Fluid Patterning in a Cavity Array for High-Throughput Screening and Biotechnological Applications

Biomedical EngineeringOver the past few decades, fundamental biological understanding has advanced significantly with the help of experimental biotechnologies. Among them, synthetic biology as a rising research field has shown its capability for building new microorganisms from the scratch. In conjunction with synthetic biology, directed evolution techniques seem to be highly useful for industrial purposes such as the over-production of chemical products such as biofuels, which are expected to resolve global energy problems. However, it still requires a high-throughput screening technique and/or compartmentalized environments for cell sorting. In this thesis, two microfluidic technologies are described. First, a novel microdroplet trapping technology is developed that utilizes the difference of specific gravity between two immiscible fluids to offer simple and easy manipulation of microdroplets for time-traceable single microorganism analysis. Second, a high-throughput screening technology is developed by patterning fluid, in which individual Escherichia coli cells can be immobilized and cultured in a cavity array format. It is noted that the cavities were coated with parylene and bonded with another parylene layer to secure chemical compatibility. In addition, it was successfully demonstrated that the two technologies hold a high potential to enable not only high-throughput screening but also many biological experiments such as detection of cell-excreted products, long-term cell incubation, cell to cell communication, and detection of target molecules via a whole cell biosensor.ope

MICROFLUIDIC APPROACHES FOR QUANTITATIVE ANALYSIS AND SCREENING OF SYNTHETICALLY ENGINEERED MICROBES

Department of Biomedical EngineeringRecently, our understanding of complex genomes, proteomes, bio-molecules, and even many metabolic pathways has been developed significantly as growing a fundamental knowledge of the biochemistry of life. These newly revealed findings have incredibly influenced in recent bio-technologies, such as synthetic biology having massive potential to solve the missing connected dots. To have better understanding of microbe, microfluidic approaches were innovatively introduced to the field with the potential to revolutionize high-throughput biological assays. In this study, suggested approach to address limitations of conventional microbiology is microfluidics integrated with synthetic biology. At first, the microbial biosensors will be introduced into microfluidic ratchet platform for a quantitative analysis of microbial bio-signal. The microfluidic device using microfabricated arrowhead-shaped ratchet structures has an intrinsic function that concentrates motile microbes in a microchamber array. Additionally, the ratchet structure provides the concentrated microbes to grow better in a continuous-feed mode. A continuous exposure of detection analytes leads the amplification of fluorescence signal from microbes in a microchamber. Therefore, it was noted that the substantial amplification of bio-signal was achieved from the microfluidic device and measured signals were analyzed in quantitative manner. As a second practical application of the microfluidic approach, for high-throughput screening (HTS) application, a fluid array will be developed by using immiscible character between water and oil for microbial incubation, analysis, selective extraction, recovery process and the demonstration of practical applications. From the characterization of the fluid array platform, HTS will be demonstrated based on two different categories: reporter-gene basis and growth complementation basis. The fluid array device showed not only demonstrations of high-throughput screening, but also advanced screening applications were also demonstrated with higher mutant library screening with 106 and C2C communication screening system. Outstanding mutants were sophisticatedly screened among 106 of a mutant library based on the hybrid type screening method. Also, the proposed C2C screening approach has enabled high-throughput compartmentalization and resulted in 10 possible mutants showing higher extracellular biomolecule secretion performance. The proposed microfluidic approaches can be practically useful combinations showing many advantages: 1) economical and reduced time requirement for real application without complex instruments, 2) facile potentials to enable a multiplex quantitative analysis in a high-throughput manner, and 3) selective, direct and convenient measurement without pre- or post-treatment of sample solutions in near future the entire processes could be fully automated. In this dissertation, different type of microfluidic devices was developed for various collaborative purposes for the bottleneck of conventional microbiology. Therefore, the microfluidic devices have knocked a new door for high-throughput screening application for synthetically engineered microorganisms and quantitative approaches for microbial biosensors. Thus, the research contributions in this doctoral dissertation are the microfluidic approaches to popularize and overcome conventional constraints from biological experimental tools by integration of total analysis system for synthetically engineered microbe cases.ope

SIMPLY PLEASING AND UNIQUELY ORIGINAL: UNCOVERING AND RE-EVALUATING THE VALUE OF SOLO PIANO WORKS BY FRANCIS POULENC

Francis Poulenc, though highly regarded by many musicians for his vocal and chamber works, is not considered one of the “mainstream” composers in the world of piano music. Though praised by many musicologists and music critics for his beautiful melodies and uniquely individual writing style, Poulenc’s solo piano works have not been a major part of the recital repertoire. While searching for some less technically-demanding recital pieces due to the recent recovery of a shoulder injury, I found hidden gems of this great composer. His solo piano works are delightfully pleasing but also shockingly eccentric, light-hearted and humorous but also warm and solemn. This fascinating juxtaposition of completely contrasting ideas led to more serious research, and I began to question why his solo piano pieces are not performed more regularly. The purpose of this project is to discuss the little-known solo piano compositions of Poulenc and to re-evaluate their significance as part of classical piano literature. Possibly eclipsed by the popularity of the music by Debussy and Ravel or even by his own vocal and larger scale works, or perhaps because of the lack of virtuosity and bold emotional expression, Poulenc’s solo piano works have largely been neglected by many pianists. Focusing mainly on three relatively early compositions, Suite en ut, Huit Nocturnes, and Suite française, I hope to emphasize the value of his solo piano music through thorough examination and musical analysis using articles, interviews, diaries, and letters written by Poulenc and his contemporaries

