Simultaneous imaging of different focal planes in fluorescence microscopy for the study of cellular dynamics in three dimensions

The imaging of cellular dynamics in three dimensions using a standard microscope is severely limited due to the fact that only one focal plane can be imaged at a given point in time. Here we present a modification of the classical microscope design with which two or more focal planes can be imaged simultaneously. This is achieved by a modification of the emission pathway of a standard microscope. The efficacy of the design is shown by imaging bead samples and an FcRn-green fluorescent protein expressing tubule that leaves a sorting endosome and subsequently exocytoses at the plasma membrane.     

A note on a system theoretic approach to a conjecture by Peller-Khrushchev

Based on the construction of infinite dimensional balanced realizations an alternative solution to the following inverse spectral problem is presented: Given a monotonically decreasing sequence of positive numbers (σ_n)_{n 1}, does there exist a Hankel operator whose sequence of singular values is (σ_n)_{n 1}?     

Homozygosity for a Novel DOCK7 Variant Due to Segmental Uniparental Isodisomy of Chromosome 1 Associated with Early Infantile Epileptic Encephalopathy (EIEE) and Cortical Visual Impairment

Early infantile epileptic encephalopathy (EIEE) is a severe neurologic and neurodevelopmental disease that manifests in the first year of life. It shows a high degree of genetic heterogeneity, but the genetic origin is only identified in half of the cases. We report the case of a female child initially diagnosed with Leber congenital amaurosis (LCA), an early-onset retinal dystrophy due to photoreceptor cell degeneration in the retina. The first examination at 9 months of age revealed no reaction to light or objects and showed wandering eye movements. Ophthalmological examination did not show any ocular abnormalities. The patient displayed mildly dysmorphic features and a global developmental delay. Brain MRI demonstrated pontine hypo-/dysplasia. The patient developed myoclonic epileptic seizures and epileptic spasms with focal and generalized epileptiform discharges on electroencephalogram (EEG) at the age of 16 months. Genetic screening for a potentially pathogenic DNA sequence variant by whole-exome sequencing (WES) revealed a novel, conserved, homozygous frameshift variant (c.5391delA, p.(Ala1798LeufsTer59)) in exon 42 of the DOCK7 gene ({"type":"entrez-nucleotide","attrs":{"text":"NM_001271999.1","term_id":"431822374","term_text":"NM_001271999.1"}}NM_001271999.1). Further analysis by SNP array (Karyomapping) showed loss of heterozygosity (LOH) in four segments of chromosome 1. WES data of the parents and the index patient (trio analysis) demonstrated that chromosome 1 was exclusively inherited from the mother. Four LOH segments of chromosome 1 alternately showed isodisomy (UPiD) and heterodisomy (UPhD). In WES data, the father was a noncarrier, and the mother was heterozygous for this DOCK7 variant. The DOCK7 gene is located in 1p31.3, a region situated in one of the four isodisomic segments of chromosome 1, explaining the homozygosity seen in the affected child. Finally, Sanger sequencing confirmed maternal UPiD for the DOCK7 variant. Homozygous or compound heterozygous pathogenic variants in the DOCK7 (dedicator of cytokinesis 7) gene are associated with autosomal recessive, early infantile epileptic encephalopathy 23 (EIEE23; OMIM #615,859), a rare and heterogeneous group of neurodevelopmental disorders diagnosed during early childhood. To our knowledge, this is the first report of segmental uniparental iso- and heterodisomy of chromosome 1, leading to homozygosity of the DOCK7 frameshift variant in the affected patient.     

Sucrose transport through maltoporin mutants of Escherichia coli

Maltoporin (LamB) and sucrose porin (ScrY) reside in the bacterialouter membrane and facilitate the passive diffusion of maltodextrinsand sucrose, respectively. To gain further insight into thedeterminants of solute specificity, LamB mutants were designedto allow translocation of sucrose, which hardly translocatesthrough wild-type LamB. Three LamB mutants were studied. (a)Based on sequence and structure alignment of LamB with ScrY,two LamB triple mutants were generated (R109D, Y118D,D121F;R109N,Y118D,D121F) to mimic the ScrY constriction. The crystalstructure of the first of these mutants was determined to be3.2 Å and showed an increased ScrY-like cross-sectionexcept for D109 that protrudes into the channel. (b) Based onthis crystal structure a double mutant was generated by truncationof the two residues that obstruct the channel most in LamB (R109A,Y118A).Analysis of liposome swelling and in vivo sugar uptake demonstratedsubstantial sucrose permeation through all mutants with thedouble alanine mutant performing best. The triple mutants didnot show a well-defined binding site as indicated by sugar-inducedion current noise analysis, which can be explained by remainingsteric interference as deduced from the crystal structure. Binding,however, was observed for the double mutant that had the obstructingresidues truncated to alanines.     

Charakterisierung des Beulverhaltens von Gaspipelines

The aim of this diploma thesis was a comparison of different possible classifications of dents in the context of experiments and FEM (finite element method) simulations. In general, denting is a frequent kind of damage in transport pipelines. Dents are, for example, caused by incorrect embedding or by third parties. Literature already provides a variety of classifications for dents, which are briefly summarized. Subsequently, necessary forces which cause dents and the resulting geometry of the pipeline after the denting process have been measured by tests. For a better understanding of the denting process, FEM simulations were performed. This thesis should improve the knowledge of the characteristic behaviour of dents.     

Imaging and Analysis of THz Surface Plasmon Polariton Waves with the Integrated Sub-wavelength Aperture Probe

We demonstrate that the integrated sub-wavelength aperture probe designed for THz near-field scanning probe microscopy can be used to map surface plasmon waves at THz frequencies. Observed near-field images of metallic patterns reveal surface plasmon waves superimposed over THz transmission images. We discuss the coupling mechanism for the surface waves and arrive to an important conclusion that the detected surface wave images represent the spatial derivative of the surface plasmon electric field. The relationship between the electric field and the measured signal is confirmed experimentally by mapping surface waves in bow-tie antennas. This study explains previously observed effects in THz near-field microscopy and provides a framework for analysis of near-field images.     

Analysis of Mitochondrial Function,Structure, and Intracellular Organization In Situ in Cardiomyocytes and Skeletal Muscles

Analysis of the function, structure, and intracellular organization of mitochondria is important for elucidating energy metabolism and intracellular energy transfer. In addition, basic and clinically oriented studies that investigate organ/tissue/cell dysfunction in various human diseases, including myopathies, cardiac/brain ischemia-reperfusion injuries, neurodegenerative diseases, cancer, and aging, require precise estimation of mitochondrial function. It should be noted that the main metabolic and functional characteristics of mitochondria obtained in situ (in permeabilized cells and tissue samples) and in vitro (in isolated organelles) are quite different, thereby compromising interpretations of experimental and clinical data. These differences are explained by the existence of the mitochondrial network, which possesses multiple interactions between the cytoplasm and other subcellular organelles. Metabolic and functional crosstalk between mitochondria and extra-mitochondrial cellular environments plays a crucial role in the regulation of mitochondrial metabolism and physiology. Therefore, it is important to analyze mitochondria in vivo or in situ without their isolation from the natural cellular environment. This review summarizes previous studies and discusses existing approaches and methods for the analysis of mitochondrial function, structure, and intracellular organization in situ.