Progressive Hypoxia-on-a-chip: An In Vitro Oxygen Gradient Model for Capturing the Effects of Hypoxia on Primary Hepatocytes in Health and Disease

Oxygen is vital to the function of all tissues including the liver and lack of oxygen, that is, hypoxia can result in both acute and chronic injuries to the liver in vivo and ex vivo. Furthermore, a permanent oxygen gradient is naturally present along the liver sinusoid, which plays a role in the metabolic zonation and the pathophysiology of liver diseases. Accordingly, here, we introduce an in vitro microfluidic platform capable of actively creating a series of oxygen concentrations on a single continuous microtissue, ranging from normoxia to severe hypoxia. This range approximately captures both the physiologically relevant oxygen gradient generated from the portal vein to the central vein in the liver, and the severe hypoxia occurring in ischemia and liver diseases. Primary rat hepatocytes cultured in this microfluidic platform were exposed to an oxygen gradient of 0.3–6.9%. The establishment of an ascending hypoxia gradient in hepatocytes was confirmed in response to the decreasing oxygen supply. The hepatocyte viability in this platform decreased to approximately 80% along the hypoxia gradient. Simultaneously, a progressive increase in accumulation of reactive oxygen species and expression of hypoxia‐inducible factor 1α was observed with increasing hypoxia. These results demonstrate the induction of distinct metabolic and genetic responses in hepatocytes upon exposure to an oxygen (/hypoxia) gradient. This progressive hypoxia‐on‐a‐chip platform can be used to study the role of oxygen and hypoxia‐associated molecules in modeling healthy and injured liver tissues. Its use can be further expanded to the study of other hypoxic tissues such as tumors as well as the investigation of drug toxicity and efficacy under oxygen‐limited conditions

Engineering Biomolecular Interfaces for Applications in Biotechnology

Protein interactions occurring through biomolecular interfaces play an important role in the circle of life. These interactions are responsible for cellular function, including RNA transcription, protein translation, cell division and cell death among many others. There are different types of interactions based on the strength and the duration of the association. Transient interactions govern most steps of the cellular metabolism, where the associations between two or more molecules are responsive to environmental cues. Among the participants of transient interactions, intrinsically disordered proteins are employed in signaling and other regulatory events within the cell. These proteins exhibit allosteric regulation and gain secondary structure when they bind other proteins or small molecules. In this doctoral thesis work, the biochemical and biophysical principals governing protein associations are investigated and using protein engineering tools, novel biomolecular interfaces are engineered, with potential applications in different areas of biotechnology. The first part of the thesis (Chapter 2) focuses on the investigation of supramolecular enzyme association among tricarboxylic acid cycle enzymes, specifically between citrate synthase and mitochondrial malate dehydrogenase. In this study, the interactions between these enzymes are examined, both among their natural and synthetically produced recombinant versions. In addition, mutational analysis of the amino acid residues at the complex interface was performed to explore the importance of the positively charged patch connecting the active sites of the enzymes. It was discovered that the channeling of the negatively charged intermediate is severely impaired upon mutation of surface residues contributing to the electrostatic channeling. This work provides an important insight into understanding the coupled reaction-transport systems and metabolon formation in general. In addition, it constitutes a great example for substrate channeling in leaky systems, which are relevant to most biological processes. The next section of the thesis (Chapter 3) focuses on an intrinsically disordered peptide, the β-roll. This peptide is isolated from the Block V repeats-in-toxin (RTX) domain of adenylate cyclase from Bordetella pertussis. It is disordered in the absence of calcium and it folds into a β-roll secondary structure composed of two parallel β-sheet faces upon binding to calcium ions. This way, the peptide can transition between its unfolded state and the β-roll structure in a reversible way. We have utilized the allosteric regulation of this domain as a tool to engineer new protein interfaces. In its folded state, the peptide has two faces, serving as binding surfaces available for interaction with other proteins. Our work involved the alteration of the residues, which form these faces upon calcium binding, via combinatorial protein design techniques. The potential of this peptide is evaluated as a cross-linking domain for hydrogel formation. By rationally engineering the two faces of the folded β-roll to contain leucine residues, we have created hydrophobic interfaces, serving as environmentally-responsive cross-linking domains. When there is no calcium, the β-roll domains remain unstructured, delocalizing the leucine rich patches. After calcium binding, the β-rolls fold and the leucine rich faces are exposed creating a hydrophobic driving force for self-assembly. This way, we showed that the β-roll peptide can function as a biomaterials building block capable of proteinaceous hydrogel formation, only in the presence of calcium. The next study (Chapter 4) demonstrates the utilization of this peptide as an alternative scaffold for biomolecular recognition applications. A library of mutant β-rolls was constructed by randomizing the amino acid residues on one of the β-sheet forming faces. Mutant peptides demonstrating an affinity for hen egg white lysozyme were selected, which was chosen as a model target molecule. The thermodynamic parameters of the interactions between the β-roll mutants and the lysozyme were quantified. Upon performing further protein engineering (e.g. concatenation of the single mutants on the DNA level), a mutant with mid-nanomolar affinity was identified. Affinity chromatography experiments showed that this mutant was capable of capturing the target, in the presence of calcium. The captured target was easily released upon removal of the calcium ions. The reversibility of the calcium binding allowed the engineered molecular interface to be controllable. Throughout this study, the β-roll peptide was explored as an allosterically-regulated protein switch for on/off biomolecular recognition, which can be mediated by simply changing the calcium concentration, allowing control over the binding behavior between molecules. The last part of the thesis (Chapter 5) expands on the calcium dependent network formation study. A hydrogel construct was genetically built by fusing the cross-linking β-roll domain and the lysozyme binding β-roll mutant, resulting in a smart biomaterial with dual-functionality. The network-assembly and target capture functions of this construct were tested by various assays including hydrogel erosion experiments. This allosterically-regulated biomaterial exhibited promising results, where calcium-dependent lysozyme entrapment within the assembled network and lysozyme capture on the hydrogel surface were demonstrated. The work presented in this thesis demonstrates different approaches to understand and engineer molecular interfaces in both natural and recombinant systems. In the future, these approaches and the knowledge gained from these studies can be further built upon for different biotechnological applications and can also be applied to other synthetic systems