High-Throughput Screening of Acyl-CoA Thioesterase I Mutants Using a Fluid Array Platform

Screening target microorganisms from a mutated recombinant library plays a crucial role in advancing synthetic biology and metabolic engineering. However, conventional screening tools have several limitations regarding throughput, cost, and labor. Here, we used the fluid array platform to conduct high-throughput screening (HTS) that identified Escherichia coli ???TesA thioesterase mutants producing elevated yields of free fatty acids (FFAs) from a large (106) mutant library. A growth-based screening method using a TetA-RFP fusion sensing mechanism and a reporter-based screening method using high-level FFA producing mutants were employed to identify these mutants via HTS. The platform was able to cover >95% of the mutation library, and it screened target cells from many arrays of the fluid array platform so that a post-analysis could be conducted by gas chromatography. The ???TesA mutation of each isolated mutant showing improved FFA production in E. coli was characterized, and its enhanced FFA production capability was confirmed

Intravitreal Bevacizumab (Avastin) as an Adjuvant for the Treatment of Posterior Scleritis

We report a case of posterior scleritis effectively managed with intravitreal bevacizumab. A 71-year-old woman was diagnosed with posterior scleritis. Although she was initially treated with systemic steroids, her clinical presentation deteriorated. She was then treated with a single intravitreal injection of bevacizumab and aqueous humor collection. The aqueous level of vascular endothelial growth factor prior to the intravitreal injection was 880.51 pg/mL, greater than that in the healthy control group (p < 0.001). One month later, the scleritis was completely resolved, and the patient remained stable during six months of follow-up. Intravitreal bevacizumab appears to be an effective adjuvant therapy for patients with posterior scleritis

Immediate implant placement in conjunction with guided bone regeneration and/or connective tissue grafts: an experimental study in canines

PURPOSE This study was conducted to assess the effect of hard and/or soft tissue grafting on immediate implants in a preclinical model. METHODS In 5 mongrel dogs, the distal roots of P2 and P3 were extracted from the maxilla (4 sites in each animal), and immediate implant placement was performed. Each site was randomly assigned to 1 of the following 4 groups: i) gap filling with guided bone regeneration (the GBR group), ii) subepithelial connective tissue grafting (the SCTG group), iii) GBR and SCTG (the GBR/SCTG group), and iv) no further treatment (control). Non-submerged healing was provided for 4 months. Histological and histomorphometric analyses were performed. RESULTS Peri-implant tissue height and thickness favored the SCTG group (height of peri-implant mucosa: 1.14 mm; tissue thickness at the implant shoulder and ±1 mm from the shoulder: 1.14 mm, 0.78 mm, and 1.57 mm, respectively; median value) over the other groups. Bone grafting was not effective at the level of the implant shoulder and on the coronal level of the shoulder. In addition, simultaneous soft and hard tissue augmentation (the GBR/SCTG group) led to a less favorable tissue contour compared to GBR or SCTG alone (height of peri-implant mucosa: 3.06 mm; thickness of peri-implant mucosa at the implant shoulder and ±1 mm from the shoulder: 0.72 mm, 0.3 mm, and 1.09 mm, respectively). CONCLUSION SCTG tended to have positive effects on the thickness and height of the peri-implant mucosa in immediate implant placement. However, simultaneous soft and hard tissue augmentation might not allow a satisfactory tissue contour in cases where the relationship between implant position and neighboring bone housing is unfavorable

Augmentation of keratinized tissue at tooth and implant sites by using autogenous grafts and collagen-based soft-tissue substitutes

AIM To investigate the effect of three treatment modalities on the gain of keratinized tissue (KT) at tooth and implant sites in dogs. MATERIALS AND METHODS In five dogs, the distal roots of the mandibular second, third and fourth premolars were extracted, while the mesial roots were maintained. After 2 months of healing, implants were placed with KT excision. After another 2 months of healing, free gingival grafts, collagen-based matrices and apically positioned flap only were applied. The height of KT was measured during implant placement, immediately before soft-tissue grafting and after 10, 30 and 60 days. RESULTS Two months after KT excision, spontaneous KT regrowth was greater at tooth sites than at implant sites (median, 2.0 mm vs. 1.1 mm). The outcomes of soft-tissue grafting at implant sites favoured the free gingival graft treatment, with a greater final median height (5.0-5.5 mm) and increase in KT (4.0-4.2 mm). Locations of the recipient sites significantly influenced KT regeneration at both tooth and implant sites. CONCLUSIONS At implant sites, the free gingival graft treatment led to higher KT regeneration. At tooth sites, however, the differences between the three treatment modalities seemed clinically irrelevant