862 research outputs found
Air Pollution and Mortality for 60 US Cities in 1960
No full textData includes measurements on mortality rate and explanatory variables(air-pollution, socio-economic and meteorological) for 60 US cities in 1960. This data was originally published in McDonald, G.C. and Schwing,R.C. (1973) 'Instabilities of regression estimates relating air pollution to mortality', Technometrics, vol.15, 463-482. It was redistributed through Carnegie Mellon University's StatLib (lib.stat.cmu.edu
RNA interference: a new tool to study gene functions in adult mammalian muscle "in vivo"
No full textRNA interference (RNAi) is a powerful method for sequence-specific posttranscriptional
gene silencing (PTGS), which allows rapid survey of gene functions using
double-stranded RNA (dsRNA). At the time when we started this work, RNAi was a
recently developed tool that had been successfully applied to many organisms, in
particular C. elegans and Drosophila, but not to any mammalian system. It was generally
doubted that RNAi would also work in mammals in vivo, because the introduction of
dsRNA can induce general shutdown of translation and apoptosis in several mammalian
cell types. One excellent model system for investigating this open question is the nervemuscle
synapse known as the neuromuscular junction (NMJ).
Characteristic for the NMJ is the precise apposition of the neurotransmitter
release machinery on the nerve terminal side and the neurotransmitter receptors on the
muscle fiber membrane. At least two mechanisms underlie the formation and
maintenance of a postsynaptic apparatus on the muscle fiber membrane. Both
mechanisms are triggered by the heparan sulfate proteoglycan agrin, which is released
by the motor neuron. First, neural agrin activates all the cellular mechanisms necessary
to assemble a fully functional postsynaptic structure including aggregates of acetylcholine
receptors (AChRs). Besides this redistribution of preexisting molecules, agrin signaling
restricts the transcription of postsynaptic proteins to myonuclei located in the NMJ. Still
little is known about the agrin signaling cascade. Therefore, once RNAi could be
developed for mammals system, it will in turn provide a unique tool to address the role of
newly identified genes in the postsynaptic differentiation, since there are no tools
available for the fast and reliable perturbation of gene function in vivo.
In the first part of this work, we investigated the potential of RNAi in perturbing
the formation and stability of postsynaptic structures in adult muscle in vivo (chapter 2
and 3). First, we used the experimental paradigm where neural agrin expressed in nonjunctional
regions of rat soleus muscle induces formation of ectopic AChR aggregates.
Knockout experiments have shown that this agrin activity requires the receptor tyrosine kinase MuSK and the AChR-associated scaffolding molecule rapsyn, but not the
cytoskeletal proteins sarcoglycan α (SGCA) and utrophin. In our experiments, we show
that co-injection of dsRNAs derived from MuSK or rapsyn perturbed agrin-induced
formation of ectopic AChR aggregates, while dsRNAs derived from SGCA or utrophin had
no significant effect. In a further step, we used RNAi to study the role of MuSK at adult
NMJs. Here, the electroporation of plasmids encoding short hairpin-based 21-bp small
interfering RNAs (siRNAs) or long hairpin dsRNAs, which allow global and sustained
perturbation of MuSK expression, leads to the disassembly of NMJs in adult mice. These
results are consistent with the finding that auto-antibodies to MuSK, which also lower the
amount of MuSK protein, cause severe forms of myasthenia gravis. In summary, these
results demonstrate for the first time the effectiveness of long dsRNA as well as siRNA in
silencing endogenous genes in adult mammalian muscle in vivo and they provide strong
evidence that continuous expression MuSK is required to maintain the NMJ.
The second part of this work aimed to establishing RNAi in adult muscle to study
the role of newly identified genes in the development of the NMJ and in the growth of
muscle fibers (chapter 4 and appendix). First, we used RNAi to perturb neural agrininduced
formation of ectopic AChR aggregates on mouse soleus muscle. We show that
electroporation of plasmids encoding short hairpin- derived siRNA for MuSK leads to the
perturbation of ectopic AChR aggregation, regardless whether agrin expression vector or
recombinant protein was applied to innervated or denervated muscles. These results
clearly show the reliability of RNAi in adult muscle in vivo and therefore set the stage for
experiments aimed to study the function of genes, whose expression is altered during the
formation postsynaptic structures. A protocol was established to identify functional siRNA
target sites in several genes. Plasmids were designed that encoded short hairpin RNAs
(shRNAs) derived from different putative effectors of the mammalian target of rapamycin
(mTOR) signaling pathway. For some candidate effectors, electroporation of the
corresponding plasmids into mouse soleus muscle leads to altered muscle fiber size.
