Along with proteins, a myriad of different molecular biomarkers, such as post-translational modifications and autoantibodies, could be used in an attempt to improve disease detection and progression. In this thesis, I build on several iterations of the proximity ligation assay to develop and apply new adaptable methods to facilitate detection of proteins, autoantibodies and post-translational modifications. In paper I, we present an adaptation of the solid-phase proximity ligation assay (SP-PLA) for the detection of post-translational modification of proteins (PTMs). The assay was adapted for the detection of two of the most commons PTMs present in proteins, glycosylation and phosphorylation, offering the encouraging prospect of using detection of PTMs in a diagnostic or prognostic capacity.  In paper II, we developed a variant of the proximity ligation assay using micro titer plate for detection and quantification of protein using optical density as readout in the fluorometer, termed PLARCA. With a detection limit considerably lower than ELISA, PLARCA detected femtomolar levels of these proteins in patient samples. In paper III, we aim to compare detection values of samples collected from earlobe capillary, venous plasma, as well as capillary plasma stored in dried plasma spots (DPS) assessed with a 92-plex inflammation panel using multiplex proximity extension assay (PEA). Despite the high variability in protein measurements between the three sample sources, we were able to conclude that earlobe capillary sampling is a suitable less invasive alternative, to venipuncture. In paper IV, we describe the application of PLARCA and proximity extension assay (PEA) for the detection of GAD65 autoantibodies (GADA). Thus, offering highly sensitive and specific autoimmunity detection.

of thesis Diagnostics of neuroinfection caused by human herpesviruses using nucleic acid amplification methods author: MUDr. Klára Labská supervisor: doc. MUDr. Vilma Marešová, CSc. In recent years, the diagnosis of neuroinfections has undergone a shift towards molecular biology methods. Our research focused on the predictive value of the capture of herpesvirus (HV) DNA in cerebrospinal fluid. In the first study, we examined the presence of DNA neurotropic herpes viruses (HSV1, HSV2, VZV and HHV6) in cerebrospinal fluid in immunocompetent patients with laboratory-confirmed tick-borne meningoencephalitis and enterovirus meningitis and meningoencephalitis. The control group consisted of patients with proven absence of an inflammation in the cerebrospinal fluid. Patients were followed for 6 months. The course of the disease and its consequences, including laboratory tests, were compared between groups of patients with and without the presence of HV DNA. In the second study, we tried to demonstrate the presence of HSV1 DNA in cerebrospinal fluid during its symptomatic reactivation in patients with purulent meningitis. In our group of immunocompetent patients with non-purulent inflammation in the cerebrospinal fluid, the proportion of HV DNA positive patients reached 7.5% (13 out of 173), we also...

Autosomal recessive polycystic kidney disease (ARPKD) is a rare severe inherited disease manifested by cystic renal disease, congenital hepatic fibrosis and dilatatation of bile ducts. The spectrum of clinical manifestations is very wide and variable, depends on the age at which the disease was manifested. In severe forms of the disease, it is possible to detect the first symptoms prenatally around the 20th week of pregnancy due to increased echogenic kidneys and the presence of oligohydramnios. The causal gene of this disease is thePKHD1 gene with protein product fibrocystin that is most likely contributing on maintaining the intracellular concentration of Ca2+ cations. The exact phatophysiology mechanism of ARPKD remains unknown. Phenotypic manifestations of this disease may overlap with mutations associated with other genes. One of the genes mimicking the ARPKD phenotype is the HNF1B gene. Mutations associated with HNF1B gene are the most common monogenic cause of developmental kidney abnormalities. HNF1B is a tissue-specific transcription factor that regulates the expression of PKHD1. In experimental part I worked on genetic analysis of the HNF1B gene in 28 patients who have not been confirmed ARPKD diagnosis by detection of 2 PKHD1 mutations. For the purposes of mutational screening, I used...

