Calcium induced calcium release (CICR) is the process mediating cardiac excitation contraction coupling (ECC). In brief, depolarisation of the plasma membrane of the cardiac myocyte leads to an influx of calcium (Ca2+) into the cytosol via the L-type voltage gated Ca2+ channels. The raised level of cytosolic Ca2+ initiates Ca2+ release from the junctional cisternae of sarcoplasmic reticulum (SR) through the opening of the ryanodine receptor 2 (RyR2). The exact mechanism of termination of CICR remains to be elucidated. It has been proposed that a drop in the luminal [Ca2+] reduces the open probability of RyR2 thereby leading to termination of CICR. It is also believed that RyR2 senses the luminal [Ca2+] through the formation of a quaternary complex with the SR proteins; calsequestrin (CSQ), triadin and junctin. However, the mechanism governing the assembly of this SR ‘luminal Ca2+ sensing complex’ is still far from being fully understood. A thorough knowledge of how this protein network is assembled is not only required for a robust understanding of the normal physiology of ECC but also for understanding the pathogenesis of disease, since disruption in luminal Ca2+ sensing is reported to lead to diastolic Ca2+ leak resulting in delayed after depolarisations (DADs), the precursor of premature beats and tachyarrhythmias. The primary focus of this thesis research was to investigate the structural basis for the formation of the luminal Ca2+ sensing complex with an emphasis on RyR, CSQ and triadin interactions. In order to achieve this goal, a protocol was developed to purify RyR2 from bovine heart employing a variety of techniques. Unfortunately this work resulted in only a partial purification of RyR2 with very low yields. However, more success was achieved with the isolation of the skeletal muscle ryanodine receptor isoform, RyR1, from sheep skeletal muscle employing sucrose gradient fractionation. The second aim of this study was to purify calsequestrin to enable investigations into its mode of interaction with the RyR. A molecular biology approach was taken and human cardiac calsequestrin (hCSQ2) was expressed as a GST tagged fusion protein and purified from E.coli BL21 (DE3) cells. A similar strategy was taken to express and purify the full-length and C-terminal luminal domain of mouse cardiac triadin isoform 1 (Trd1). However, this proved unsuccessful. A range of biochemical and biophysical techniques was next employed to examine whether the ryanodine receptor associated with hCSQ2 in the absence of triadin. It was found that purified RyR1 bound to immobilised GST-hCSQ2 through co-precipitation experiments suggesting a direct interaction between the two proteins. Further studies using surface plasmon resonance (SPR) also showed that immobilised hCSQ2 bound both RyR1 and RyR2. These findings were then developed by experiments employing quartz crystal microbalance and dissipation (QCM-D) monitoring. The data from QCM-D was also found to support a direct interaction of RyR1 (closed state) with both Ca2+ free and Ca2+ bound hCSQ2. Isolation of a RyR1 (closed state) and hCSQ2 complex (in the absence of Ca2+) was achieved using a sucrose cushion with an aliquot of the sample examined by transmission electron microscopy (TEM) using both negative staining and cryo-electron microscopy methods. The raw images of the complex suggested a direct interaction between RyR1 and CSQ2 in agreement with the data described above. However, intriguingly the hCSQ2 appeared to form strands of protein linking adjacent RyR molecules. These images, therefore, may suggest a possible role for hCSQ2 in a putative RyR coupled-gating mechanism. Another aspect of this research work was to optimise and employ a [3H] ryanodine binding assay to investigate how the channel activity of RyR1 and RyR2 within the SR preparations was regulated by hCSQ2 and triadin. Neither removal of endogenous CSQ from the SR membranes nor the addition of the recombinant hCSQ2, after removal of endogenous protein, modified the channel activity. However, interestingly, a synthesized domain of triadin (Trd KEKE motif) was found to enhance the channel activity as indicated by an increased [3H] ryanodine binding to both RyR1 and RyR2. In conclusion, the results from this thesis work provide evidence for a direct interaction between RyR and hCSQ2 and suggest a stimulatory role of a domain of triadin upon the activity of both isoforms of the ryanodine receptor.

Calcium aluminate phases are important parts of Ordinary Portland cement and Alumina cement. Various calcium aluminate hydrates originate during the hydration of calcium alumina phases. Their origin depends on the conditions of hydration. In the diploma thesis was investigated hydration of four calcium aluminate phases – CA, CA2, C12A7, C3A under the conditions of four pH – 6, 9, 11, 12,65. Calcium aluminate phases were prepared from CaCO3 and Al2O3 by clinkering in solid phase in laboratory oven. The phases were hydrated for 48 hours. Kinetics of the hydration was investigated by calorimetry. Calcium aluminate hydrates were identified by XRD and DTA.

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Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Abstract—This paper will detail the process and methods to the problem with having an Offline Certificate Authority that can still be accessed remotely. Specifically, to update CRL on the server when the need arises without bringing the entire CA online. This has been managed via local access in the past but to ease the use a need for remote management has arisen. This paper will explain how this problem was solved with the use of a Data diode to prevent the CA to be fully online. A Data Diode will only allow traffic in one direction keeping any data from leaking from the CA while still making it available for specific uploads, in this case CRL files that handle the revocation of certificates issued by the CA. This will allow for more up to date lists when the server is brought online for the issuance of certificates once per year. This paper will try to detail the steps that need to be taken in order to set up an edge server that allows the transmission of files with the use of UDP.  Keywords—CRL Content Revocation List, CA Certificate Authority, Data Diode, Offline Server, UDP user Datagram Protocol.

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