprosthesis for the metacarpophalangeal joint
No full textRheumatoid arthritis is a painful and debilitating disease which often afflicts the key joint of the hand, the metacarpophalangeal joint. In the worst cases the diseased joint has to be replaced with an artificial joint or prosthesis. The development of the Durham metacarpophalangeal prosthesis as it was taken from prototypes through to production samples, is described in this thesis. Testing of several Durham prostheses to over 70 million cycles has been carried out on a finger function simulator and consistent wear factors of the order of 0.4 x 10(^-) (^6)mm(^3)/Nm have been measured. These wear factors for the prosthesis were also significantly lower than any found previously. Production samples of the prosthesis have been manufactured together with appropriate surgical instrumentation. Tests of the prosthesis material, cross-linked polyethylene, rubbing against itself, have been undertaken on reciprocating pin on plate rigs and again show total wear factors of the order of 0.4 x lO(^-6)mm(^3)/Nm. Interestingly, it was found that pin wear was very much less than plate wear. The pin on plate tests were extended to include ultra-high molecular weight polyethylene (UHMWPE) rubbing against UHMWPE, as well as both polyethylenes against hard counterfaces and the results are reported. A new finger function simulator has been designed, manufactured and a validation test undertaken. Having written the necessary protocol, in conjunction with clinicians and the prosthesis manufacturer, ethical approval was obtained from the local research ethics committee and the Medical Devices Agency, to permit implantation of the prosthesis in human subjects. Lastly a hand strength measurement device for pre and post operation assessment of patients has been developed and manufactured
Method of Detecting and Targeting Mutations in Cancer
No full textWhile there are many differences between tumor and non-tumor cells, the basic underlying distinction is in the DNA. Tumor cells harbor mutations, at least some of which are not present in non-tumor cells. Thus, a method of directly targeting cells containing specific mutations has potential for detection or treatment of cancer without the toxicity associated with more indirect approaches. Also, as mutations are a necessary component of malignancy, such a method is potentially applicable to all tumors. 

I propose a method by which several recently developed techniques can be utilized in a novel way to accomplish the goal of directly targeting mutations for cancer detection and therapy. The model can be summarized as follows: (1) Determine potential target mutations present in tumor cells but not non-tumor cells. (2) Construct molecules that will bind to DNA at the sites of mutation, but will not bind to DNA in normal cells. And, as a consequence of the molecules binding to the mutation, the cells will be destroyed. (3) Deliver these molecules to all cells (or at least all tumor cells). I hypothesize that such molecules can now be constructed using sequence-specific DNA binding proteins (such as customized zinc-finger DNA binding proteins) fused to transcriptional activator domains (such as VP16) and reporter or toxin genes. The necessary genes can be linked to the DNA binding proteins utilizing a recently described method based on expressed protein ligation. 

Mutation-independent treatment of autosomal dominant Retinitis Pigmentosa (adRP)
No full textViral-mediated gene therapy holds great promise for the treatment of severe inherited retinal diseases, such as Retintitis Pigmentosa (RP), which is caused by mutations in genes preferentially expressed in photoreceptor cells. The availability of vectors derived from the small adeno-associated virus (AAV) which efficiently and stably transduce the retina of animal models after intraocular administration strongly support the possibility to develop novel strategies for the treatment of such severe retinal degenerations otherwise incurable thus far.
The main goals of my PhD project were:
- generate artificial transcription repressors (ZFPs) targeted to the human rhodopsin promoter to silence at the transcriptional level the rhodopsin gene;
- assess the efficacy of the treatment and the impact on the disease progression in the RP mouse model.
Retinitis pigmentosa is by far the most studied inherited retinal disease. It is clinically and genetically heterogeneous recognizing autosomal recessive (arRP), autosomal dominant (adRP), X-linked, and digenic patterns of inheritance. More than 30 diseases genes have been identified so far and 12 of these have been associated with (adRP), representing between 15% and 35% of all cases. Despite recent success of the gene-based complementation approach for genetic recessive traits, the development of therapeutic strategies for gain-of-function mutations poses great challenges. General therapeutic principles to correct these genetic defects mostly rely on post-transcriptional gene regulation (RNA silencing). Engineered zinc finger protein (ZFP)-based-repression of transcription may represent a novel and alternative mutation independent therapeutic approach for treating gain-of-function mutations, but proof-of-concept of this use is still lacking. In my PhD project we used a novel strategy to treat adRP based on zinc-finger-based artificial transcription factors (ZF-ATFs). These molecules can be engineered to silence genes carrying gain-of-function mutations that cause toxic effects into the cell where they are expressed. We generated ten artificial transcriptional repressors targeted to the human Rhodopsin which is the gene most commonly associated with adRP (20–30% of cases) with more than 150 mutations identified throughout its sequence, representing the most commonly mutated gene in RP.
We characterized in vitro the ability of artificial transcriptional repressors to bind specifically the human rhodopsin promoter in order to exert a specific transcriptional control and we selected two out of ten functional zinc-finger-based repressors of rhodopsin. One of this was selected as the most efficient and was enclosed in an AAV2/8 for in vivo experiments. We demonstrated that the selected artificial zinc-finger-based repressors (ZFRs) resulted in a robust transcriptional repression of hRHO impacting disease progression in a mouse model of adRP over-expressing the P347S mutation.
