The Development Of Platforms For Inhibiting RHAMM-HA Interactions & The Development Of An Optical Probe For Measuring Glomerular Filtration Rate

Carbohydrates are a class of molecule occurring widely in the body. Their presence has varied biological implications, generating clinical interest regarding their impact on disease prognosis. This thesis will investigate the development of chemical entities surrounding two carbohydrates, hyaluronan and inulin. The Receptor for hyaluronan mediated motility (RHAMM) is one of several receptors for hyaluronan (HA), a polysaccharide that, when fragmented, has pro-angiogenic and inflammatory properties. RHAMM expression is tightly regulated during homeostasis but increases in response to cellular stress, including during injury or disease states. HA-RHAMM interactions stimulate the Ras-ERK-Mek pathway to promote cell motility, differentiation, and proliferation. Specific inhibition of HA-RHAMM interactions could have significant therapeutic potential. Chapters 2 and 3 explore two platforms for disrupting HA-RHAMM interactions. Chapter 2 discusses development of a 62-amino acid chemically synthesized truncated RHAMM protein, 7 kDa RHAMM, for use in screening novel therapeutics. This mini-protein exhibited similar secondary structure, bioactivity, and HA-binding capabilities as the full-length protein, and binds known RHAMM-binding peptides with similar affinities as recombinant RHAMM. This suggests that it is a suitable replacement for the difficult-to-obtain recombinant version. Chapter 3 discusses the development of double stapled RHAMM peptide mimetics as therapeutic anti-inflammatory agents. The peptides were evaluated for secondary structure, HA-binding capability, and inflammation-related bioactivity. The lead compound blocked 27.2% and 52% of induced inflammation in culture and in vivo, respectively. The lead peptide was further optimized to improve metabolic stability while maintaining secondary structure and HA-binding affinity, improving therapeutic efficacy. Glomerular filtration rate (GFR) is a measure of kidney function and a prognostic indicator of chronic kidney disease. Filtration of the polysaccharide inulin is the gold standard for measuring GFR clinically, as it is neither reabsorbed nor secreted by the kidneys; however, this method is laborious and invasive. Chapter 3 explores the development of a near-infrared dye-labeled inulin, Cy7.5-inulin conjugate, as an optical probe to accurately and non-invasively measure GFR by transcutaneous pulse dye densitometer. The conjugate was characterized by different analytical techniques, and is stable under in vivo conditions. The probe was successfully used in a pig model to accurately measure GFR non-invasively

Building a hurricane risk map for continental Portugal based on loss data from hurricane Leslie

A complete model to analyse and predict future losses in the property portfolio of an insurance company due to hurricanes is proposed. A novel statistical model, in which weather data is not required, is considered. Climate data may not be reliable, or may be difficult to deal with or to obtain, hence we reconstruct the storm behaviour through the registered claims and respective losses. The model is calibrated using the loss data of the property portfolio of the insurance company Fidelidade, from hurricane Leslie, which hit the center of continental Portugal in October 2018. Several scenarios are simulated and risk maps are built. The simulated scenarios can be used to compute risk premiums per risk class in the portfolio. These can be used to adjust the policy premiums accounting for a storm risk. The risk map of the company also depends on its portfolio, namely its exposure, providing a hurricane risk management tool for the insurance company.info:eu-repo/semantics/publishedVersio

Recombinant protein expression by targeting pre-selected chromosomal loci

<p>Abstract</p> <p>Background</p> <p>Recombinant protein expression in mammalian cells is mostly achieved by stable integration of transgenes into the chromosomal DNA of established cell lines. The chromosomal surroundings have strong influences on the expression of transgenes. The exploitation of defined loci by targeting expression constructs with different regulatory elements is an approach to design high level expression systems. Further, this allows to evaluate the impact of chromosomal surroundings on distinct vector constructs.</p> <p>Results</p> <p>We explored antibody expression upon targeting diverse expression constructs into previously tagged loci in CHO-K1 and HEK293 cells that exhibit high reporter gene expression. These loci were selected by random transfer of reporter cassettes and subsequent screening. Both, retroviral infection and plasmid transfection with eGFP or antibody expression cassettes were employed for tagging. The tagged cell clones were screened for expression and single copy integration. Cell clones producing > 20 pg/cell in 24 hours could be identified. Selected integration sites that had been flanked with heterologous recombinase target sites (FRTs) were targeted by Flp recombinase mediated cassette exchange (RMCE). The results give proof of principle for consistent protein expression upon RMCE. Upon targeting antibody expression cassettes 90-100% of all resulting cell clones showed correct integration. Antibody production was found to be highly consistent within the individual cell clones as expected from their isogenic nature. However, the nature and orientation of expression control elements revealed to be critical. The impact of different promoters was examined with the tag-and-targeting approach. For each of the chosen promoters high expression sites were identified. However, each site supported the chosen promoters to a different extent, indicating that the strength of a particular promoter is dominantly defined by its chromosomal context.</p> <p>Conclusion</p> <p>RMCE provides a powerful method to specifically design vectors for optimized gene expression with high accuracy. Upon considering the specific requirements of chromosomal sites this method provides a unique tool to exploit such sites for predictable expression of biotechnologically relevant proteins such as antibodies.</p

