MicroRNA-132 is a physiological regulator of hematopoietic stem cell function and B-cell development

MicroRNAs are a class of small non-coding RNAs that negatively regulate gene expression. Several microRNAs have been implicated in altering hematopoietic cell fate decisions. Importantly, deregulation of many microRNAs can lead to deleterious consequences in the hematopoietic system, including the onset of cancer, autoimmunity, or a failure to respond effectively to infection. As such, microRNAs fine-tune the balance between normal hematopoietic output and pathologic consequences. In this work, we explore the role of two microRNAs, miR-132 and miR-125b, in regulating hematopoietic stem cell (HSC) function and B cell development. In particular, we uncover the role of miR-132 in maintaining the appropriate balance between self-renewal, differentiation, and survival in aging HSCs by buffering the expression of a critical transcription factor, FOXO3. By maintain this balance, miR-132 may play a critical role in preventing aging-associated hematopoietic conditions such as autoimmune disease and cancer. We also find that miR-132 plays a critical role in B cell development by targeting a key transcription factor, Sox4, that is responsible for the differentiation of pro-B cells into pre-B cells. We find that miR-132 regulates B cell apoptosis, and by delivering miR-132 to mice that are predisposed to developing B cell cancers, we can inhibit the formation of these cancers and improve the survival of these mice. In addition to miR-132, we uncovered the role of another critical microRNA, miR-125b, that potentiates hematopoietic stem cell function. We found that enforced expression of miR-125b causes an aggressive myeloid leukemia by downregulation of its target Lin28a. Importantly, miR-125b also plays a critical role in inhibiting the formation of pro-B cells. Thus, we have discovered two microRNAs with important roles in regulating normal hematopoiesis, and whose dregulation can lead to deleterious consequences such as cancer in the aging hematopoietic system. Both miR-132 and miR-125b may therefore be targeted for therapeutics to inhibit age-related immune diseases associated with the loss of HSC function and cancer progression.

Structure-function studies of serine hydrolases: synthesis of a gene for ∝-lytic protease and the purification and characterization of a mutant β-lactamase

The author has constructed a synthetic gene for ∝-lytic protease. Since the DNA sequence of the protein is not known, the gene was designed by using the reverse translation of ∝-lytic protease's amino acid sequence. Unique restriction sites are carefully sought in the degenerate DNA sequence to aid in future mutagenesis studies. The unique restriction sites are designed approximately 50 base pairs apart and their appropriate codons used in the DNA sequence. The codons used to construct the DNA sequence of ∝-lytic protease are preferred codons in E-coli or used in the production of β-lactamase. Codon usage is also distributed evenly to ensure that one particular codon is not heavily used. The gene is essentially constructed from the outside in. The gene is built in a stepwise fashion using plasmids as the vehicles for the ∝-lytic oligomers. The use of plasmids allows the replication and isolation of large quantities of the intermediates during gene synthesis. The ∝-lytic DNA is a double-stranded oligomer that has sufficient overhang and sticky ends to anneal correctly in the vector. After six steps of incorporating ∝-lytic DNA, the gene is completed and sequenced to ensure that the correct DNA sequence is present and that no mutations occurred in the structural gene.

β-lactamase is the other serine hydrolase studied in this thesis. The author used the class A RTEM-1 β- lactamase encoded on the plasmid pBR322 to investigate the roll of the conserved threonine residue at position 71. Cassette mutagenesis was previously used to generate all possible amino acid substitutions at position 71. The work presented here describes the purification and kinetic characterization of a T71H mutant previously constructed by S. Schultz. The mutated gene was transferred into plasmid pJN for expression and induced with IPTG. The enzyme is purified by column chromatography and FPLC to homogeneity. Kinetic studies reveal that the mutant has lower k_(cat) values on benzylpenicillin, cephalothin and 6-aminopenicillanic acid but no changes in k_m except for cephalothin which is approximately 4 times higher. The mutant did not change siginificantly in its pH profile compared to the wild-type enzyme. Also, the mutant is more sensitive to thermal denaturation as compared to the wild-type enzyme. However, experimental evidence indicates that the probable generation of a positive charge at position 71 thermally stabilized the mutant.

