Structural characterization and distribution of zebrafish germ plasm during interphase and mitosis

In zebrafish, germ cells are responsible for transmitting the genetic information from one generation to the next. During the first cleavages of zebrafish embryonic development, a specialized part of the cytoplasm known as germ plasm, is responsible of committing four blastomeres to become the progenitors of all germ cells in the forming embryo. Much is known about how the germ plasm is spatially distributed in early stages of primordial germ cell development, a process described to be dependant on microtubules and actin. However, little is known about how the material is inherited after it reorganizes into a perinuclear location, or how is the symmetrical distribution regulated in order to ensure proper inheritance of the material by both daughter cells. It is also not clear whether there is a controlled mechanism that regulates the number of granules inherited by the daughter cells, or whether it is a random process. We describe the distribution of germ plasm material from 4hpf to 24hpf in zebrafish primordial germ cells using Vasa protein as marker. Vasa positive material appears to be conglomerate into 3 to 4 big spherical structures at 4hpf. While development progresses, these big structures become smaller perinuclear granules that reach a total number of approximately 30 at 24hpf. We investigated how this transformation occurs and how the minus-end microtubule dependent motor protein Dynein plays a role in this process. Additionally, we describe specific colocalization of microtubules and perinuclear granules during interphase and more interestingly, during all different stages of cell division. We show that distribution of granules follow what seems to be a regulated distribution: during cells division, daughter cells inherit an equal number of granules. We propose that due to the permanent colocalization of microtubular structures with germinal granules during interphase and cell division, a coordinated mechanism between these structures may ensure proper distribution of the material among daughter cells. Furthermore, we show that exposure to the microtubule-depolymerizing drug nocodazole leads to disassembly of the germ cell nuclear lamin matrix, chromatin condensation, and fusion of granules to a big conglomerate, revealing dependence of granular distribution on microtubules and proper nuclear structure.

Development of mechanical methods for cell-tray propagation and field transplanting of dwarf napiergrass (Pennisetum purpureum Schumach.)

Since dwarf napiergrass (Pennisetum purpureum Schumach.) must be propagated vegetatively due to lack of viable seeds, root splitting and stem cuttings are generally used to obtain true-to-type plant populations. These ordinary methods are laborious and costly, and are the greatest barriers for expanding the cultivation area of this crop. The objectives of this research were to develop nursery production of dwarf napiergrass in cell trays and to compare the efficiency of mechanical versus manual methods for cell-tray propagation and field transplanting. After defoliation of herbage either by a sickle (manually) or hand-mowing machine, every potential aerial tiller bud was cut to a single one for transplanting into cell trays as stem cuttings and placed in a glasshouse over winter. The following June, nursery plants were trimmed to a 25–cm length and transplanted in an experimental field (sandy soil) with 20,000 plants ha^(−1) either by shovel (manually) or Welsh onion planter. Labour time was recorded for each process. The manual defoliation of plants required 44% more labour time for preparing the stem cuttings (0.73 person-min. stemcutting^(−1)) compared to using hand-mowing machinery (0.51 person-min. stem-cutting^(−1)). In contrast, labour time for transplanting required an extra 0.30 person-min. m^(−2) (14%) using the machinery compared to manual transplanting, possibly due to the limited plot size for machinery operation. The transplanting method had no significant effect on plant establishment or plant growth, except for herbage yield 110 days after planting. Defoliation of herbage by machinery, production using a cell-tray nursery and mechanical transplanting reduced the labour intensity of dwarf napiergrass propagation.

