Neuroblastoma (NB) is one of the most common extracranial cancers found in children under the age of five. The cause of NB is not well understood, about 2% of the cases have been linked to rare germline mutations in the anaplastic lymphoma kinase (ALK) gene. However, NB is thought to be mainly caused by genetic mutation at the early stages of development. Clinically, NB can be grouped into three risk groups: low, intermediate and high-risk disease. The survival rate of patients with high-risk NB is less than 50% of the diagnosed cases. Survival rates emphasizes the necessity for future NB diagnostic therapy. One potential study area is miRNA, studies have demonstrated both prognostic and predictive usefulness to therapies. MiRNA is a single-stranded RNA that is 18-24 nucleotides long. Its function is to regulate numerous cellular activities, and to act as tumor suppressors or oncogenes. Genetic anomalies such as MYCN amplification and 11q deletion cause NB by disrupting the expression patterns of certain miRNAs. In this experiment the miRNA, hsa-miR-708-5p, was examined in three genetically diverse NB cell lines; NB69 without MYCN amplification and 11q deletion, SKNBE with MYCN amplification, and Kelly with a chromosome 11q deletion, the cell lines were used to see if the expression levels of hsa-miR-708-5p differed. The expression level of hsa-miR-708-5p, was assessed using qPCR; variation in gene expression was identified between the cell lines. Therefore, miR-708-5p could be a viable option when looking at gene expression of hsa-miR-708-5p for future diagnostic or prognostic in NB.
Sclerotinia sclerotiorum is a plant pathogenic fungus that causes Sclerotinia stem rot in oilseed rape. In Sweden, the disease causes severe crop loss that varies by year. Previous studies have shown a relationship between the proportion of infected petals and disease incidence in infected fields in places with high humidity levels before and during flowering. In this study, the aim was to develop a technique to detect S. sclerotiorum and other fungi pathogens in the petals of oilseed rape from naturally infected fields by using nanopore sequencing from Oxford Nanopore Technologies. DNA was extracted from the petals of oilseed rape and subsequently amplified by performing PCR after optimizing the optimal annealing temperature. Using the forward primer ITS1catta and the reverse primer ITS4ngsUni, these primers targeted the ITS region, which is used as a marker for the identification of fungi. The resulting Amplicon concentrations varied. Five amplicon PCR samples were selected for MinION sequencing. These samples were selected since they had the best purity levels. Finally, bioinformatic analysis was done with Kraken2 and the Pavian tool and compared with UNITE databases. The result showed hundreds of thousands of reads were recovered from the Ascomycota and Basidiomycota fungi divisions; S. sclerotiorum was observed in one field sample; other Sclerotiniaceae species like Dumontinia tuberosa, Botrytis cinerea, and Sclerotinia bulborum were detected in two fields; and many other fungal pathogen species affecting rapeseed crops in Sweden were successfully detected. MinION was successful in identifying S. sclerotiorum and other plant pathogens.
Inflammation is a biological defence mechanism applied by living organisms against foreign invaders. In the response to DAMPs and PAMPs, organisms use inflammatory multi-protein complexes to fight the attackers. The most studied inflammasome proteins are NLRP3, ASC and Caspase-1. This study is aimed at understanding the role of NEK7 protein in the NLRP3 inflammasome’s activation, using CRISPR/Cas9 system. To determine the effect of CRISPR/Cas9 and transfection, mRNA expression was analyzed. The results obtained suggest that neither the transfection nor the NEK7 protein knockout have sufficiently worked. This study could not experimentally establish that NEK7 triggers NLRP3 inflammasome activation because ELISA was not conducted to verify the levels of cytokines emitted, due to there being no statistical differences between the samples. Above all, the research question in this thesis project was not answered because the instability of the ACTB reference gene negatively influenced the results. However, previous related studies conclude that NEK7 plays a crucial role in the activation of the NLRP3 inflammasome.
Pseudokirchneriella subcapitata is a sickle-shaped freshwater green microalga that is normally found in unicellular form. It is the best known and most frequently used species of ecotoxicological bioindicator because of its high growth rate and sensitivity to toxicants. Metronidazole (MTZ) is a routinely used nitroimidazole antibiotic that has caused environmental issues owing to incorrect use. A toxicity test was performed in order to understand the relationship between the MTZ concentrations and response at a physiological level. The study found a growth percentage of (0, 4.8571, 4.5714, -15.1429, -37.1429 %) accordingly. The changes on the transcriptomic level were tested by performing a RT-qPCR. Using ∆∆Ct method to compare the treated samples with low and high MTZ concentration against the control sample. The study found that Exposure to MTZ at the low and high concentrations gave rise to 1.45 fold upregulated pcna gene expression that was differentially expressed in control R. subcapitata. The high group of samples in the high group were clearly distinguishable from those in the control and low treatment groups.
Viral-bacterial coinfection is a major and life-threatening issue, and it ranks among the primary causes of mortality. Current monotherapies that only target either bacteria or viruses, are deemed insufficient for treating coinfection and may be associated with undesirable consequences. Therefore, the development of novel treatment approaches is necessary, preferably with dual effects against both viruses and bacteria. This study sought to assess the effectiveness of PLNC8 αβ, in specifically targeting both KUNV and S. aureus infection in an in vitro coinfection model using human keratinocyte cells. The LDH cytotoxicity test was performed to evaluate the peptide’s effectiveness in reducing cytotoxicity that is individually caused by S. aureus, KUNV, as well as their coinfection. Additionally, ELISA was utilized to quantify the levels of inflammatory cytokines, namely CXCL8, and IL-6. The results demonstrated that both forms of the PLNC8 αβ effectively decreased the infection-induced cellular cytotoxicity. The D- PLNC8 αβ exhibited superior efficacy compared to the L- PLNC8 αβ, since the latter was more susceptible to enzymatic degradation, resulting in the loss of its functionality. Furthermore, both forms of PLNC8 αβ effectively modulated the levels of inflammatory cytokine and restored cellular viability. In addition, the peptide substantially reduced the number of bacterial colonies in both S. aureus infection and coinfection. Based on these findings D- PLNC8 αβ possesses a dual antimicrobial action and could be further characterized and validated as a promising therapeutic agent against viral-bacterial coinfection.
