The Sertoli cell ectoplasmic specialization is a specialized domain of the calcium-dependent Sertoli-spermatid adherens junction. Structurally abnormal or absent Sertoli ectoplasmic specializations are associated with spermatid sloughing and subsequent oligospermia in conditions associated with reduced fertility potential, although the junctional strength between these cells is not known. Adjudin is a potential male contraceptive agent thought to interrupt testicular binding dynamics of adherens junctions, resulting in controlled spermatid sloughing.It was hypothesized that the mechanism of action of Adjudin, pertinent to its putative contraceptive effect, is the disruption of the Sertoli cell-spermatid junction. This was tested in vitro using primary isolates of germ cells and both primary and immortal Sertoli cells.This dissertation presents the examination of Sertoli-germ cell interactions in three parts, which address the overall aims of this dissertation project: (1) measurement of the junctional strength between Sertoli cells and spermatids in vitro, (2) determination of the efficacy of sk Sertoli cell lines in Sertoli-germ cell binding studies in vitro, and (3) assessment of Adjudin as a potential male contraceptive, by measuring the junctional binding strength between Sertoli cells and spermatids exposed to this chemical in vitro.For the first time, the strength of the Sertoli-spermatid junction has been measured, using a micropipette pressure transducing system (MPTS). Results reported in this dissertation demonstrate that the junctional strength between Sertoli cells and germ cells can be measured in vitro, support long held speculations regarding Sertoli-spermatid junctional interactions, and provide a technology to test proposed mechanisms of junctional binding dynamics between cells of the seminiferous epithelium (Chapter 2). Although the sk cell lines initially expressed mRNA for the FSH receptor, coculture results determined that these cell lines have limited value for investigating Sertoli-germ cell binding dynamics in vitro (Chapter 3). By utilizing the MPTS and primary cell isolates, Adjudin was determined to reduce the junctional strength between Sertoli cells and step-8 spermatids. In conclusion, results support the use of Adjudin as a potential reversible male contraceptive agent by a mechanism which alters the adhesion properties between the step-8 spermatid and the Sertoli cell (Chapter 4).

Nanocrystalline (NC) metals with averaged grain size smaller than 100 nm have shown promising mechanical properties such as higher hardness and toughness than conventional coarse-grained metals. Unlike conventional metals in which the deformation is controlled by dislocation activities, the microstructural evolution in NC metals is mainly dominated by grain rotation and stress-driven grain boundary motion (SDGBM) due to the high density of grain boundaries (GBs). SDGBM is thus among the most studied modes of microstructural evolution in NC materials with particular interests on their fundamental atomistic mechanisms. In the first part of this thesis, molecular dynamics simulations were used to investigate the influences of Triple Junctions (TJs) on SDGBM of symmetric tilt GBs in copper by considering a honeycomb NC model. TJs exhibited asymmetric pinning effects to the GB migration and the constraints by the TJs and neighboring grains led to remarkable non-linear GB motion in directions both parallel and normal to the applied shear. Based on these findings, a generalized model for SDGBM in NC Cu was proposed. In the second part, the interaction of SDGBM with crack, voids and precipitates was investigated. It was found that depending on the GB structure, material type and temperature, there is a competition between different atomistic mechanisms such as crack healing, recrystallization and GB decohesion. It is hoped that the findings of this work could clarify the micro-mechanisms of various experimental phenomena such as grain refinement in metals during severe plastic deformation, which can be used to design optimized route of making stabilized bulk NC metals. / February 2016

A mechanically controllable break junction setup was designed, constructed and characterized. The mechanically controllable break junction technique is commonly used for measurement of quantum conductance of metals and single molecule conductance. The technique relies on resistance to external vibrations disrupting the atomic or molecular junctions formed and should be in a low electronic noise environment. Through a series of experiments the setup was found to have high mechanical stability and low electronic noise. The quantum conductance of gold was measured repeatedly and a histogram was plotted showing good agreement with the literature. The results indicate that with modifications, the setup can be used to measure the conductance of single molecule junctions and single molecule thermoelectric properties.

The function and evolution of introns have been topics of great interest since introns were discovered in the 1970s. Introns that interrupt protein-coding regions have the most obvious potential to affect coding sequences; therefore, their evolution have been studied most intensively. Splicing of introns within untranslated regions does not contribute directly to the diversity of proteins, yet ~35% of human transcripts contain introns within the 5' untranslated region (UTR). The evolution and possible functions of 5'UTR introns (5UIs) remain largely unexplored. Here we undertook a genome-wide functional analysis of 5UIs. Our main results are as follows: First, the distribution of these introns in the human genome is nonrandom. While genes with regulatory roles are enriched in having 5UIs, genes encoding proteins that are targeted to the endoplasmic reticulum and mitochondria are surprisingly depleted of these introns. Second, we offered and supported a model whereby gene encoding secretory and nuclear-encoded mitochondrial proteins share a common regulatory mechanism at the level of mRNA export, which is dependent on the absence of 5'UTR introns. Specifically, the upstream element in a given transcript, be it an intron or RNA elements near the 5' end of coding sequences (CDS), dictates the mRNA export pathway used. Finally, we discovered a strong correlation between existence of 5'UTR introns and sequence features near the 5' end of CDS. We developed an integrated machine-learning framework that can predict absence of 5UIs using solely the sequence near the 5' end of CDS. Our model achieved >80% accuracy when validated against nuclear-encoded mitochondrial transcripts. Specific RNA elements predictive of 5UI absence are found in ~40% of human transcripts spanning a wide spectrum of functions. By analyzing hundreds of large-scale datasets, we functionally characterized the transcripts with these RNA elements; revealing their association with translational regulation. These RNA elements were bound by proteins interacting with the Exon Junction Complex in vivo suggesting a molecular mechanism that links these elements to their downstream effects in mRNA export and translational regulation. While some 5'UTR introns might be evolving neutrally, our results, taken together, suggest that complex evolutionary forces are acting on this distinct class of introns.