Single cell proteomics microchip to profile immune function, with applications in stem cell biology, translational disease mechanism study and clinical therapeutics monitoring

In response to infection or tissue dysfunction, immune cells develop into highly heterogeneous repertoires with diverse functions. Capturing the full spectrum of these functions requires analysis of large numbers of effector molecules from single cells. However, currently only 3-5 functional proteins can be measured from single cells. We developed a single cell functional proteomics approach that integrates a microchip platform with multiplex cell purification. This approach can quantitate 20 proteins from >5,000 phenotypically pure single cells simultaneously. With a 1-million fold miniaturization, the system can detect down to ~100 molecules and requires only ~104 cells. Single cell functional proteomic analysis finds broad applications in basic, translational and clinical studies. In the three studies conducted, it yielded critical insights for understanding clinical cancer immunotherapy, inflammatory bowel disease (IBD) mechanism and hematopoietic stem cell (HSC) biology.

To study phenotypically defined cell populations, single cell barcode microchips were coupled with upstream multiplex cell purification based on up to 11 parameters. Statistical algorithms were developed to process and model the high dimensional readouts. This analysis evaluates rare cells and is versatile for various cells and proteins. (1) We conducted an immune monitoring study of a phase 2 cancer cellular immunotherapy clinical trial that used T-cell receptor (TCR) transgenic T cells as major therapeutics to treat metastatic melanoma. We evaluated the functional proteome of 4 antigen-specific, phenotypically defined T cell populations from peripheral blood of 3 patients across 8 time points. (2) Natural killer (NK) cells can play a protective role in chronic inflammation and their surface receptor – killer immunoglobulin-like receptor (KIR) – has been identified as a risk factor of IBD. We compared the functional behavior of NK cells that had differential KIR expressions. These NK cells were retrieved from the blood of 12 patients with different genetic backgrounds. (3) HSCs are the progenitors of immune cells and are thought to have no immediate functional capacity against pathogen. However, recent studies identified expression of Toll-like receptors (TLRs) on HSCs. We studied the functional capacity of HSCs upon TLR activation. The comparison of HSCs from wild-type mice against those from genetics knock-out mouse models elucidates the responding signaling pathway.

In all three cases, we observed profound functional heterogeneity within phenotypically defined cells. Polyfunctional cells that conduct multiple functions also produce those proteins in large amounts. They dominate the immune response. In the cancer immunotherapy, the strong cytotoxic and antitumor functions from transgenic TCR T cells contributed to a ~30% tumor reduction immediately after the therapy. However, this infused immune response disappeared within 2-3 weeks. Later on, some patients gained a second antitumor response, consisted of the emergence of endogenous antitumor cytotoxic T cells and their production of multiple antitumor functions. These patients showed more effective long-term tumor control. In the IBD mechanism study, we noticed that, compared with others, NK cells expressing KIR2DL3 receptor secreted a large array of effector proteins, such as TNF-α, CCLs and CXCLs. The functions from these cells regulated disease-contributing cells and protected host tissues. Their existence correlated with IBD disease susceptibility. In the HSC study, the HSCs exhibited functional capacity by producing TNF-α, IL-6 and GM-CSF. TLR stimulation activated the NF-κB signaling in HSCs. Single cell functional proteome contains rich information that is independent from the genome and transcriptome. In all three cases, functional proteomic evaluation uncovered critical biological insights that would not be resolved otherwise. The integrated single cell functional proteomic analysis constructed a detail kinetic picture of the immune response that took place during the clinical cancer immunotherapy. It revealed concrete functional evidence that connected genetics to IBD disease susceptibility. Further, it provided predictors that correlated with clinical responses and pathogenic outcomes.