Modelling and simulation of a photovoltaic fuel cell hybrid system

A stand-alone power system is an autonomous system that supplies electricity to the user load without being connected to the electric grid. This kind of decentralized system is frequently located in remote and inaccessible areas. It is essential for about one third of the world population which are living in developed or isolated regions and have no access to an electricity utility grid. The most people live in remote and rural areas, with low population density, lacking even the basic infrastructure. The utility grid extension to these locations is not a cost effective option and sometimes technically not feasible. The purpose of this thesis is the modelling and simulation of a stand-alone hybrid power system, referred to as “hydrogen Photovoltaic-Fuel Cell (PVFC) hybrid system”. It couples a photovoltaic generator (PV), an alkaline water electrolyser, a storage gas tank, a proton exchange membrane fuel cell (PEMFC), and power conditioning units (PCU) to give different system topologies. The system is intended to be an environmentally friendly solution since it tries maximising the use of a renewable energy source. Electricity is produced by a PV generator to meet the requirements of a user load. Whenever there is enough solar radiation, the user load can be powered totally by the PV electricity. During periods of low solar radiation, auxiliary electricity is required. An alkaline high pressure water electrolyser is powered by the excess energy from the PV generator to produce hydrogen and oxygen at a pressure of maximum 30bar. Gases are stored without compression for short- (hourly or daily) and long- (seasonal) term. A proton exchange membrane (PEM) fuel cell is used to keep the system’s reliability at the same level as for the conventional system while decreasing the environmental impact of the whole system. The PEM fuel cell consumes gases which are produced by an electrolyser to meet the user load demand when the PV generator energy is deficient, so that it works as an auxiliary generator. Power conditioning units are appropriate for the conversion and dispatch the energy between the components of the system. No batteries are used in this system since they represent the weakest when used in PV systems due to their need for sophisticated control and their short lifetime. The model library, ISET Alternative Power Library (ISET-APL), is designed by the Institute of Solar Energy supply Technology (ISET) and used for the simulation of the hybrid system. The physical, analytical and/or empirical equations of each component are programmed and implemented separately in this library for the simulation software program Simplorer by C++ language. The model parameters are derived from manufacturer’s performance data sheets or measurements obtained from literature. The identification and validation of the major hydrogen PVFC hybrid system component models are evaluated according to the measured data of the components, from the manufacturer’s data sheet or from actual system operation. Then, the overall system is simulated, at intervals of one hour each, by using solar radiation as the primary energy input and hydrogen as energy storage for one year operation. A comparison between different topologies, such as DC or AC coupled systems, is carried out on the basis of energy point of view at two locations with different geographical latitudes, in Kassel/Germany (Europe) and in Cairo/Egypt (North Africa). The main conclusion in this work is that the simulation method of the system study under different conditions could successfully be used to give good visualization and comparison between those topologies for the overall performance of the system. The operational performance of the system is not only depending on component efficiency but also on system design and consumption behaviour. The worst case of this system is the low efficiency of the storage subsystem made of the electrolyser, the gas storage tank, and the fuel cell as it is around 25-34% at Cairo and 29-37% at Kassel. Therefore, the research for this system should be concentrated in the subsystem components development especially the fuel cell.

Functional genomics of the Drosophila melanogaster X chromosome and the role of DWnt5 during development

The collection of X chromosome insertions (PX) lethal lines, which was isolated from a screen for essential genes on the X chromosome, was characterized by means of cloning the insertion sites, mapping the sites within genomic DNA and determination of the associated reporter gene expresssion patterns. The established STS flanking the P element insertion sites were submitted to EMBL nucleotide databases and their in situ data together with the enhancer trap expression patterns have been deposited in the FlyView database. The characterized lines are now available to be used by the scientific community for a detailed analysis of the newly established lethal gene functions. One of the isolated genes on the X chromosome was the Drosophila gene Wnt5 (DWnt5). From two independent screens, one lethal and three homozygous viable alleles were recovered, allowing the identification of two distinct functions for DWnt5 in the fly. Observations on the developing nervous system of mutant embryos suggest that DWnt5 activity affects axon projection pattern. Elevated levels of DWNT5 activity in the midline cells of the central nervous system causes improper establishment and maintenance of the axonal pathways. Our analysis of the expression and mutant phenotype indicates that DWnt5 function in a process needed for proper organization of the nervous system. A second and novel function of DWnt5 is the control of the body size by regulation of the cell number rather than affecting the size of cells. Moreover, experimentally increased DWnt5 levels in a post-mitotic region of the eye imaginal disc causes abnormal cell cycle progression, resulting in additional ommatidia in the adult eye when compared to wild type. The increased cell number and the effects on the cell cycle after exposure to high DWNT5 levels is the result of a failure to downregulate cyclin B and therefore the unsuccessful establishment of a G1 arrest.

The role of Ken&Barbie in JAK/STAT signalling during development of Drosophila melanogaster