Few things are more fascinating than finding new interactions between previously unrelated pathways. Glycogen synthase kinase-3 (GSK-3), a ubiquitous kinase initially known for its role in regulating glycogen metabolism, has recently been found to be an indispensable regulator of the TLR4-mediated inflammatory response. GSK-3 inhibition exhibits potent anti-inflammatory effects by acting on both arms of the inflammatory response, reducing the secretion of pro-inflammatory cytokines, and promoting the production of anti-inflammatory cytokines. Tumor Necrosis Factor-α (TNF-α) is among the most important inflammatory cytokines. Aberrant TNF-α expression is associated with various inflammatory conditions, including sepsis and cancer. Thus, understanding the mechanisms regulating TNF-α production could reveal potential therapeutic strategies for TNF-α-associated diseases. Consequently, this study aimed to examine the effect of GSK-3 inhibition on TLR4-induced TNF-a production by THP-1 macrophages. THP-1 macrophages were stimulated with LPS and nigericin in the presence and absence of GSK-3 inhibitor, and TNF-α protein and mRNA levels were evaluated by ELISA and Real-time PCR, respectively. GSK-3 inhibition significantly attenuated TNF-α protein levels in a dose-dependent manner, whereas TNF-α mRNA levels remained unaffected, reflecting a possible post-transcriptional modulation of TNF-α biosynthesis by GSK-3. However, more comprehensive research is needed to elucidate the precise contribution of GSK-3 to TNF-α biosynthesis and to identify novel therapeutic mechanisms to alleviate inflammatory diseases associated with abnormal TNF-α production.
Antibiotics are considered a type of antimicrobial that particularly has an impact on bacteria or fungi in humans and animals. The widespread use of common antibiotics, combined with the fact that the majority of active antibiotics and their metabolites are water-soluble, results in persistent pollution in aquatic environments, as well as a potential threat to ecosystems. Moreover, there are inadequate ecotoxicological data on many antibiotics, such as azithromycin, which has been quantified at elevated levels in the aquatic system. Raphidocelis subcapitata is a globally distributed green alga that is commonly used as a model species for evaluating chemical toxicity due to the availability of a sequenced genome and its rapid growth, which allows assessing chemical effects across many generations. the aim of this project is to provide an insight on genotoxicity for R. subcapitata and study the effects of azithromycin antibiotic on algae, on both growth rate and molecular levels by determining gene expression levels, specifically, its effect related to chlorophyll pigments,biosynthesis, and DNA replication levels. In order to do that, toxicity test according to OECD guidelines for 7 days, photosynthetic pigment extraction and qRT-PCR were utilized. In the present study, an EC50 of 24 µg/L was obtained, while low risk in the Swedish water streams was indicated, significant induction in Chlorophyll a and b at high concentrations while no effects on carotenoids were observed, no significant difference in pcna and cyt P450 at LOEC and lower concentrations was obtained. This might suggests testing higher concentrations in upcoming research.
Inflammation is the body's response to infection or injury and is mediated by the innate immune system. The NLRP3 inflammasome is a multi-protein complex that is a major contributor to many inflammatory disorders. Emerging evidence suggests the involvement of the Endoplasmic reticulum stress with the NLRP3 inflammasome. The endoplasmic reticulum stress is a series of stress signals that can activate the unfolded protein response and usually accompanies inflammation and eventually causes cell death. Recently, a localized endoplasmic reticulum micro-protein called the chloride clic like-1 channel was found to be involved in the endoplasmic reticulum homeostasis. Recent evidence suggests the involvement of endoplasmic reticulum stress in the inflammation pathways of the NLRP3 inflammasome. The relationship between the ER and the NLRP3 inflammasome has not been clearly described. This study aimed at investigating the expression levels of the microprotein CLCC1 to shed a light on the relationship between the endoplasmic reticulum stress and the NLRP3 inflammasome. The expression levels of CLCC1 were analyzed by qPCR in cultured monocytes under different time points of Lipopolysachaaride immuno-stimulation. The stability of expression in candidate reference genes was investigated for normalization purposes. This study reported the regulation of CLCC1 as a novel finding under prolonged LPS exposure of monocytes and stable reference genes such as GUSB and ACTB were identified. The relationship between CLCC1 and NLRP3 inflammasome priming by LPS indicated that CLCC1 is regulated and may be involved in the inflammatory mechanisms of endoplasmic reticulum stress and NLRP3 inflammasome inflammatory diseases, contributing to a potential therapeutic target in the endoplasmic reticulum and inflammasome related diseases.
