A biochemical analysis into the co-translational folding of a G protein-coupled receptor; GPR35

The folding and targeting of membrane proteins poses a major challenge to the cell, as they must remain insertion competent while their highly hydrophobic transmembrane (TM) domains are transferred from the ribosome, through the aqueous cytosol and into the lipid bilayer. The biogenesis of a mature membrane protein takes place through the insertion and integration into the lipid bilayer. A number of TM proteins have been shown to gain some degree of secondary structure within the ribosome tunnel and to retain this conformation throughout maturation. Although studies into the folding and targeting of a number of membrane proteins have been carried out to date, there is little information on one of the largest class of eukaryotic membrane proteins; the G-protein-coupled receptors (GPCRs). This project studies the early folding events of the human ortholog of GPR35. To analyse the structure of the 1st TM domain, intermediates were generated and assessed by the biochemical method of pegylation (PEG-MAL). A structurally-similar microbial opsin (Bacterioopsin) was also used to investigate the differences in the early protein folding within eukaryotic and prokaryotic translation systems. Results showed that neither the 1st TM domain of GPR35 nor Bacterioopsin were capable of compacting in the ribosome tunnel before their N-terminus reached the ribosome exit point. The results for this assay remained consistent whether the proteins were translated in a eukaryotic or prokaryotic translation system. To examine the communication mechanism between the ribosome, the nascent chain and the protein targeting pathway, crosslinking experiments were carried out using the homobifunctional lysine cross-linker BS3. Specifically, the data generated here show that the nascent chain of GPR35 reaches the ribosomal protein uL23 in an extended conformation and interacts with the SRP protein as it exits the ribosome tunnel. This confirms the role of SRP in the co-translational targeting of GPR35. Using these methods insights into the early folding of GPCRs has been obtained. Further experiments using site-directed mutagenesis to reduce hydrophobicity in the 1st TM domain of GPR35, highlighted the mechanisms by which GPCRs are targeted to the endoplasmic reticulum. Confirming that hydrophobicity within the signal anchor sequence is essential of SRP-dependent targeting. Following the successful interaction of the nascent GPR35 and SRP, GPR35 is successfully targeted to ER membranes, shown here as dog pancreas microsomes (DPMs). Glycosylation of the GPR35 N-terminus was used to determine nascent chain structure as it is inserted into the ER membrane. These glycosylation experiments confirm that TM1 has obtained its compacted state whilst residing in the translocon. Finally, a site-specific cross-linking approach using the homobifunctional cysteine cross-linker, BMH, was used to study the lateral integration of GPR35 into the ER. Cross-linking of GPR35 TM1 and TM2 could be detected adjacent to a protein of ~45kDa, believed to be Sec61α. The loss of this adduct, as the nascent chain extends, showed the lateral movement of GPR35 TM1 from the translocon was dependent on the subsequent synthesis of TM2.

Microfluidic Encapsulation of Pickering Oil Microdroplets into Alginate Microgels for Lipophilic Compound Delivery

Alginate microgels are widely used as delivery systems in food, cosmetics, and pharmaceutical industries for encapsulation and sustained release of hydrophilic compounds and cells. However, the encapsulation of lipophilic molecules inside these microgels remains a great challenge because of the complex oil-core matrix required. The present study describes an original two-step approach allowing the easy encapsulation of several oil microdroplets within alginate microgels. In the first step, stable oil microdroplets were formed by preparing an oil-in-water (O/W) Pickering emulsion. To stabilize this emulsion, we used two solid particles, namely the cotton cellulose nanocrystals (CNC) and calcium carbonate (CaCO3). It was observed that the surface of the oil microdroplets formed was totally covered by a CNC layer, whereas CaCO3 particles were adsorbed onto the cellulose layer. This solid CNC shell efficiently stabilized the oil microdroplets, preventing them from undesired coalescence. In the second step, oil microdroplets resulting from the Pickering emulsion were encapsulated within alginate microgels using microfluidics. Precisely, the outermost layer of oil microdroplets composed of CaCO3 particles was used to initiate alginate gelation inside the microfluidic device, following the internal gelation mode. The released Ca2+ ions induced the gel formation through physical cross-linking with alginate molecules. This innovative and easy to carry out two-step approach was successfully developed to fabricate monodisperse alginate microgels of 85 pm in diameter containing around 12 oil microdroplets of 15 mu m in diameter. These new oil-core alginate microgels represent an attractive system for encapsulation of lipophilic compounds such as vitamins, aroma compounds or anticancer drugs that could be applied in various domains including food, cosmetics, and medical applications.

