Collagen-like block copolymers with tunable design : production in yeast and functional characterisation

Animal-derived collagen and gelatin have been extensively used in the past decades for several pharmaceutical and biomedical applications. However, there is need for collagen-based materials with predictable and tailorable properties. The aim of this thesis is the design and microbial production of gel forming non-hydroxylated collagen-like proteins. Recombinant protein expression and protein engineering are used to develop collagen-like polymers with defined composition, structure, and tunable physical-chemical properties. The possibility of using these proteins as controlled release systems is also explored, as well as the set-up of efficient and scalable production procedures using P. pastorisas a microbial factory. In chapter 2 we describe the genetic design, recombinant production and preliminary characterisation of a new class of ABA triblock copolymers forming thermosensitive gels with highly controllable and predictable properties. Gel formation is obtained by combining proline-rich collagen-inspired (Pro-Gly-Pro)9 end-blocks (T), which have triple helix-forming ability, with highly hydrophilic random coil blocks (Pn or Rn) defining the distance between the trimer forming end-blocks. We report the secreted production in yeast at several g/l of two such non-hydroxylated ~42 kDa triblock copolymers, TP4T and TR4T. The dynamic elasticity (storage modulus) of the gels from these collagen-inspired triblock copolymers was comparable to animal gelatin with a similar content of triple helices. In favourable contrast to traditional gelatin, the dynamic elasticity of the new material, in which only one single (well-defined) type of cross links is formed, is independent of the thermal history of the gel. The novel hydrogels have a ~37 °C melting temperature. However, the thermostability of the hydrogels formed by these polymers can be tailored by changing the number of (Pro-Gly-Pro) repeats. The concept allows to produce custom-made precision gels for biomedical applications. In chapter 3 it was shown that small, but tailored changes in the length of the mid-block of the collagen-inspired triblock copolymers results in significant changes in the viscoelastic properties of the hydrogels. We compared 4 different triblock copolymers, differing only in their mid-block size or mid-block amino sequence. The shorter versions, i.e. TP4T and TR4T, had mid-blocks made of ~400 amino acids, and their longer counterparts, i.e. TP8T and TR8T, ~800 amino acids. These results obtained indicate that the elastic properties of the network are not only a function of concentration and temperature but also of polymer length. The experimental results were well described by an analytical model that was based on classical gel theory and accounted for the particular molecular structure of the gels, and the presence of loops and dangling ends. These results suggest that, by controlling the structure of the present type of hydrogel-forming polymers through genetic engineering their physical-chemical properties can be predicted, and tailored in order to match a specific application In chapter 4 we explored the potential of hydrogels from collagen-inspired triblock co-polymers as drug delivery systems. We studied the erosion and protein release kinetics of two of these hydrogel-forming polymers, i.e. TR4T and TR8T, differing only in their mid-block length (mid-block molecular weights ~37 kDa and ~73 kDa). By varying polymer length and concentration, the elastic properties of the hydrogels as well as their mesh size, swelling and erosion behaviour can be tuned. We show that the hydrogel networks are highly dense and that the decrease of gel volume is mainly the result of surface erosion, which in turn depends on both temperature and initial polymer concentration. In addition, we show that the release kinetics of an entrapped protein is governed by a combined mechanism of erosion and diffusion. The prevalence of one or the other is strongly dependent on polymer concentration. Most importantly, the encapsulated protein was quantitatively released demonstrating that these hydrogels offer great potential as drug delivery systems. The development of efficient large-scale production processes can be a critical factor in whether or not a relevant pharmaceutical material is available in sufficient amounts to be used for application studies and eventually enter human clinical trials and the marketplace. In chapter 5 we describe the development of a pilot-scale process for the fermentation and purification of five collagen-inspired triblock copolymers (TP4T, TR4T, TP8T, TR8T and TP12T) with molecular weights ranging from ~42 kDa to ~114 kDa. P. pastoris strains were grown in a 140 liter bioreactor using a three-phase fermentation process. The fermentation culture reached high cell densities, and all proteins were efficiently expressed and secreted into the fermentation medium at a concentration of ~700-800 mg/l of cell free broth. The downstream processing principles elaborated previously at lab-scale were successfully adapted to the larger scale and resulted in 80-95 % recovery. The purified proteins were intact and showed a similar performance to those obtained using lab-scale procedures. The good productivity and efficient downstream processing (DSP) shown in this study provides a promising perspective towards a potential further scale-up to industrial production of these proteins. In chapter 6 some of the results obtained in the thesis are highlighted and suggestions for further research are given. The contents of this thesis provide a good starting point for future development of this novel class of hydrogel forming collagen-like proteins. <br/

A Unified Experimental/Theoretical Description of the Ultrafast Photophysics of Single and Double Thionated Uracils