Legendre pairs of lengths ℓ≡0\ell\equiv0 (mod 5)

Compression of complementary sequences has been proved to be a valuable tool to discover several new complementary sequences of various kinds in the past decade. Compression based search algorithms encompass a two-stage process, that typically involves the computation of several candidate compression sequences, followed by the computationally expensive decompression phase. In this paper we show how to shorten the former phase in the case of Legendre pairs of lengths $\ell\equiv0$ (mod 5), using a plausible conjecture, supported by overwhelming numerical evidence. This is achieved by significantly decreasing the number of candidate compression sequences by using a sums-of-squares Diophantine equation. We verify our conjecture for all odd values from 3 to 17. In particular, this allows us to exhibit the first known examples of Legendre Pairs of length 85, which has been the smallest open length. We also find Legendre pairs of the open length 87 by assuming a balanced compression. As a consequence, there remains eleven lengths less than 200 for which the question of existence of a Legendre pair remains open

Design of Experiments for Screening

The aim of this paper is to review methods of designing screening experiments, ranging from designs originally developed for physical experiments to those especially tailored to experiments on numerical models. The strengths and weaknesses of the various designs for screening variables in numerical models are discussed. First, classes of factorial designs for experiments to estimate main effects and interactions through a linear statistical model are described, specifically regular and nonregular fractional factorial designs, supersaturated designs and systematic fractional replicate designs. Generic issues of aliasing, bias and cancellation of factorial effects are discussed. Second, group screening experiments are considered including factorial group screening and sequential bifurcation. Third, random sampling plans are discussed including Latin hypercube sampling and sampling plans to estimate elementary effects. Fourth, a variety of modelling methods commonly employed with screening designs are briefly described. Finally, a novel study demonstrates six screening methods on two frequently-used exemplars, and their performances are compared