Innate immunity is the immune systems rapid responses to infection after being attacked by a pathogen. Inflammatory responses are activated by the detection of pathogen-associated molecular patterns and danger-associated molecular patterns through pattern recognition receptors on inflammatory cells. NLRs are activated by intracellular PAMPs which warn cells of damage and have a major role in initiating the innate inflammatory responses as well as the development of infectious and inflammatory diseases. NLRP3 is a very large multiprotein complex and is the most studied inflammasome. The NLRP3 Inflammasome follows a two-signal model for activation, signal one forms the NLRP3 complex and signal two activates the inflammasome. NLRP3 initiates an inflammatory form of cell death called pyroptosis and triggers the release of pro-inflammatory cytokines IL-1β and IL-18. The miR-200 family has five members, miR-200a, miR-200b and miR-429 located on chromosome 1 and miR-200c and miR-141 located on chromosome 12. In this study, THP-1 cells were differentiated with PMA then stimulated with LPS and ATP. Various time samples were collected and isolated to obtain miRNA. Two-step RT-qPCR was then performed to quantitively monitor the changes in miRNA-200 family expression levels. The purpose of this study was to observe how miRNA-200 family expression levels change in stimulated THP-1 cells as the NLRP3 inflammasome is activated. This became a pilot study as all biological replicates could not be analyzed, miR-200 family is showing a potential response to the activation of the NLRP3 inflammasome and they should be investigated further.
Obesity leading to Type-2-diabetes is a major health issue all over the world. Obesity characterized by expansion of adipose tissue, in particular white adipose tissue (WAT) which controls the metabolic physiology in the body by secreting proteins like adiponectin and resistin. Adiponectin has a protective role against diabetes development whereas resistin causes insulin resistance. Protein folding, maturation and translocation is performed by the Endoplasmic reticulum using calcium ions. The calcium homeostasis is maintained by calcium pumps and channels, chief of this is sarco-/endoplasmic reticulum (SR/ER) Ca2+ ATPase pump (SERCA) which restores calcium back to the ER. To study the effect of SERCA2 on adiponectin and resistin secretion in different adipose tissue depots using an adipocyte specific tamoxifen-inducible SERCA2 Knock-out mice, short term secretion experiments were performed. Chemical inhibition of SERCA2 and ER stress was performed in in-vitro experiments using adipocyte like 3T3-L1 cell line. The experiments revealed that SERCA2 dysfunction led to decrease in adiponectin and resistin secretion in normal and stimulant conditions in both male and female mice. In-vitro experiments revealed that ER stress led to misfolded protein accumulation affecting exocytotic events of adiponectin containing vesicles. Therapeutic agents can be formulated to tackle the SERCA2 dysfunction and to maintain calcium homeostasis by identifying these key mechanisms for diabetes and related metabolic disorders.
A current trend utilizing the biomedical approach in the field of wound care is focused on the increased potential to develop wound healing materials designed to address specific types of wounds or underlying pathologies to achieve improved healing. The work presented in this thesis evaluates the blood response to wood-derived nanocellulose functionalized with a peptide, with the ultimate aim of characterizing the material as a potential wound dressing for chronic wound care. The material was evaluated based on the response toward the innate immune system. These interactions between the material and blood were studied using an in vitro whole blood loop model, and then, the coagulation and complement system activation markers were quantified using enzyme-linked immunosorbent assays. The platelet count and the levels of the thrombin-antithrombin complex reported for the material showed no activation of the coagulation cascade whereas there was an activation caused in the complement system showing higher levels of C3a and s-C5b9 components as compared to the controls. The observations obtained from this interdisciplinary project can be considered as a stepping stone toward the need for further analysis of the material in advanced wound care applications. This can be achieved by targeting the specific phases of the wound healing process in order to promote effective wound management.
Sepsis presents a complex challenge, requiring detailed investigation into its immunopathology, diagnosis and treatment. Among 258 promising biomarkers for sepsis diagnosis, miRNAs are emerging as valuable components to be incorporated in a multimarker panel for sepsis assessment when assessing bodily fluid samples. The aim of this project was to compare manual and semi-robotic techniques for extracting total RNA, including miRNA, from healthy human blood plasma samples. The comparison focused on hands-on time (HOT), turn-around time (TAT), and the absolute quantification of the candidate miRNA, miR-Seps5. The study utilized two extraction methods to isolate total RNA, including miRNA. The emphasis was on detecting and quantifying the novel candidate miRNA, miR-Seps5, using the Two-Tailed RT-qPCR technique and absolute quantification. This approach effectively amplified synthetic and circulating miR-Seps5 from 100 μl of healthy human blood plasma. No-significant difference was found between two methods of extraction on the basis of TAT and data retrieved from two-tailed RT-qPCR while HOT was significantly lower for semi-robotic extraction method as compared to manual extraction method indicating Qiacube to be a time efficient method. Linear standard curve and melt curve analysis provided insights into the specificity of Two-tailed RT-qPCR assay but unspecified amplification in non-template control (NTC) requires further investigation. The quantified values of miR-Seps5 in both spiked and non-spiked samples showed inconsistency. Considering these inconsistencies, further analysis is required to assess the reliability of the RT-qPCR assay and miR-Seps5 before considering it as a suitable biomarker in a future multimarker panel for sepsis diagnosis.
