Development, characterization and application of momentum dominated jet microfluidics

Resto, Pedro J The University of Wisconsin - Madison 2012 해외박사(DDOD)

Microfluidics is the scientific field that deals with the study and control of small volumes of liquid. Microfluidics has found its way into many areas of science including biotechnology, the life sciences, defense, public health and pharmacology. One of the most important aspects of microfluidics is the ability to move and control liquid at the micro scale. The laws of physics at the micro scale are different from the laws of physics at the macro scale. For example, the macro scale is dominated by momentum, turbulence and gravity whereas surface tension, inter-atomic forces, capillary action, viscosity, laminar flow, diffusion, fluidic resistance and surface area to volume ratios dominate the micro scale. The research community has taken advantage of these micro scale forces to perform tasks not possible at the macro scale. However, there exists a transition zone between the micro and macro scale where both inertia and viscosity of the fluid are finite, between Stokes flow and inviscid flow, so that inertia or viscosity cannot be discounted. This thesis work is on the characterization of jet microfluidics; this is the control of liquid at the micro scale using inertia. We define two flow regimes: inertia enhanced passive pumping and inertia actuated flow. We then apply our microfluidic technology to electrophysiology where we develop of a new high-throughput drug discovery tool using jet microfluidics and patch clamping.

Chromosome loss ZUO1/MPP11 regulates levels of an essential yeast kinetochore protein (Ctf13p) and may contribute to the progression of head and neck squamous cell cancer

Resto, Vicente Andres The Johns Hopkins University 2000 해외박사(DDOD)

Kinetochore assembly and function are essential for segregation of chromosomes. In order to study the kinetochore, a genetic screen was designed to identify mutations in new kinetochore components that are synthetically lethal with a mutation in <italic>CTF13</italic>, a gene encoding for an essential centromere binding protein. Synthetic lethal mutants were also screened for increased chromosome loss (ctf phenotype). Three mutants displayed both synthetic lethality and ctf phenotypes and one, <italic>ksl-3</italic>, was chosen for further study. In isolation, the <italic>ksl-3</italic> mutation causes a cold sensitive phenotype and increased rates of missegregation of a non-essential marker chromosome. Upon cloning and sequencing, the mutated gene proved to be <italic> ZUO1</italic>, a non-essential gene that encodes a putative Z-DNA binding protein of unknown function. Sequencing of the mutant genomic locus revealed an amber nonsense mutation. A role for <italic>ZUO1</italic> in kinetochore function was further explored by preparing double mutants containing a <italic>zuo1</italic> null allele in combination with mutant alleles of genes encoding known kinetochore proteins (<italic>CTF13, NDC10, CBF1</italic>) or selected cis-acting mutations in the three conserved centromere elements (CDE I, II, and III). Genetic analysis of a <italic>zuo1</italic> null allele showed synthetic lethality with <italic> ctf13–30</italic>, synthetic slow growth with <italic>ndc10–42 </italic> and <italic>cbf1-Δ1</italic>, synergistic chromosome loss with <italic>ndc10–42</italic> and <italic>CDEII-Δ31</italic>, and suppression of the chromosome loss phenotype of the <italic>CDEI-3G</italic> mutation. Furthermore, kinetochore integrity is weakened in a <italic>zuo1 </italic> null mutant as assayed by relaxation of a centromere DNA-mediated transcriptional block <italic>in vivo</italic>. To examine whether Zuo1p formed part of the yeast kinetochore complex, an <italic>in vitro</italic> gel retardation assay that assesses the CBF3 centromere-binding activity was used to probe for the presence of Zuo1p. The latter revealed that Zuo1p was not a part of the CBF3 complex and that a <italic>zuo1</italic> null mutant did not qualitatively affect bandshift activity. Immunolocalization experiments showed Zuo1p is present in the cytoplasm as well as the nucleus, thus suggesting an indirect role in kinetochore function. Since <italic>CTF13</italic> has been shown to be dosage sensitive, we examined the levels of Ctf13p in a <italic>zuo1</italic> null strain as compared to wild type. Ctf13 protein was not detectable in the former. We show this effect is, at least in part, due to decreased <italic>CTF13</italic> mRNA levels. Based on these data, we present a model that suggests Zuo1p is important in the transcriptional regulation of <italic>CTF13</italic>. In addition, bioinformatic techniques were used to identify <italic> MPP11</italic> as a human homologue of <italic>ZUO1</italic>. <italic>MPP11 </italic> was mapped to the cytogenetic interval 7q22–7q31.1. Interestingly, this genomic interval is frequently deleted in numerous cancer types. We thus analyzed primary head and neck squamous cell tumors (HNSCC) for loss of heterozygosity/allelic imbalance (LOH/AI) at the <italic>MPP11</italic> genomic locus. Thirty eight percent of tumors examined displayed LOH/AI involving the <italic>MPP11</italic> genomic locus. Mutation analysis of <italic>MPP11</italic> in the latter samples did not identify any inactivating mutations. However, immunohistochemical staining of primary tumor sections revealed a tumor-specific high level of expression of Mpp11p. FISH analysis done on cell lines identified increased chromosome 7 copy number with a concomitant increase in <italic>MPP11</italic> copy number. These results suggest an oncogenic role for <italic>MPP11</italic> in HNSCC.

Modeling Human Epiblast Morphogenesis

Resto, Agnes M University of Michigan ProQuest Dissertations & Th 2023 해외박사(DDOD)

The development of the human embryo is arguably the most complex process that we could care to study. In this process, the developing embryo must undergo proliferation, reorganization, lineage diversification, and dozens of cell fate specification events. During this time, a myriad of events are happening in parallel at the cell level, each one setting the foundation for the emergence of increasingly complex tissues of increasingly complex function. Understanding the mechanisms guiding these processes is pivotal not only for embryogenesis-related applications in fertility and development, but also for regenerative medicine applications such as the development of organ replacements.In this dissertation, I propose an integrative approach to the study of morphogenesis and patterning, specifically in the context of stem cell-based models of human development. Firstly, I present a novel machine learning-assisted imaging pipeline that permits the careful characterization of cell-level events occurring in our in vitro model of epiblast cyst morphogenesis. Secondly, I present a novel agent-based model (ABM)-genetic algorithm (GA) framework for the generation of models of morphogenesis. The framework was first tested to determine its ability to generate structures of desired patterns. It was then applied for the generation of models that plausibly capture mechanisms at work during epiblast cyst morphogenesis and symmetry breaking. With preliminary in silico experiments, I showed that the framework was able to output models that partially captured the effect of initial cell number on final cyst composition. I further showed that correct structure formation was heavily impacted by just a few model parameters. Combined with in vitro experimentation, these tools have the potential to shed light into the mechanisms guiding growth, movement, and cell fate specification in in vitro models of human development.