The social implications of Covid-19 on Communities

This report reviews the social implications of Covid-19 on communities in the UK. It is organised into five principal parts, with questions which have guided the narrative of our enquiry as follows: The nature of community ● How has community support mobilised in response to Covid-19? ● What are the implications of how community support has mobilised on: ○ Social infrastructure and its role in delivering urgent intervention, and longer- term policy led, priorities ○ Our understanding of the role of community in times of acute, and increasingly protracted, crisis ● How has/should the relationship between residents, volunteers, community-based organisations, special issue charities, service providers and local authorities be(en) reconfigured in response to the crisis? Changing places? ● How has Covid-19 affected understanding of ‘place-based community’ in the UK context? ● How has Covid-19 changed the nature of communities (in terms of both their demographic composition, spatial and geopolitical parameters, and the ways in which they are defined/measured through policy, practice and research)? Inequalities ● How has Covid-19 affected inequalities between different demographic groups within communities (e.g. shielding) ● How has Covid-19 affected inequalities between different communities characterised by different demographic groups? Social cohesion ● How has C19 affected different people’s sense of community? ● How do different ideas of cohesion include/exclude certain groups? ● Why do different groups of people feel more/less connected? ● How does trust/compliance with regulations relate to community cohesion? Trust ● What does the nature of volunteering tell us about differences within/across communities, and the relationship between people and the state? ● How have the effects of Covid-19 and the resilience it has produced in local communities been distributed according to different local environments, infrastructures and economies? ● How have wider issues of trust relating to the media, government and science changed from a community perspective

A Microfluidic Patterned Model of Non-Alcoholic Fatty Liver Disease: Applications to Disease Progression and Zonation

Non-alcoholic fatty liver disease (NAFLD) and its progressive form non-alcoholic steatohepatitis (NASH) affect 25% of the world population. NAFLD is predicted to soon become the main cause of liver morbidity and transplantation. The disease is characterized by a progressive increase of lipid accumulation in hepatocytes, which eventually induce fibrosis and inflammation, and can ultimately cause cirrhosis and hepatic carcinoma. Here, we created a patterned model of NAFLD on a chip using free fatty acid gradients to recapitulate a spectrum of disease conditions in a single continuous liver tissue. We established the NAFLD progression via quantification of intracellular lipid accumulation and transcriptional levels of fatty acid transporters and NAFLD pathogenesis markers. We then used this platform to create oxygen driven steatosis zonation mimicking the sinusoidal lipid distribution on a single continuous tissue and showed that this fat zonation disappears under progressed steatosis, in agreement with in vivo observations and recent computational studies. While we focus on free fatty acids and oxygen as the drivers of NAFLD, the microfluidic platform here is extensible to simultaneous use of other drivers

Exploiting Symmetry in Integer Convex Optimization using Core Points

We consider convex programming problems with integrality constraints that are invariant under a linear symmetry group. To decompose such problems we introduce the new concept of core points, i.e., integral points whose orbit polytopes are lattice-free. For symmetric integer linear programs we describe two algorithms based on this decomposition. Using a characterization of core points for direct products of symmetric groups, we show that prototype implementations can compete with state-of-the-art commercial solvers, and solve an open MIPLIB problem.Comment: 15 pages; small changes according to suggestions of a referee; to appear in Operations Research Letter

Evaluation of Clinical and Immunological Alterations Associated with ICF Syndrome

Purpose: Immunodeficiency with centromeric instability and facial anomalies (ICF) syndrome is a rare autosomal recessive combined immunodeficiency. The detailed immune responses are not explored widely. We investigated known and novel immune alterations in lymphocyte subpopulations and their association with clinical symptoms in a well-defined ICF cohort. Methods: We recruited the clinical findings from twelve ICF1 and ICF2 patients. We performed detailed immunological evaluation, including lymphocyte subset analyses, upregulation, and proliferation of T cells. We also determined the frequency of circulating T follicular helper (cTFH) and regulatory T (Treg) cells and their subtypes by flow cytometry. Results: There were ten ICF1 and two ICF2 patients. We identified two novel homozygous missense mutations in the ZBTB24 gene. Respiratory tract infections were the most common recurrent infections among the patients. Gastrointestinal system (GIS) involvements were observed in seven patients. All patients received intravenous immunoglobulin replacement therapy and antibacterial prophylaxis; two died during the follow-up period. Immunologically, CD4+ T-cell counts, percentages of recent thymic emigrant T cells, and naive CD4+ T decreased in two, five, and four patients, respectively. Impaired T-cell proliferation and reduced CD25 upregulation were detected in all patients. These changes were more prominent in CD8+ T cells. GIS involvements negatively correlated with CD3+ T-, CD3+CD4+ T-, CD16+CD56+ NK-cell counts, and CD4+/CD8+ T-cell ratios. Further, we observed expanded cTFH cells and reduced Treg and follicular regulatory T cells with a skewing to a TH2-like phenotype in all tested subpopulations. Conclusion: The ICF syndrome encompasses various manifestations affecting multiple end organs. Perturbed T-cell responses with increased cTFH and decreased Treg cells may provide further insight into the immune aberrations observed in ICF syndrome