These preliminary results are consistent with several reported findings, which indicate
that the mTOR signaling pathway is a central controller of muscle fiber atrophy and
hypertrophy. The efficiently induced RNAi in those experiments demonstrates that our protocol is useful for identifying siRNA targets. In summary, these results demonstrate
that we have successfully established RNAi as a fast and reliable gene knockdown
method in muscle fibers of mammals. This method will be important for future
investigation of gene functions in adult mammalian muscle in vivo
Target-controlled infusion of propofol in dogs - Evaluation of four targets for induction of anaesthesia
No full textFour groups of 20 dogs were anaesthetised by means of target-controlled infusions of propofol designed to achieve 2.5 μg/ml, 3.0 μg/m., 3.5 μg/ml or 4.0 μg/ml of propofol in blood. The dogs' pulse rate and respiratory rate were recorded before premedication and induction, immediately after endotracheal intubation and three and five minutes later (times 0, 3 and 5, respectively), and their arterial blood pressure was recorded oscillometrically just before induction and at times 0, 3 and 5. The targets of 2.5, 3.0,3.5 and 4.0 μg/ml resulted in the successful induction of anaesthesia in 13 (65 per cent), 16 (80 per cent), 20 (100 per cent) and 20 (100 per cent) of the dogs, respectively. The incidence of postindudion apnoea was 0 (0 per cent), one (5 per cent), two (10 per cent) and eight (40 per cent) at time 5 for groups 2.5, 3.0, 3.5 and 4.0 μg/ml, respectively, and its incidence at time 5 was significantly higher in the 4.0 μg/ml group (P<0.05) than in the other groups. In all the groups there was a significant (P<0.05) decrease in blood pressure between just before induction and the later measurements. Although there were no statistically significant differences between the groups in terms of inducing anaesthesia at a specific target, a target of 3.5 μg/ml appears to ensure a successful induction of anaesthesia without a significant increase in the incidence of apnoea
Pressure and volume controlled mechanical ventilation in anaesthetized pregnant sheep
No full textOptimal mechanical ventilation of the pregnant ewe during anaesthesia is of vital importance for maintaining fetal viability. This study aimed to compare peak inspiratory pressure (PIP), oxygenation and cardiovascular parameters with pressure-control (PCV) or volume-control (VCV) mechanical ventilation of anaesthetized pregnant sheep. Twenty ewes at 110 days gestation underwent general anaesthesia in dorsal recumbency for fetal surgery in a research setting. All the sheep were mechanically ventilated; one group with PCV (n=10) and another with VCV (n=10) to maintain normocapnia. PIP, direct arterial blood pressure, heart rate, arterial pH and arterial oxygen tension were recorded. PIP was lower in the PCV group (P<0.001). Arterial oxygen tension was higher in the PCV group (P=0.013). Mean and diastolic pressures were lower in the PCV group (P=0.029 and P=0.047, respectively). Both VCV and PCV provide adequate oxygenation of pregnant sheep anaesthetized in dorsal recumbency, though PCV may provide superior oxygenation at a lower PIP
Target-controlled infusion of propofol combined with variable rate infusion of remifentanil for anaesthesia of a dog with patent ductus arteriosus