Function of microRNA-146a and NF-κB in physiologic and pathologic hematopoiesis

During inflammation and infection, hematopoietic stem and progenitor cells (HSPCs) are stimulated to proliferate and differentiate into mature immune cells, especially of the myeloid lineage. MicroRNA-146a (miR-146a) is a critical negative regulator of inflammation. Deletion of the gene encoding miR-146a—expressed in all blood cell types—produces effects that appear as dysregulated inflammatory hematopoiesis, leading to a decline in the number and quality of hematopoietic stem cells (HSCs), excessive myeloproliferation, and, ultimately, to exhaustion of the HSCs and hematopoietic neoplasms. Six-week-old deleted mice are normal, with no effect on cell numbers, but by 4 months bone marrow hypercellularity can be seen, and by 8 months marrow exhaustion is becoming evident. The ability of HSCs to replenish the entire hematopoietic repertoire in a myelo-ablated mouse also declines precipitously as miR-146a-deficient mice age. In the absence of miR-146a, LPS-mediated serial inflammatory stimulation accelerates the effects of aging. This chronic inflammatory stress on HSCs in deleted mice involves a molecular axis consisting of upregulation of the signaling protein TRAF6 leading to excessive activity of the transcription factor NF-κB and overproduction of the cytokine IL-6. At the cellular level, transplant studies show that the defects are attributable to both an intrinsic problem in the miR-146a-deficient HSCs and extrinsic effects of miR-146a-deficient lymphocytes and non-hematopoietic cells. This study has identified a microRNA, miR-146a, to be a critical regulator of HSC homeostasis during chronic inflammatory challenge in mice and has provided a molecular connection between chronic inflammation and the development of bone marrow failure and myeloproliferative neoplasms. This may have implications for human hematopoietic malignancies, such as myelodysplastic syndrome, which frequently displays downregulated miR-146a expression.

Mathematical study of complex networks : brain, internet, and power grid

The dissertation is concerned with the mathematical study of various network problems. First, three real-world networks are considered: (i) the human brain network (ii) communication networks, (iii) electric power networks. Although these networks perform very different tasks, they share similar mathematical foundations. The high-level goal is to analyze and/or synthesis each of these systems from a “control and optimization” point of view. After studying these three real-world networks, two abstract network problems are also explored, which are motivated by power systems. The first one is “flow optimization over a flow network” and the second one is “nonlinear optimization over a generalized weighted graph”. The results derived in this dissertation are summarized below.

Brain Networks: Neuroimaging data reveals the coordinated activity of spatially distinct brain regions, which may be represented mathematically as a network of nodes (brain regions) and links (interdependencies). To obtain the brain connectivity network, the graphs associated with the correlation matrix and the inverse covariance matrix—describing marginal and conditional dependencies between brain regions—have been proposed in the literature. A question arises as to whether any of these graphs provides useful information about the brain connectivity. Due to the electrical properties of the brain, this problem will be investigated in the context of electrical circuits. First, we consider an electric circuit model and show that the inverse covariance matrix of the node voltages reveals the topology of the circuit. Second, we study the problem of finding the topology of the circuit based on only measurement. In this case, by assuming that the circuit is hidden inside a black box and only the nodal signals are available for measurement, the aim is to find the topology of the circuit when a limited number of samples are available. For this purpose, we deploy the graphical lasso technique to estimate a sparse inverse covariance matrix. It is shown that the graphical lasso may find most of the circuit topology if the exact covariance matrix is well-conditioned. However, it may fail to work well when this matrix is ill-conditioned. To deal with ill-conditioned matrices, we propose a small modification to the graphical lasso algorithm and demonstrate its performance. Finally, the technique developed in this work will be applied to the resting-state fMRI data of a number of healthy subjects.