Targeting DNA has the advantage over proteins for cancer remediation because of the fewer copies of the ligands required for the desired therapeutic effect. Traditionally, covalent DNA binders like alkylating agents have been used to induce genetic instability through the formation of DNA lesions and strand breaks, leading to cellular apoptosis. The primary drawback of this treatment is the non-specific binding that affects both cancerous and non-cancerous cells. G-quadruplexes are the DNA secondary structures that are present in abundance near the promoter regions of the oncogenes and are involved in the regulation of their activities. A ligand-mediated stabilization of G-quadruplexes in the promoter regions and down-regulation of the associated oncogenes have been validated. In contrast to alkylating agents, G-quadruplex ligands induce genetic stabilization through non-covalent interactions. They can be designed to interact specifically with G-quadruplex DNA over duplex DNA, which reduce side effects arising from the off-targeting. G-quadruplex ligands invariably have the large planar aromatic moiety to interact with G-quadruplexes through π- π stacking interactions. For determining the size effect of the aromatic moiety on stabilization of G-quadruplexes, a series of ligands were synthesized by conjugating nucleobases or 1,10-phenanthroline with an aminoglycoside, neomycin. The resulting conjugates increased the binding affinity synergistically and enabled us to study the effect of the stacking moiety required for G-quadruplex stabilization. Nucleobase-neomycin conjugates did not show stabilization stabilize of human telomeric G-quadruplex. 1,10-Phenanthroline-neomycin conjugate (7b) on the other hand binds to human telomeric G-quadruplex with a Ka of (8.92.4)×108 M-1 and inhibits telomerase activity at 1.56 µM probably through G-quadruplex stabilization. Moving forward, we further enlarged the aromatic moiety by tethering two 1,10-phenantholine molecules together through a five-atom linker. The resulting molecule (2-Clip-phen) was conjugated with various amino-containing side chains. 2-Clip-phen derivatives showed at least 30 times weaker binding to duplex DNA over G-quadruplex DNA. In addition, compounds showed a preference for the antiparallel G-quadruplex conformation over parallel and hybrid G-quadruplex conformations, as shown in the CD spectroscopy studies. Ligands 11 and 13 induced the formation of an antiparallel G-quadruplex from random coils and stabilize it to 60 oC (Tm) in a salt-free condition. Mass spectrometry study showed the formation of a two-tetrad G-quadruplex with the 2-Clip-phen ligand. Docking study showed that the ligand interacts most favorably with antiparallel G-quadruplex conformation, which is supported further by the larger thermal stabilization effect on antiparallel G-quadruplex compared with other G-quadruplex conformations. Our study suggests that 2-Clip-phen can be used as a scaffold for designing G-quadruplex binding ligands that preferentially bind to antiparallel G-quadruplexes, which has never been reported before.

<p dir="ltr">The necessity for on-farm, point-of-need (PON) nucleic acid amplification tests (NAATs) arises from the prolonged turnaround times and high costs associated with traditional laboratory equipment. This thesis aims to address these challenges by developing devices and a user-interface application designed for the efficient, accurate, and rapid detection of <i>Bacteroidales</i> as an indicator of fecal contamination on fresh produce farms.</p><p dir="ltr">In pursuit of this, I collaborated with lab members to engineer a Field-Applicable Rapid Microbial Loop-mediated isothermal Amplification Platform, FARM-LAMP. This device is portable (164 x 135 x 193 mm), energy-efficient (operating under 20 W), achieves the target 65°C with ± 0.2°C fluctuations, and is compatible with paper-based biosensors for loop-mediated isothermal amplification (LAMP). Subsequently, I led the fabrication of the microfluidic Field-Applicable Sampling Tool, FAST, designed to deliver high-throughput (10 samples per device), equal flow-splitting of fluids to paper-based biosensors, eliminating the need for a laboratory or extensive training. FARM-LAMP achieved 100% concordance with standard lab-based tests when deployed on a commercial lettuce farm and FAST achieved an average accuracy of 89% in equal flow-splitting and 70% in volume hydration.</p><p dir="ltr">A crucial aspect of device development is ensuring that results are easily interpretable by users. To this end, I developed a Python-based image analysis codebase to quantify sample positivity for fecal contamination, ranging from 0% (no contamination) to nearly 100% (definite contamination) and the concentration of field samples. It utilizes calculus-based mathematics, such as first and second derivative analysis, and incorporates image analysis techniques, including hue, saturation, and value (HSV) binning to a sigmoid function, along with contrast limited adaptive histogram equalization (CLAHE). Additionally, I developed a preliminary graphical user interface in Python that defines a prediction model for the concentration of <i>Bacteroidales</i> based on local weather patterns.</p><p dir="ltr">This thesis encompasses hardware development for on-field quantification and the creation of a preliminary user-interface application to assess fecal contamination risk on fresh produce farms. Integrating these devices with a user-interface application allows for rapid interpretation of results on-farm, aiding in the effective development of strategies to ensure safety in fresh produce operations.</p>

科学研究費補助金 研究種目:基盤研究(B)(2) 課題番号:08455251 研究代表者:福和 伸夫 研究期間:1996-1998年度

No description available.

