The actinin family of actin crosslinking proteins: natural functions and potential applications in synthetic biology

Actinin and spectrin proteins are members of the Spectrin Family of Actin Crosslinking Proteins. The importance of these proteins in the cytoskeleton is demonstrated by the fact that they are common targets for disease causing mutations. In their most prominent roles, actinin and spectrin are responsible for stabilising and maintaining the muscle architecture during contraction, and providing shape and elasticity to the red blood cell in circulation, respectively. To carry out such roles, actinin and spectrin must possess important mechanical and physical properties. These attributes are desirable when choosing a building block for protein-based nanoconstruction. In this study, I assess the contribution of several disease-associated mutations in the actinin-1 actin binding domain that have recently been linked to a rare platelet disorder, congenital macrothrombocytopenia. I investigate the suitability of both actinin and spectrin proteins as potential building blocks for nanoscale structures, and I evaluate a fusion-based assembly strategy to bring about self-assembly of protein nanostructures. I report that the actinin-1 mutant proteins display increased actin binding compared to WT actinin-1 proteins. I find that both actinin and spectrin proteins exhibit enormous potential as nano-building blocks in terms of their stability and ability to self-assemble, and I successfully design and create homodimeric and heterodimeric bivalent building blocks using the fusion-based assembly strategy. Overall, this study has gathered helpful information that will contribute to furthering the advancement of actinin and spectrin knowledge in terms of their natural functions, and potential unnatural functions in protein nanotechnology.

The Power of the Synthetic Control Method

The synthetic control method (SCM) is a new, popular method developed for the purpose of estimating the effect of an intervention when only one single unit has been exposed. Other similar, unexposed units are combined into a synthetic control unit intended to mimic the evolution in the exposed unit, had it not been subject to exposure. As the inference relies on only a single observational unit, the statistical inferential issue is a challenge. In this paper, we examine the statistical properties of the estimator, study a number of features potentially yielding uncertainty in the estimator, discuss the rationale for statistical inference in relation to SCM, and provide a Web-app for researchers to aid in their decision of whether SCM is powerful for a specific case study. We conclude that SCM is powerful with a limited number of controls in the donor pool and a fairly short pre-intervention time period. This holds as long as the parameter of interest is a parametric specification of the intervention effect, and the duration of post-intervention period is reasonably long, and the fit of the synthetic control unit to the exposed unit in the pre-intervention period is good.

A Kaleidoscope Perspective: Change in the Semantics and Structure of Facets and Isolates in Analytico-Synthetic Classification

Examines the limitations of the dynamic theory of classification in accommodating the changes and rapid growth of new topics in the universe of knowledge. Change in an analytico-synthetic scheme for classification is much more a web of connections and mapping these changes is a complex process. Suggests that there is need for exploration of this complexity for both improving systems, and revisiting our theory.

Synthetic studies towards the total synthesis of hexacyclinic acid

In the first chapter of this thesis, published works found in the literature about hexacyclinic acid and FR182877 are reported and commented. A quick summary of the previous work done in the Prunet group is also described. In the second and third chapter, a more detailed account of the work undertaken during this PhD was given. Firstly, syntheses of two ABC tricycles incorporating tert-butyl and (trimethylsilyl)ethyl esters were undertaken. These syntheses include two key steps previously developed in the group, a diastereoselective Michael addition and a Snider cyclisation. Multiple conditions for the hydrolysis of the esters were attempted but none of them gave the desired product. The main part of this work is focused on the synthesis of a CDEF model and in particular about the development of the key step, the formation of a nine-membered ring. Several DEF fragments were synthesised in short synthetic sequences and as single isomers. Six different synthetic pathways were developed in total and a novel method, a Michael/elimination reaction, was found to be a very efficient way to close the desired medium-size ring. From the nine-membered ring, regioselective reduction and palladiumcatalysed allylic substitution led to the formation of the CDF tricycle. Final steps of the synthesis were fruitless and led only to decomposition. A synthesis of a chiral C-ring was also developed during this PhD. II Finally, another project was undertaken, not related to hexacyclinic acid. Methodology developed in the group for the diastereoselective formation of trisubstituted alkenes employing a temporary silicon-tethered ring-closing metathesis was extended to homoallylic alcohols. The first steps of the method were similar to the previous methodology but the end-game had to be modified in favour of an oxidation/reduction sequence to successfully obtain the desired products with the correct geometry. In the fourth chapter, procedures and analytical data for the synthesised compounds previously described are reported.

Synthetic portfolio for event studies: Estimating the effects of volatility call auctions

We propose a method denoted as synthetic portfolio for event studies in market microstructure that is particularly interesting to use with high frequency data and thinly traded markets. The method is based on Synthetic Control Method and provides a robust data driven method to build a counterfactual for evaluating the effects of the volatility call auctions. We find that SMC could be used if the loss function is defined as the difference between the returns of the asset and the returns of a synthetic portfolio. We apply SCM to test the performance of the volatility call auction as a circuit breaker in the context of an event study. We find that for Colombian Stock Market securities, the asynchronicity of intraday data reduces the analysis to a selected group of stocks, however it is possible to build a tracking portfolio. The realized volatility increases after the auction, indicating that the mechanism is not enhancing the price discovery process.

