Tripeptidyl peptidse II (TPPII) is an enzyme forming a complex of 4,5 MDa. It is located in the cytosol and participates in protein turnover where it degrades oligopeptides to smaller peptides and tripeptides. Moreover, some of its products might take part in antigen presentation through MHC-class 1. High expression of the enzyme is believed to correlate with tumor progression. A decreased concentration seems to contribute to increased susceptibility to infection. The aim of this project was to develop an enzyme linked immusorbent assay (ELISA) specific for TPPII and compare it to two commercial kits. TPPII was detected through western blot and activity assay to ensure which samples were containing the enzyme. Samples with purified enzyme from different species, erythrocyte lysate and plasma were used to develop an indirect ELISA as well as compare all the assays and evaluate which one performed the best. The commercial kits were based on sandwich and competitive techniques. It was observed that the developed ELISA was able to detect the purified enzyme of different concentrations whereas the commercial kits could not. On the other hand, the commercial kits were able to generate their own standard curve which leads to suspicion that these might be non-specific to TPPII. Though, all assays did detect the enzyme in erythrocyte lysate. This study presents that it is possible to develop an ELISA specific for TPPII using an indirect technique which also performed better than commercial kits.

Fungals are opportunistic pathogens that can cause invasive infections primarily among immunocompromised individuals. Traditionally, fungi have been identified by culturing on agar plates and using morphological criteria. Molecular detection methods such as polymerase chain reaction (PCR) and sequencing is faster and superior at identifying fungi at species level compared to culturing and microscopic examination. The aim of this project was to identify fungi at the species level by sequencing the internal transcribed spacer (ITS) region of different control strains and patient samples. This was accomplished using a nested PCR followed by Nanopore sequencing. Two different workflows using different databases were created. One workflow created a consensus sequence which was followed by a BLAST search. The other workflow used an Emu program and mapped reads against the UNITE database. The result was compared to identification previously achieved. Amplification was successful in 28 out of 30 positive samples. The BLAST workflow managed to identify nine out of eleven samples, but was difficult to interpret. The Emu workflow was only concordant with previous identification in 20 out of 28 species in sequenced samples and failed to identify 4 previously identified fungi at the species level, but was easier to interpret and could identify several species from a mixed culture. Amplifying and sequencing of the ITS region can in several cases provide accurate identification, but the method of extraction, choice of DNA polymerase and choice of database need to be considered for successful amplification and accurate species identification before implementation as a diagnostic method.

The prevalence of diabetes is sufficiently high to be classified as an epidemic, and 20-40% of these patients are expected to develop diabetic nephropathy, a leading cause of end-stage renal failure. Studies have identified a correlation between diabetic nephropathy and hypoxia in renal tissue in human studies. Increased oxygen consumption has been associated with the proton transport protein, uncoupling protein-2 (UCP-2), which uncouples the mitochondria. Previous research has reported elevated levels of UCP-2 in diabetic renal tissue. Consequently, it is crucial to determine how different preservation methods affect the detectability of UCP-2 in renal tissue for clinical applications. This study aimed to evaluate the effectiveness of Western blotting for detecting UCP-2 in snap frozen, fresh untreated, formalin-fixed, methyl carnoy-fixed, and RNA later-preserved rat kidneys. Preliminary trials were conducted to identify the optimal antibody combinations, followed by testing on various preserved tissues. The antibodies produced non-reproducible, unspecific, and unselective results. Additionally, technical challenges, such as gels adhering to membranes and low protein concentrations in some samples, rendered the results inconclusive. Further investigations are necessary to explore additional antibodies and variables that may influence the detection of UCP-2 in differently preserved tissues. Overall, this study highlights the complexity and challenges in developing reliable protocols for UCP-2 detection in preserved renal tissue, indicating that significant optimization is still required for consistent results.