Seroprofiling of Antibodies Against Endemic Human Coronaviruses and Severe Acute Respiratory Syndrome Coronavirus 2 in a Human Immunodeficiency Virus Cohort in Lesotho: Correlates of Antibody Response and Seropositivity

BACKGROUND: Serological data on endemic human coronaviruses (HCoVs) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in southern Africa are scarce. Here, we report on (1) endemic HCoV seasonality, (2) SARS-CoV-2 seroprevalence, and (3) correlates of SARS-CoV-2 seropositivity and strength of SARS-CoV-2 and endemic HCoV serological responses among adults living with human immunodeficiency virus (HIV). METHODS: Plasma samples were collected from February 2020 to July 2021 within an HIV cohort in Lesotho. We used the AntiBody CORonavirus Assay (ABCORA) multiplex immunoassay to measure antibody responses to endemic HCoV (OC43, HKU1, NL63, and 229E) and SARS-CoV-2 antigens. RESULTS: Results for 3173 samples from 1403 adults were included. Serological responses against endemic HCoVs increased over time and peaked in winter and spring. SARS-CoV-2 seropositivity reached >35% among samples collected in early 2021 and was associated with female sex, obesity, working outside the home, and recent tiredness or fever. Positive correlations were observed between the strength of response to endemic HCoVs and to SARS-CoV-2 and between older age or obesity and the immunoglobulin G response to SARS-CoV-2. CONCLUSIONS: These results add to our understanding of the impact of biological, clinical, and social/behavioral factors on serological responses to coronaviruses in southern Africa

Seroprofiling of antibodies against endemic human coronaviruses and SARS-CoV-2 in an HIV cohort in Lesotho: correlates of antibody response and seropositivity.

BACKGROUND Serological data on endemic human coronaviruses (HCoVs) and SARS-CoV-2 in southern Africa are scarce. Here, we report on i) endemic HCoV seasonality, ii) SARS-CoV-2 seroprevalence, and iii) predictive factors for SARS-CoV-2 seropositivity and strength of SARS-CoV-2 and HCoV serological response during a 17-month period at the start of the COVID-19 pandemic among adults living with HIV. METHODS Plasma samples were collected from February 2020 to July 2021 within an outpatient HIV cohort in Lesotho. We used the ABCORA multiplex immunoassay to measure antibody responses to endemic HCoV (OC43, HKU1, NL63, and 229E) and SARS-CoV-2 antigens. RESULTS Results of 3'173 samples from 1'403 adults were included. Serological responses against endemic HCoVs increased over time and peaked in winter/spring. SARS-CoV-2 seropositivity reached >35% among samples collected in early 2021 and was associated with female sex (p = 0.004), obesity (p < 0.001), working outside the home (p = 0.02), and recent tiredness (p = 0.005) or fever (p = 0.007). Positive correlations were observed between the strength of response to endemic HCoVs and to SARS-CoV-2, and between older age or obesity and the IgG response to SARS-CoV-2. CONCLUSIONS These results add to our understanding of the impact of biological, clinical, and social/behavioural factors on serological responses to coronaviruses in southern Africa

The HDF-North SCUBA Super-map II: Multi-wavelength properties

We present radio, optical and X-ray detected counterparts to the sub-mm sources found using SCUBA in the Hubble Deep Field North region (GOODS-N). A new counterpart identification statistic is developed to identify properties of galaxies detected at other wavelengths that can be used to aid counterpart identification. We discriminate between criteria that can be used to pre-select sub-mm bright objects, and those that identify the counterpart to a known sub-mm object. Optically faint galaxies detected in the deepest 1.4 GHz radio continuum maps are the only effective way of pre-selecting SCUBA galaxies, and radio sources are the best way to identify counterparts to known sub-mm detections. Looking at radio spectral indices, only the steeper sources (indicative of star formation) are detected in the sub-mm. Although we find several X-ray identifications, we show that deep Chandra images do not contribute to counterpart identifications, since in all cases they are already detected in the more easily obtained VLA radio maps. We also find find no evidence for clustering between Chandra and SCUBA sources in this field. For a known SCUBA position, the reddest source tends to be the correct association, although we can find no cut on colour, magnitude, or clustering property that efficiently pre-selects for SCUBA sources. 15 micron ISO sources are statistically detected by SCUBA, but the limiting mid-IR flux is not low enough to provide useful constraints. We present postage stamp strips for each SCUBA detection in separate bands from X-ray to radio, providing direct visual evidence that approximately half of the sub-mm sources in this field remain unidentified, despite an abundance of deep multi-wavelength data.Comment: Accepted for publication in MNRAS. High resolution version available at http://www.submm.caltech.edu/~borys/paper