Communication Networks: Congestion control techniques aim to adjust the transmission rates of competing users in the Internet in such a way that the network resources are shared efficiently. Despite the progress in the analysis and synthesis of the Internet congestion control, almost all existing fluid models of congestion control assume that every link in the path of a flow observes the original source rate. To address this issue, a more accurate model is derived in this work for the behavior of the network under an arbitrary congestion controller, which takes into account of the effect of buffering (queueing) on data flows. Using this model, it is proved that the well-known Internet congestion control algorithms may no longer be stable for the common pricing schemes, unless a sufficient condition is satisfied. It is also shown that these algorithms are guaranteed to be stable if a new pricing mechanism is used.

Electrical Power Networks: Optimal power flow (OPF) has been one of the most studied problems for power systems since its introduction by Carpentier in 1962. This problem is concerned with finding an optimal operating point of a power network minimizing the total power generation cost subject to network and physical constraints. It is well known that OPF is computationally hard to solve due to the nonlinear interrelation among the optimization variables. The objective is to identify a large class of networks over which every OPF problem can be solved in polynomial time. To this end, a convex relaxation is proposed, which solves the OPF problem exactly for every radial network and every meshed network with a sufficient number of phase shifters, provided power over-delivery is allowed. The concept of “power over-delivery” is equivalent to relaxing the power balance equations to inequality constraints.

Flow Networks: In this part of the dissertation, the minimum-cost flow problem over an arbitrary flow network is considered. In this problem, each node is associated with some possibly unknown injection, each line has two unknown flows at its ends related to each other via a nonlinear function, and all injections and flows need to satisfy certain box constraints. This problem, named generalized network flow (GNF), is highly non-convex due to its nonlinear equality constraints. Under the assumption of monotonicity and convexity of the flow and cost functions, a convex relaxation is proposed, which always finds the optimal injections. A primary application of this work is in the OPF problem. The results of this work on GNF prove that the relaxation on power balance equations (i.e., load over-delivery) is not needed in practice under a very mild angle assumption.

Generalized Weighted Graphs: Motivated by power optimizations, this part aims to find a global optimization technique for a nonlinear optimization defined over a generalized weighted graph. Every edge of this type of graph is associated with a weight set corresponding to the known parameters of the optimization (e.g., the coefficients). The motivation behind this problem is to investigate how the (hidden) structure of a given real/complex valued optimization makes the problem easy to solve, and indeed the generalized weighted graph is introduced to capture the structure of an optimization. Various sufficient conditions are derived, which relate the polynomial-time solvability of different classes of optimization problems to weak properties of the generalized weighted graph such as its topology and the sign definiteness of its weight sets. As an application, it is proved that a broad class of real and complex optimizations over power networks are polynomial-time solvable due to the passivity of transmission lines and transformers.

Characterization of the human SCF ubiquitin ligases - structure, function, and regulation

The SCF ubiquitin ligase complex of budding yeast triggers DNA replication by cata lyzi ng ubiquitination of the S phase CDK inhibitor SIC1. SCF is composed of several evolutionarily conserved proteins, including ySKP1, CDC53 (Cullin), and the F-box protein CDC4. We isolated hSKP1 in a two-hybrid screen with hCUL1, the human homologue of CDC53. We showed that hCUL1 associates with hSKP1 in vivo and directly interacts with hSKP1 and the human F-box protein SKP2 in vitro, forming an SCF-Iike particle. Moreover, hCUL1 complements the growth defect of yeast CDC53^(ts) mutants, associates with ubiquitination-promoting activity in human cell extracts, and can assemble into functional, chimeric ubiquitin ligase complexes with yeast SCF components. These data demonstrated that hCUL1 functions as part of an SCF ubiquitin ligase complex in human cells. However, purified human SCF complexes consisting of CUL1, SKP1, and SKP2 are inactive in vitro, suggesting that additional factors are required.

