Analysis of the transcriptional program governing meiosis and gametogenesis in yeast and mammals

During meiosis a competent diploid cell replicates its DNA once and then undergoes two consecutive divisions followed by haploid gamete differentiation. Important aspects of meiotic development that distinguish it from mitotic growth include a highly increased rate of recombination, formation of the synaptonemal complex that aligns the homologous chromosomes, as well as separation of the homologues and sister chromatids during meiosis I and II without an intervening S-phase. Budding yeast is an excellent model organism to study meiosis and gametogenesis and accordingly, to date it belongs to the best studied eukaryotic systems in this context. Knowledge coming from these studies has provided important insights into meiotic development in higher eukaryotes. This was possible because sporulation in yeast and spermatogenesis in higher eukaryotes are analogous developmental pathways that involve conserved genes. For budding yeast a huge amount of data from numerous genome-scale studies on gene expression and deletion phenotypes of meiotic development and sporulation are available. In contrast, mammalian gametogenesis has not been studied on a large-scale until recently. It was unclear if an expression profiling study using germ cells and testicular somatic control cells that underwent lengthy purification procedures would yield interpretable results. We have therefore carried out a pioneering expression profiling study of male germ cells from Rattus norvegicus using Affymetrix U34A and B GeneChips. This work resulted in the first comprehensive large-scale expression profiling analysis of mammalian male germ cells undergoing mitotic growth, meiosis and gametogenesis. We have identified 1268 differentially expressed genes in germ cells at different developmental stages, which were organized into four distinct expression clusters that reflect somatic, mitotic, meiotic and post-meiotic cell types. This included 293 yet uncharacterized transcripts whose expression pattern suggests that they are involved in spermatogenesis and fertility. A group of 121 transcripts were only expressed in meiotic (spermatocytes) and postmeiotic germ cells (round spermatids) but not in dividing germ cells (spermatogonia), Sertoli cells or two somatic control tissues (brain and skeletal muscle). Functional analysis reveals that most of the known genes in this group fulfill essential functions during meiosis, spermiogenesis (the process of sperm maturation) and fertility. Therefore it is highly possible that some of the �30 uncharacterized transcripts in this group also contribute to these processes. A web-accessible database (called reXbase, which was later on integrated into GermOnline) has been developed for our expression profiling study of mammalian male meiosis, which summarizes annotation information and shows a graphical display of expression profiles of every gene covered in our study. In the budding yeast Saccharomyces cerevisiae entry into meiosis and subsequent progression through sporulation and gametogenesis are driven by a highly regulated transcriptional program activated by signal pathways responding to nutritional and cell-type cues. Abf1p, which is a general transcription factor, has previously been demonstrated to participate in the induction of numerous mitotic as well as early and middle meiotic genes. In the current study we have addressed the question how Abf1p transcriptionally coordinates mitotic growth and meiotic development on a genome-wide level. Because ABF1 is an essential gene we used the temperature-sensitive allele abf1-1. A phenotypical analysis of mutant cells revealed that ABF1 plays an important role in cell separation during mitosis, meiotic development, and spore formation. In order to identify genes whose expression depends on Abf1p in growing and sporulating cells we have performed expression profiling experiments using Affymetrix S98 GeneChips comparing wild-type and abf1-1 mutant cells at both permissive and restrictive temperature. We have identified 504 genes whose normal expression depends on functional ABF1. By combining the expression profiling data with data from genome-wide DNA binding assays (ChIPCHIP) and in silico predictions of potential Abf1p-binding sites in the yeast genome, we were able to define direct target genes. Expression of these genes decreases in the absence of functional ABF1 and whose promotors are bound by Abf1p and/or contain a predicted binding site. Among 352 such bona fide direct target genes we found many involved in ribosome biogenesis, translation, vegetative growth and meiotic developement and therefore could account for the observed growth and sporulation defects of abf1-1 mutant cells. Furthermore, the fact that two members of the septin family (CDC3 and CDC10 ) were found to be direct target genes suggests a novel role for Abf1p in cytokinesis. This was further substantiated by the observation that chitin localization and septin ring formation are perturbed in abf1-1 mutant cells

Multivalent interaction and selectivities in selectin binding of functionalized gold colloids decorated with carbohydrate mimetics

Colloidal gold particles with functionalized organic shells were applied as novel selectin binders. The ligand shell was terminated with different monocyclic carbohydrate mimetics as simplified analogs of the sLe(x) unit found in biological selectin ligands. The multivalent presentation of the sulfated selectin binding epitopes on the gold particles led to extremely high binding affinities towards L- and P-selectin and IC(50) values in the subnanomolar range. Depending on the ring size of the sulfated carbohydrate mimetic, its substitution pattern and its configuration, different selectivities for either L-selectin or P-selectin were obtained. These selectivities were not found for gold particles with simple acyclic sulfated alcohols, diols and triols in the ligand shell. In addition, the influence of the particle size and the thickness of the hydrophobic organic shell were systematically investigated