Cryptosporidium is a parasite that causes gastrointestinal issues such as diarrhoea and stomach pain. The main transmission routes are through contaminated water or food, between humans and from animal to humans. Cryptosporidium infects through oocysts which contain four sporozoites that releases when entering a host and can continue to breed inside the body. Cryptosporidium can cause massive outbreaks if established in a water source used for drinking water. To prevent and detect an outbreak it´s important to trace the transmission back to the source. The GP60-gene is used to identify and subtype Cryptosporidium and is a very useful tool during contact tracing. The purpose of this study was to identify species and subtype of Cryptosporidium using nanopore sequencing. In this study the GP60-gene was amplified using a Nested PCR protocol and then sequenced using nanopore sequencing. The sequences acquired where then used to make a search in BLAST to identify the species. The GP60 subtyping method uses the hypervariable region on the GP60-gene. A series of tandem repeats are used to identify the subtype. In this study seven positive Cryptosporidium faeces samples were amplified and sequenced. Nanopore sequencing was possible for five of the seven samples with C. parvum identified in four of these samples. Targeting the GP60-gene to determine species and subtype works well for the most common human pathogen species of Cryptosporidium. Further optimization is required before the method can be implemented för diagnostic use.

As pollinators bumble bees play the crucial role of contributing to propagation of flowering plants in favour of food production as well as biodiversity. Over the course of a few decades bumble bees have seen a remarkable decline, with contributing factors being climate change, pesticides and pathogens such as viruses and parasites. In Sweden, commercially reared bumble bees are bought for the purpose of pollination in fruit and berry plantations. However, these reared bumble bees are a suspected contributor to a spillover of pathogens to wild bees in the same area. The aim of the study was to determine the prevalence of five viruses and five parasites in wild bumble bees nearby commercially reared bumble bees and to determine seasonal variation in pathogen distribution. qPCR was used for analysis of Acute bee paralysis virus, Deformed wing virus, Slow bee paralysis virus, Black queen cell virus and Sacbrood virus as well as the parasites Crithidia bombi, Apicystis bombi, Nosema bombi, Sphaerularia bombi and Locustacarus buchneri. The results showed a statistically significant, 4,8 times higher prevalence of A. bombi nearby commercially reared bumble bees in greenhouses compared to control landscapes. The results were also compared to pathogen prevalences in bumble bees caught in June the same year, showing a significantly higher prevalence in a majority of the parasites. It also showed a decrease in all viruses except Black queen cell virus, where the decrease might be explained by RNA degradation.

To reduce the risk of transfusion related complications, blood components should, according to European guidelines, contain less than 1 x 106 leukocytes per unit. To verify that these guidelines are upheld, residual leukocytes are measured in randomly selected blood components as means of quality control. At Uppsala University Hospital, the method currently used for this is flow cytometry (FCM). However, the hospital recently purchased a new instrument, ADAM rWBC2, for this purpose. The aim of this study was to verify ADAM rWBC2 as a replacement method for FCM and investigate whether the type of test tube chosen for the instrument (EDTA or micro test tube) would affect the leukocyte concentration. To conduct the study, 30 red blood cell units (RBCs), 30 platelet units (PLTs) and 30 plasma units were analyzed on both the ADAM rWBC2 instrument and with FCM. In addition to this, each RBC and PLT unit was allocated into both an EDTA tube and a micro test tube before analysis on the ADAM rWBC2 instrument. Results from both methods and tubes were compared using statistical analysis. The results from ADAM rWBC2 tended to be higher than the results from FCM, and the difference turned out to be statistically significant (p<0,001). No significant difference could be detected between the results from the different test tubes. The assessment is that ADAM rWBC2 will replace FCM for quality control of residual leukocytes in blood components. According to the results, the type of test tube used does not affect the leukocyte concentration.

<p dir="ltr">As the world becomes increasingly reliant on technology, the role of software and systems in everyday life has increased exponentially. From mobile applications to critical infrastructure systems, software runs at the core of most modern systems. However, with this widespread usage comes the increased risk of cyber threats silently embedded in these systems. As software systems grow in complexity and scale, vulnerabilities become more difficult to detect and mitigate. The growing number of cyberattacks in recent years highlights the importance of not only building functional systems but also ensuring they are secure from the development stages. This emphasizes the need for a strong focus on secure coding practices as a vital component of both the software development process and education. Every computer engineering or computer science student is required to take programming courses as part of their curriculum. These courses teach fundamental programming aspects and skills, but lack the educational material about writing secure code. Many vulnerabilities that are present in software systems are caused by human error, and are introduced in code. This makes it imperative that students must be introduced to secure coding practices and general cybersecurity awareness while they are learning a new programming language. In this research we focus on the development of educational modules for secure software development and secure networking. A total of six secure coding modules were created, and a total of four secure networking modules were created. These modules provide clarity on a variety of vulnerabilities that may be introduced in code, such as lack of input validation, integer overflow, SQL injection, and SlowHTTP attacks. The module are designed as supplemental work that is performed concurrently with the regular curriculum, reinforcing the general information with security aspects. The goal of the modules are to increase the general cybersecurity awareness in students, and teach them how to mitigate to common vulnerabilities in code.</p>