The assembly, form, and function of the heart is regulated by complex mechanical signals originating from intrinsic and extrinsic sources, such as the cytoskeleton and the extracellular matrix. During development, mechanical forces influence the self-assembly of highly organized ventricular myocardium. However, mechanical overload induces maladaptive remodeling of tissue structure and eventual failure. Thus, mechanical forces potentiate physiological or pathological remodeling, depending on factors such as frequency and magnitude. We hypothesized that mechanical stimuli in the form of microenvironmental stiffness, cytoskeletal architecture, or cyclic stretch regulate cell-cell junction formation and cytoskeletal remodeling during development and disease. To test this, we engineered cardiac tissues in vitro and quantified structural and functional remodeling over multiple spatial scales in response to diverse mechanical perturbations mimicking development and disease. We first asked if the mechanical microenvironment impacts tissue assembly. To investigate this, we cultured two-cell cardiac µtissues on flexible substrates with tunable stiffness and monitored cell-cell junction formation over time. As myocytes transitioned from isolated cells to interconnected µtissues, focal adhesions disassembled near cell-cell interfaces and mechanical forces were transmitted almost completely through cell-cell junctions. However, µtissues cultured on stiff substrates mimicking fibrotic microenvironments retained focal adhesions near the cell-cell interface, potentially to reinforce the cell-cell junction in response to excessive forces generated by myofibrils in stiff microenvironments. Intercellular electrical conductance between myocytes was measured as a function of connexin 43 immunosignal and the length-to-width ratio of cell pairs. We observed that conductance was correlated to connexin 43 immunosignal and cell pair length-to-width ratio, indicating that tissue architecture can affect electrical coupling. The impact of mechanical overload was also determined by applying chronic cyclic stretch to engineered cardiac tissues. Stretch activated gene expression patterns characteristic of pathological remodeling, including up-regulation of focal adhesion genes, and impacted sarcomere alignment and myocyte shape. Furthermore, chronic cyclic stretch altered intracellular calcium cycling in a manner similar to heart failure and decreased contractile stress generation, suggestive of maladaptive remodeling. In summary, we show that the assembly, form, and function of cardiac tissue is sensitive to a wide range of mechanical cues that emerge during physiological and pathological growth. / Engineering and Applied Sciences

Transport through nanostructures is studied at the many-body level using exact diagonalization and nonequilibrium Green's functions. Organic molecular junctions are a particular focus because of their technological promise. Work is presented regarding: (1) A π-electron model of organic molecular junctions developed using effective field theory; (2) series transmission and transmission node structure in interacting systems; (3) the effect of interactions on quantum interference and thermoelectricity in polycyclic junctions; and (4) nanoscale transport calculations using self-consistent statistical ensembles.

This thesis is concerned with employing the statistics of charge transfer in a conductor as a tool for quantum measurement. The physical systems studied are electronic devices made by nanoscale manufacturing techniques. In this context quantum measurement appears not as a postulate, but as physical process. In this thesis I am considering a quantum system, in particular a qubit or a nanomechanical resonator, interacting with a tunnel junction. The effect of coupling a quantum system to a tunnel junction is twofold: The state of the quantum system will be changed and there will be information about the quantum system in the statistics of charge transfer of the tunnel junction. As the first example a quantum measurement process of a qubit is considered. A common description of the system and charge dynamics is found by introducing a new quantity, the charge specific density matrix. By deriving and solving a Markovian master equation for this quantity the measurement process is analyzed. The measurement is shown to be a dynamical process, where correlations between the initial state of the qubit and the number of charges transferred in the tunnel junction arise on a typical timescale, the measurement time. As another example of a quantum system a nanomechanical oscillator is considered. It is found, that the biased tunnel junction, acting as a non-equilibrium environment to the oscillator, increases the temperature of the oscillator from its thermal equilibrium value. The current in the junction is modulated by the interaction with the oscillator, but the influence vanishes for bias voltages smaller than the oscillator frequency. For an asymmetric junction and non-vanishing oscillator momentum a current is shown to flow through the junction even at zero bias. The current noise spectrum induced by the oscillator in the tunnel junction consists of a noise floor and a peaked structure with peaks at zero frequency, the oscillator frequency and double the oscillator frequency. The peak heights are dependent on the coupling strength between oscillator and junction, the occupation number of the oscillator, the bias voltage and the junction temperature. I show how the peak height can be used as a measure of the oscillator temperature, demonstrating that the noise of a tunnel junction can be used for electronic thermometry of a nanomechanical oscillator.