Extracellular vesicles (EV’s) are small lipid bilayer vesicles that are generated by almost all kind of cells in a body. EV’s are considered as one of the key intercellular messengers regulating cell signalling mechanisms. Earlier studies have shown that, in metabolic diseases like diabetes and obesity, as well as during hypertension and neurodegenerative disease there is increased production and secretion of EV’s. Secretion mechanism of EV’s is yet unknown. The aim of this study was to investigate the EV production and secretion mechanism in type-2-diabetes and in parallel to EV studies, measurement of SST secretion, to elucidate how it is influenced by EV’s. Tetraspanin was used to label EV’s and the efficiency was evaluated by using TIRFM and considering how many exosomes they label per cell and how well they express. Further, the EV marker were exploited in studying trafficking events of EV’s at the plasma membrane. This included EV approach to PM through docking/visiting, and EV loss from PM through undocking. EV labelling showed that CD63 and CD151 were two efficient markers for live-cell imaging by TIRF microscopy (TIRFM). Trafficking analysis of EV’s showed that number of visiting events were significantly higher compared to docking and undocking events. To know how many of total EV’s in a cell are ready to fuse with plasma membrane, rate of displacement of EV’s was monitored. This showed, small fraction of EV’s were slow-moving, probably docked at the PM while rest EV’s were fast-moving, either visiting or undocking EV’s. Docked EV’s fuse with plasma membrane. SST secretion from δ-cells was studied using pancreatic islets. There are no currently reliable means to measure δ-cells SST secretion. Commercially available antibodies against SST were evaluated compared to antibodies developed in the lab. Efficiency of the antibodies was studied by analyzing number of δ-cells and their distribution in an islet. The results showed that the antibodies against SST that were developed in the lab have a higher efficiency compared to the commercially available antibodies in δ-cells in an islets and tissue. These antibodies were used for staining δ-cells in type-2 diabetic vs healthy islets. Decrease in number of δ-cells in diabetic islets was observed. Therefore, these developed antibodies can be used for future hormone secretion studies.
Malaria is a life threatening parasite disease caused and transmitted by infected female anopheles mosquito. However, the parasite, Plasmodium falciparum, has become resistant to most anti malarial drugs, such as chloroquine, which contributes to fever and anaemia because of its ability to digest the haemoglobin in the red blood cells. The aims of this project were to establish whether “Bac to Bac” Baculoviral Expression System is suitable for expression of pfmdr 1 gene and for purification of the pgh 1 protein. The pfmdr 1 gene encodes an ABC transporter protein, pgh 1, fixed in the cell membrane of the Plasmodium falciparuum gut, which assist in elimination of drug compounds. Furthermore, “Bac to Bac” Baculoviral Expression System uses vectors with histidine tags to clone the pfmdr 1 gene and subsequently transform these into DH10Bac cells to produce the recombinant bacmid DNA. Since pfmdr 1 gene is an AT-rich sequence, PCR was optimized, by lowering the annealing and extension temperature to 47Co and 66Co respectively. The results show that “Bac to Bac” Baculoviral Expression System can be used to express the pfmdr 1 gene, though further experiments has to be performed.
Escherichia coli is an important model organism in scientific research. Different strains can carry different genetic properties, and gene knockouts are used to study effects on phenotype. Deletion of the aroE gene in the MG1655 and ATCC 25922 strains of Escherichia coli, coding for an essential enzyme in the biosynthesis pathway for aromatic amino acids, resulted in auxotrophy. Subsequent selection for prototrophy revealed that the auxotrophic phenotype could be suppressed at a frequency of approximately 10-8 in ATCC 25922 but not in MG1655. The aim of this study was to identify the mutation(s) that suppressed the phenotype of the aroE gene deletion and determine why suppression occurred in ATCC 25922, but not in MG1655. Independently selected mutants were analysed by whole genome sequencing, but no obvious genetic alterations were identified. This prompted an evaluation of the phenotypic stability of the selected mutants. Mutants were growth in rich medium then tested for auxotrophy, which revealed that the mutant phenotype was highly unstable. While the initial research question could not be answered, both the frequency of occurrence and instability of the selected mutant phenotype, are important clues. Genetic instability is associated both with gene duplication, for which no evidence was found in the genome sequence, and epigenetic changes, which are not revealed by standard DNA sequencing. In conclusion, an auxotrophy bypass mechanism has been shown to exist in ATCC 25922 with no obvious genotypic source. Further experimentation, including methylome sequencing, might provide more leads.
Anaplastic lymphoma kinase (Alk) is a receptor tyrosine kinase primarily expressed within the brain, recognized for its pivotal role as an oncogenic driver across various cancers, including neuroblastoma. Recent studies have expanded the current understanding of Alk's function, extending beyond its oncogenic properties to encompass developmental and metabolic functions, suggesting its involvement as a modulator of body weight. Notably, Alk's gain-of-function in Drosophila has been linked to a significant reduction in pupal size. This study aimed to elucidate the signaling pathway between the brain and fat body governed by Alk and identify the molecular players involved using Drosophila as a model organism. Transcriptomic analyses were employed to identify genes upregulated in the fat body under conditions of Alk gain-of-function. Using the Gal4/UAS system, specific genes were manipulated in the fat body based on their upregulation in Alk gain-of-function mutants. Immunofluorescence techniques were utilized to visualize morphological changes in the fat body and the distribution of lipid droplet distribution, which were then quantified and compared to the Alk gain-of-function phenotype. The results demonstrate that Alk gain-of-function mutation disrupts fat body physiology, resulting in an abnormal phenotype. Several genes emerged as potential candidates involved in fat body metabolism, with mthl8 notably upregulated. Targeted expression of mthl8 led to a phenotype like the Alk gain-of-function fat body phenotype, highlighting mthl8 as a potential key player in the Alk-mediated signaling pathway between the brain and fat body. This study provides mechanistic insights into the role of Alk in metabolism.
