ORGANIZATION OF AN INSPECTION BODY FOR INSPECTING PRESSURE EQUIPMENT

S pričetkom veljavnosti Pravilnika o pregledovanju in preizkušanju opreme pod tlakom je aprila 2004 prešla kontrola opreme pod tlakom v obratovanju z državnih organov na Organe za periodične preglede — Kontrolne organe. Osnovni namen izvajanja periodičnih kontrol je zagotoviti varno obratovanje opreme pod tlakom tekom celotne dobe uporabe. Za izpolnjevanje teh zahtev mora imeti kontrolni organ na razpolago določene prostore in opremo. Razpolagati mora z zadostnim številom osebja, ki mora biti strokovno usposobljeno za izvajanje kontrol. Imeti mora izdelan sistem kakovosti, v katerem so predpisani organizacijski postopki, kontrolni postopki, navodila za delo z opremo ter obrazci za izdajo poročil in potrdil o kontrolah. Tema diplomske naloge je organizacija kontrolnega organa za kontrolo opreme pod tlakom v skladu z zahtevami zgoraj navedenega pravilnika in standarda SIST EN ISO/IEC 17020:2004.With the Regulation on examination and testing of pressure equipment, coming into force in April 2004, the control of pressure equipment in operation passed from national authorities to the Bodies for periodic inspection - the Inspection Body. The primary purpose of carrying out periodic control is to ensure the safe operation of equipment under pressure during the entire period of use. In order to meet with these requirements, the Inspection Body must have at it’s disposal certain premises and equipment. It must have a sufficient number of qualified staff members to carry out checks. It must have a made out quality system that prescribes: organizational procedures, control procedures, instructions for work with the equipment and forms to issue reports and certificates of controls. The topic of this diploma work is the organization of the Inspection Body for the inspection of pressure equipment in accordance with the requirements of the above regulations and the standard SIST EN ISO / IEC 17020:2004

Cysteine Cathepsins and Their Extracellular Roles: Shaping the Microenvironment

For a long time, cysteine cathepsins were considered primarily as proteases crucial for nonspecific bulk proteolysis in the endolysosomal system. However, this view has dramatically changed, and cathepsins are now considered key players in many important physiological processes, including in diseases like cancer, rheumatoid arthritis, and various inflammatory diseases. Cathepsins are emerging as important players in the extracellular space, and the paradigm is shifting from the degrading enzymes to the enzymes that can also specifically modify extracellular proteins. In pathological conditions, the activity of cathepsins is often dysregulated, resulting in their overexpression and secretion into the extracellular space. This is typically observed in cancer and inflammation, and cathepsins are therefore considered valuable diagnostic and therapeutic targets. In particular, the investigation of limited proteolysis by cathepsins in the extracellular space is opening numerous possibilities for future break-through discoveries. In this review, we highlight the most important findings that establish cysteine cathepsins as important players in the extracellular space and discuss their roles that reach beyond processing and degradation of extracellular matrix (ECM) components. In addition, we discuss the recent developments in cathepsin research and the new possibilities that are opening in translational medicine

The Tumor Proteolytic Landscape: A Challenging Frontier in Cancer Diagnosis and Therapy

In recent decades, dysregulation of proteases and atypical proteolysis have become increasingly recognized as important hallmarks of cancer, driving community-wide efforts to explore the proteolytic landscape of oncologic disease. With more than 100 proteases currently associated with different aspects of cancer development and progression, there is a clear impetus to harness their potential in the context of oncology. Advances in the protease field have yielded technologies enabling sensitive protease detection in various settings, paving the way towards diagnostic profiling of disease-related protease activity patterns. Methods including activity-based probes and substrates, antibodies, and various nanosystems that generate reporter signals, i.e., for PET or MRI, after interaction with the target protease have shown potential for clinical translation. Nevertheless, these technologies are costly, not easily multiplexed, and require advanced imaging technologies. While the current clinical applications of protease-responsive technologies in oncologic settings are still limited, emerging technologies and protease sensors are poised to enable comprehensive exploration of the tumor proteolytic landscape as a diagnostic and therapeutic frontier. This review aims to give an overview of the most relevant classes of proteases as indicators for tumor diagnosis, current approaches to detect and monitor their activity in vivo, and associated therapeutic applications.ISSN:1422-006

Fast profiling of protease specificity reveals similar substrate specificities for cathepsins K, L and S

Proteases are important effectors of numerous physiological and pathological processes. Reliable determination of a protease's specificity is crucial to understand protease function and to develop activity-based probes and inhibitors. During the last decade, various proteomic approaches for profiling protease substrate specificities were reported. Although most of these approaches can identify up to thousands of substrate cleavage events in a single experiment, they are often time consuming and methodologically challenging as some of these approaches require rather complex sample preparation procedures. For such reasons their application is often limited to those labs that initially introduced them. Here, we report on a fast and simple approach for proteomic profiling of protease specificities (fast profiling of protease specificity (FPPS)), which can be applied to complex protein mixtures. FPPS is based on trideutero-acetylation of novel N-termini generated by the action of proteases and subsequent peptide fractionation on Stage Tips containing ion-exchange and reverse phase chromatographic resins. FPPS can be performed in 2 days and does not require extensive fractionation steps. Using this approach, we have determined the specificity profiles of the cysteine cathepsins K, L and S. We further validated our method by comparing the results with the specificity profiles obtained by the N-terminal combined fractional diagonal chromatography method. This comparison pointed to almost identical substrate specificities for all three cathepsins and confirmed the reliability of the FPPS approach. All MS data have been deposited in the ProteomeXchange with identifiers PXD001536 and PXD001553 (; )

Proteomic identification of cysteine cathepsin substrates shed from the surface of cancer cells

Extracellular cysteine cathepsins are known to drive cancer progression, but besides degradation of extracellular matrix proteins little is known about their physiological substrates and thus the molecular mechanisms they deploy. One of the major mechanisms used by other extracellular proteases to facilitate cancer progression is proteolytic release of the extracellular domains of transmembrane proteins or ectodomain shedding. Here we show using a mass spectrometry-based approach that cathepsins L and S act as sheddases and cleave extracellular domains of CAM adhesion proteins and transmembrane receptors from the surface of cancer cells. In cathepsin S-deficient mouse pancreatic cancers, processing of these cathepsin substrates is highly reduced, pointing to an essential role of cathepsins in extracellular shedding. In addition to influencing cell migration and invasion, shedding of surface proteins by extracellular cathepsins impacts intracellular signaling as demonstrated for regulation of Ras GTPase activity, thereby providing a putative mechanistic link between extracellular cathepsin activity and cancer progression. The MS data is available via ProteomeXchange with identifier PXD002192