Assessing immunogenicity barriers of the HIV-1 envelope trimer

Understanding the balance between epitope shielding and accessibility on HIV-1 envelope (Env) trimers is essential to guide immunogen selection for broadly neutralizing antibody (bnAb) based vaccines. To investigate the antigenic space of Env immunogens, we created a strategy based on synthetic, high diversity, Designed Ankyrin Repeat Protein (DARPin) libraries. We show that DARPin Antigenicity Analysis (DANA), a purely in vitro screening tool, has the capability to extrapolate relevant information of antigenic properties of Env immunogens. DANA screens of stabilized, soluble Env trimers revealed that stronger trimer stabilization led to the selection of highly mutated DARPins with length variations and framework mutations mirroring observations made for bnAbs. By mimicking heterotypic prime-boost immunization regimens, DANA may be used to select immunogen combinations that favor the selection of trimer-reactive binders. This positions DANA as a versatile strategy for distilling fundamental antigenic features of immunogens, complementary to preclinical immunogenicity testing

Comparing the Toxicological Responses of Pulmonary Air–Liquid Interface Models upon Exposure to Differentially Treated Carbon Fibers

In recent years, the use of carbon fibers (CFs) in various sectors of industry has been increasing. Despite the similarity of CF degradation products to other toxicologically relevant materials such as asbestos fibers and carbon nanotubes, a detailed toxicological evaluation of this class of material has yet to be performed. In this work, we exposed advanced air–liquid interface cell culture models of the human lung to CF. To simulate different stresses applied to CF throughout their life cycle, they were either mechanically (mCF) or thermo-mechanically pre-treated (tmCF). Different aspects of inhalation toxicity as well as their possible time-dependency were monitored. mCFs were found to induce a moderate inflammatory response, whereas tmCF elicited stronger inflammatory as well as apoptotic effects. Furthermore, thermal treatment changed the surface properties of the CF resulting in a presumed adhesion of the cells to the fiber fragments and subsequent cell loss. Triple-cultures encompassing epithelial, macrophage, and fibroblast cells stood out with an exceptionally high inflammatory response. Only a weak genotoxic effect was detected in the form of DNA strand breaks in mono- and co-cultures, with triple-cultures presenting a possible secondary genotoxicity. This work establishes CF fragments as a potentially harmful material and emphasizes the necessity of further toxicological assessment of existing and upcoming advanced CF-containing materials

Stepwise Conformational Stabilization of a HIV-1 Clade C Consensus Envelope Trimer Immunogen Impacts the Profile of Vaccine-Induced Antibody Responses.

Stabilization of the HIV-1 Envelope glycoprotein trimer (Env) in its native pre-fusion closed conformation is regarded as one of several requirements for the induction of neutralizing antibody (nAb) responses, which, in turn, will most likely be a prerequisite for the development of an efficacious preventive vaccine. Here, we systematically analyzed how the stepwise stabilization of a clade C consensus (ConC) Env immunogen impacts biochemical and biophysical protein traits such as antigenicity, thermal stability, structural integrity, and particle size distribution. The increasing degree of conformational rigidification positively correlates with favorable protein characteristics, leading to optimized homogeneity of the protein preparations, increased thermal stability, and an overall favorable binding profile of structure-dependent broadly neutralizing antibodies (bnAbs) and non-neutralizing antibodies (non-nAbs). We confirmed that increasing the structural integrity and stability of the Env trimers positively correlates with the quality of induced antibody responses by the immunogens. These and other data contribute to the selection of ConCv5 KIKO as novel Env immunogens for use within the European Union's H2020 Research Consortium EHVA (European HIV Alliance) for further preclinical analysis and phase 1 clinical development

Action-based flood forecasting for triggering humanitarian action

Too often, credible scientific early warning information of increased disaster risk does not result in humanitarian action. With financial resources tilted heavily towards response after a disaster, disaster managers have limited incentive and ability to process complex scientific data, including uncertainties. These incentives are beginning to change, with the advent of several new Forecast-based Financing systems that provide funding based on a forecast of an extreme event. Given the changing landscape, here we demonstrate a method to select and use appropriate forecasts for specific humanitarian disaster prevention actions, even in a data-scarce location. This action-based forecasting methodology takes into account the parameters of each action, such as action lifetime, when verifying a forecast. Forecasts are linked with action based on an understanding of (1) the magnitude of previous flooding events and (2) the willingness to act "in vain" for specific actions. This is applied in the context of the Uganda Red Cross Society Forecast-based Financing pilot project, with forecasts from the Global Flood Awareness System (GloFAS). Using this method, we define the "danger level" of flooding, and we select the probabilistic forecast triggers that are appropriate for specific actions. Results from this methodology can be applied globally across hazards, and fed into a financing system that ensures that automatic, pre-funded early action will be triggered by forecasts