The need for modern transportation systems together with the high demand for sustainable pavements under applied loads have led to a great deal of research on concrete pavements worldwide. Development of finite element techniques enabled researchers to analyse the concrete pavement under a combination of axle group loadings and environmental effects. Consequently, mechanistic approaches for designing of concrete pavements were developed based on results of finite element analyses. However, unpredictable failure modes of concrete pavements associated with expensive maintenance and rehabilitation costs have led to the use of empiricalmechanistic approach in concrete pavement design. Despite progressive knowledge of concrete pavement behaviour under applied loads, concrete pavements still suffer from deterioration due to crack initiation and propagation, indicating the need for further research. Cracks can be related to fatigue of the concrete and/or erosion of materials in sub-layers. Although longitudinal, midedge and corner cracks are the most common damage modes in concrete pavements, Austroads method for concrete pavement design was developed based on traditional mid-edge bottom-up transverse cracking introduced by Packard and Tayabji (1985). Research presented in this thesis aims to address the most common fatigue related distresses in concrete pavements. It uses comprehensive finite element models and analyses to determine the structural behaviour of concrete pavements under vehicular loads and environmental effects. Results of this research are supported by laboratory tests and an experimental field test. Results of this research indicate that the induced tensile stresses within the concrete pavement are significantly affected by vehicle speed, differential temperature gradient and loss of moisture content. Subsequently, the interaction between the above mentioned factors and concrete damage modes are discussed. Typical dynamic amplifications of different axle groups are presented. A new fatigue test setup is also developed to take into consideration effects of pavement curvature on fatigue life of the concrete. Ultimately, results of the research presented in this thesis are employed to develop a new guide for designing concrete pavements with zero maintenance of fatigue damage.

The bullwhip effect is defined as an upstream amplification of demand variability and has received interest within multinational companies for decades. As early as in the 1950’s, Forrester (1958) discussed what is today known as the bullwhip effect, which has a negative impact on the customer service, costs, and inventory investment in a supply chain (Lee et al., 1997). Even though the bullwhip effect has been noticed in various industries, the consequences, in form of decreased availability and increased costs the further up the supply chain the bullwhip goes, still remain. The employees at Volvo Group Service Market Logistics suspect that their supply chain has been affected by the bullwhip effect and want to know if it is correct and subsequently know why it has occurred. Therefore, this master’s thesis highlights the root causes of the bullwhip effect and presents strategies to mitigate it. To understand how the bullwhip effect affected the Volvo Group Service Market Logistics’ supply chain, the purpose was formulated as follow: The purpose of this study is to identify events in the Volvo Group Service Market Logistics’ supply chain where the bullwhip effect has occurred, its root causes, and how to reduce or eliminate the bullwhip effects.  The studied flow was from the Central Distribution Center (CDC) in Ghent, to the Regional Distribution Center (RDC) in Brazil, to the Dealers associated to the RDC in Brazil, and the customers. Data was collected from each node and events were studied to find bullwhip events. After sorting out the part numbers that passed the criteria for bullwhip events, the amount of data had to be reduced even more. A couple of different conditions were applied which resulted in four suitable bullwhip events. Thereafter, the authors conducted interviews with Logistics Managers at each node of the supply chain to find the root causes of the bullwhip effect in each studied event.  Among the several found root causes, lack of information transparency was the most frequent occurring root cause, found in three out of four studied bullwhip events. Insufficient communication and lack of information sharing cause bullwhip effects, and the authors found that improved communication both between and within the nodes will contribute to better planning, and consequently avoided bullwhip effects. Other root causes found were issues with the ordering system, lack of learning and experience, neglected lead times, fear of empty stock, price fluctuations, and phase-out of the spare part.  To reduce or eliminate the bullwhip effect, the focus was on mitigating the root causes since the root causes create opportunities for the bullwhip effect to occur. Four suggestions were given with suitable mitigation strategies found in the literature, where the four suggestions were sales campaigns, prepare for boosts, keep track of manually placed orders, and ordering system and Logistics Manager behavioural issues. The suggestions could then be connected to the different found root causes. The stated suggestions and mitigation strategies focused on mitigating the root causes in a long-term perspective and consequently the bullwhip effect itself.