Hidrógeis de Poli(N-Vinil 2-Pirrolidona) com nano argilas laponite RD sinterizados por irradiação gama

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Połączenia krzyżowe pochodnych cysteiny z aldehydowymi adduktami nukleozydów adeninowych

Wydział Chemii

Protein disulfide isomerase as a target for besnoitiosis therapy : molecular characterization and studies of its role in infection and host immune response

Tese de Doutoramento em Ciências Veterinárias na especialidade de Sanidade Animal

Desenvolvimento e caraterização de hidrogéis físicos e químicos a partir de polissacarídeos naturais

Dissertação de Mestrado, Engenharia Biológica, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2016

Oligodeoxynucleotide synthesis using protecting groups and a linker cleavable under non-nucleophilic conditions

Oligodeoxynucleotides (ODNs) containing latent electrophilic groups can be highly useful in antisense drug development and many other applications such as chemical biology and medicine, where covalent cross-linking of ODNs with mRNA, protein and ODN is required. However, such ODN analogues cannot be synthesized using traditional technologies due to the strongly nucleophilic conditions used in traditional deprotection/cleavage process. To solve this long lasting and highly challenging problem in nucleic acid chemistry, I used the 1,3-dithian-2-yl-methoxycarbonyl (Dmoc) function to protect the exo-amino groups on the nucleobases dA, dC and dG, and to design the linker between the nascent ODN and solid support. These protecting groups and linker are completely stable under all ODN synthesis conditions, but can be readily cleaved under non-nucleophilic and nearly neutral conditions. As a result, the new ODN synthesis technology is universally useful for the synthesis of electrophilic ODNs. The dissertation is mainly comprised of two portions. In the first portion, the development of the Dmoc-based linker for ODN synthesis will be described. The construction of the dT-Dmoc-linker required a total of seven steps to synthesize. The linker was then anchored to the solid support―controlled pore glass (CPG). In the second portion, the syntheses of Dmoc-protected phosphoramidites ODN synthesis monomers including Dmoc-dC-amidite, Dmoc-dA-amidite, Dmoc-dG-amidite are described. The protection of dC and dA with 1,3-dithian-2-yl-methyl 4-nitrophenyl carbonate proceeded smoothly giving Dmoc-dC and Dmoc-dA in good yields. However, when the same acylation procedure was applied for the synthesis of Dmoc-dG, very low yield was obtained. This problem was later solved using a highly innovative and environmentally benign procedure, which is expected to be widely useful for the acylation of the exo-amino groups on nucleoside bases. The reactions to convert the Dmoc-protected nucleosides to phosphoramidite monomers proceeded smoothly with high yields. Using the Dmoc phosphoramidite monomers dA, dC, dG and the commercially available dT, and the Dmoc linker, four ODN sequences were synthesized. In all cases, excellent coupling yields were obtained. ODN deprotection/cleavage was achieved by using non-nucleophilic oxidative conditions. The new technology is predicted to be universally useful for the synthesis of ODNs containing one or more electrophilic functionalities.

Effects of plasticizing and crosslinking on the mechanical and barrier properties of coatings based on blends of starch and poly(vinyl alcohol)