No full textAn 18-month-old Lurcher was anaesthetized for surgical ligation of a patent ductus arteriosus using a target-controlled infusion (TCI) of propofol and a variable rate infusion of remifentanil. Before anaesthesia, radiographic and echocardiographic examination indicated that the dog had left-sided congestive heart failure and impaired left ventricular systolic function. Ramipril and furosemide were administered pre-operatively. Following pre-anaesthetic medication with morphine, 0.5 mg kg-1, by intramuscular injection, and pre-oxygenation, remifentanil was infused for 5 minutes at 0.2 μg kg-1 minute-1, followed by induction of anaesthesia using intravenous propofol administered by TCI, set at a target concentration of 3.5 μg mL-1 of propofol in blood. Tracheal intubation was performed and 100% oxygen delivered through a non-rebreathing (Bain) system and then a circle system in the operating theatre. Anaesthesia was maintained with propofol and remifentanil, adjusted according to clinical requirements. Peri-operative analgesia consisted of intercostal bupivacaine nerve block, with meloxicam, morphine and remifentanil
Maternal and fetal arterial blood gas data during general anaesthesia for caesarean delivery of preterm twin lambs
No full textMuch remains to be understood with regards the effects of prolonged anaesthesia on maternal and fetal haemodynamics and oxygenation. With the aim of improving anaesthetic management of pregnant sheep undergoing recovery surgery under anaesthesia, paired maternal and fetal arterial blood samples were collected during caesarean delivery of twin preterm lambs to document the blood gas status of the ewe and fetus. Twenty-one Merino twin pregnant ewes at 126 (+/- 1) days of gestation were anaesthetized for caesarean delivery of their fetuses. Arterial blood samples were collected from the radial artery of the ewe and umbilical artery of the fetus at the point of delivery. There was a significant difference between maternal PaCO2 and end-tidal CO2 and alveolar and arterial PaO2, indicating ventilation perfusion mismatch. Interestingly, the ewes were anaemic but the fetuses were not. These data underscore the need to undertake further work to determine the optimal anaesthetic regimen for twin pregnant ewes at different gestational ages in a biomedical research setting
Agrin promotes acetylcholine receptor clustering at the mammalian neuro-muscular junction by PI3K/GSK3ß²-mediated regulation of +TIPs and microtubule capture
No full textNeurotransmission an der neuromuskulären Endplatte von Säugetieren
erfolgt durch Ausschüttung von Acetylcholin (ACh) aus motorischen Nerven
in den synaptischen Spalt und verursacht letztendlich Muskelkontraktion im
angesteuerten Muskel. Der Signalstoff Agrin, welcher ebenfalls vom
Motornerv sekretiert wird, ist hauptverantwortlich für die postsynaptische
Differenzierung der Muskelfasern und bewirkt die räumliche Konzentration
der Rezeptoren für den Neurotransmitter Acetylcholin (AChR) in einem
kleinen - meist zentralen - Areal der Faser unterhalb des Ansatzpunkt der
Motornervendigung. Nach intensiver Beforschung sind die Endeffekte von
Agrin auf Muskelfasern gut dokumentiert und viele involvierte Moleküle
bekannt. Trotzdem ist die Rolle des subsynaptischen Mikrotubuli-Zytoskeletts
beim Transport, bei der Membraninsertion und bei der Konzentrierung von
AChR-Molekülen wenig erforscht und die zugrundeliegenden molekularen
Mechanismen, welche ein Mikrotubuli-Netzwerk unterhalb der Synapse
etablieren, sind grossteils unbekannt. Im Zuge meiner Doktorarbeit
untersuchte ich daher neue Aspekte der agrin-induzierten biochemischen
Signalübertragung im Muskel und deren zellbiologischen Konsequenzen auf
das Mikrotubuli-Zytoskelett.
Gerichteter intrazellulärer Vesikel-Transport erfolgt generell entlang von
polaren Cytoskelettstrukturen, ein grosser Teil davon entlang von Mikrotubuli
- kurzlebige Cytoskelettfilamente, deren dynamischeres „Plus-Ende“
beständig zwischen Wachsen und Schrumpfen wechselt. Bestimmte Stimuli
jedoch können über Signaltransduktions-kaskaden dazu führen, dass die
schnell wachsenden Plus-Enden der Mikrotubuli mithilfe assoziierter Proteine
(+TIP-Proteine) längerfristig an die Innenseite der Zellmembran oder an das
Actinzytoskelett binden und dort verankert werden („Microtubule Capture“).