<p dir="ltr">Online learning has become a staple in contemporary education, requiring new ways to display and promote experiences. Virtual labs can be used to give learners a way to enter a lab environment without the need for expensive equipment or materials. Examples of possible virtual labs include common drying and sterilization operations, more specifically a fluidized bed dryer and heat exchanger system. These processes incorporate numerous material properties and engineering design to describe and simulate them. The models being developed include various material characteristics and their corresponding effects for fluid flow, heat transfer, mass transfer, and reaction kinetics. Computer simulations of the systems were developed using data from numerous sources regarding product physical and chemical properties and the engineering relationships of fluid flow, reaction kinetics, and heat transfer for fluidized beds, plate heat-exchangers, and cylindrical pipes. This data was integrated into standard and accepted engineering and kinetic models to predict the system response with extra data gathered to estimate Maillard reactions for crystal malt using a fluidized bed with two temperature treatments. The heat exchanger system has models in place to evaluate an orange juice concentrate, a milk concentrate, and a carboxymethyl cellulose solution. The developed simulations show that the outputted data can be used to evaluate trends and fit models that match up to the literature expectations, with some tolerances to account for simplifications and assumptions. Roasted crystal malt color analysis shows rates of 0.33 to 0.34 EBC per minute and total EBC units after two hours of 72 and 96 EBC for temperature treatments of 125C and 142C, respectively. The non-reliance of temperature for the rates follows available data in literature with only the overall EBC color changing between temperatures. Further work to expand the scope of the models can help enhance their results and usability by including dynamics, accessory systems, or corrections to data and assumptions.</p>

Staphylococci are bacterial species where some strains can be found in the human microbiome. If the bacteria infect other body parts it could cause mild to life-threatening infections. The infection should be treated with an effective antibiotic where the bacteria strain’s antibiotic sensibility should be tested. One of the most trustworthy laboratory methods is the broth microdilution (BMD) method. BMD is based on exposing bacteria to different concentrations of different antibiotics to determine the minimum inhibitory concentration (MIC) a specific bacteria strain has, and thus the kind of antibiotic the bacteria strain is most susceptible to. A sensititre plate with 96 wells is used containing freeze-dried antibiotics in varied concentrations. Therefore, the microbiologic laboratory at Gävle Hospital wants to implement BMD as a routine analyzing method where a so-called SEMSE7 sensititre plate verification is needed. The verification is essential to ensure the plate’s performance and ability to give reliable results which was the purpose of this study. Obtained results were compared to reference laboratory values, where 100 % accordance regarding ± one dilution step was obtained of all MIC-values (90 % limit) and 94,3 % accordance regarding the SIR-system (90 % limit). Contrariwise, one very major error, and one major error were obtained which means 5,7 % where the limit was at 3 %. Furthermore, concerning the plate’s performance 97,3 % accordance was obtained regarding ± one dilution step (90 % limit). Most verification requirements were fulfilled but completion is needed before implementing BMD as a routine analysis method.

Amyloidosis is a disease caused by misfolding of proteins that accumulate in different organs in the body. One of the most common forms of amyloidosis is Transthyretin amyloidosis (ATTR-amyloidosis). Diagnostic of amyloidosis is done by Congo red staining and immunohistochemistry where high-affinity monoclonal antibodies are preferred. For treatment to be effective it is necessary to obtain the diagnosis at an early stage. The aim of this study was to produce monoclonal antibodies against transthyretin for diagnostic purposes of ATTR-amyloidosis. To produce monoclonal antibodies, the spleen from a mouse immunized with transthyretin was collected. Antibody producing cells from the spleen were fused with myeloma cells deficient of their own antibody production. After 2 weeks culture in HAT selection media cells were screened for antibody production with ELISA and immune-histochemistry. Recombinant transtyretin (TTR) was produced and used as antigen in the ELISA. All 65 hybridomas tested were negative on ELISA. Three out of 25 hybridomas tested in immunohistochemistry showed visible staining against muscle cells in tissue from patients with TTR amyloid deposits. No antibody could be produced that detected ATTR-amyloidosis deposits.