Development of Microfluidic Devices with the Use of Nanotechnology to Aid in the Analysis of Biological Systems Including Membrane Protein Separation, Single Cell Analysis, and Genetic Markers

Computation technology has dramatically changed the world around us; you can hardly find an area where cell phones have not saturated the market, yet there is a significant lack of breakthroughs in the development to integrate the computer with biological environments. This is largely the result of the incompatibility of the materials used in both environments; biological environments and experiments tend to need aqueous environments. To help aid in these development chemists, engineers, physicists and biologists have begun to develop microfluidics to help bridge this divide. Unfortunately, the microfluidic devices required large external support equipment to run the device. This thesis presents a series of several microfluidic methods that can help integrate engineering and biology by exploiting nanotechnology to help push the field of microfluidics back to its intended purpose, small integrated biological and electrical devices. I demonstrate this goal by developing different methods and devices to (1) separate membrane bound proteins with the use of microfluidics, (2) use optical technology to make fiber optic cables into protein sensors, (3) generate new fluidic devices using semiconductor material to manipulate single cells, and (4) develop a new genetic microfluidic based diagnostic assay that works with current PCR methodology to provide faster and cheaper results. All of these methods and systems can be used as components to build a self-contained biomedical device.

Surface markers of regionalization in the vertebrate nervous system

In order to identify new molecules that might play a role in regional specification of the nervous system, we generated and characterized monoclonal antibodies (mAbs) that have positionally-restricted labeling patterns.

The FORSE-1 mAb was generated using a strategy designed to produce mAbs against neuronal cell surface antigens that might be regulated by regionally-restricted transcription factors in the developing central nervous system (CNS). FORSE-1 staining is enriched in the forebrain as compared to the rest of the CNS until E18. Between E11.5-E13.5, only certain areas of the forebrain are labeled. There is also a dorsoventrally-restricted region of labeling in the hindbrain and spinal cord. The mAb labels a large proteoglycan-like cell-surface antigen (>200 kD). The labeling pattern of FORSE-1 is conserved in various mammals and in chick.

To determine whether the FORSE-1 labeling pattern is similar to that of known transcription factors, the expression of BF-1 and Dlx-2 was compared with FORSE-1. There is a striking overlap between BF-1 and FORSE-1 in the telencephalon. In contrast, FORSE-1 and Dlx-2 have very different patterns of expression in the forebrain, suggesting that regulation by Dlx-2 alone cannot explain the distribution of FORSE-1. They do, however, share some sharp boundaries in the diencephalon. In addition, FORSE-1 identifies some previously unknown boundaries in the developing forebrain. Thus, FORSE-1 is a new cell surface marker that can be used to subdivide the embryonic forebrain into regions smaller than previously described, providing further complexity necessary for developmental patterning.

I also studied the expression of the cell surface protein CD9 in the developing and adult rat nervous system. CD9 is implicated in intercellular signaling and cell adhesion in the hematopoetic system. In the nervous system, CD9 may perform similar functions in early sympathetic ganglia, chromaffin cells, and motor neurons, all of which express the protein. The presence of CD9 on the surfaces of Schwann cells and axons at the appropriate time may allow the protein to participate in the cellular interactions involved in myelination.

Anatomical and developmental patterns of interleukin-2 gene expression in the mouse: analysis of IL-2 expressing cells and partial characterization of interactions involved in mediating IL-2 gene expression in vivo

Interleukin-2 (IL-2) is an important mediator in the vertebrate immune system. IL-2 is a potent growth factor that mature T lymphocytes use as a proliferation signal and the production of IL-2 is crucial for the clonal expansion of antigen-specific T cells in the primary immune response. IL-2 driven proliferation is dependent on the interaction of the lymphokine with its cognate multichain receptor. IL-2 expression is induced only upon stimulation and transcriptional activation of the IL-2 gene relies extensively on the coordinate interaction of numerous inducible and constitutive trans-acting factors. Over the past several years, thousands of papers have been published regarding molecular and cellular aspects of IL-2 gene expression and IL-2 function. The vast majority of these reports describe work that has been carried out in vitro. However, considerably less is known about control of IL-2 gene expression and IL-2 function in vivo.

To gain new insight into the regulation of IL-2 gene expression in vivo, anatomical and developmental patterns of IL-2 gene expression in the mouse were established by employing in situ hybridization and immunohistochemical staining methodologies to tissue sections generated from normal mice and mutant animals in which T -cell development was perturbed. Results from these studies revealed several interesting aspects of IL-2 gene expression, such as (1) induction of IL-2 gene expression and protein synthesis in the thymus, the primary site of T-cell development in the body, (2) cell-type specificity of IL-2 gene expression in vivo, (3) participation of IL-2 in the extrathymic expansion of mature T cells in particular tissues, independent of an acute immune response to foreign antigen, (4) involvement of IL-2 in maintaining immunologic balance in the mucosal immune system, and (5) potential function of IL-2 in early events associated with hematopoiesis.

