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Background: There is great interest to engineer in vitro models that allow the study of complex biological processes of the microvasculature with high spatiotemporal resolution. Microfluidic systems are currently used to engineer microvasculature in vitro, which consists of perfusable microvascular networks (MVNs). These are formed through spontaneous vasculogenesis and exhibit the closest resemblance to physiological microvasculature. Unfortunately, under standard culture conditions and in the absence of co-culture with auxiliary cells as well as protease inhibitors, pure MVNs suffer from a short-lived stability. Methods: Herein, we introduce a strategy for stabilization of MVNs through macromolecular crowding (MMC) based on a previously established mixture of Ficoll macromolecules. The biophysical principle of MMC is based on macromolecules occupying space, thus increasing the effective concentration of other components and thereby accelerating various biological processes, such as extracellular matrix deposition. We thus hypothesized that MMC will promote the accumulation of vascular ECM (basement membrane) components and lead to a stabilization of MVN with improved functionality. Results: MMC promoted the enrichment of cellular junctions and basement membrane components, while reducing cellular contractility. The resulting advantageous balance of adhesive forces over cellular tension resulted in a significant stabilization of MVNs over time, as well as improved vascular barrier function, closely resembling that of in vivo microvasculature. Conclusion: Application of MMC to MVNs in microfluidic devices provides a reliable, flexible and versatile approach to stabilize engineered microvessels under simulated physiological conditions.

Background There is great interest to engineer in vitro models that allow the study of complex biological processes of the microvasculature with high spatiotemporal resolution. Microfluidic systems are currently used to engineer microvasculature in vitro, which consists of perfusable microvascular networks (MVNs). These are formed through spontaneous vasculogenesis and exhibit the closest resemblance to physiological microvasculature. Unfortunately, under standard culture conditions and in the absence of co-culture with auxiliary cells as well as protease inhibitors, pure MVNs suffer from a short-lived stability. Methods Herein, we introduce a strategy for stabilization of MVNs through macromolecular crowding (MMC) based on a previously established mixture of Ficoll macromolecules. The biophysical principle of MMC is based on macromolecules occupying space, thus increasing the effective concentration of other components and thereby accelerating various biological processes, such as extracellular matrix deposition. We thus hypothesized that MMC will promote the accumulation of vascular ECM (basement membrane) components and lead to a stabilization of MVN with improved functionality. Results MMC promoted the enrichment of cellular junctions and basement membrane components, while reducing cellular contractility. The resulting advantageous balance of adhesive forces over cellular tension resulted in a significant stabilization of MVNs over time, as well as improved vascular barrier function, closely resembling that of in vivo microvasculature. Conclusion Application of MMC to MVNs in microfluidic devices provides a reliable, flexible and versatile approach to stabilize engineered microvessels under simulated physiological conditions.

In soft robotics, the successful development of soft robots involves careful designing that can benefit from current technologies. The use of Finite Element Method (FEM) software and additive manufacturing is essential to optimize the design before fabrication and to facilitate the process. Therefore, we present the design of a 3D printed low-pressure soft pneumatic actuator (SPA) with 3 DoF and a material characterization method to simulate the behaviour of the system. In attempt to define a suitable material modelling method and its reliability to simulate actuator behaviours, we introduce a characterization method and corroborate its efficiency through the evaluation of the performance using the FEM and preliminary tests of the actuator performance. The purpose of this article is to help future projects to effectively simulate the behaviour of 3D printed soft pneumatic actuators to improve the design before fabrication. Throughout the description of the process to effectively fabricate a functional SPA.

Glucuronosyl diacylglycerides (GlcAGroAc2) are functionally important glycolipids and membrane anchors for cell wall lipoglycans in the Corynebacteria. Here we describe the complete synthesis of distinct acyl-isoforms of GlcAGroAc2 bearing both acylation patterns of (R)-tuberculostearic acid (C19:0) and palmitic acid (C16:0) and their mass spectral characterization. Collision-induced fragmentation mass spectrometry identified characteristic fragment ions that were used to develop “rules” allowing the assignment of the acylation pattern as C19:0 (sn-1), C16:0 (sn-2) in the natural product from Mycobacterium smegmatis, and the structural assignment of related C18:1 (sn-1), C16:0 (sn-2) GlcAGroAc2 glycolipids from M. smegmatis and Corynebacterium glutamicum. A synthetic hydrophobic octyl glucuronoside was used to characterize the GDP-mannose-dependent mannosyltransferase MgtA from C. glutamicum that extends GlcAGroAc2. This enzyme is an Mg2+/Mn2+-dependent metalloenzyme that undergoes dramatic activation upon reduction with dithiothreitol.

Glucuronosyl diacylglycerides (GlcAGroAc2) are functionally important glycolipids and membrane anchors for cell wall lipoglycans in the Corynebacteria. Here we describe the complete synthesis of distinct acyl-isoforms of GlcAGroAc2 bearing both acylation patterns of (R)-tuberculostearic acid (C19:0) and palmitic acid (C16:0) and their mass spectral characterization. Collision-induced fragmentation mass spectrometry identified characteristic fragment ions that were used to develop “rules” allowing the assignment of the acylation pattern as C19:0 (sn-1), C16:0 (sn-2) in the natural product from Mycobacterium smegmatis, and the structural assignment of related C18:1 (sn-1), C16:0 (sn-2) GlcAGroAc2 glycolipids from M. smegmatis and Corynebacterium glutamicum. A synthetic hydrophobic octyl glucuronoside was used to characterize the GDP-mannose-dependent mannosyltransferase MgtA from C. glutamicum that extends GlcAGroAc2. This enzyme is an Mg2+/Mn2+-dependent metalloenzyme that undergoes dramatic activation upon reduction with dithiothreitol.

Mycobacterium tuberculosis, the causitive agent of tuberculosis, possesses a complex cell wall containing mannose-rich glycophospholipids termed phosphatidylinositol mannosides (PIMs), lipomannan (LM), and lipoarabinomannan (LAM). These glycophospholipids are believed to play important roles in cell wall function and hostpathogen interactions. Synthetic PIM/LM/LAM substructures are useful biochemical tools to delineate and dissect the fine details of mannose glycophospholipid biosynthesis and their interactions with host cells. The first part of this thesis describes the efficient synthesis of a series of azidooctyl di- and trimannosides possessing the following glycan structures: alpha-Man-1,6-alpha-Man, alpha-Man-1,6-alpha-Man-1,6-alpha-Man, alpha-Man-1,2-alpha-Man-1,6-alpha-Man and 2,6-di-(alpha-Man)-alpha-Man. Highlights of the synthesis include the use of nonbenzyl protecting groups compatible with the azido group and preparation of the branched trisaccharide structure 2,6-di-(alpha-Man)-alpha-Man through a double glycosylation of a 3,4-butanediacetal-protected mannoside. The hydrophobic azidooctyl aglycon of these compounds confers useful lipophilicity allowing simple extraction of enzymatic products from reaction mixtures. The azidooctyl groups of these synthetic mannans were elaborated to fluorescent neoglycoconjugates and haptens for the study of mycobacterial glycolipid biosynthesis and antigenicity. The second part of this thesis describes the use of synthetic mycobacterial glycolipid antigens in defining a new population of natural killer T (NKT) cells. NKT cells are a small subpopulation of T lymphocytes that recognize glycolipid antigens when presented by the antigen presenting protein CD1d. The most widely studied subset of NKT cells are termed type I NKT cells and are characterized by the expression of an invariant T cell receptor Valpha14-alpha-chain in mice. When activated, NKT cells produce significant amounts of pro-inflammatory TH1 cytokines and