This study examines the use of DNA methylation as an application in forensic science, namely age estimation and individual identification. DNA methylation is an epigenetic marker that assists forensic analyses by providing information about biological age and tissue types. A meta-analysis methodology that integrated data from 15 research papers was utilized to evaluate the precision and reliability of age predictions based on DNA methylation. The findings indicate that DNA methylation is a valid method for age determination, which is especially useful when traditional forensic methods are insufficient on degraded biological samples. The outcomes suggest a high overall effect, with an effect size (r) of 0.96, and a 95% confidence interval of 0.94 to 0.97. Given the variations in sample sizes and methodologies across the publications, slight differences in the MAD values were expected. Nevertheless, these values remained within a comparable range. The results imply that DNA methylation markers are highly effective for age estimate, particularly in genes KLF14 and ELOVL2 where there is a strong association between CpG sites and age. In conclusion, DNA methylation is a practical and essential technique for establishing age and identifying individuals. The study emphasizes how DNA methylation can improve forensic procedures and age estimation accuracy, potentially aiding in the resolution of criminal cases.
Nowadays, sepsis has become a major healthcare problem. Its variance of symptoms and the lack of time to act makes it greatly difficult to treat. An early diagnosis using biomarkers, particularly miRNA, could potentially increase the patient’s prognosis as well as reduce the use of antibiotics for the treatment. The lack of method optimization for miRNA extraction and quantification calls for investigation prior to the construction of a multi-biomarker panel for sepsis diagnosis. The aim of this project was to examine and compare manual and semi-automatic extraction methodologies through the small RNA quantity and RNA quality, as well as test the detection and quantification abilities of the novel technique, two-tailed RT-qPCR. 30 extractions have been performed, their extracted elutions have been subjected to quality and quantity control and detection and absolute quantification through the two-tailed RT-qPCR. The results show no significant differences between the quantity and quality of the RNA extracted using both methods. Time management, on the contrary, reported significant differences between the two methods. On the other hand, the two-tailed RT-qPCR successfully amplified the miRNA candidate from as little as 100 µL of healthy plasma. The absolute quantification showed the miRNA candidate’s low concentration in plasma. Moreover, the qPCR efficiency was irregular during the project which may alert of contamination or unspecific primers. However, the melt curve showed a single amplicon which suggests great specificity. The detection and quantification of the miRNA candidate have been successful, though further investigation is recommended.
Neuroblastoma (NB) is the most frequent extracranial solid tumour in childhood. The clinical diagnosis of NB is difficult due to the age of the patient and the vague appearance of the symptoms. Moreover, there are two groups of aggressive NBs, one with MYCN amplification and the other with an 11q deletion. Some genes could be a candidate suppressor for NB, e.g., the DLG2 gene that resides within the 11q-deleted region. The DLG2 gene has a large number of exons and multiple isoforms depending on the alternative splicing process. Moreover, these isoforms can include the L27 domain or not. This study aimed to analyse, by applying bioinformatic tools, if isoform 2, which does not have L27 domain, could be a candidate suppressor for this disease. RNA-seq samples from different human cell lines were collected from NCBI and a quality analysis was performed. The filtered samples were run in R and Python programs to do a visualization of the exon expression level and the prediction of Rsubread for exon-exon junctions. The results showed that isoform 2 of DLG2 gene was not expressed in the samples of NB, which is a promising result for being a candidate suppressor of NB. Furthermore, the prediction of exon-exon junctions by Rsubread was confirmed to be very accurate. In conclusion, this study shows that isoform 2 of DLG2 gene could be a candidate tumour suppressor in NB that could, in the future, be used as a target to help to detect earlier the presence of NB and increase the life expectancy of children who suffer from this disease.
A constant flow of various pathogens enters the respiratory system on daily basis through the involuntary mechanism of breathing. Respiratory viral infections are common yet can be fatal in vulnerable populations. Respiratory syncytial virus (RSV) is one of the first and most common viruses that the human population acquire in the first two years of life. Despite the ability of most infants to recover from a RSV infection, many require hospitalization and, in few cases, die from such an infection. The pattern of seasonality of respiratory viruses also applies to RSV. In this work the temperature dependence of infectivity was studied in Hep-2 cells infected with RSV that had been incubated with bronchoalveolar lavage (BAL) fluid. The results indicate a temperature dependence of infectivity. Inhibition of the viral infectivity was observed at three different temperatures 37 ̊C, 40 ̊C and 42 ̊C. The inhibition appears to be linked to the appearance of large agglutinates that appear to reduce the infectivity of RSV. Such a study found that viral neutralization is dependent on a temperature-dependent agglutination reaction. The causality of agglutination formation requires further investigation in order to conclusively confirm the immunological component(s) of this reaction, and how temperature is contributing to this reaction.
Many of the processes that regulate male fertility are intricate and subfertility strikes hard against both couples trying to concieve and cattle farmers where the fertility of males used for artificial insemination is the single most important factor viewed in relative economic terms. A recently published study by Fagerlind and collegues showed that seven microRNA sequences differed significantly in expression between bulls with moderate and high fertility.
In order to study the effect these might have on fertility, a model organism is needed. The present study aim to assess if the fruit fly Drosophila Melanogaster could be used as such. It have served science for over a century, is cheap to grow and has a short generation time. A secondary objective of the present study was to elucidate if any of the observed microRNAs was expressed at a higher concentration at a specific life stage of the fly. Samples from eggs, the three larval stages and adult males and females were collected. Subsequently, after conversion into cDNA with primers for miR-34, miR-1249, miR-148b and miR-15b, the microRNA concentrations were evaluated with Quantitative Real-Time PCR. Three out of four microRNA sequenses showed expression in the fly and for one of them, miR-34, a marked difference in expression between the developmental stages could be observed, but not confirmed statistically due to the low number of samples. This result enables further studies on these sequences and their role in male fertility.