Extensive analysis of IL-2 mRNA accumulation and protein production in the murine thymus at various stages of development established the existence of two classes of intrathymic IL-2 producing cells. One class of intrathymic IL-2 producers was found exclusively in the fetal thymus. Cells belonging to this subset were restricted to the outermost region of the thymus. IL-2 expression in the fetal thymus was highly transient; a dramatic peak ofiL-2 mRNA accumulation was identified at day 14.5 of gestation and maximal IL-2 protein production was observed 12 hours later, after which both IL-2 mRNA and protein levels rapidly decreased. Significantly, the presence of IL-2 expressing cells in the day 14-15 fetal thymus was not contingent on the generation of T-cell receptor (TcR) positive cells. The second class of IL-2 producing cells was also detectable in the fetal thymus (cells found in this class represented a minority subset of IL-2 producers in the fetal thymus) but persist in the thymus during later stages of development and after birth. Intrathymic IL-2 producers in postnatal animals were located in the subcapsular region and cortex, indicating that these cells reside in the same areas where immature T cells are consigned. The frequency of IL-2 expressing cells in the postnatal thymus was extremely low, indicating that induction of IL-2 expression and protein synthesis are indicative of a rare activation event. Unlike the fetal class of intrathymic IL-2 producers, the presence of IL-2 producing cells in the postnatal thymus was dependent on to the generation of TcR+ cells. Subsequent examination of intrathymic IL-2 production in mutant postnatal mice unable to produce either αβ or γδ T cells showed that postnatal IL-2 producers in the thymus belong to both αβ and γδ lineages. Additionally, further studies indicated that IL-2 synthesis by immature αβ -T cells depends on the expression of bonafide TcR αβ-heterodimers. Taken altogether, IL-2 production in the postnatal thymus relies on the generation of αβ or γδ-TcR^+ cells and induction of IL-2 protein synthesis can be linked to an activation event mediated via the TcR.

With regard to tissue specificity of IL-2 gene expression in vivo, analysis of whole body sections obtained from normal neonatal mouse pups by in situ hybridization demonstrated that IL-2 mRNA^+ cells were found in both lymphoid and nonlymphoid tissues with which T cells are associated, such as the thymus (as described above), dermis and gut. Tissues devoid of IL-2 mRNA^+ cells included brain, heart, lung, liver, stomach, spine, spinal cord, kidney, and bladder. Additional analysis of isolated tissues taken from older animals revealed that IL-2 expression was undetectable in bone marrow and in nonactivated spleen and lymph nodes. Thus, it appears that extrathymic IL-2 expressing cells in nonimmunologically challenged animals are relegated to particular epidermal and epithelial tissues in which characterized subsets of T cells reside and thatinduction of IL-2 gene expression associated with these tissues may be a result of T-cell activation therein.

Based on the neonatal in situ hybridization results, a detailed investigation into possible induction of IL-2 expression resulting in IL-2 protein synthesis in the skin and gut revealed that IL-2 expression is induced in the epidermis and intestine and IL-2 protein is available to drive cell proliferation of resident cells and/or participate in immune function in these tissues. Pertaining to IL-2 expression in the skin, maximal IL-2 mRNA accumulation and protein production were observed when resident Vγ_3^+ T-cell populations were expanding. At this age, both IL-2 mRNA^+ cells and IL-2 protein production were intimately associated with hair follicles. Likewise, at this age a significant number of CD3ε^+ cells were also found in association with follicles. The colocalization of IL-2 expression and CD3ε^+ cells suggests that IL-2 expression is induced when T cells are in contact with hair follicles. In contrast, neither IL-2 mRNA nor IL-2 protein were readily detected once T-cell density in the skin reached steady-state proportions. At this point, T cells were no longer found associated with hair follicles but were evenly distributed throughout the epidermis. In addition, IL-2 expression in the skin was contingent upon the presence of mature T cells therein and induction of IL-2 protein synthesis in the skin did not depend on the expression of a specific TcR on resident T cells. These newly disclosed properties of IL-2 expression in the skin indicate that IL-2 may play an additional role in controlling mature T-cell proliferation by participating in the extrathymic expansion of T cells, particularly those associated with the epidermis.