The precise structure of the adult Drosophila eye results from a coordinated process of cell sorting, differentiation and selective cell death in the retinal epithelium. Mutations in the gene echinus cause supernumerary pigment cells due to insufficient cell death. This study reports the identification of echinus and the characterization of its role in Drosophila retinal development. Using a combination of deletion mapping, gene expression analysis and genomic sequencing, echinus was cloned and several alleles were sequenced. echinus encodes a ~180kDa protein containing an ubiquitin hydrolase domain at its N-terminus and a polyglutamine tract at its C-terminus. echinus is expressed in the retina during pupal development and mutants of echinus have decreased levels of apoptosis during several stages of retinal development. Defects in the cell sorting process that precedes cell death are also observed in echinus loss-of-function mutants and echinus overexpression can cause defects in ommatidial rotation and the morphology of cone cells. echinus is a positive regulator of DE-cadherin and Enabled accumulation in adherens junctions of retinal epithelial cells. Genetic interactions were observed between echinus and the genes wingless, enabled and expanded. An immunofluorescence assay in Drosophila S2 cell cultured demonstrated that Echinus localizes to intracellular vesicles that do not appear to be endocytic in nature, and the C-terminal region of Echinus was shown to be necessary for this association. A protein interaction screen using an immunoprecipitation and mass spectrometry approach identified interactions between Echinus and the vesicle coat protein Clathrin, the scaffolding protein RACK1 and the casein kinase I epsilon (Dco). Co-immunoprecipitation additionally identified an interaction between Echinus and Enabled. This work has revealed echinus to be an important regulator of cell sorting and adherens junction formation in the developing retina and has identified multiple interactions between echinus and enabled, a regulator of the actin cytoskeleton.

This thesis investigates the origin of spontaneous activity in the uterus. This organ does not show any activity until shortly before delivery, where fast and efficient contractions are generated. The aim of this work is to provide insight into the origin of spontaneous oscillations and into the transition from asynchronous to synchronized activity in the pregnant uterus. One intriguing aspect in the uterus is the absence of any pacemaker cell. The organ is composed of muscular cells, which are excitable, and connective cells, whose behavior is purely passive; None of these cells, taken in isolation, spontaneously oscillates. We develop an hypothesis based on the observed strong increase in the electrical coupling between cells in the last days of pregnancy. The study is based on a mathematical model of excitable cells, coupled to each other on a regular lattice, and to a fluctuating number of passive cells, consistent with the known structure of the uterus. The two parameters of the model, the coupling between excitable cells, and between excitable and passive cells, grow during pregnancy.Using both a model based on measured electrophysiological properties, and a generic model of excitable cell, we demonstrate that spontaneous oscillations can appear when increasing the coupling coefficients, ultimately leading to coherent oscillations over the entire tissue. We study the transition towards a coherent regime, both numerically and semi-analytically, using the simple model of excitable cells. Last, we demonstrate that, the realistic model reproduces irregular action potential propagation patterns as well as the bursting behavior, observed in the in-vitro experiments.

Šiame darbe pateikiama ląstelių plyšinės jungties Markovo modelių sudarymo metodika, naudojant Markovo procesus, apimanti būsenų grafų generavimą, stacionariųjų tikimybių skaičiavimą ir plyšinės jungties laidumo priklausomybės nuo įtampos skaičiavimus. Darbe aprašomi skirtingi plyšinės jungties modeliai. Kiekvienas modelis turi savo koneksinų būsenų grafus, kuriais remiantis yra simuliuojama plyšinės jungties laidžio priklausomybė nuo įtampos. Kiekvienas koneksinas gali būti aprašomas dviejomis būsenomis: „O“ – atvira, „C“ - uždara ir trijomis būsenomis: „O“ – atvira, „C“ – uždara, „D“ – visiškai uždara. Remiantis sumodeliuotais modeliais, buvo sukurta programinė įranga leidžianti grafiškai pavaizduoti modelių būsenų grafus, simuliuoti modelius ir gauti simuliacijos rezultatus. Taipogi buvo realizuota programinės įrangos realizacija į kitas sistemas. / In this paper methology of composing Markov preocess models of gap junction cells is introduced. This methology contains state graphs generation, computing of stationary probabilities and computing of the conductance of the gap junction dependence on a voltage. In this paper different gap junction models are presented. Every model has it‘s own connexin state graphs, on which the conductance of the gap junction dependence on a voltage simulation is based. Every connexin can have two different state scenarios: first scenario where two connexin model is based on two states „O“ – open or „C“ – closed and second scenario where three connexin model is based on three states „O“ – open, „C“ – closed and „D“ – deep closed. The computer programs based on these models where created, which allows user graphically see the models state graphs, simulate models and get the needed results. Also these programs are integrated into more difficult systems and into other libraries.