Evidence that Meningeal Mast Cells Can Worsen Stroke Pathology in Mice

Stroke is the leading cause of adult disability and the fourth most common cause of death in the United States. Inflammation is thought to play an important role in stroke pathology, but the factors that promote inflammation in this setting remain to be fully defined. An understudied but important factor is the role of meningeal-located immune cells in modulating brain pathology. Although different immune cells traffic through meningeal vessels en route to the brain, mature mast cells do not circulate but are resident in the meninges. With the use of genetic and cell transfer approaches in mice, we identified evidence that meningeal mast cells can importantly contribute to the key features of stroke pathology, including infiltration of granulocytes and activated macrophages, brain swelling, and infarct size. We also obtained evidence that two mast cell-derived products, interleukin-6 and, to a lesser extent, chemokine (C-C motif) ligand 7, can contribute to stroke pathology. These findings indicate a novel role for mast cells in the meninges, the membranes that envelop the brain, as potential gatekeepers for modulating brain inflammation and pathology after stroke

Impact of prototyping resource environments and timing of awareness of constraints on idea generation in product design

Research and development laboratories in universities and firms around the world try to maximize innovation with a limited set of resources. However, questions remain about the influence of resource constraints on idea generation in early-stage product design. Multiple embedded case studies were conducted with engineering students and faculty at two university campuses in Mexico. Students developed sketches for products that would satisfy an open-ended design problem in a constrained-resource setting, where the variables were the timing of when information about these constraints was revealed, and the regular prototyping environment of the student. The evidence suggests that the timing of awareness of constraints can have an impact on design outcomes, but that this effect varies depending on the designer's regular prototyping resource environment.MIT International Science and Technology InitiativeLegatum Center for Development & Entrepreneurship (Massachusetts Institute of Technology)National Science Foundation (U.S.) (Award CMMI-1130791

Gene Annotation and Drug Target Discovery in Candida albicans with a Tagged Transposon Mutant Collection

Candida albicans is the most common human fungal pathogen, causing infections that can be lethal in immunocompromised patients. Although Saccharomyces cerevisiae has been used as a model for C. albicans, it lacks C. albicans' diverse morphogenic forms and is primarily non-pathogenic. Comprehensive genetic analyses that have been instrumental for determining gene function in S. cerevisiae are hampered in C. albicans, due in part to limited resources to systematically assay phenotypes of loss-of-function alleles. Here, we constructed and screened a library of 3633 tagged heterozygous transposon disruption mutants, using them in a competitive growth assay to examine nutrient- and drug-dependent haploinsufficiency. We identified 269 genes that were haploinsufficient in four growth conditions, the majority of which were condition-specific. These screens identified two new genes necessary for filamentous growth as well as ten genes that function in essential processes. We also screened 57 chemically diverse compounds that more potently inhibited growth of C. albicans versus S. cerevisiae. For four of these compounds, we examined the genetic basis of this differential inhibition. Notably, Sec7p was identified as the target of brefeldin A in C. albicans screens, while S. cerevisiae screens with this compound failed to identify this target. We also uncovered a new C. albicans-specific target, Tfp1p, for the synthetic compound 0136-0228. These results highlight the value of haploinsufficiency screens directly in this pathogen for gene annotation and drug target identification

De novo Assembly of a 40 Mb Eukaryotic Genome from Short Sequence Reads: Sordaria macrospora, a Model Organism for Fungal Morphogenesis

Filamentous fungi are of great importance in ecology, agriculture, medicine, and biotechnology. Thus, it is not surprising that genomes for more than 100 filamentous fungi have been sequenced, most of them by Sanger sequencing. While next-generation sequencing techniques have revolutionized genome resequencing, e.g. for strain comparisons, genetic mapping, or transcriptome and ChIP analyses, de novo assembly of eukaryotic genomes still presents significant hurdles, because of their large size and stretches of repetitive sequences. Filamentous fungi contain few repetitive regions in their 30–90 Mb genomes and thus are suitable candidates to test de novo genome assembly from short sequence reads. Here, we present a high-quality draft sequence of the Sordaria macrospora genome that was obtained by a combination of Illumina/Solexa and Roche/454 sequencing. Paired-end Solexa sequencing of genomic DNA to 85-fold coverage and an additional 10-fold coverage by single-end 454 sequencing resulted in ∼4 Gb of DNA sequence. Reads were assembled to a 40 Mb draft version (N50 of 117 kb) with the Velvet assembler. Comparative analysis with Neurospora genomes increased the N50 to 498 kb. The S. macrospora genome contains even fewer repeat regions than its closest sequenced relative, Neurospora crassa. Comparison with genomes of other fungi showed that S. macrospora, a model organism for morphogenesis and meiosis, harbors duplications of several genes involved in self/nonself-recognition. Furthermore, S. macrospora contains more polyketide biosynthesis genes than N. crassa. Phylogenetic analyses suggest that some of these genes may have been acquired by horizontal gene transfer from a distantly related ascomycete group. Our study shows that, for typical filamentous fungi, de novo assembly of genomes from short sequence reads alone is feasible, that a mixture of Solexa and 454 sequencing substantially improves the assembly, and that the resulting data can be used for comparative studies to address basic questions of fungal biology