Finally, regarding IL-2 expression and protein synthesis in the gut, IL-2 producing cells were found associated with the lamina propria of neonatal animals and gut-associated IL-2 production persisted throughout life. In older animals, the frequency of IL-2 producing cells in the small intestine was not identical to that in the large intestine and this difference may reflect regional specialization of the mucosal immune system in response to enteric antigen. Similar to other instances of IL-2 gene expression in vivo, a failure to generate mature T cells also led to an abrogation of IL-2 protein production in the gut. The presence of IL-2 producing cells in the neonatal gut suggested that these cells may be generated during fetal development. Examination of the fetal gut to determine the distribution of IL-2 producing cells therein indicated that there was a tenfold increase in the number of gut-associated IL-2 producers at day 20 of gestation compared to that observed four days earlier and there was little difference between the frequency of IL-2 producing cells in prenatal versus neonatal gut. The origin of these fetally-derived IL-2 producing cells is unclear. Prior to the immigration of IL-2 inducible cells to the fetal gut and/or induction of IL-2 expression therein, IL-2 protein was observed in the fetal liver and fetal omentum, as well as the fetal thymus. Considering that induction of IL-2 protein synthesis may be an indication of future functional capability, detection of IL-2 producing cells in the fetal liver and fetal omentum raises the possibility that IL-2 producing cells in the fetal gut may be extrathymic in origin and IL-2 producing cells in these fetal tissues may not belong solely to the T lineage. Overall, these results provide increased understanding of the nature of IL-2 producing cells in the gut and how the absence of IL-2 production therein and in fetal hematopoietic tissues can result in the acute pathology observed in IL-2 deficient animals.

Multi-model inference ranking and applications to physics at the Large Hadron Collider

In the measurement of the Higgs Boson decaying into two photons the parametrization of an appropriate background model is essential for fitting the Higgs signal mass peak over a continuous background. This diphoton background modeling is crucial in the statistical process of calculating exclusion limits and the significance of observations in comparison to a background-only hypothesis. It is therefore ideal to obtain knowledge of the physical shape for the background mass distribution as the use of an improper function can lead to biases in the observed limits. Using an Information-Theoretic (I-T) approach for valid inference we apply Akaike Information Criterion (AIC) as a measure of the separation for a fitting model from the data. We then implement a multi-model inference ranking method to build a fit-model that closest represents the Standard Model background in 2013 diphoton data recorded by the Compact Muon Solenoid (CMS) experiment at the Large Hadron Collider (LHC). Potential applications and extensions of this model-selection technique are discussed with reference to CMS detector performance measurements as well as in potential physics analyses at future detectors.

Storm track response to perturbations in climate

This thesis advances our understanding of midlatitude storm tracks and how they respond to perturbations in the climate system. The midlatitude storm tracks are regions of maximal turbulent kinetic energy in the atmosphere. Through them, the bulk of the atmospheric transport of energy, water vapor, and angular momentum occurs in midlatitudes. Therefore, they are important regulators of climate, controlling basic features such as the distribution of surface temperatures, precipitation, and winds in midlatitudes. Storm tracks are robustly projected to shift poleward in global-warming simulations with current climate models. Yet the reasons for this shift have remained unclear. Here we show that this shift occurs even in extremely idealized (but still three-dimensional) simulations of dry atmospheres. We use these simulations to develop an understanding of the processes responsible for the shift and develop a conceptual model that accounts for it.

We demonstrate that changes in the convective static stability in the deep tropics alone can drive remote shifts in the midlatitude storm tracks. Through simulations with a dry idealized general circulation model (GCM), midlatitude storm tracks are shown to be located where the mean available potential energy (MAPE, a measure of the potential energy available to be converted into kinetic energy) is maximal. As the climate varies, even if only driven by tropical static stability changes, the MAPE maximum shifts primarily because of shifts of the maximum of near-surface meridional temperature gradients. The temperature gradients shift in response to changes in the width of the tropical Hadley circulation, whose width is affected by the tropical static stability. Storm tracks generally shift in tandem with shifts of the subtropical terminus of the Hadley circulation.

We develop a one-dimensional diffusive energy-balance model that links changes in the Hadley circulation to midlatitude temperature gradients and so to the storm tracks. It is the first conceptual model to incorporate a dynamical coupling between the tropical Hadley circulation and midlatitude turbulent energy transport. Numerical and analytical solutions of the model elucidate the circumstances of when and how the storm tracks shift in tandem with the terminus of the Hadley circulation. They illustrate how an increase of only the convective static stability in the deep tropics can lead to an expansion of the Hadley circulation and a poleward shift of storm tracks.

The simulations with the idealized GCM and the conceptual energy-balance model demonstrate a clear link between Hadley circulation dynamics and midlatitude storm track position. With the help of the hierarchy of models presented in this thesis, we obtain a closed theory of storm track shifts in dry climates. The relevance of this theory for more realistic moist climates is discussed.

A computer-aided investigation of motion detection units in the fly

This work deals with two related areas: processing of visual information in the central nervous system, and the application of computer systems to research in neurophysiology.

