Vibrio vulnificus, a natural inhabitant of brackish and estuarine environments, is a fatal opportunistic human pathogen with a mortality rate of over 50%. Unlike for other pathogenic Vibrio species, and despite its high mortality rate, there is no conclusive approach to indicate the pathogenic potential of V. vulnificus isolates from the environment. To this day, a single gene encoding a hemolysin, vvh, has been used to detect V. vulnificus, and one phenotype - indole production, has been used to assess the pathogenic potential and classification of a given strain. In this study, we use genomic-based approaches to identify distinct phenotypes that can characterize V. vulnificus strains with highest pathogenic potential (cluster 1 – C1), and to determine novel genes that can be used to accurately detect and identify V. vulnificus strains from natural reservoirs. Our phenotypic analyses indicate that strains from C1 utilize a more diverse range of carbon sources compared to strains less likely to emerge as pathogenic (cluster 2 – C2). We found that C1 and C2 prefer to inhabit different niches leading to behavioral separation. Physiological adaptations like motility in the presence of mucin and growth at different salinities indicate markedly different lifestyles for these clusters. These phenotypes can thus be used as markers to predict the pathogenic potential of unknown V. vulnificus isolates from the environment based on their clusters. Furthermore, we identified six candidate genes that can distinctly discriminate between the two clusters and are more sensitive in detecting V. vulnificus compared to existing typing techniques. The high degree of resolution offered by this simple, reproducible approach can thus be used to identify V. vulnificus strains from natural reservoirs, as validated in our study using environmental isolates from oysters and water.

Prostate cancer is the second highest cause of cancer related deaths among men. According to the diagnostic pathway for prostate cancer, a prostate biopsy is performed if an individual showed signs of lesions through high prostate-specific antigen (PSA) concentration or suggestive digital rectal exam (DRE) results. The core biopsy mechanism involves inserting a beveled needle into the organ and removing a cylindrical fragment of tissue. Many factors affect the histological quality of the sample, including fragmentation, needle deflection, and needle insertion velocity. If a biopsy core is not clinically viable, an alternative core will need to be taken, resulting in increased patient trauma and potential risk of infection. Many of these relevant factors are impacted by sources of friction in the system. Prior studies have examined methods of decreasing the friction of the interactions between different components of the biopsy system to reduce the negative effects on histological sample quality. While scenarios have been explored that examine reducing the friction between the needle and tissue through sharpening and polishing techniques, the friction introduced by the needle guide in template guided core prostate biopsies has not been analyzed in the decades since its development. This study aims to introduce the biopsy guide as an additional source of friction which can be optimized to reduce friction force, while proposing and testing several configurations of the needle guide that would reduce the friction force of the system. A Finite Element Analysis (FEA) was conducted using SIMULA Abaqus modeling software, and the simulation was correlated with a derived equation that estimated friction force according to material properties. The study demonstrated that configurations for the internal surface of the needle guide which provided decreased contact surface compared to the control needle guide resulted in lower friction force between the needle and guide. Conditions which had contact points oriented parallel to the direction of insertion had the lowest recorded friction force. This suggests that the traditional biopsy needle guide may be optimized to introduce less friction force by reducing the contact area between the needle and guide inner surface. This has application in reducing the number of passes required to obtain a histologically viable core specimen, and therefore reducing the opportunity for patients to develop infection.

Sepsis is aggressive and severe inflammatory body response to an infection and is considered to be one of the most common death causes in patients. The current diagnosis of sepsis is not fast enough to help those who get sepsis, due to its fast progression. The current golden standard for sepsis diagnosis is blood culturing. However, the biggest downside of it is the long time. Research is now focused on finding a faster way to diagnose sepsis on early stages. The most promising one tends to be the usage of biomarkers. Today, there are 260 defined sepsis biomarkers, however, only few of them are clinically used. Among them, C-reactive protein, and procalcitonin. Another potential biomarker could be miRNAs. The research about that today is at early stage. To use miRNAs as biomarkers, they need to be quantified. One way to quantify miRNAs is the two-tailed RT-qPCR method together with absolute quantification. This study focused on evaluating the best extraction method of small RNA for later quantification of specific miRNA. The blood plasma from healthy donors was divided into spiked and non-spiked samples, where the synthetic miRSeps-3 served as a spike-in positive controls. All samples were extracted using two methods, manual and robotic with Qiacube (Qiagen). Absolute quantification was applied to quantify miRNA in all samples. The successful results indicated that the two-tailed RT-qPCR was sensitive enough. More optimization is required for the methods, however, the whole method has a good potential to become helpful for clinical usage in the future

Interventions for influenza virus infections are essential to minimize the worldwide annual morbidity, mortality, and economic loss caused by this highly contagious respiratory pathogen. Establishment of universal, long-lasting protection against epidemic and pandemic strains of the virus can potentially eradicate the necessity of annual reformulation and readministration of low-efficacious seasonal vaccines, increasing pandemic preparedness. The protective potential of Type 1 T helper (TH1)-polarized memory CD4+ T cells against Influenza A virus (IAV) infection and generation of secondary memory populations following viral clearance are well-characterized. To assess the potential of CD4+ T memory cells as a candidate for adoptive immunotherapy, here we validated and optimized cryopreserved IAV-specific memory CD4+ T cell-mediated protection against infection and evaluated their potential for subsequent memory formation. Donor-derived in vitro-generated memory CD4+ T cells were transferred into IAV-infected naïve mice following cryopreservation of these cells for 6-12 months and overnight activation with gamma-chain cytokines, interleukin (IL)-7 and IL-7+IL-2. Results showed that cytokine-cultured cryopreserved memory CD4+ T cells, compared to their non-cultured counterparts, controlled viral titer in the lung at the peak infection phase, decreased morbidity, expedited recovery, and formed increased secondary memory cells in the lung, the primary site of infection, including lung tissue-resident memory (TRM) CD4+ T cells. Phenotypic and functional analysis confirmed that donor-derived secondary memory CD4+ T cells retain a TH1-phenotype and produce cytokines associated with protection against IAV. These observations support that the protectiveness and memory-forming potential of host- and/or donor-derived memory CD4? T cells can be preserved and harnessed for future use. This T-cell based adoptive immunotherapy addresses some of the current challenges of available preventative and therapeutic options, such as low vaccine efficacy, availability of only early treatment drugs, lack of immunity against pandemic strains and effective memory cell generation.