Subsequently, mammalian SCF ubiquitin ligases were shown to regulate various physiological processes by targeting important cellular regulators, like lĸBα, β-catenin, and p27, for ubiquitin-dependent proteolysis by the 26S proteasome. Little, however, is known about the regulation of various SCF complexes. By using sequential immunoaffinity purification and mass spectrometry, we identified proteins that interact with human SCF components SKP2 and CUL1 in vivo. Among them we identified two additional SCF subunits: HRT1, present in all SCF complexes, and CKS1, that binds to SKP2 and is likely to be a subunit of SCF5^(SKP2) complexes. Subsequent work by others demonstrated that these proteins are essential for SCF activity. We also discovered that COP9 Signalosome (CSN), previously described in plants as a suppressor of photomorphogenesis, associates with CUL1 and other SCF subunits in vivo. This interaction is evolutionarily conserved and is also observed with other Cullins, suggesting that all Cullin based ubiquitin ligases are regulated by CSN. CSN regulates Cullin Neddylation presumably through CSNS/JAB1, a stochiometric Signalosome subunit and a putative deneddylating enzyme. This work sheds light onto an intricate connection that exists between signal transduction pathways and protein degradation machinery inside the cell and sets stage for gaining further insights into regulation of protein degradation.

Edge function method applied to thin plates resting on Winkler foundation

The Edge Function method formerly developed by Quinlan⁽²⁵⁾ is applied to solve the problem of thin elastic plates resting on spring supported foundations subjected to lateral loads the method can be applied to plates of any convex polygonal shapes, however, since most plates are rectangular in shape, this specific class is investigated in this thesis. The method discussed can also be applied easily to other kinds of foundation models (e.g. springs connected to each other by a membrane) as long as the resulting differential equation is linear. In chapter VII, solution of a specific problem is compared with a known solution from literature. In chapter VIII, further comparisons are given. The problems of concentrated load on an edge and later on a corner of a plate as long as they are far away from other boundaries are also given in the chapter and generalized to other loading intensities and/or plates springs constants for Poisson's ratio equal to 0.2

Sequence-function relationships in E. coli transcriptional regulation

Understanding how transcriptional regulatory sequence maps to regulatory function remains a difficult problem in regulatory biology. Given a particular DNA sequence for a bacterial promoter region, we would like to be able to say which transcription factors bind there, how strongly they bind, and whether they interact with each other and/or RNA polymerase, with the ultimate objective of integrating knowledge of these parameters into a prediction of gene expression levels. The theoretical framework of statistical thermodynamics provides a useful framework for doing so, enabling us to predict how gene expression levels depend on transcription factor binding energies and concentrations. We used thermodynamic models, coupled with models of the sequence-dependent binding energies of transcription factors and RNAP, to construct a genotype to phenotype map for the level of repression exhibited by the lac promoter, and tested it experimentally using a set of promoter variants from E. coli strains isolated from different natural environments. For this work, we sought to ``reverse engineer'' naturally occurring promoter sequences to understand how variations in promoter sequence affects gene expression. The natural inverse of this approach is to ``forward engineer'' promoter sequences to obtain targeted levels of gene expression. We used a high precision model of RNAP-DNA sequence dependent binding energy, coupled with a thermodynamic model relating binding energy to gene expression, to predictively design and verify a suite of synthetic E. coli promoters whose expression varied over nearly three orders of magnitude.

However, although thermodynamic models enable predictions of mean levels of gene expression, it has become evident that cell-to-cell variability or ``noise'' in gene expression can also play a biologically important role. In order to address this aspect of gene regulation, we developed models based on the chemical master equation framework and used them to explore the noise properties of a number of common E. coli regulatory motifs; these properties included the dependence of the noise on parameters such as transcription factor binding strength and copy number. We then performed experiments in which these parameters were systematically varied and measured the level of variability using mRNA FISH. The results showed a clear dependence of the noise on these parameters, in accord with model predictions.

Finally, one shortcoming of the preceding modeling frameworks is that their applicability is largely limited to systems that are already well-characterized, such as the lac promoter. Motivated by this fact, we used a high throughput promoter mutagenesis assay called Sort-Seq to explore the completely uncharacterized transcriptional regulatory DNA of the E. coli mechanosensitive channel of large conductance (MscL). We identified several candidate transcription factor binding sites, and work is continuing to identify the associated proteins.