Certain classes of interneurons in the brain and optic lobes of the blowfly Calliphora phaenicia were previously shown to be sensitive to the direction of motion of visual stimuli. These units were identified by visual field, preferred direction of motion, and anatomical location from which recorded. The present work is addressed to the questions: (1) is there interaction between pairs of these units, and (2) if such relationships can be found, what is their nature. To answer these questions, it is essential to record from two or more units simultaneously, and to use more than a single recording electrode if recording points are to be chosen independently. Accordingly, such techniques were developed and are described.

One must also have practical, convenient means for analyzing the large volumes of data so obtained. It is shown that use of an appropriately designed computer system is a profitable approach to this problem. Both hardware and software requirements for a suitable system are discussed and an approach to computer-aided data analysis developed. A description is given of members of a collection of application programs developed for analysis of neuro-physiological data and operated in the environment of and with support from an appropriate computer system. In particular, techniques developed for classification of multiple units recorded on the same electrode are illustrated as are methods for convenient graphical manipulation of data via a computer-driven display.

By means of multiple electrode techniques and the computer-aided data acquisition and analysis system, the path followed by one of the motion detection units was traced from open optic lobe through the brain and into the opposite lobe. It is further shown that this unit and its mirror image in the opposite lobe have a mutually inhibitory relationship. This relationship is investigated. The existence of interaction between other pairs of units is also shown. For pairs of units responding to motion in the same direction, the relationship is of an excitatory nature; for those responding to motion in opposed directions, it is inhibitory.

Experience gained from use of the computer system is discussed and a critical review of the current system is given. The most useful features of the system were found to be the fast response, the ability to go from one analysis technique to another rapidly and conveniently, and the interactive nature of the display system. The shortcomings of the system were problems in real-time use and the programming barrier—the fact that building new analysis techniques requires a high degree of programming knowledge and skill. It is concluded that computer system of the kind discussed will play an increasingly important role in studies of the central nervous system.

The structure elucidation of a proposed intermediate in the stereoselective synthesis of dl-desoxypodocarpic acid

The hydroxyketone C-3, an intermediate in the stereo-selective total synthesis of dl-Desoxypodocarpic acid (ii), has been shown by both degradative and synthetic pathways to rearrange in the presence of base to diosphenol E-1 (5-isoabietic acid series). The exact spatial arrangements of the systems represented by formulas C-3 and E-1 have been investigated (as the p-bromobenzoates) by single-crystal X-ray diffraction analyses. The hydroxyketone F-1, the proposed intermediate in the rearrangement, has been synthesized. Its conversion to diosphenol E-1 has been studied, and a single-crystal analysis of the p-bromobenzoate derivative has been performed. The initially desired diosphenol C-6 has been prepared, and has been shown to be stable to the potassium t-butoxide rearrangement conditions. Oxidative cleavage of diosphenol E-1 and subsequent cyclization with the aid of polyphosphoric acid has been shown to lead to keto acid I-2 (benzobicyclo [3.3.1] nonane series) rather than keto acid H-2 (5-isoabietic acid series).

Conditioned responses in the reticular formation

Unit activity was recorded from the midbrain and pons of 40 freely moving rats in an appetitive classical conditioning situation. Responses to auditory stimuli were observed from 100 units before and during a conditioning procedure in which presentation of food occurred 1 sec after the onset of the auditory stimulus. Conditioned unit responses (i.e., spike rate accelerations or decelerations) were considered to be positive when 1) no similar responses appeared prior to conditioning, and 2) latencies were equal to or less than those of sensory responses derived from the inferior colliculus. Such short latency conditioned unit responses were recorded from 11 probes located in the mid-lateral pert of the ventral region of the brain stem. This region was differentiated from paramedian, far lateral and dorsal parts of the brain stem reticular formation. Conditioned unit responses of considerably longer latencies were recorded from 76 probe located in these other regions. Among the longer latency responses interesting differences appeared in experiments conducted after the first conditioning series was completed. With additional training, units in the "reticular activating system" of midbrain and pons tended to yield stabilized responses in the early portion of the CS-US interval closely related in time to the orientation responses evoked by the CS. In contrast, the responses of units in the limbic midbrain tended to stabilize in the later part of the CS-US interval closely related in time to preparatory responses tied to the US. During extinction when the auditory stimulus was no longer followed by presentation of food, many of the responses were reduced to their pre-conditioning levels. However, there was a tendency for units which had displayed short latency responses on the first conditioning day to be more resistant to extinction than units which had displayed longer latency conditioned responses. The data were interpreted as indicating a local correlate of learning in the reticular formation of midbrain end pons and a separation of the midbrain system into at least two areas: 1) the classical "reticular activating system" related to orienting reactions, and 2) the limbic midbrain areas related to drives and rewards. Because the ventral and mid-lateral area with very short latency conditioned responses was not clearly tied to either of these; it was considered as possibly representing a third division.