דניאל מאַרלין׃ דרײַ לידער

Long noncoding RNAs (lncRNAs) have been described in cell lines and various whole tissues, but lncRNA analysis of development in vivo is limited. Here, we comprehensively analyze lncRNA expression of the adult mouse subventricular zone neural stem cell lineage. We utilize complementary genome-wide techniques including RNA-seq, RNA CaptureSeq, and ChIP-seq to associate specific lncRNAs with neural cell types, developmental processes, and human disease states. By integrating data from chromatin state maps, custom microarrays, and FACS purification of the subventricular zone lineage, we stringently identify lncRNAs with potential roles in adult neurogenesis. shRNA-mediated knockdown of two such lncRNAs, Six3os and Dlx1as, indicate roles for lncRNAs in the glial-neuronal lineage specification of multipotent adult stem cells. Using our lncRNA pipeline, we identify Pinky (Pnky) as a long non-coding RNA (lncRNA) that regulates the transition between NSCs and neurogenic progenitors. Pnky is expressed in NSC populations and becomes downregulated during neurogenesis. shRNA-mediated knockdown of Pnky expands the pool of neurogenic progenitors, increasing neurogenesis. Pinky is conserved and expressed in NSCs of the developing mouse and human cortex. In embryonic mouse brain, Pnky knockdown accelerates neurogenesis and depletes this embryonic NSC population. Pnky physically interacts with PTBP1, a regulator of mRNA splicing, and Pnky depletion leads to the differential splicing of PTBP1 targets. These data thus identify Pinky as a conserved lncRNA that regulates a critical stage of neurogenesis from NSCs in the adult and embryonic brain. Taken together, our data forms the foundation of a resource for the study of lncRNAs in development and disease. In depth study of the novel lncRNA Pnky highlights the utility of this resource for the identification of lncRNAs that play key roles in neural lineage progression.

Providing effective healthcare to underrepresented populations is a major challenge worldwide due to environmental, economic, and social disparities. Both developing countries and low income rural/urban areas in the US tend to have reduced healthcare access which limits the frequency and quality of medical screenings as well as treatment options. This has led to an increased effort in the development of point-of-care devices which emphasize, immediate and onsite results. One device that has the potential to fulfill these requirements is the lateral-flow immunoassay (LFA). While the LFA has been successful in the form of the over-the-counter pregnancy test, it still does have some disadvantages that limit its ability to fully replace sophisticated laboratory-based assays. Throughout this thesis, I will be discussing the development of cutting-edge technologies to address the two major disadvantages of the LFA which are its poor quantitative characteristics and low sensitivity.Cerebrospinal fluid (CSF) leaks are a common complication of numerous procedures in Otolaryngology. These leaks are relatively difficult to diagnose and the current laboratory-based tests to do so have a turn-around time that is unacceptable for rapid clinical decision making. We addressed this problem by developing the first rapid diagnostic test kit for the detection of CSF leaks. In Chapter 2, we describe the development of a barcode-style LFA for the semi-quantitative detection of beta-trace protein, which is present in CSF at high concentrations. This work was expanded upon in Chapter 3, where we created a complete, standalone rapid diagnostic test kit composed of our barcode-style LFA, a collection swab, dilution buffers, disposable pipettes, and instructions. Clinical studies demonstrated excellent predictive capabilities of this kit in distinguishing patient samples containing CSF from those that did not. In some cases, a more quantitative readout than what the barcode-style LFA can achieve is required. In Chapter 4, we developed a new technique to introduce an even more quantitative readout to the LFA without the need of electronic readers. This technology was applied to the therapeutic drug monitoring of the cardiac glycoside digoxin. Digoxin capture and binding occurs on a modified LFA test strip which utilizes platinum nanozyme probes that possess catalase-like activity. This test strip is combined with a reaction that controls the anisotropic etching of gold nanorods to produce a wide range of visible color hues dependent on the initial digoxin concentration in the sample which allowed for naked-eye quantification. We then shifted to the development of technologies to improve the detection limit and sensitivity of the LFA. Our lab previously discovered the phenomenon of an aqueous two-phase system (ATPS) separating on paper, which allowed for the seamless integration of biomarker preconcentration and detection on the LFA. In Chapter 5, we have extended the functionality of an ATPS separating on paper to automate the sequential delivery of signal enhancement reagents in addition to concentrating biomarkers. The timing of reagent delivery was controlled by changing the initial composition of the ATPS and a mathematical model was developed to help predict the ATPS flow behavior. We applied this technology to automate biomarker preconcentration and nanozyme signal enhancement on the LFA, resulting in a 30-fold improvement in detection limit over the conventional LFA when detecting Escherichia coli, all while maintaining a single application step. In late 2019, a new virus known as SARS-CoV-2 began to sweep across the globe, leading to the COVID-19 pandemic. The development of highly sensitive and easily deployable rapid diagnostic tests is a vital aspect of managing this pandemic. In Chapter 6, we discuss ongoing efforts to develop a highly sensitive, nanozyme signal enhanced LFA for the rapid detection of the SARS-CoV-2 nucleocapsid protein. A 3D-printed casing was designed to store the test strip and all reagents in a way that maintains user friendly operation by eliminating liquid handling steps. Besides developing highly sensitive diagnostics, the ability to deploy these tests quickly and in a highly scalable manner is crucial for being able to manage a pandemic. The work described in Chapter 7 highlights the integration of novel fibronectin-based antibody mimetics, called monobodies, into the LFA for the detection of the nucleocapsid protein of SARS-CoV-2. These monobodies are advantageous over monoclonal antibodies in that they are rapidly developed and produced recombinantly in Escherichia coli which is more affordable and scalable than monoclonal antibody production.