Sepsis is an irregular systemic response to an infection, in which a pathogen or some of its component(s)reaches the bloodstream of the host or sterile tissue, triggering a disproportionate immune reaction. The first three hours are critical in the diagnosis of sepsis, in order to ensure an effective treatment with less impact on the patient. Culture-dependent diagnosis is the present standard procedure which can take up to several days. Metagenomics Next Generation Sequencing (mNGS) is a culture independent diagnostics method which could be used to identify the presence of pathogens from DNA extracted from human whole blood enabling a more effective treatment procedure of infected patients. The aim of this research was to utilize the sequencing data obtained with the MinION Nanopore sequencing device, in order to systematize its use as a tool for the early detection of sepsis; furthermore, determine if this technology is effective to use on DNA extracted from whole blood. The main research question of this thesis focused on whether the MinION Nanopore sequencing is a reliable tool for the early detection of sepsis. Whole blood samples from healthy donors was spiked with bacteria and DNA was extracted and sequenced with MinION device. The sequencing results were interpreted with the MinKNOW v2.0 software, through the application What’s In My Pot (WIMP). Also, the web tool PATRIC 3.6.12. and KRAKEN2 algorithm. The reads from the taxonomic family where the bacteria belong to was analyzed, presuming the bacterial DNA was present in the DNA extracted but the genus was not detected. According to the KRAKEN2 and WIMP analysis, the bacteria used to spike the whole blood samples was detected up to the taxonomic family level. Thus, confirming the presence of the spiked bacteria in the purified DNA samples.
Sepsis is a preventable yet life threatening condition, resulting from body response to infection. Time is crucial in sepsis diagnosis since deterioration in patients’ health can occur rapidly. Blood culturing is the gold standard for diagnosis, along with clinical assessment. The discovery of miRNA in biofluids as a biomarker, founded the way for extensive research on its capabilities. MiRNA showed promises in diagnosing, assessing outcome and reporting sepsis progression. Since being delicate to handle while present in biofluid, the need was uttermost to find an effective way for miRNA isolation and detection, to facilitate developing multi-marker panel that help diagnosing sepsis, more efficiently than blood culturing. The current study aimed at using manual and robotic (QIAcube) methods, with MiRNeasy Serum/Plasma Advanced (Qiagen) as kit and protocol, to extract miRNA from human plasma samples. Plasma was either spiked with synthetic miR-223 to act as a positive control, or non-spiked. Once extraction was done, quality-quantity assessment was conducted using Qubit and Nanodrop. Two-tailed RT-qPCR (TATAA Biocenter) was used for miRNA quantification. QIAcube showed better results in quantity, hands-on and turn-around time compared to manual extraction, while better purity was scored for the manual method. While amplification appeared in all spiked samples, absolute quantification detected miRNA in some of the non-spiked samples. The study verified using the extraction kit with 100 μl of plasma is effective for miRNA extraction. Although faced with difficulties, absolute quantification using two-tailed RT-qPCR demonstrates its success in detecting lowly expressed miRNA. Future studies are needed for more optimized verification.
Pharmaceuticals and their metabolites are present in the environment, with levels expected to rise in the future. The effects on off-target organisms are still mostly unknown and of increasing concern, as are combination cocktail effects. Microalgae are of importance to ecosystems due to being primary producers and responsible for approximately half the global photosynthetic activity. Additionally, in some algal species a detoxification ability has been uncovered, linked to the enzymatic metabolism of cytochrome P450. However, the molecular mechanisms involved in the xenobiotic metabolism of algae are poorly studied, especially regarding cocktail effects. The aim of this study was to investigate the cocktail effect on growth inhibition and gene expression in Raphidocelis subcapitata, from environmentally relevant levels of pharmaceuticals. This was achieved by performing a growth inhibition test, according to the OECD 201 guidelines. Four of the most prescribed pharmaceuticals in Sweden, paracetamol, metoprolol, omeprazole and diclofenac were tested as a cocktail and compared to metoprolol exposure. Test concentrations ranged from naturally occurring levels up to that of a tenfold. In addition to optimization of RNA extraction, a transcriptomic analysis was performed on cocktail treated groups to evaluate expression of cytochrome P450. For all concentrations, metoprolol exposure resulted in algal growth inhibition, while cocktail exposure surprisingly promoted growth. Here the lowest and medium concentrations tested yielded a downregulation of the gene, while the highest concentration elicited an upregulation. The prospect of predicting cocktail toxicity is discussed, likewise the possibility of drug-drug interactions in microalgae due to cocktail exposure.
Via the membrane trafficking system, proteins and macromolecules dispense in different pathways into cells. Moreover, it transports proteins inside and outside of cells. AP3 and LAMP1 are crucial in the biogenesis of lysosomes and sorting the cargo proteins in the trafficking membrane. The result of immunoprecipitation and mass spectrometry proteomics that Paul Manna carried out showed that the RABL6 protein has a relation to AP3. RABL6 has been known as a proto-oncogene previously, and this is the first time that RABL6 may have a function in trafficking membrane. This study aimed to find the association of RABL6 with AP3 and the function of RABL6 via the distribution of LAMP1. HeLa cell line was transfected by a plasmid containing RABL6/GFP. The cells transfected by RABL6/GFP plasmids and wild-type cells were fixed and prepared for immunostaining. After immunostaining, confocal microscopy was used to show the interaction of AP3 and RABL6 and the distribution of LAMP1. In addition, HeLa cells were knockout for RABL6 to show the function of AP3 in RABL6 knockout cells. Optimizing immunostaining, the ratio of 1:100 for RABL6 antibody with PFA fixing shows the best result. The colocalization of RABL6 and AP3 was calculated in cells transfected by RABL6/GFP plasmid and wild-type cells and interpreted the association to each other. The knockout RABL6 cells were unsuccessful, although some differences were observed in the size of the cells. The distribution of LAMP1 in wild-type cells and RABL6 cells transfected by RABL6/GFP plasmid displayed statistical differences. In overexpressed cells, LAMP1 showed more intensity. In conclusion, the RABL6 is involved in trafficking membrane with AP3 and LAMP1.