Gas-sensing properties of V2O5-nanofibers and carbon nanotubes

In this thesis, the synthesis and characterization of V2O5-nanostructures, as well as the gas sensing properties of V2O5-nanofibers and carbon nanotubes have been investigated. Various modifications of the nanowires have been successfully employed in order to achieve an improved sensitivity and selectivity of the sensors to specific analytes. The V2O5-nanofibers have been synthesized via solution-based chemistry. Two modifications of the standard synthesis route have been attempted in order to reduce the time required to grow fibers of sufficient length. The first modification utilizes silver ions, which allowed for a ten-fold increase in growth speed. In order to determine the role of the silver in the synthesis, energy dispersive X-ray (EDX) analysis has been performed, which revealed the presence of silver-clusters attached to the V2O5-nanofibers, as well as silver incorporated into the fibers. The second modification is based on hydrothermal synthesis performed at 180oC, which yielded VOx-nanobelts rather than V2O5-nanofibers. Most striking is their appearance in the shape of a boomerang with a reproducible angle of 96o. The origin of the kinked structure, as determined with transmission electron microscopy (TEM) and selected area electron diffraction (SAED), was found to be twinning of the crystalline material along the [130]-direction. Raman spectroscopy and temperature-dependent electrical transport measurements on the V2O5-materials revealed their close similarity. In all cases, the electrical transport was found to be dominated by a hopping-like conduction. To assess the gas sensing properties of V2O5-nanofibers and carbon nanotubes, network samples have been investigated at room temperature, using the change of resistance as sensor signal. For the detection of ammonia with the V2O5-nanofibers, the change of resistance has been ascribed to charge transfer. Through evaporation of gold onto the V2O5-nanofibers the sensor response could be improved by a factor of seven. The slower desorption of ammonia from the gold-modified fibers, as compared to the unmodified material, is attributed to a stronger binding of ammonia to gold. The detection of butylamine with V2O5-nanofibers has been studied due to the application relevance, as this compound is produced in rotten food. By investigating different contact configurations, the sensor response (~500 %) has been found to originate from intercalation of the analyte between electrode and fiber, resulting in an increased contact resistance. Modifying networks of V2O5-nanofibers via deposition of an ultrathin layer of palladium rendered the sensors highly sensitive to hydrogen, resulting in responses of more than 100,000 %. The sensor mechanism, as elucidated through a combined study using Raman spectroscopy and temperature-dependent electrical transport measurements, involved atomic hydrogen, formed within the palladium layer. Its reaction with oxygen ions in the V2O5-lattice leads to oxygen deficiencies and hence the formation of additional polarons, which combine to less mobile bipolarons, as apparent from an increase in the hopping activation energy. Besides V2O5-nanofibers, the sensor properties of carbon nanotubes have been studied. Their gas sensing behavior has been interpreted in terms of charge transfer between the analyte molecules and the p-type semiconducting nanotubes present in network samples. In the specific case of ammonia exposure, the nanotube sensors do not recover completely to their original resistance, but equilibrate at an increased value. This observation has been accounted for by an adapted Langmuir isotherm, which assumes reversible and irreversible adsorption sites on the nanotube surface. The presence of irreversible sites, which require temperatures up to 500 K for the desorption of ammonia, could be experimentally confirmed by thermal desorption spectroscopy (TDS). To render carbon nanotubes sensitive to hydrogen, palladium nanoparticles were electrodeposited. This method offers the advantage of restricting the modification to nanotubes which are contacted to the electrodes, while isolated nanotubes and substrate are not affected. Palladium-modified nanotubes prepared in this fashion showed good responses to hydrogen at room temperature

Theoretical and experimental investigations on the influence of the microscopic anisotropic material properties of gear teeth on their macroscopic behavior

Innovative cold rolling technologies are important future production methods to gain more resource efficiency producing gears. Through current research at the Fraunhofer Institute for Machine Tools and Forming Technology (IWU), it is possible to produce running gears for use in vehicle transmissions by this method. The paper contends theoretical and experimental investigations on cold rolled gears to determine their properties in detail. The theoretical principles of material anisotropy following cold forming processes as well as the results of investigations on gear teeth are discussed. Different calculation methodologies to compute the efficient elastic behavior of an anisotropic material area are compared by applying them to measurement data of an Electron Backscatter Diffraction (EBSD). Furthermore, methods to simulate the rolling process are described and applied to the investigated gear. It is shown that the production method can have an effect on the elastic material behavior of gear teeth