Dieser Vorgang stabilisiert die betreffenden Mikrotubuli und verlängert ihre
Halbwertszeit – die stabilisierten Microtubuli dienen der Zelle in weiterer
Folge als gerichtete Transportrouten.
In meiner Doktorarbeit konnte ich zeigen, dass die Acetylcholinrezeptorreiche
postsynaptische Zentralregion der Muskelfasern von einem dichten
Netzwerk von Microtubuli umgeben ist, einige dieser Microtubuli sind zudem
durch post-translationale Modifikationen weiter stabilisiert. Weiters konnte
ich demonstrieren, dass der Botenstoff Agrin im Muskel eine biochemische
Signaltransduktions-kaskade auslöst, welche die PI3-Kinase aktiviert und an
deren Ende GSK3β durch Phosphorylierung lokal inaktiviert wird.
Da viele der zuvor erwähnten +TIP-Proteine, zum Beispiel CLASP2, durch
GSK3β negativ reguliert sind, ist eine lokalisierte Inaktivierung von GSK3β
zentrale Voraussetzung dafür, dass +TIP-Proteine an Mikrotubuli Plus-Enden
binden können und somit zur Etablierung von stabilen Mikrotubuli-Filamenten
beitragen. Nach agrin-induzierter Inaktivierung von GSK3β bindet CLASP2 an
Mikrotubuli Plus-Enden und befestigt diese am Zellkortex in der AChR-reichen
Region von Myotuben in Wechselwirkung mit LL5β - welches von PI3K zur
synaptischen Membran rekrutiert wird. Die betreffenden Mikrotubuli werden
dadurch stabilisiert und können der Zelle als intrazelluläre Transportrouten
für postsynaptisches Material wie etwa AChRs und Strukturproteine dienen.
Funktionsverändernde Mutationen beteiligter Moleküle sowie
pharmakologische Eingriffe in den PI3K/GSK3β-Signalweg zeigten allesamt
Effekte auf die lokale Konzentrierung von AChRs, die mit oben angeführter
Kausalkette konsistent sind: Die Depolymerisierung von Microtubuli, die
Hemmung der PI3-Kinase, der Verlust des Clasp2-Gens, die shRNAvermittelte
Unterdrückung der LL5β-Expression sowie die Hyperaktivierung
von GSK3β führten jeweils zu reduzierter Grösse der AChR-Ansammlungen,
während die Hemmung von GSK3β die Grösse der AChR-Ansammlungen
erhöhte. Zusammengefasst trägt agrin-induzierte und +TIP-vermittelte
Befestigung von Mikrotubuli an der subsynaptischen Membran zur fokalen
Insertion und zum lokalen Konzentration von AChRs bei.
Abstract
Neurotransmission at the neuromuscular synapse of mammals occurs after
secretion of acetylcholine (ACh) from the motor nerve into the synaptic cleft
and ultimately results in contraction of the target muscle. The signaling
molecule agrin, which is also secreted by the motoneuron, is the main
organizer of postsynaptic differentiation and induces the clustering of
receptors for the neurotransmitter acetylcholine (AChR) in a small – mostly
central – area of the myofiber directly underneath the motoneuron terminal
bouton. After intense research, the end results of agrin-induced
differentiation are well documented and many involved molecules are known.
Nevertheless, the role of the subsynaptic microtubule (MT) cytoskeleton in
the process of AChR transport, insertion and clustering and the molecular
mechanisms of the establishment of such a microtubule network are poorly
defined. During my thesis, I analyzed new aspects of agrin-induced
biochemical signaling and the cell biological consequences for the
microtubule cytoskeleton.
Targeted intracellular transport of vesicles generally occurs along polar
cytoskeletal structures, a major part along microtubules – cytoskeletal
filaments that are normally short-lived with a highly dynamic “plus-end”,
which alternates between periods of growing and shrinking. However, certain
stimuli induce signal transduction cascades which result in the “capture” of
microtubule plus-ends at the cell cortex by interaction of plus-end binding
proteins (+TIPS) with factors that localize to the cortex or the actin
cytoskeleton. This event stabilizes microtubules and can drastically increase
their lifetime – affected microtubule then serve the cell as stable, directional
transport tracks.