Photoinduced processes in thiouracil derivatives have lately attracted considerable attention due to their suitability for innovative biological and pharmacological applications. Here, sub-20 fs broadband transient absorption spectroscopy in the near-UV are combined with CASPT2/MM decay path calculations to unravel the excited-state decay channels of water solvated 2-thio and 2,4-dithiouracil. These molecules feature linear absorption spectra with overlapping ππ* bands, leading to parallel decay routes which we systematically track for the first time. The results reveal that different processes lead to the triplet states population, both directly from the ππ* absorbing state and via the intermediate nπ* dark state. Moreover, the 2,4-dithiouracil decay pathways is shown to be strongly correlated either to those of 2- or 4-thiouracil, depending on the sulfur atom on which the electronic transition localizes

Coronary computed tomography angiography-adapted Leaman score as a tool to noninvasively quantify total coronary atherosclerotic burden

To describe a coronary computed tomography angiography (CCTA)-adapted Leaman score (CT-LeSc) as a tool to quantify total coronary atherosclerotic burden with information regarding localization, type of plaque and degree of stenosis and to identify clinical predictors of a high coronary atherosclerotic burden as assessed by the CT-LeSc. Single center prospective registry including a total of 772 consecutive patients undergoing CCTA (Dual-source CT) from April 2011 to March 2012. For the purpose of this study, 581 stable patients referred for suspected coronary artery disease (CAD) without previous myocardial infarction or revascularization procedures were included. Pre-test CAD probability was determined using both the Diamond-Forrester extended CAD consortium method (DF-CAD consortium model) and the Morise score. Cardiovascular risk was assessed with the HeartScore. The cut-off for the 3rd tercile (CT-LeSc ≥8.3) was used to define a population with a high coronary atherosclerotic burden. The median CT-LeSc in this population (n = 581, 8,136 coronary segments evaluated; mean age 57.6 ± 11.1; 55.8 % males; 14.6 % with diabetes) was 2.2 (IQR 0-6.8). In patients with CAD (n = 341), the median CT-LeSc was 5.8 (IQR 3.2-9.6). Among patients with nonobstructive CAD, most were classified in the lowest terciles (T1, 43.0 %; T2, 36.1 %), but 20.9 % were in the highest tercile (T3). The majority of the patients with obstructive CAD were classified in T3 (78.2 %), but 21.8 % had a CT-LeSc in lower terciles (T1 or T2). The independent predictors of a high CT-LeSc were: Male sex (OR 1.73; 95 % CI 1.04-2.90) diabetes (OR 2.91; 95 % CI 1.61-5.23), hypertension (OR 2.54; 95 % CI 1.40-4.63), Morise score ≥16 (OR 1.97; 95 % CI 1.06-3.67) and HeartScore ≥5 (OR 2.42; 95 % CI 1.41-4.14). We described a cardiac CT adapted Leaman score as a tool to quantify total (obstructive and nonobstructive) coronary atherosclerotic burden, reflecting the comprehensive information about localization, degree of stenosis and type of plaque provided by CCTA. Male sex, hypertension, diabetes, a HeartScore ≥5 % and a Morise score ≥16 were associated with a high coronary atherosclerotic burden, as assessed by the CT-LeSc. About one fifth of the patients with nonobstructive CAD had a CT-LeSc in the highest tercile, and this could potentially lead to a reclass

Hydrogels of collagen-inspired telechelic triblock copolymers for the sustained release of proteins

We studied the release of entrapped protein from transient gels made of thermosensitive, collagen-inspired ABA triblock copolymers with tailorable properties and with mid blocks of two different lengths (~ 37 kDa and ~ 73 kDa). These polymers were produced as heterologous proteins in recombinant yeast. By varying polymer length and concentration, the elastic properties of the hydrogels as well as their mesh size, swelling and erosion could be tuned. Whereas the volume of the investigated dense networks decreased in time as a result of temperature- and polymer concentration-dependent surface erosion, the release of entrapped protein was governed by a combination of gel erosion and protein diffusion. The prevalence of one or the other was strongly dependent on polymer concentration. Most importantly, the encapsulated protein was quantitatively released, which demonstrates that these hydrogels offer great potential as drug delivery systems

Secreted production of collagen-inspired gel-forming polymers with high thermal stability in Pichia pastoris