Synthetic polymers blend used in the production of high activated carbon for pesticides removals from liquid phase

For the activated carbon (AC) production, we used the most common industrial and consumer solid waste, namely polyethyleneterephthalate (PET), alone or blended with other synthetic polymer such polyacrylonitrile (PAN). By mixing PET, with PAN, an improvement in the yield of the AC production was found and the basic character and some textural and chemical properties were enhanced. The PET–PAN mixture was subjected to carbonisation, with a pyrolysis yield of 31.9%, between that obtained with PET (16.9%) or PAN (42.6%) separately. The AC revealed a high surface area (1400, 1230 and 1117 m2 g−1) and pore volume (0.46, 0.56 and 0.50 cm3 g−1), respectively, for PET, PAN and PET–PAN precursors. Selected ACs were successfully tested for 4- chloro-2-methylphenoxyacetic acid (MCPA) and diuron removal from the liquid phase, showing a higher adsorption capacity (1.7 and 1.2 mmol g−1, respectively, for MCPA and diuron) and good fits with the Langmuir (PET) and Freundlich equation (PAN and PET–PAN blend). With MCPA, the controlling factor to the adsorption capacity was the porous volume and the average pore size. Concerning diuron, the adsorption was controlled essentially by the external diffusion. A remarkable result is the use of different synthetic polymers wastes, as precursors for the production of carbon materials, with high potential application on the pesticides removals from the liquid phase.

Activated Carbons Produced from Mixtures of Synthetic Polymers by Physical and Chemical Activation: Application on MCPA Removal

The production of AC was achieved using the most common industrial and consumer solid waste, namely PET, alone or blended with other synthetic polymer such PAN. The PET-PAN mixture (1:1 W/W %) was subjected to carbonization, with a pyrolysis yield off 31.9%, between that obtained with PET (16.9%) or PAN (42.6%) separately. By mixing PET, as a raw material, with PAN (different ratio), an improvement in the final yield of the AC production, for the same activation time, with CO2, was found.

Activated Carbons Produced from Mixtures of Synthetic Polymers by Physical and Chemical Activation: Application on MCPA Removal

The production of AC was achieved using the most common industrial and consumer solid waste, namely PET, alone or blended with other synthetic polymer such PAN. The PET-PAN mixture (1:1 W/W %) was subjected to carbonization, with a pyrolysis yield off 31.9%, between that obtained with PET (16.9%) or PAN (42.6%) separately. By mixing PET, as a raw material, with PAN (different ratio), an improvement in the final yield of the AC production, for the same activation time, with CO2, was found.

Production of activated carbons from single and mixtures of synthetic polymers

The production of activated carbons (ACs) involves two main steps: the carbonization of the carbonaceous of raw materials at temperatures below 1073 K in the absence of oxygen and the activation had realized at the temperature up to 1173 but the most useful temperature at 1073 K. In our study we used the most common industrial and consumer solid waste, namely PET, alone or blended with other synthetic polymer PAN. By mixing the two polymers in different ratios, an improvement of the yield of the AC production was found and some textural properties were enhanced by comparison with the AC prepared using each polymer separately. When all the samples were exposed through the carbonization process with a pyrolysis the mixture of PAN-PET (1:1w/w) yield around 31.9%, between that obtained with PET (16.9%) or PAN (42.6%) separately. The combine activation, with CO2 at 1073 K, allow ACs with a lower burn-off degree isothermally, when compared with those attained with PET or PAN alone, but with similarly chemicals or textural properties. The resultant ACs are microporous in their nature, as the activation time increase, the PET-PAN mixture AC are characterized by a better developed porous structure, when associated with the AC prepared from PAN. The AC prepared from PET-PAN mixture are characterized by basic surface characteristics, with a pHpzc around 10.5, which is an important characteristic for future applications on acidic pollutants removals from liquid or gaseous phase. In this study we had used the FTIR methods to determine the main functional groups in the surface of the activated carbons. The adsorbents prepared from PAN fibres presents an IR spectrum with similar characteristics to those obtained with PET wastes, but with fewer peaks and bands with less intensity, in particular for the PAN-8240 sample. This can be reflected by the stretching and deformation modes of NH bond in the range 3100 – 3300 cm-1 and 1520 – 1650 cm-1, respectively. Also, stretching mode associated to C–N, C=N, can contributed to the profile of IR spectrum around 1170 cm-1, 1585 – 1770 cm-1. And the TGA methods was used to study the loses of the precursors mass according to the excessive of the temperature. The results showed that, there were different decreasing of the mass of each precursors. PAN degradation started at almost 573 K and at 1073 K, PAN preserve more than 40% of the initial mass. PET degradation started at 650 K, but at 1073 K, it has lost 80% of the initial mass. However, the mixture of PET-PAN (1:1w/w) showed a thermogravimetric profile between the two polymers tested individually, with a final mass slightly less than 30%. From a chemical point of view, the carbonisation of PET mainly occurs in one step between 650 and 775 K.

Fusarium head blight evaluation and genetic stability in synthetic hexaploid wheats.

2016