digitalisering@umu

Detection of circulating tumor cells (CTC) in peripheral blood is indicative of early recognition of tumor progression and such an important biomarker for early diagnosis, staging, monitoring and prognosis of cancer. However, CTC are found in very low concentrations and reliable isolation of these rare cells is challenging. Microfluidics enables precise manipulation of fluids and cells and is ideal for cell sorting methods for clinical diagnostics. The thesis contributes towards the development of microfluidic based CTC isolation methods from peripheral blood. The methods are based on size and immunoaffinity. The first part of the thesis describes the phenomenon of inertial focusing for size based cell separation at high throughputs. In paper 1, we demonstrate continuous filtration of leukocytes from diluted blood, with an efficiency of 78% at a flow rate of 2.2ml/min. In the paper 2, separation of total and subpopulation of leukocytes with a purity of 86% for granulocytes and 91% for lymphocytes is demonstrated. Furthermore, cancer cells spiked into whole blood could be separated at a yield of 88%. Finally, in paper 3 and 4 we unravel parts of the unexplored elasto-inertial microfluidics and was utilized to precisely focus the cells, as part of an integrated optofluidic micro flow cytometer device, capable to simultaneously measure fluorescence and scattering of cells and particles at a rate of 2500 particles/sec (paper 4). Second part of the thesis focuses on acoustophoresis. In (paper 5), a multifunctional acoustic microdevice was developed for isolation of cancer cells from red blood cells with a separation efficiency of 92.4% and trapping efficiency of 93%. In (paper 6), microbubbles activated acoustic cell sorter was developed for affinity based cell separation. As a proof of principle, cancer cells in a suspension were separated at an efficiency of 75%. In the third part, using cellulose nano fibrils (paper 7), we demonstrate efficiently capture and release of cancer cells at a release efficiency of 95%. Finally, a novel, single step self-assembly of spider silk proteins is introduced inside microfluidic channels for effective capture of cancer cells with 85% capture efficiency and subsequent release of captured cells with 95% release efficiency (paper 8). The novel recombinant silk modified microfluidic device was validated using pancreatic cancer patients. In summary, we have developed different microfluidic based isolation technologies for the capture and characterization of CTC. / <p>QC 20170321</p>

Omi, also known as HtrA2, is a mammalian pro-apoptotic mitochondrial protein and a member of the HtrA (high temperature requirement A) family of serine proteases. Omi promotes the caspase-dependent apoptotic pathway through cleavage of IAPs (inhibitor of apoptosis proteins); this cleavage inactivates IAPs and facilitates caspase activity. Omi's proteolytic activity is necessary and essential for its pro-apoptotic function. This study is aimed to further understand the role of Omi in the cytoplasm by using the yeast two-hybrid system to identify novel Omi interactors/substrates. A HeLa (cervical carcinoma cell line) cDNA library was screened using Omi as a "bait" protein. One of the proteins indentified in this screen as a strong Omi interactor was the S5a protein and was selected for further analysis. S5a is a soluble cytosolic mammalian protein and a component of the proteasome's 19S regulatory subunit. The proteasome is a large cytosolic protein complex responsible for the controlled degradation of damaged or denatured cellular proteins. Further characterization of the interaction through an in vitro proteolytic assay demonstrated that Omi can cleaves recombinant S5a protein. This data suggests that S5a is a bona fide substrate of Omi that is degraded upon induction of apoptosis. It also provides a new mechanism that leads to the inactivation of the proteasome during cell death.

Neuroblastoma is a form of embryonal neuroendocrine tumor that arises from neural crest progenitor cells, which can differentiate into multiple cell types. It is caused by genetic abnormalities such as MYCN oncogene amplification and 11q deletion, both of which can deregulate neuroblastoma suppressor genes located on chromosome 11q. MiRNAs are noncoding RNAs with an average length of 22 nucleotides. By binding to the 3′ untranslated regions of target messenger RNA, the RISC represses protein synthesis. The aim of this experimental project was to determine the effects of MYCN amplification or 11q deletion on the expression level of the miRNA, hsa-miR-34b-3p in neuroblastoma. The total RNA was extracted from the neuroblastoma cell lines, NB69 without MYCN amplification and 11q deletion, Kelly with 11q deletion and SK-NBE(2) with MYCN amplification. cDNAs were generated from the miRNA, hsa-miR-34b-3p located on chromosome 11q and the reference gene, RNU6B. The cDNAs were amplified and quantified by qPCR. The qPCR data were analysed using the comparative Ct method, Kruskal-Wallis test with multiple comparisons to determine whether or not hsa-miR-34b-3p was significantly differentially expressed in Kelly and SK-N-BE(2) compared to NB69. The results showed that hsa-miR-34b-3pwas significantly downregulated in the cell lines, Kelly and SK-N-BE(2) compared to NB69. In conclusion, the findings of this study showed that hsa-miR-34b-3p is downregulated in Kelly and SK-N-BE(2) compared to NB69, and call for further research to investigate its clinical potential in the therapy of neuroblastoma.

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No description available.