In the last decades, intensive research has been carried out in order to replace oil-based polymers with bio-based polymers due to growing environmental concerns. So far, most of the barrier materials used in food packaging are petroleum-based materials. The purpose of the barrier is to protect the packaged food from oxygen, water vapour, water and fat. The mechanical and barrier properties of coatings based on starch-plasticizer and starch-poly(vinyl alcohol) (PVOH)-plasticizer blends have been studied in the work described in this thesis. The plasticizers used were glycerol, polyethylene glycol and citric acid. In a second step, polyethylene coatings were extruded onto paperboard pre-coated with a starch-PVOH-plasticizer blend. The addition of PVOH to the starch increased the flexibility of the film. Curing of the film led to a decrease in flexibility and an increase in tensile strength. The flexibility of the starch-PVOH films was increased more when glycerol or polyethylene glycol was added than citric acid. The storage modulus of the starch-PVOH films containing citric acid increased substantially at high temperature. It was seen that the addition of polyethylene glycol or citric acid to the starch-PVOH blend resulted in an enrichment of PVOH at the surface of the films. Tensile tests on the films indicated that citric acid acted as a compatibilizer and increased the compatibility of the starch and PVOH in the blend. The addition of citric acid to the coating recipe substantially decreased the water vapour transmission rate through the starch-PVOH coated paperboard, which indicated that citric acid acts as a cross-linker for starch and/or PVOH. The starch-PVOH coatings containing citric acid showed oxygen-barrier properties similar to those of pure PVOH or of a starch-PVOH blend without plasticizer when four coating layers were applied on a paperboard. The oxygen-barrier properties of coatings based on a starch-PVOH blend containing citric acid indicated a cross-linking and increase in compatibility of the starch-PVOH blends. Polyethylene extrusion coating on a pre-coated paperboard resulted in a clear reduction in the oxygen transmission rate for all the pre-coating formulations containing plasticizers. The addition of a plasticizer to the pre-coating reduced the adhesion of polyethylene to pre-coated board. Polyethylene extrusion coating gave a board with a lower oxygen transmission rate when the paperboard was pre-coated with a polyethylene-glycol-containing formulation than with a citric-acid-containing formulation. The addition of polyethylene glycol to pre-coatings indicated an increase in wetting of the pre-coated paperboard by the polyethylene melt, and this may have sealed the small defects in the pre-coating leading to low oxygen transmission rate. The increase in brittleness of starch-PVOH films containing citric acid at a high temperature seemed to have a dominating effect on the barrier properties developed by the extrusion coating process. 

Estudio molecular en dos hermanas afectadas por ictiosis congénita autosómica recesiva : descripción de una nueva mutación causal en TGM1

Se describe la variante homocigota c.320-2A>G de TGM1 en dos hermanas con ictiosis congénita autosómica recesiva. El clonaje de los transcritos generados por esta variante permitió identificar tres mecanismos moleculares de splicing alternativos.

Cross-Lingual knowledge discovery : Chinese-to-English article linking in Wikipedia

In this paper we examine automated Chinese to English link discovery in Wikipedia and the effects of Chinese segmentation and Chinese to English translation on the hyperlink recommendation. Our experimental results show that the implemented link discovery framework can effectively recommend Chinese-to-English cross-lingual links. The techniques described here can assist bi-lingual users where a particular topic is not covered in Chinese, is not equally covered in both languages, or is biased in one language; as well as for language learning.

Perturbative analysis of the triply differential cross section and circular dichroism in photo-double-ionization of He

We extend application of our lowest-order perturbative approach (in electron-electron correlation) for analysis of photo-double-ionization (PDI) of He [A.Y. Istomin et al., J. Phys. B 35, L543 (2002)] to excess energies up to 450 eV and to analysis of circular dichroism. We find that account of electron correlation in the final state to first order provides predictions for the triply differential cross section and circular dichroism that are in reasonable agreement with absolute data for excess energies up to 80 eV. For an excess energy of 450 eV, account of electron correlation in both initial and final states is necessary and the predicted triply differential cross sections are in agreement with absolute data only for large mutual ejection angles. We find that at excess energies of a few tens of eV, the PDI is dominated by the "virtual" knock-out mechanism, while the "direct" (on-shell) knock-out process gives only small contributions for large mutual ejection angles. As a result, we conclude that the circular dichroism effect at these energies originates from the nonzero electron Coulomb phase shifts.

Cross-Lingual Linking of News Stories using ESA

In this paper, we describe our approach for Cross-Lingual linking of Indian news stories, submitted for Cross-Lingual Indian News Story Search (CL!NSS) task at FIRE 2012. Our approach consists of two major steps, the reduction of search space by using di�erent features and ranking of the news stories according to their relatedness scores. Our approach uses Wikipedia-based Cross-Lingual Explicit Semantic Analysis (CLESA) to calculate the semantic similarity and relatedness score between two news stories in di�erent languages. We evaluate our approach on CL!NSS dataset, which consists of 50 news stories in English and 50K news stories in Hindi.