Measurement of the neutron (^3He) spin structure function at low Q^2 : a connection between the Bjorken and Drell-Hearn-Gerasimov sum rules

The spin dependent cross sections, σT_1/2 and σT_3/2 , and asymmetries, A_∥ and A_⊥ for ³He have been measured at the Jefferson Lab's Hall A facility. The inclusive scattering process ³He(e,e)X was performed for initial beam energies ranging from 0.86 to 5.1 GeV, at a scattering angle of 15.5°. Data includes measurements from the quasielastic peak, resonance region, and the deep inelastic regime. An approximation for the extended Gerasimov-Drell-Hearn integral is presented at a 4-momentum transfer Q² of 0.2-1.0 GeV².

Also presented are results on the performance of the polarized ³He target. Polarization of ³He was achieved by the process of spin-exchange collisions with optically pumped rubidium vapor. The ³He polarization was monitored using the NMR technique of adiabatic fast passage (AFP). The average target polarization was approximately 35% and was determined to have a systematic uncertainty of roughly ±4% relative.

Binding Studies of Neuronal Nicotinic Acetylcholine Receptors Expressing Unnatural Amino Acids

Nicotinic acetylcholine receptors are pentameric ligand-gated ion channels mediating fast synaptic transmission throughout the peripheral and central nervous systems. They have been implicated in various processes related to cognitive functions, learning and memory, arousal, reward, motor control and analgesia. Therefore, these receptors present alluring potential therapeutic targets for the treatment of pain, epilepsy, Alzheimer’s disease, Parkinson’s disease, Tourette’s syndrome, schizophrenia, anxiety, depression and nicotine addiction. The work detailed in this thesis focuses on binding studies of neuronal nicotinic receptors and aims to further our knowledge of subtype specific functional and structural information.

Chapter 1 is an introductory chapter describing the structure and function of nicotinic acetylcholine receptors as well as the methodologies used for the dissertation work described herein. There are several different subtypes of nicotinic acetylcholine receptors known to date and the subtle variations in their structure and function present a challenging area of study. The work presented in this thesis deals specifically with the α4β2 subtype of nicotinic acetylcholine receptor. This subtype assembles into 2 closely related stoichiometries, termed throughout this thesis as A3B2 and A2B3 after their respective subunit composition. Chapter 2 describes binding studies of select nicotinic agonists on A3B2 and A2B3 receptors determined by whole-cell recording. Three key binding interactions, a cation-π and two hydrogen bonds, were probed for four nicotinic agonists, acetylcholine, nicotine, smoking cessation drug varenicline (Chantix®) and the related natural product cytisine.

Results from the binding studies presented in Chapter 2 show that the major difference in binding of these four agonists to A3B2 and A2B3 receptors lies in one of the two hydrogen bond interactions where the agonist acts as the hydrogen bond acceptor and the backbone NH of a conserved leucine residue in the receptor acts as the hydrogen bond donor. Chapter 3 focuses on studying the effect of modulating the hydrogen bond acceptor ability of nicotine and epibatidine on A3B2 receptor function determined by whole-cell recording. Finally, Chapter 4 describes single-channel recording studies of varenicline binding to A2B3 and A3B2 receptors.

Structure-Function Studies of Nicotinic Acetylcholine Receptors Using Selective Agonists and Positive Allosteric Modulators

This dissertation primarily describes chemical-scale studies of nicotinic acetylcholine receptors (nAChRs) in order to better understand ligand-receptor selectivity and allosteric modulation influences during receptor activation. Electrophysiology coupled with canonical and non-canonical amino acids mutagenesis is used to probe subtle changes in receptor function.