Pharmaceuticals are an important class of pollutants in aquatic ecosystems. Detected concentration are typically in the range 1 ng/L – 1 μg/L. Traditional wastewater treatment does not provide a complete removal of these contaminants; hence, they may have a negative impact on the environment. In addition, microalgae are an ecologically-meaningful target group of species for bioindication purposes as well as primary production and oxygen supply. The present work aimed to investigate the effect of Ibuprofen on the green alga Pseudokirchneriella subcapitata. Algal cultures were exposed to five different concentrations of the drug (5, 15, 45, 135, 405 mg/L) for four days. Absorbance measured at 680 nm was determined every day and obtained data were transformed into cell concentration (cells/mL) by a previously prepared calibration curve. Specific growth rate, generation time, percent inhibition and effective concentration were calculated. Moreover, one way ANOVA with Tukey’s test were applied to observe differences between groups and time periods. Based on this study, all the cultures treated with Ibuprofen had a growth inhibition as well as presenting a lag phase. Increasing the Non-Steroidal Anti-Inflammatory drug (NSAID) concentration reduced the growth rate and consequently, increased the percent inhibition in a concentration-dependent manner. According to this report, new research should be focused on the development of hybrid systems for degradation and removal of pharmaceuticals. NSAID pollution may lead to a reduction in the diversity and number of functional groups of eukaryotic algae. Finally, more research should be devoted to the toxicity of drugs in a variety of test organisms and development of reliable methods for toxicity test at low and chronic exposures to achieve more realistic conclusions.
Trimethylaminuria is an autosomal recessive disorder characterized by a decreased oxidation capacity of trimethylamine to trimethylamine-N-oxide in the liver. The condition is diagnosed by estimating concentrations of trimethylamine and trimethylamine-N-oxide in human urine and then evaluating their respective creatinine ratios and the oxidation efficiency percentage. Values previously retrieved with liquid chromatography-tandem mass spectrometry with electrospray ionization but not the novel ionization interface UniSpray. Thus, this project aims to initiate the development of a liquid chromatography-tandem mass spectrometry diagnostic method for trimethylaminuria with UniSpray ionization. The analytes were extracted from urine with a liquid-liquid extraction method and separated with hydrophilic interaction liquid chromatography using an isocratic profile with 5 mM of ammonium formate in water and methanol and multiple reaction monitoring. Overall, the mean coefficient of variation and recovery percentage from trimethylamine spiked urine samples were lower than expected, whereas the intra-precision for trimethylamine-N-oxide was acceptable. Three urine samples had estimated oxidation percentages, but only one had derived comparable creatinine ratios to an external laboratory. Due to the inadequacy of comparative data and the precision and recovery percentage deviations, the results presented in this report need cautious interpretation. Future development of the method could include manual tuning, reconsidering the calibration curve and reference values, and comparisons to the extraction method. Although there are apparent discrepancies in the precision and reliability of the derived trimethylamine and trimethylamineN-oxide concentrations, the initial steps in the pursuit of a liquid chromatography-tandem mass spectrometry diagnostic method for trimethylaminuria provide a practical foundation to continue the development.
Infectious diseases caused by human papillomavirus (HPV) are among the most common sexually transmitted diseases in the world. Currently, all countries of the WHO Eastern Mediterranean Region (EMRO) except the United Arab Emirates and Libya do not have a national vaccination program including the HPV vaccine. Cervical cancer risk can be reduced through the use of prophylactic HPV vaccines. Hence, the aim of this study was to examine the severity of HPV-16/18 infection in cervical cancer through a systematic review and to evaluate the effectiveness of vaccines against HPV-16/18 variants to prevent cervical cancer via a meta-analysis. Both the systematic review and meta-analysis contain nine relevant studies with 66154 and 78308 cervical cancer participants respectively. Statistical analyses were performed using pooled odds ratios (OR) with 95% confidence intervals (95% CI). Publication bias was examined using the funnel plot graph. The findings stated that overall 70% of cervical cancer was attributed to either HPV 16 or HPV 18. Heterogeneity for this meta-analysis was found to be I2= 80% with a p-value<0.01 and overall OR (odds ratio) was 0.09 (95% CI= 0.04-0.20) for the random effect model. The lower odds ratio (less than 1) indicated fewer occurrences of cervical cancer in the HPV 16/18 vaccinated group than in the unvaccinated individuals. The overall vaccination efficiency was found to be 91% from the odds ratio ((1-0.09)x100=91). Thus, the present findings support that a prophylactic vaccine against HPV16/18 prevents the severity of HPV-associated cervical cancer.
Sepsis is one of the leading causes of patient mortality and is caused by an inflammatory response to infection. Identifying sepsis at an early stage can improve treatment methods and have a major positive impact on health conditions. The aim of this study was to compare two methods of total RNA extraction, manual vs robotic.
The comparison was done with hands-on time (HOT), turn-around time (TAT) and absolute quantification. The main focus was to extract total RNA including miRNA, miR-Seps3, a candidate for future sepsis diagnosis. The method utilized 100 ul of healthy human blood plasma for manual and robotic extraction method as input volume followed by two-tailed RT-qPCR.