The data obtained support the use of ZFP-mediated silencing as a potentially relevant therapeutic strategy to treat gain of function mutations
EXPERIMENTAL EVALUATION OF DEWAR VOLUME AND COLD FINGER SIZE IN A STIRLING CRYOCOOLER LIQUID AIR ENERGY STORAGE (LAES) SYSTEM
No full textThis paper uses an experimental approach to evaluate two design characteristics for a liquid air energy storage (LAES) and generation system as part of the verification and validation of system component design for a microgrid power system. The LAES subsystem evaluated utilized a Stirling engine–based cryocooler that employs a cold finger placed into Dewar, which allows the pumping of heat out of a Dewar. As the heat is pumped out, the air temperature in the Dewar cools to below the condensation point and the air in the Dewar liquifies and is stored in the Dewar. Using a design of experiments, the cold finger surface area and Dewar volume were evaluated to determine the criticality and significance of changing their dimensions on the total liquid air production mass and average liquid air production rate during the experiments. This analysis found that changing the surface area of the cryocooler cold finger was a statistically significant design characteristic that affected total liquid air production and average production rate while changing the volume of the Dewar was not statistically significant. Additional responses relative to the time when the first gram of liquid air was produced and the minimum cold tip temperature that the cryocooler was able to achieve provided additional insight into design characteristics that can be used to inform the engineer when making design tradeoffs for specific microgrid operational environments.Approved for public release. Distribution is unlimited.Lieutenant Colonel, United States Arm
Structural studies on protein scaffolds related to muscle physiology and disease : the titin filament, its associated component MuRF-1 and nuclear LAP2[alpha]
No full textThe titin molecule has a length of overµm and functions as a colossal protein scaffold in the muscle sarcomere. Up to 90% of its total mass is composed of repetitive immunoglobulin (Ig) and fibronectin (FnIII) domains that form linear tandems interspersed by unique sequences, among them a Ser/Thr kinase domain located at its C-terminus. The distinct pattern of Ig and FnIII motifs N-terminal to the kinase domain is conserved in other „giant kinases“ and invertebrate titin homologues. In vertebrate titin, it is involved in the specific recruitment of the ubiquitin ligase MuRF-1 to the filament. MuRF-1 is involved in the pathological atrophy of skeletal and cardiac muscle. We have determined the crystal structure of titin A168-A170 comprising two Ig and one FnIII domains and established its binding to MuRF-1 in solution. We analysed the structure with the aim to understand the interdomain relationships between repetitive Ig and FnIII subunits in titin as well as to shed light into the molecular determinants that confer specificity to ligand binding on the scaffold and in particular in the M-line interface to MuRF-1. A168-A170 shows an extended, rigid architecture. Its surface displays a shallow groove along its full length as well as a unique loop protrusion, both features conceivably mediating MuRF-1 binding. Moreover, our ITC data show that binding occurs with high affinity between residues 166-315 of MuRF-1. These data suggest that A168-A170 is of interest to attempt therapeutic inhibition of MuRF-1-mediated muscle turnover. In addition we have elucidated the structure of the B-box domain of MuRF-1 to further investigate the role of MuRF-1 in homo- and hetero-oligomeric interactions at the M-line region. We found that MuRF-1 B-box adopts a RING-finger-like fold and exists in a dimeric state in solution. The domain possesses characteristic surface properties that are likely to mediate interactions of MuRF-1 with other sarcomeric components that are important in MuRF-1 function at the M-line. Finally, we have also carried out the biophysical characterization of the nuclear adaptor protein LAP2! that interacts with the nuclear lamina scaffold. Conceptually, LAP2! and the nuclear lamina are closely related systems to MuRF-1 and titin. This characterization, whose ultimate finality is to understand the interaction of LAP2! with lamin A/C establishes now the basis for a future structure elucidation.
This work illustrates how scaffold protein systems, which are structural skeletons
composed of multiple repetitive units, can become functionalized by the recruitment of
specific shuttle proteins to their surface. Specific binding in such systems involves
steric factors as well as the evolution of unique sequence inserts at defined locations.
Recruited proteins often act as adaptors that, in turn, attract other cellular components.
They often result in large, heterogeneous molecular assemblies that amplify the
physiological response. In the case of titin, the potential formation of a signalosome
assembly at its M-line, surrounding a kinase domain, is thought to mediate mechanotransduction
pathways involved in the regulation of myofibril turn-over and, thereby, in
the adaptative remodelling of muscle to mechanical load
A variant in LIN28B is associated with 2D:4D finger-length ratio, a putative retrospective biomarker of prenatal testosterone exposure
No full textThe ratio of the lengths of an individual's second to fourth digit (2D:4D) is commonly used as a noninvasive retrospective biomarker for prenatal androgen exposure. In order to identify the genetic determinants of 2D:4D, we applied a genome-wide association approach to 1507 11-year-old children from the Avon Longitudinal Study of Parents and Children (ALSPAC) in whom 2D:4D ratio had been measured, as well as a sample of 1382 12- to 16-year-olds from the Brisbane Adolescent Twin Study. A meta-analysis of the two scans identified a single variant in the LIN28B gene that was strongly associated with 2D:4D (rs314277: p = 4.1 108) and was subsequently independently replicated in an additional 3659 children from the ALSPAC cohort (p = 1.53 106). The minor allele of the rs314277 variant has previously been linked to increased height and delayed age at menarche, but in our study it was associated with increased 2D:4D in the direction opposite to that of previous reports on the correlation between 2D:4D and age at menarche. Our findings call into question the validity of 2D:4D as a simplistic retrospective biomarker for prenatal testosterone exposure
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