The first half of this dissertation focuses on differential agonist selectivity of α4β2-containing nAChRs. The α4β2 nAChR can assemble in alternative stoichiometries as well as assemble with other accessory subunits. Chapter 2 identifies key structural residues that dictate binding and activation of three stoichiometry-dependent α4β2 receptor ligands: sazetidine-A, cytisine, and NS9283. These do not follow previously suggested hydrogen-bonding patterns of selectivity. Instead, three residues on the complementary subunit strongly influence binding ability of a ligand and receptor activation. Chapter 3 involves isolation of a α5α4β2 receptor-enriched population to test for a potential alternative agonist binding location at the α5 α4 interface. Results strongly suggest that agonist occupation of this site is not necessary for receptor activation and that the α5 subunit only incorporates at the accessory subunit location.

The second half of this dissertation seeks to identify residue interactions with positive allosteric modulators (PAMs) of the α7 nAChR. Chapter 4 focuses on methods development to study loss of potentiation of Type I PAMs, which indicate residues vital to propagation of PAM effects and/or binding. Chapter 5 investigates α7 receptor modulation by a Type II PAM (PNU 120596). These results show that PNU 120596 does not alter the agonist binding site, thus is relegated to influencing only the gating component of activation. From this, we were able to map a potential network of residues from the agonist binding site to the proposed PNU 120596 binding site that are essential for receptor potentiation.

Organization and function of the phage Lambda chromosome

The process of prophage integration by phage λ and the function and structure of the chromosomal elements required for λ integration have been studied with the use of λ deletion mutants. Since att^φ, the substrate of the integration enzymes, is not essential for λ growth, and since att^φ resides in a portion of the λ chromosome which is not necessary for vegetative growth, viable λ deletion mutants were isolated and examined to dissect the structure of att^φ.

Deletion mutants were selected from wild type populations by treating the phage under conditions where phage are inactivated at a rate dependent on the DNA content of the particles. A number of deletion mutants were obtained in this way, and many of these mutants proved to have defects in integration. These defects were defined by analyzing the properties of Int-promoted recombination in these att mutants.

The types of mutants found and their properties indicated that att^φ has three components: a cross-over point which is bordered on either side by recognition elements whose sequence is specifically required for normal integration. The interactions of the recognition elements in Int-promoted recombination between att mutants was examined and proved to be quite complex. In general, however, it appears that the λ integration system can function with a diverse array of mutant att sites.

The structure of att^φ was examined by comparing the genetic properties of various att mutants with their location in the λ chromosome. To map these mutants, the techniques of heteroduplex DNA formation and electron microscopy were employed. It was found that integration cross-overs occur at only one point in att^φ and that the recognition sequences that direct the integration enzymes to their site of action are quite small, less than 2000 nucleotides each. Furthermore, no base pair homology was detected between att^φ and its bacterial analog, att^B. This result clearly demonstrates that λ integration can occur between chromosomes which have little, if any, homology. In this respect, λ integration is unique as a system of recombination since most forms of generalized recombination require extensive base pair homology.

An additional study on the genetic and physical distances in the left arm of the λ genome was described. Here, a large number of conditional lethal nonsense mutants were isolated and mapped, and a genetic map of the entire left arm, comprising a total of 18 genes, was constructed. Four of these genes were discovered in this study. A series of λdg transducing phages was mapped by heteroduplex electron microscopy and the relationship between physical and genetic distances in the left arm was determined. The results indicate that recombination frequency in the left arm is an accurate reflection of physical distances, and moreover, there do not appear to be any undiscovered genes in this segment of the genome.

Studies on the role of histones in the structure and function of chromatin

Studies on the dissociation of histones from chromatin by increasing concentrations of sodium deoxycholate (DOC) have shown that histrone II is removed at lowest concentrations of DOC, while slightly higher concentrations remove histones III and IV. Still higher concentrations remove histone I.

The complete separation of chromatin and ¹⁴C-DOC by sucrose sedimentation indicated that the binding of DOC to chromatin is readily and completely reversible.

The dissociation of histones from chromatin by increasing concentrations of related cholanic acids and some of their conjugated derivatives were studied. The results suggested that the driving force for the interaction between the cholanic acid anion and histones is the lowering of the activity coefficient of the cholanic acid anion which occurs when it is partially removed from solution by interaction with hydrophobic regions of the positively charged histones.