The “new cinema” was a loose film movement in the Philippines in the 1970s-80s known for combining nationalist and social themes with melodramatic form. Film scholarship in the Philippines has traditionally historicized the “new cinema” through an affirmative, national lens. More recent approaches problematize the nationalist frame through critical transnationalism, discourse analysis, cultural memory studies, and affect and genre theory. Yet, these studies overlook the ideological relation between cinema and the global Left. This dissertation emphasizes national cinema as a historically specific ideology. It situates the “new cinema” within shifting regimes of global capital accumulation through cinema’s politicization—that is, in terms of political ideas and aesthetic conceptions on the Left. This progressive discourse was committed to the political and economic autonomy of the Philippine nation yet regarded the artist’s freedom with ambivalence. The same ambivalence characterized the “new cinema’s” opposition to Hollywood—with which it was always face-to-face. In this respect, national cinema was not an idée fixe but a battleground where the nation-state was both abettor and enemy.

This dissertation asks: what are the roots of debates on political art in the Philippines? What was at stake in the conflict between the “new cinema” and the filmmaking industry built by the preceding generation? What was the aesthetic self-understanding of independent filmmakers and collectives during democratization? After the 1986 revolution, in what sense did the media cartel inherit the “new cinema’s” nationalist and pedagogical concerns? It concludes that while a nationalist self-understanding led to novel aesthetic results, a progressive narrative limited the “new cinema’s” critical interpretation.