Cell-cell interactions during embryonic development are crucial in the co-ordination of growth, differentiation and maintenance of many different cell types. To achieve this co-ordination each cell must properly translate signals received from neighbouring cells, into spatially and temporally appropriate developmental responses. A surprisingly limited number of signal pathways are responsible for the differentiation of enormous variety of cell types. As a result, pathways are frequently 'reused' during development. Thus, in mammals the JAK/STAT pathway is required during early embryogenesis, mammary gland formation, hematopoiesis and, finally, plays a pivotal role in immune response. In the canonical way, the JAK/STAT pathway is represented by a transmembrane receptor associated with a Janus kinase (JAK), which upon stimulation by an extra-cellular ligand, phosphorylates itself, the receptor and, finally, the signal transducer and activator of transcription (STAT) molecules. Phosphorylated STATs dimerise and translocate to the nucleus where they activate transcription of target genes. The JAK/STAT pathway has been conserved throughout evolution, and all known components are present in the genome of Drosophila melanogaster. Besides hematopoietic and immunity functions, the pathway is also required during development for processes including embryonic segmentation, tracheal morphogenesis, posterior spiracle formation etc. This study describes Drosophila Ken&Barbie (Ken) as a selective regulator of JAK/STAT signalling. ken mutations identified in a screen for modulators of an eye overgrowth phenotype, caused by over-expression of the pathway ligand unpaired, also interact genetically with the pathway receptor domeless (dome) and the transcription factor stat92E. Over-expression of Ken can phenocopy developmental defects known to be caused by the loss of JAK/STAT signalling. These genetic interactions suggest that Ken may function as a negative regulator of the pathway. Ken has C-terminal Zn-finger domain, presumably for DNA binding, and N-terminal BTB/POZ domain, often found in transcriptional repressors. Using EGFP-fused construct expressed in vivo revealed nuclear accumulation of Ken. Therefore, it is proposed that Ken may act as a suppresser of STAT92E target genes. An in vitro assay, termed SELEX, determined that Ken specifically binds to a DNA sequence, with the essential for DNA recognition core overlapping that of STAT92E. This interesting observation suggests that not all STAT92E sites may also allow Ken binding. Strikingly, when effects of ectopic Ken on the expression of putative JAK/STAT pathway target genes were examined, only a subset of the genes tested, namely vvl, trh and kni, were down-regulated by Ken, whereas some others, such as eve and fj, appeared to be unresponsive. Further analysis of vvl, one of the genes susceptible to ectopic Ken, was undertaken. In the developing hindgut, expression of vvl is JAK/STAT pathway dependent, but remains repressed in the posterior spiracles, despite the stimulation of STAT92E by Upd in their primordia. Importantly, ken is also expressed in the developing posterior spiracles. Strikingly, up-regulation of vvl is observed in these tissues in ken mutant embryos. These imply that while ectopic Ken is sufficient to repress the expression of vvl in the hindgut, endogenous Ken is also necessary to prevent its activation in the posterior spiracles. It is therefore conceivable that ectopic vvl expression in the posterior spiracles of the ken mutants may be the result of de-repression of endogenous STAT92E activity. Another consequence of these observations is a fine balance that must exist between STAT92E and Ken activities. Apparently, endogenous level of Ken is sufficient to repress vvl, but not other, as yet unidentified, JAK/STAT pathway targets, whose presumable activation by STAT92E is required for posterior spiracle development as the embryos mutant for dome, the receptor of the pathway, show severe spiracle defects. These defects are also observed in the embryos mis-expressing Ken. Though it is possible that the posterior spiracle phenotype caused by higher levels of Ken results from a JAK/STAT pathway independent activity, it seems to be more likely that Ken acts in a dosage dependent manner, and extra Ken is able to further antagonise JAK/STAT pathway target genes. While STAT92E binding sites required for target gene expression have been poorly characterised, the existence of genome data allows the prediction of candidate STAT92E sites present in target genes promoters to be attempted. When a 6kb region containing the putative regulatory domains flanking the vvl locus are examined, only a single potential STAT92E binding site located 825bp upstream of the translational start can be detected. Strikingly, this site also includes a perfect Ken binding sequence. Such an in silico observation, though consistent with both Ken DNA binding assay in vitro and regulation of STAT92E target genes in vivo, however, requires further analysis. The JAK/STAT pathway is implicated in a variety of processes during embryonic and larval development as well as in imago. In each case, stimulation of the same transcription factor results in different developmental outcomes. While many potential mechanisms have been proposed and demonstrated to explain such pleiotropy, the present study indicates that Ken may represent another mechanism, with which signal transduction pathways are controlled. Ken selectively down-regulates a subset of potential target genes and so modifies the transcriptional profile generated by activated STAT92E - a mechanism, which may be partially responsible for differences in the morphogenetic processes elicited by JAK/STAT signalling during development.

Molecular mechanisms controlling the precision of chemokine guided cell migration

By the end of the first day of embryonic development, zebrafish primordial germ cells (PGCs) arrive at the site where the gonad develops. In our study we investigated the mechanisms controlling the precision of primordial germ cell arrival at their target. We found that in contrast with our expectations which were based on findings in Drosophila and mouse, the endoderm does not constitute a preferred migration substrate for the PGCs. Rather, endoderm derivatives are important for later stages of organogenesis keeping the PGC clusters separated. It would be interesting to investigate the precise mechanism by which endoderm controls germ cell position in the gonad. In their migration towards the gonad, zebrafish germ cells follow the gradient of chemokine SDF-1a, which they detect using the receptor CXCR4b that is expressed on their membrane. Here we show that the C-terminal region of CXCR4b is responsible for down-regulation of receptor activity as well as for receptor internalization. We demonstrate that receptor molecules unable to internalize are less potent in guiding germ cells to the site where the gonad develops, thereby implicating chemokine receptor internalization in facilitating precision of migration during chemotaxis in vivo. We demonstrate that while CXCR4b activity positively regulates the duration of the active migration phases, the down-regulation of CXCR4b signalling by internalization limits the duration of this phase. This way, receptor signalling contributes to the persistence of germ cell migration, whereas receptor down-regulation enables the cells to stop and correct their migration path close to the target where germ cells encounter the highest chemokine signal. Chemokine receptors are involved in directing cell migration in different processes such as lymphocyte trafficking, cancer and in the development of the vascular system. The C-terminal domain of many chemokine receptors was shown to be essential for controlling receptor signalling and internalization. It would therefore be important to determine whether the role for receptor internalization in vivo as described here (allowing periodical corrections to the migration route) and the mechanisms involved (reducing the level of signalling) apply for those other events, too.