Previously, we have shown that gel-forming triblock proteins, consisting of random coil middle blocks and trimer-forming (Pro-Gly-Pro)(9) end blocks, are efficiently produced and secreted by the yeast Pichia pastoris. These end blocks had a melting temperature (T (m) ) of ~41°C (at 1.1¿mM of protein). The present work reveals that an increase of T (m) to ~74°C, obtained by extension of the end blocks to (Pro-Gly-Pro)(16) , resulted in a five times lower yield and partial endoproteolytic degradation of the protein. A possible cause could be that the higher thermostability of the longer (Pro-Gly-Pro)(16) trimers leads to a higher incidence of trimers in the cell, and that this disturbs secretion of the protein. Alternatively, the increased length of the proline-rich (Pro-Gly-Pro)( n ) domain may negatively influence ribosomal translation, or may result in, for example, hydrophobic aggregation or membrane-active behavior owing to the greater number of closely placed proline residues. To discriminate between these possibilities, we studied the production of molecules with randomized end blocks that are unable to form triple helices. The codon- and amino acid composition of the genes and proteins, respectively, remained unchanged. As these nontrimerizing molecules were secreted intact and at high yield, we conclude that the impaired secretion and partial degradation of the triblock with (Pro-Gly-Pro)(16) end blocks was triggered by the occurrence of intracellular triple helices. This degradation was overcome by using a yapsin 1 protease disruptant, and the intact secreted polymer was capable of forming self-supporting gels of high thermal stability

Precision gels from collagen-inspired triblock copolymers

Gelatin hydrogels find broad medical application. The current materials, however, are from animal sources, and their molecular structure and thermal properties cannot be controlled. This study describes recombinant gelatin-like polymers with a general design that inherently offers independent tuning of the cross-link density, melting temperature, and biocompatibility of the gel. The polymers contain small blocks with thermoreversible trimerization capacity and defined melting temperature, separated by hydrophilic nontrimerizing blocks defining the distance between the knot-forming domains. As an example, we report the secreted production in yeast at several g/L of two nonhydroxylated 42 kDa triblock copolymers with terminal trimerizing blocks. Because only the end blocks formed cross-links, the molecular architecture of the gels is much more defined than that of traditional gelatins. The novel hydrogels had a 37 °C melting temperature, and the dynamic elasticity was independent of the thermal history. The concept allows to produce custom-made precision gels for biomedical applications

Influence of molecular size on gel-forming properties of telechelic collagen-inspired polymers

We studied the influence of molecular size on the formation of transient networks by telechelic protein polymers with 2.3 kDa collagen-like triple helix-forming end-blocks and much longer random coil mid-blocks. We compared triblock copolymers with mid-blocks of 400 and 800 amino acids (37 and 73 kDa, respectively) and two different amino acid sequences, all of which were secreted to high concentration by recombinant yeast cells. At the same molar concentration of protein and crosslink-forming end-blocks, the storage modulus of the longer polymers was higher than that of the shorter polymers. Differences in storage modulus values were also observed for the polymers with mid-blocks of the same amino acid composition but different amino sequence, which correlated to differences in the measured hydrodynamic radius of the mid-blocks. The melting temperature of the triple helices was the same for both larger and smaller polymers; however, the elastic properties of the gels were lost at lower temperature for the smaller polymers than for the larger polymers. Using an analytical model based on classical gel theory and accounting for the well-defined multiplicity of the network, we could ascribe these differences to the lower probability of the longer chains to form intramolecular loops

Distribuição de Biomphalaria straminea ao Sul da Região Neotropical, Brasil Distribution of Biomphalaria straminea in the Southern Neotropical Region of Brazil

Objetivou-se atualizar os conhecimentos sobre a área colonizada pela Biomphalaria straminea e de alguns dos determinantes envolvidos na dispersão dessa espécie hospedeira intermediária de Schistosoma mansoni. Foram examinados 10.616 exemplares de caramujos procedentes de 76 localidades do Estado de São Paulo (Brasil), e realizado levantamento dos registros de ocorrência da espécie disponíveis na literatura especializada. Ficou demonstrada a expansão dos domínios territoriais de B. straminea na região, ressaltando que na parte superior da bacia hidrográfica do rio Paraná, a disseminação dos caramujos mostra estreita relação com o aproveitamento de longos trechos de rios para a navegação fluvial. Dados os riscos epidemiológicos associados à propagação desses transmissores da esquistossomose, ressalta-se a necessidade da manutenção do controle e vigilância da endemia na região.<br>A careful anatomical revision of 10,616 preserved specimens of snails from 76 localities of the State of S. Paulo, Brazil, was made with a view better to determining the geographical distribution of Biomphalaria straminea in the Neotropical Region of Southern Brazil. The analysis has shown that previous determinations were correct. The study was then complemented with a survey of information from the literature about distribution of the species. The distribution pattern of the species has expanded greatly over the last few years, perhaps an account of the construction of new dams, and the navigation system in the upper Paraná Basin. Epidemiological data have shown that B. straminea is a good host to S. mansoni. Continuous schistosomiasis control must be exercised so as to prevent the further expansion of the disease

Ultrafast intersystem crossing in 4-thiothymidine proceeds through a vibrational coherently accessed dark intermediate state

4-Thiothymidine 20 fs broadband Transient Transmission Spectroscopy results and analysis provide evidence that along the photoexcited relaxation pathway, the intersystem crossing originates primarily from a dark intermediate state, which is vibrationally coherent accessible