This work was performed as Northern California Marine Sport Fish Survey, Dingell-Johnson Project F-12-R. The State of California is responsible for regulating, protecting, enhancing, and assessing the fish and game resources of California. Estimating the existing and potential take of marine resources is one of the tasks of the Marine Resources Operations unit of the Department of Fish and Game. Basic life history information on important species, total catch estimates by species, and enumeration of boats, angler effort, and facilities are compiled and published by this unit. Commercial catch records are gathered, compiled, and published for each year, and give data by month and port of landing. All commercial landings are recorded by species or major groupings at the time of sale to fish buyers. These landings are submitted monthly to the Terminal Island statistical unit. The sport catch, however, has not been assessed except for the partyboat catch which is recorded on logs. An ever-increasing demand placed on marine resources by expanding commercial and sport operations has necessitated a more complete evaluation of all segments of the sport fishery. Popular species such as king salmon,[1] striped bass, and lingcod were known to be important in central and northern California. Information on catch of all species was needed so research funds could be allocated more intelligently and recommendations could be made as to future marine sportfishing improvements projects. Upon completion of the Ocean Salmon Study (Dingell-Johnson project F-12-R) the project was amended to assess total sport catch and effort from Oregon to Point Arguello. Although a routine annual tabulation of catch and effort, as has been conducted on partyboat and commercial catches, could not be perpetuated, one "base" year's data were gathered. Thus, future surveys could reveal trends of effort and catch for any or all segments of the sport fishery. Short-cut methods could be adopted using basic data gathered during the base year survey. The objectives of this assessment survey, the Northern California Marine Sport Fish Survey, were: (1) To estimate the total average annual fishing effort, in angler days, for each fishing method (i.e. party boat, skiff, pier, skindiving, and shore fishing, including surf casting, rocky shore fishing, fishing from jetties, and surf netting). (2) To estimate the total catch by numbers and species for each fishing method. (3) To estimate the total catch by weight for certain fisheries. (This objective was added to the project in 1959.) (4) To compute catch-per-unit-of-effort values for each fishing method. (5) To record length composition of all species taken by partyboat fishermen, skiff fishermen, and skindivers. (6) To enumerate and describe all marine sportfishing facilities. (7) To develop sampling techniques and fish identification guides. All catch and effort data were grouped by monthly intervals at each port or fishing locality, and then summarized into yearly totals for each unit of area. Originally, the objectives included an economic survey of the sport fishery, including amount of money spent per trip, distance traveled, etc., such as was conducted in Florida (Ellis, Rosen, and Moffett, 1958). Also, a survey of San Pablo and San Francisco bays was included. The first 6 months of shore coverage, wherein all fishing methods were sampled, revealed the usual array of ensuing problems, and led to re-evaluation of objectives and methodology. The economic survey was discontinued because, though valuable, it was not essential in a biological approach to management. In addition to estimates of annual average effort and catch, biological information, usable in setting up research programs and as "base year" information, was needed. Since the total shoreline of the many bays of the study area was extensive, only skiff facilities, partyboat operations, and certain piers were included. Shore fishing, skindiving, and fishing from small piers were not surveyed in bays and estuaries. Night surf netting and night pier fishing were not included because the project was unable to provide coverage for night work. The need to collect several series of data per fishing method precluded simultaneous survey of all fishing methods. A complete study in 1 year covering all methods was highly desirable, but our limited budget would not permit such an approach. Thus, pier, shore (primarily surf casting), and surf net fishing were surveyed in 1958; skiff fishing in 1959; and partyboat, rocky shore fishing, and skindiving in 1960–61. Some series' of data were collected throughout the 3 years to make it possible to evalute yearly variations in catch and effort. No exceptional physical environmental changes, which may have materially altered biological relationships between species, occurred during the survey. The data from several consecutive years have been collated to represent average annual estimates for all parameters. Had this study been conducted during the 3 previous years (1955–1957), the sudden warming of ocean waters in 1957, and concurrent influx of southern forms, would have presented unique problems in treating the data in this way. From 1958–1961 there was gradual cooling and a return to a more normal physical environment, with consequent reduced occurrences of southern forms in central and northern California, such as barracuda which, in 1957, ranged as far north as Canada. Results of this survey have been summarized in three different ways: (i) as an administrative report, which included a complete set of typed raw data tables along with a narrative description of each fishery and methods used at each locality; (ii) as this report, which includes much of the original data, but in several cases annual summaries have been given in lieu of monthly summaries, and length frequency data are summarized; (iii) as four fishing maps covering 10 counties from Oregon to Santa Barbara County, Calif. These maps list all fishing facilities, public camping sites, and areas where shore fishing, skindiving, clamming, abalone picking, and surf netting are conducted. Also included are details as to the best times of year to fish, gear and bait to use, and notes on life history and behavior of certain fishes.

A lack of access to proper disease screening and diagnostics continues to plague low-resource countries. As a result, diseases that have long been forgotten in affluent areas still cause an enormous, yet preventable burden in areas without adequate medical systems. In those countries, only the centralized healthcare facilities have the luxury of modern diagnostic equipment, along with the expert personnel required to use that equipment. Advances in consumer technologies, particularly the mobile phone, offer the potential to address the lack of access to healthcare in low-resource regions by creating new data collection and interpretation methods. One example with potential to improve disease diagnosis is the mobile phone microscope. By adding lenses to a conventional mobile phone camera, controlling sample illumination and motion, and automatically interpreting images, mobile phone microscopes have been shown to detect the presence of a pathogens and guide treatment decisions.

This thesis advances the state-of-the-art in mobile phone microscopy by introducing new applications, new contrast modalities, and resolution enhancements. First, I give an overview of the current prototype implementation of darkfield and fluorescence microscopy on a mobile phone microscopy platform developed by the Fletcher Lab, detailing the physical design of the system along with the assumptions and disadvantages associated with that design. I also show results of the microscope's initial on-site imaging of Fusarium at various banana plantations in the Philippines. Next, I introduce a new illumination scheme to overcome key limitations of the current implementation. I show that by using an LCD screen as the light source, it is possible to finely tune the illumination in a way that simultaneously captures uniform brightfield, darkfield, and phase contrast without the need for larger standard microscope optics. Finally, I take advantage of the smartphone's onboard image stabilizer to displace the sample image relative to the camera sensor. I use this displacement to demonstrate a simple dithering algorithm that requires no additional hardware while more than doubling the microscope's resolution. By combining these new methods, mobile phone microscopes of the future have the potential to provide high-quality disease diagnoses at the point-of-care.