The role of histones in the structure of chromatin has been studied by comparing the effects of selective removal of histones from chromatin by increasing concentrations of DOC with those caused by NaCl (removes histone I at lowest concentrations, while higher concentrations remove histones II, III, and IV). Properties studied included thermal denaturation, sedimentation velocity, flow dichroism, relaxation times of molecules oriented in a flow field, and the irreversible disruption of a 130 S, cross-linked component of sheared chromatin. The data indicated that none of the structural or chemical parameters with which these properties are correlated show a dependence on the presence of one particular histone fraction.

The template activity (ability to prime a 0.2 M KC1 DNA-dependent RNA synthesis system catalyzed by E. coli RNA polymerase) increases from that of native chromatin (approximately 25 per cent of that pure DNA) to that of pure DNA in a fashion which shows a nearly linear relationship to the amount of histone coverage of the template. The precipitability of partially dehistonized chromatin samples in 0.15 M NaCl shows a large dependence on the presence of histone I.

Part I: Latent heat of vaporization of n-decane. Part II: Heat and mass transfer from a cylinder placed in a turbulent air stream - Sherwood and Frössling numbers as a function of free stream turbulence level and Reynolds number

Part I

The latent heat of vaporization of n-decane is measured calorimetrically at temperatures between 160° and 340°F. The internal energy change upon vaporization, and the specific volume of the vapor at its dew point are calculated from these data and are included in this work. The measurements are in excellent agreement with available data at 77° and also at 345°F, and are presented in graphical and tabular form.

Part II

Simultaneous material and energy transport from a one-inch adiabatic porous cylinder is studied as a function of free stream Reynolds Number and turbulence level. Experimental data is presented for Reynolds Numbers between 1600 and 15,000 based on the cylinder diameter, and for apparent turbulence levels between 1.3 and 25.0 per cent. n-heptane and n-octane are the evaporating fluids used in this investigation.

Gross Sherwood Numbers are calculated from the data and are in substantial agreement with existing correlations of the results of other workers. The Sherwood Numbers, characterizing mass transfer rates, increase approximately as the 0.55 power of the Reynolds Number. At a free stream Reynolds Number of 3700 the Sherwood Number showed a 40% increase as the apparent turbulence level of the free stream was raised from 1.3 to 25 per cent.

Within the uncertainties involved in the diffusion coefficients used for n-heptane and n-octane, the Sherwood Numbers are comparable for both materials. A dimensionless Frössling Number is computed which characterizes either heat or mass transfer rates for cylinders on a comparable basis. The calculated Frössling Numbers based on mass transfer measurements are in substantial agreement with Frössling Numbers calculated from the data of other workers in heat transfer.

Connectivity and Function of the Primate Insula

The insula is a mammalian cortical structure that has been implicated in a wide range of low- and high-level functions governing one’s sensory, emotional, and cognitive experiences. One particular role of this region is considered to be processing of olfactory stimuli. The ability to detect and evaluate odors has significant effects on an organism’s eating behavior and survival and, in case of humans, on complex decision making. Despite such importance of this function, the mechanism in which olfactory information is processed in the insula has not been thoroughly studied. Moreover, due to the structure’s close spatial relationship with the neighboring claustrum, it is not entirely clear whether the connectivity and olfactory functions attributed to the insula are truly those of the insula, rather than of the claustrum. My graduate work, consisting of two studies, seeks to help fill these gaps. In the first, the structural connectivity patterns of the insula and the claustrum in a non-human primate brain is assayed using an ultra-high-quality diffusion magnetic resonance image, and the results suggest dissociation of connectivity — and hence function — between the two structures. In the second study, a functional neuroimaging experiment investigates the insular activity during odor evaluation tasks in humans, and uncovers a potential spatial organization within the anterior portion of the insula for processing different aspects of odor characteristics.