During my thesis, I could show that the AChR-rich postsynaptic membrane
encompasses a dense subsynaptic MT network, with some MTs further
stabilized by post-translational modifications. In addition, I could
demonstrate that agrin induces PI3 kinase signaling in muscle, which
ultimately inactivates GSK3β by phosphorylation.
Since many of the former mentioned +TIP proteins are negatively regulated
by GSK3β, localized inactivation of GSK3β is crucial to enable +TIP-mediated
microtubule capture and establishment of stable transport tracks at sites with
elevated protein requirements such as the postsynaptic membrane. +TIP
proteins, in particular Clasp2, then capture MTs at the cell cortex within
AChR-rich sites by interacting with the membrane PIP3-sensor LL5β and thus
establish intracellular transport routes for focal transport of postsynaptic
material like AChRs and structural proteins towards the synapse.
Mutants of involved molecules as well as pharmacologic manipulation of the
PI3K/GSK3β-axis displayed effects on the clustering of AChRs, which are fully
consistent with the aforementioned chain of events: Microtubule
depolymerization, inhibition of PI3K, loss of the clasp2 gene, shRNAmediated
repression of LL5β expression and hyper-activation of GSK3β lead
to reduced AChR clustering, while inhibition of GSK3β elevates AChR
clustering. Taken together, agrin-induced and +TIP-mediated MT capture at
the subsynaptic muscle membrane contributes to focal insertion and
clustering of AChRs at the neuromuscular junction
Clinical and functional studies of autoimmune disorders of neuromuscular transmission
No full textInherited and acquired disorders of the neuromuscular junction are an important cause of muscle weakness and fatigability. In this thesis I focus on the autoimmune disorders of neuromuscular transmission. Myasthenia Gravis (MG) is the most common of these diseases and is typically caused by antibodies against the post-synaptic acetylcholine receptor. Lambert Eaton Myasthenic Syndrome (LEMS) is a pre-synaptic disorder typically caused by antibodies against voltage gated calcium channels (VGCC). With regard to LEMS, my main aim was to gain a more complete understanding of the pathomechanisms of the disease. To date, the direct effect of LEMS IgG on presynaptic neurotransmitter release had not been investigated in detail. I examined how LEMS IgG affects neurotransmitter release by imaging action potential dependent vesicle exocytosis using a fluorescent dye. I found that LEMS IgG significantly inhibited the rate of synaptic vesicle release but this effect was lost in synapses from a Cacna1a knockout mouse. These data provide direct evidence that LEMS is caused by impaired neurotransmitter release due to an effect on P/Q-type VGCCs. With regard to MG, I studied the long-term outcome of patients with thymomatous and non-thymomatous MG after thymectomy and found that in general the outcome was favourable in the majority of patients with 34% of patients achieving complete stable remission. I also reviewed the long-term outcome of patients after a severe exacerbation of MG requiring ITU admission. Despite the significant mortality associated with severe exacerbations of MG, it was found that specialised neuro-intensive care was associated with a good long-term prognosis in the majority of patients. There were no significant differences in outcome in those with early or late onset MG. Overall the data presented in this thesis provide new insights into the pathomechanisms of LEMS IgG and provide new information regarding the long-term outcome of patients with MG
Credit Card Security
No full textAuthor: Anup G.C.
Year: 2013
Subject of thesis: Credit Card Security
Number of pages: 36+2
Credit Card is a widely used electronic chip for easy transactions. The main purpose of the report was to show the security measures of transaction by credit cards. The purpose was to give information about credit cards and how they were introduced. The thesis reportcontained the types of card theft with examples and sited the various protocols used for online transactions. The aim of the thesis project was to conclude whether the card security provided by the banks is safe enough.
The thesis report contained information about many online resources as well as liable books. Many news articles were also considered while writing the report for the card theft records.
The thesis report described both the positive and negative aspects of the protocols used for card securities. Result showed that misuse and complicated processes of protocols has led the identity theft to transactions. To conclude, although many security measures are implemented for the secure transactions, credit card fraud activities are happening in a weekly basis
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