Protemic characterization of peripheral blood mononuclear cells

Peripheral blood mononuclear cells (PBMCs) play quite diverse and important roles in monitoring immune homeostasis. Thus, these subset of blood cells may provide access to potential physiological relevant biomolecules, namely proteins. For this reason, PBMCs represent a promising biological sample in scientific research, particularly as a source of potential biological markers discovery of the most diverse diseases. Prior studies of proteomic characterization of PBMCs from healthy individuals lack either the identification of a large number of proteins or its quantification in a way that is compatible with the search of potential biomarker candidates. Therefore, this study aimed to provide a comprehensive PBMCs proteome characterisation as well as to create a SWATH library. It was also evaluated if by using the BD Vacutainer® CPT™ tubes for PBMCs isolation, it would be possible to identify a larger number of immunologically relevant proteins in comparison to plasma samples. The enrichment test assay revealed that it is possible to identify more immune-related proteins from isolated PBMCs than from plasma. Moreover, the majority of the quantified proteins with an “immune system” GO term assigned is present in higher amounts in PBMCs samples. 2D LC-MS/MS proved to be the best approach to use in qualitative analysis of PBMCs and in the construction of a SWATH library, since it resulted in an increase of both identified and quantified proteins (66.3% and 16.9%, respectively) in comparison to 1D LC-MS/MS. A total of 2071 proteins were identified and it was possible to quantify 922 different proteins among six distinct samples. From these proteins, 445 were commom between all individuals. In conclusion, this work provides a comprehensive PBMCs proteome dataset that will be useful in further studies that focus on the search for potential biological markers of various pathologies in these cells. Additionally, SWATH-MS proved to be a reproducible and effective acquisition method to quantify PBMCs proteins.

Integrin traffic and signalling – from plasma membrane to endosomes

Integrins are the main cell surface receptors by which cells adhere to the surrounding extracellular matrix (ECM). Cells regulate integrin-mediated adhesions by integrin endo/exocytic trafficking or by altering the integrin activation status. Integrin binding to ECM-components induces several intracellular signalling cascades, which regulate almost every aspect of cell behaviour from cell motility to survival, and dysregulation of integrin traffic or signalling is associated with cancer progression. Upon detachment, normal cells undergo a specialised form of programmed cell death namely anoikis and the ECM-integrin -mediated activation of focal adhesion kinase (FAK) signalling at the cell surface has been considered critical for anoikis suppression. Integrins are also constantly endocytosed and recycled back to the plasma membrane, and so far the role of integrin traffic in cancer has been linked to increased adhesion site turnover and cell migration. However, different growth factor receptors are known to signal also from endosomes, but the ability of integrins to signal from endosomes has not been previously studied. In this thesis, I demonstrate for the first time that integrins are signalling also from endosomes. In contrast to previous believes, integrin-induced focal adhesion kinase (FAK) signalling occurs also on endosomes, and the endosomal FAK signalling is critical for anoikis suppression and for cancer related processes such as anchorage-independent growth and metastasis. Moreover, we have set up a new integrin trafficking assay and demonstrate for the first time in a comprehensive manner that active and inactive integrins undergo distinct trafficking routes. Together these results open up new horizons in our understanding of integrins and highlight the fundamental connection between integrin traffic and signalling.