Development of thermo-analytical prediction and classification models for food in thermal devices using a multi sensor system and artificial neural networks

Die thermische Verarbeitung von Lebensmitteln beeinflusst deren Qualität und ernährungsphysiologischen Eigenschaften. Im Haushalt ist die Überwachung der Temperatur innerhalb des Lebensmittels sehr schwierig. Zudem ist das Wissen über optimale Temperatur- und Zeitparameter für die verschiedenen Speisen oft unzureichend. Die optimale Steuerung der thermischen Zubereitung ist maßgeblich abhängig von der Art des Lebensmittels und der äußeren und inneren Temperatureinwirkung während des Garvorgangs. Das Ziel der Arbeiten war die Entwicklung eines automatischen Backofens, der in der Lage ist, die Art des Lebensmittels zu erkennen und die Temperatur im Inneren des Lebensmittels während des Backens zu errechnen. Die für die Temperaturberechnung benötigten Daten wurden mit mehreren Sensoren erfasst. Hierzu kam ein Infrarotthermometer, ein Infrarotabstandssensor, eine Kamera, ein Temperatursensor und ein Lambdasonde innerhalb des Ofens zum Einsatz. Ferner wurden eine Wägezelle, ein Strom- sowie Spannungs-Sensor und ein Temperatursensor außerhalb des Ofens genutzt. Die während der Aufheizphase aufgenommen Datensätze ermöglichten das Training mehrerer künstlicher neuronaler Netze, die die verschiedenen Lebensmittel in die entsprechenden Kategorien einordnen konnten, um so das optimale Backprogram auszuwählen. Zur Abschätzung der thermische Diffusivität der Nahrung, die von der Zusammensetzung (Kohlenhydrate, Fett, Protein, Wasser) abhängt, wurden mehrere künstliche neuronale Netze trainiert. Mit Ausnahme des Fettanteils der Lebensmittel konnten alle Komponenten durch verschiedene KNNs mit einem Maximum von 8 versteckten Neuronen ausreichend genau abgeschätzt werden um auf deren Grundlage die Temperatur im inneren des Lebensmittels zu berechnen. Die durchgeführte Arbeit zeigt, dass mit Hilfe verschiedenster Sensoren zur direkten beziehungsweise indirekten Messung der äußeren Eigenschaften der Lebensmittel sowie KNNs für die Kategorisierung und Abschätzung der Lebensmittelzusammensetzung die automatische Erkennung und Berechnung der inneren Temperatur von verschiedensten Lebensmitteln möglich ist.

Effect of rosemary (Rosmarinus officinalis) extract on weight, hematology and cell-mediated immune response of newborn goat kids

This study aimed at evaluating the effects of different levels of rosemary (Rosmarinus officinalis) extract on growth rate, hematology and cell-mediated immune response in Markhoz newborn goat kids. Twenty four goat kids (aged 7 +/- 3 days) were randomly allotted to four groups with six replicates. The groups included: control, T1, T2 and T3 groups which received supplemented-milk with 0, 100, 200 and 400mg aqueous rosemary extract per kg of live body weight per day for 42 days. Body weights of kids were measured weekly until the end of the experiment. On day 42, 10 ml blood samples were collected from each kid through the jugular vein. Cell-mediated immune response was assessed through the double skin thickness after intradermal injection of phyto-hematoglutinin (PHA) at day 21 and 42. No significant differences were seen in initial body weight, average daily gain (ADG) and total gain. However, significant differences in globulin (P <0.05), and white blood cells (WBC) (P <0.001) were observed. There were no significant differences in haemoglobin (Hb), packed cell volume (PCV), red blood cells (RBC), lymphocytes and neutrophils between the treatments. Skin thickness in response to intra dermal injection of PHA significantly increased in the treated groups as compared to the control group at day 42 (P< 0.01) with the T3 group showing the highest response to PHA injection. In conclusion, the results indicated that aqueous rosemary extract supplemented-milk had a positive effect on immunity and skin thickness of newborn goat kids.