Reverse genetics is an innovative molecular biology tool that enables the manipulation of viral genomes at the cDNA level in order to generate particular mutants or artificial viruses. The reverse genetics system for the influenza virus is arguably one of the best illustrations of the potential power of this technology. This reverse genetics system is the basis for the ability to regularly adapt influenza vaccines strains. Today, reverse genetic systems have been developed for many animal RNA viruses. Selection-free reverse genetics systems have been developed for the members of the Reoviridae family including, African horsesickness virus, bluetongue virus and orthoreovirus. This ground-breaking technology has led to the generation of valuable evidence regarding the replication and pathogenesis of these viruses. Unfortunately, extrapolating either the plasmid-based or transcript-based reverse genetics systems to rotavirus has not yet been successful. The development of a selection-free rotavirus reverse genetics system will enable the systematic investigation of poorly understood aspects of the rotavirus replication cycle and aid the development of more effective vaccines, amongst other research avenues. This study investigated the importance of co-expressed rotavirus proteins in the development of a selection-free rotavirus reverse genetics system. The consensus sequences of the rotavirus strains Wa (RVA/Human-tc/USA/WaCS/1974/G1P[8]) and SA11 (RVA/Simian-tc/ZAF/SA11/1958/G3P[2]) where used to design rotavirus expression plasmids. The consensus nucleotide sequence of a human rotavirus Wa strain was determined by sequence-independent cDNA synthesis and amplification combined with next-generation 454® pyrosequencing. A total of 4 novel nucleotide changes, which also resulted in amino acid changes, were detected in genome segment 7 (NSP3), genome segment 9 (VP7) and genome segment 10 (NSP4). In silico analysis indicated that none of the detected nucleotide changes, and consequent amino acid variations, had any significant effect on viral structure. Evolutionary analysis indicated that the sequenced rotavirus WaCS was closely related to the ParWa and VirWa variants, which were derived from the original 1974 Wa isolate. Despite serial passaging in animals, as well as cell cultures, the Wa genome seems to be stable. Considering that the current reference sequence for the Wa strain is a composite sequence of various Wa variants, the rotavirus WaCS may be a more appropriate reference sequence. The rotavirus Wa and SA11 strains were selected for plasmid-based expression of rotavirus proteins, under control of a T7 promoter sequence, due to the fact that they propagate well in MA104 cells and the availability of their consensus sequences. The T7 RNA polymerase was provided by a recombinant fowlpox virus. After extensive transfection optimisation on a variety of mammalian cell lines, MA104 cells proved to be the best suited for the expression rotavirus proteins from plasmids. The expression of rotavirus Wa and SA11 VP1, VP6, NSP2 and NSP5 could be confirmed with immunostaining in MA104 and HEK 293H cells. Another approach involved the codon-optimised expression of the rotavirus replication complex scaffold in MA104 cells under the control of a CMV promoter sequence. This system was independent from the recombinant fowlpox virus. All three plasmid expression sets were designed to be used in combination with the transcript-based reverse genetics system in order to improve the odds of developing a successful rotavirus reverse genetics system. Rotavirus transcripts were generated using transcriptively active rotavirus SA11 double layered particles (DLPs). MA104 and HEK293H cells proved to be the best suited for the expression of rotavirus transcripts although expression of rotavirus VP6 could be demonstrated in all cell cultures examined (MA104, HEK 293H, BSR and COS-7) using immunostaining. In addition, the expression of transcript derived rotavirus VP1, NSP2 and NSP5 could be confirmed with immunofluorescence in MA104 and HEK 293H cells. This is the first report of rotavirus transcripts being translated in cultured cells. A peculiar cell death pattern was observed within 24 hours in response to transfection of rotavirus transcripts. This observed cell death, however does not seem to be related to normal viral cytopathic effect as no viable rotavirus could be recovered. In an effort to combine the transcript- and plasmid systems, a dual transfection strategy was followed where plasmids encoding rotavirus proteins were transfected first followed, 12 hours later, by the transfection of rotavirus SA11 transcripts. The codon- optimised plasmid system was designed as it was postulated that expression of the DLP-complex (VP1, VP2, VP3 and VP6), the rotavirus replication complex would form and assist with replication and/or packaging. Transfecting codon- optimized plasmids first noticeably delayed the mass cell death observed when transfecting rotavirus transcripts on their own. None of the examined coexpression systems were able to produce a viable rotavirus. Finally, the innate immune responses elicited by rotavirus transcripts and plasmid-derived rotavirus Wa and SA11 proteins were investigated. Quantitative RT-PCR (qRT-PCR) experiments indicated that rotavirus transcripts induced high levels of the expression of the cytokines IFN- α1, IFN-1β, IFN-λ1 and CXCL10. The expression of certain viral proteins from plasmids (VP3, VP7 and NSP5/6) was more likely to stimulate specific interferon responses, while other viral proteins (VP1, VP2, VP4 and NSP1) seem to be able to actively suppress the expression of certain cytokines. In the light of these suppression results, specific rotavirus proteins were expressed from transfected plasmids to investigate their potential in supressing the interferon responses provoked by rotavirus transcripts. qRT-PCR results indicated that cells transfected with the plasmids encoding NSP1, NSP2 or a combination of NSP2 and NSP5 significantly reduced the expression of specific cytokines induced by rotavirus transcripts. These findings point to other possible viral innate suppression mechanisms in addition to the degradation of interferon regulatory factors by NSP1. The suppression of the strong innate immune response elicited by rotavirus transcripts might well prove to be vital in the quest to better understand the replication cycle of this virus and eventually lead to the development of a selection-free reverse genetics system for rotavirus. / PhD (Biochemistry), North-West University, Potchefstroom Campus, 2014