FAM49 - A novel regulator of the protrusive behaviour and motility of cells

Most eukaryotic cell motility relies on plasma membrane protrusions, which depend on the actin cytoskeleton and its tight regulation. The SCAR/WAVE complex, a pentameric assembly comprising SCAR/WAVE, Nap1, CYFIP/Pir121, Abi and HSPC300, is a key driver of actin-based protrusions such as pseudopods. SCAR/WAVE is thought to activate the Arp2/3 complex, a crucial actin nucleator, after being itself activated by upstream signals such as active Rac1. Despite recent progress on the study of the SCAR/WAVE complex, its regulation is still incompletely understood, with Nap1’s role being particularly enigmatic. Upon screening for potential Nap1 binding partners in the social amoeba Dictyostelium discoideum – a well established model organism in the study of the actin cytoskeleton and cell motility – we found FAM49, a ~36 kDa protein of unknown function which is highly conserved in Metazoa (animals) and evolutionarily closer species such as D. discoideum. Interestingly, D. discoideum’s FAM49 and its homologs contain a DUF1394 domain, which is also predicted in CYFIP/Pir121 proteins and most likely involved in their direct binding to active Rac1, which in turn contributes to SCAR/WAVE’s activation. FAM49’s unknown role, apparent high degree of conservation and potential connections to SCAR/WAVE and Rac1 persuaded us to start investigating its function and biological relevance in D. discoideum, leading to the work presented in this thesis. Several pieces of our data collectively support a function for FAM49 in modulating the protrusive behaviour, and ultimately motility, of D. discoideum cells, as well as a regulatory link between FAM49 and Rac1. FAM49’s involvement in protrusion regulation was first hinted at by our observation that GFP-tagged FAM49 is enriched in pseudopods. The possibility of a link with Rac1 was then strengthened by two additional observations: first, pseudopodial GFP-FAM49 is substantially co-enriched with active Rac, both showing fairly comparable spatio-temporal accumulation dynamics; second, when dominant-active (G12V) Rac1 is expressed in cells, it triggers the recruitment and persistent accumulation of GFP-FAM49 at the plasma membrane, where both become highly co-enriched. We subsequently determined that fam49 KO cells differ from wild-type cells in the way they protrude and move, as assessed in under-agarose chemotaxis assays. In particular, our data indicate that fam49 KO cells tend to display a lower degree of global protrusive activity, their protrusions extend more slowly and are less discrete, and the cells end up moving at lower speeds and with higher directional persistence. This phenotype was substantially rescued by FAM49 re-expression. While re-expressing FAM49 in fam49 KO cells we generated putative FAM49 overexpressor cells; compared to wild-type cells, they displayed atypically thin pseudopods and what seemed to be an excessively dynamic, and perhaps less coordinated, protrusive behaviour. Additional data in our study suggest that pseudopods made by fam49 KO cells are still driven by SCAR/WAVE, which is clearly not being replaced by WASP (as is now known to be the case in D. discoideum cells lacking a functional SCAR/WAVE complex). Nonetheless, the peculiar dynamics of those pseudopods imply that SCAR/WAVE’s activity is regulated differently when FAM49 is lost, though it remains to be determined how. This thesis is the first report of a dedicated study on FAM49 and lays the foundation for future research on it.

Preparo de amostras citológicas de efusões, lavados (broncoalveolar e peritoneal) e líquor pelo método “plasma-tromboplastina”

Dissertação (mestrado)—Universidade de Brasilia, Faculdade de Ciências da Saúde, Programa de Pós-Graduação em Ciências da Saúde, 2016.

Clustering of Rab11 vesicles in influenza A virus infected cells creates hotspots containing the 8 viral ribonucleoproteins

Influenza A virus is an important human pathogen causative of yearly epidemics and occasional pandemics. The ability to replicate within the host cell is a determinant of virulence, amplifying viral numbers for host-to-host transmission. This process requires multiple rounds of entering permissive cells, replication, and virion assembly at the plasma membrane, the site of viral budding and release. The assembly of influenza A virus involves packaging of several viral (and host) proteins and of a segmented genome, composed of 8 distinct RNAs in the form of viral ribonucleoproteins (vRNPs). The selective assembly of the 8-segment core remains one of the most interesting unresolved problems in virology. The recycling endosome regulatory GTPase Rab11 was shown to contribute to the process, by transporting vRNPs to the periphery, giving rise to enlarged cytosolic puncta rich in Rab11 and the 8 vRNPs. We recently reported that vRNP hotspots were formed of clustered vesicles harbouring protruding electron-dense structures that resembled vRNPs. Mechanistically, vRNP hotspots were formed as vRNPs outcompeted the cognate effectors of Rab11, the Rab11-Family-Interacting-Proteins (FIPs) for binding, and as a consequence impair recycling sorting at an unknown step. Here, we speculate on the impact that such impairment might have in host immunity, membrane architecture and viral assembly.

Controlled surface nanotopography and oxygen plasma treatment of PEEK to improve cellular response

Poly(aryl-ether-ether-ketone) (PEEK) is a semi crystalline polymer which exhibits properties that make it an attractive choice for use as an implant material. It displays natural radiolucency, and MRI compatibility, as well as good chemical and sterilization resistance, both of which make it of particular interest in orthopaedic implants. However, PEEK has demonstrated poor cellular adhesion both in vitro and in vivo. This is problematic as implant surfaces that do not develop a layer of adhesive cells are at risk of undergoing fibrous encapsulation, which in turn leads to lack of a strong interface between the implant device and the patient tissue, which can in turn lead to failure of the implant and revision surgery . As incorporating nanotopography into a polymer surface has been demonstrated to be able to direct the differentiation behaviour of stem cells, a possible solution to PEEKs underlying issues with poor cellular response would be to incorporate specific nanoscale topography into the material surface through injection moulding, and then analysing if this is a viable method for addressing PEEKs issues with cellular response. In addition to nanoscale topography, the experimental PEEK surfaces were treated with oxygen plasma to address the underlying cytophobicity of the material. As this type of treatment has been documented to be capable of etching the PEEK surface, experiments were carried out to quantify the effect of this treatment, both on the ability of cells to adhere to the PEEK surface, as well as the effect it has upon the nanotopography present at the PEEK surface. The results demonstrated that there were a range of plasma treatments which would significantly improve the ability of cells to adhere to the PEEK surface without causing unacceptable damage to the nanotopography. Three different types of cells with osteogenic capacity were tested with the PEEK surfaces to gauge the ability of the topography to alter their behaviour: SAOS-2, osteoprogenitors and 271+ MSCs. Due to PEEKs material properties (it is non transparent, exhibits birefringence and is strongly autofluorescent) a number of histological techniques were used to investigate a number of different stages that take place in osteogenesis. The different cell types did display slightly different responses to the topographies. The SAOS-2 cells cultured on surfaces that had been plasma treated for 2 minutes at 200W had statistically significantly higher levels of von Kossa staining on the NSQ surface compared to the planar surface, and the same experiment employing alizarin red staining, showed a statistically significantly lower level of staining on the SQ surface compared to the planar surface. Using primary osteoprogenitor cells designed to look into if whether or not the presence of nanotopography effected the osteogenic response of these cells, we saw a lack of statistically significant difference produced by the surfaces investigated. By utilising HRP based immunostaining, we were able to investigate, in a quantitative fashion, the production of the two osteogenic markers osteopontin and osteocalcin by cells. When stained for osteocalcin, the SQ nanotopography had total percentage of the surface with stained material, average area and average perimeter all statistically significantly lower than the planar surface. For the cells that were stained for osteopontin, the SQ nanotopgraphy had a total percentage of the surface with stained material, average area and average perimeter all highly statistically significantly lower than those of the planar surface. Additionally, for this marker the NSQ nanotopography had average areas and average perimeters that were highly significantly higher than those of the planar surface. There were no significant differences for any of the values investigated for the 271+ MSC’s When plasma treatment was varied, the SAOS-2 cells demonstrated an overall trend i.e. increasing the energy of plasma treatment in turn leads to an increase in the overall percentage of staining. A similar experiment employing stem cells isolated from human bone marrow instead of SAOS-2 cells showed that for polycarbonate surfaces , used as a control, mineralization is statistically significantly higher on the NSQ nanopattern compared to the planar surface, whereas on the PEEK surfaces we observe the opposite trend i.e. the NSQ nanotopography having a statistically significantly lower amount of mineralization compared to the planar surface at the 200W 2min and 30W 1min plasma treatments. The standout trend from the PEEK results in this experiment was that the statistically significant differences on the PEEK substrates were clustered around the lower energy plasma treatments, which could suggest that the plasma treatment disrupted a function of the nanotopograhy which is why, as the energy increases, there are less statistically significant differences between the NSQ nanotopography and the Planar surface This thesis documents the response of a number of different types of cells to specific nanoscale topographies incorporated into the PEEK surface which had been treated with oxygen plasma. It outlines the development of a number of histological methods which measure different aspects of osteogenesis, and were selected to both work with PEEK, and produce quantitative results through the use of Cell Profiler. The methods that have been employed in this body of work would be of interest to other researchers working with this material, as well as those working with similarly autofluorescent materials.