No significant difference was found in terms of TAT for manual and robotic extraction while a significant difference was found in terms of HOT between two methods of extraction. No significant difference was observed between spiked manual extractions and spiked robotic extraction on the basis of absolute quantification. Amplification in No-Template control (NTC) compromised the reliability of the qPCR-primers by indicating potential contamination or nonspecific amplification. The results from non-spiked extractions were untrustworthy due to NTC amplification. Both methods were effective in extracting total RNA and no significant difference was observed between two methods. Which concludes that each method can be chosen corresponding to the experimental requirements. qPCR assay requires optimization to deal with the issue of NTC amplification.
Sepsis is a potentially fatal emergency medical condition that reflects the presence of the body’s systematic inflammation. Around 260 biomarkers have been determined to sepsis. The gold standard of blood culturing has remained the best technique for finding sepsis etiology up to this data. However, some vital drawbacks, such as laboriousness, have encouraged a global attempt to find new techniques. This study aimed to optimize a method from which earlier sepsis diagnosis compared to the gold standard could be resulted. DNA was extracted from both spiked and non-spiked whole blood samples. After quality control of the DNA elutions, library preparation and nanopore sequencing using the MinION device were carried out. Basecalling and demultiplexing were done using Guppy GPU and barcoded FASTQ-files were analyzed using What’s-In-My-Pot and Kraken2 taxonomy classification programs. Three different DNA extraction methods were compared from which the second and third methods opted as the optimized methods. Although spiked species were not found in the used taxonomy classification databases, their respective families were spotted. Kraken2 program indicated a relationship between the read percentage of the families and the spiking level of initial blood samples. On the other hand, What’s-In-My-Pot did not show such a trend and only the highest spiking concentration had indicated the families within the reads. A possible justification for not finding the species within the reads is the patchiness of the two databases. Despite the failure in determining the species within FASTQ-files, the whole experiment has gathered valuable experiences for future studies.
Sepsis is a life-threatening organ disfunction, which is caused by a dysfunctional immuneresponse and develops when an infection overwhelms the body’s defense mechanism and causesand uncontrolled inflammatory response. Biomarkers have a great impact on helping diagnosisand treatments of sepsis. The biomarkers, like miRNA, are needed for both more accurate andquicker diagnosis of sepsis in patients. The future diagnostics are looking at other types ofbiomarkers, e.g. miRNA, but low amounts of miRNA are present in biofluids and make itchallenging to quantify. A new methodology is needed which is both accurate and does notrequire a lot of fluid. The aim of this project was to identify which kit of two kits and which oftwo volumes of plasma would lead to the highest concentration of miRNA and highest quality ofmiRNA extracted. This was quantified by using two different volumes, 100 μl and 200 μl, andextracting the two volumes with both exoRNeasy Serum/Plasma midi kit (Qiagen) and TotalRNA Purification kit (Norgen). There was no statistical difference between median miRNAconcentrations between the two volumes within the Qiagen kit. However, the mean miRNAconcentration (0.833 ng/μl) obtained from the Norgen kit (100 μl plasma starting volume) wasstatistically higher than the mean miRNA concentration (0.570 ng/μl) obtained from the samekit with 200 μl, p = 0.033. The optimal kit and volume of this study is the Norgen kit with 100 μl.Further studies are needed to verify these results.
Each hour´s delay in administering antibiotics has been shown to result in a 9% increase in the odds of mortality in sepsis cases. It is thus evident that the development of a diagnostic method that ensures an early time to diagnosis of sepsis is essential. MiRNAs have shown promise with regards to diagnostic capabilities concerning sepsis, with differential expression of circulatory miRNAs seen during various diseased states. MiRNA can be quantified directly from a blood plasma sample, greatly decreasing the time to diagnosis, as the requirement for culturing is eliminated. Quantification of miRNA by means of qPCR has proven rather challenging, due to their short length. A solution might be two-tailed RT-qPCR, a method which utilizes a two-tailed RT primer. The aim of the project was to optimize the extraction and quantification of miRNAs from minimal amounts of human blood plasma samples, as to create a standardized and reproduceable method for measuring biomarker miRNAs within human blood plasma. In this study, a significant difference between manual and semi-automated extraction of miRNA from plasma with regards to A260/A280 ratios (p = 0.00) was observed. It was also found that a correlation exists between A260/A280 ratios and miR-seps6 quantified, using the two-tailed RT-qPCR method. This method has shown to be effective at amplifying circulating miR-seps6 arising from 100 µL of human blood plasma. A linear standard curve, constructed from synthetic miR-seps 6 produced optimal amplification efficiencies, and the melt curve indicated a single product, which correlates with good specificity. As successful detection and amplification of miR-seps 6 had been achieved during this study, the next phase of the project can be initiated, where it will be attempted to detect miR-seps 6 from plasma stored in a human biological material bank (biobank).
Inflammation is the body's natural defense reaction and is known since ancient times. The inflammation is divided into two main phases, acute and chronic inflammation dependent on the process and cellular mechanisms of the inflammation. Inflammation has become to be an important field in research by biomedical research where it is included in many cellular processes thus being phagocytosis, chemotaxis, mitosis, and cell differentiation. Inflammasomes are pro-inflammatory intracellular multimeric protein complexes that introduce the activation of pro-inflammatory cytokines, such as interleukin-1β and interleukin-18, upon trigger by PAMPs and DAMPs signals. The most studied inflammasome is the NLRP3 inflammasome that is activated by various trigger signals, like DAMPs, ATP, uric acid crystals and amyloid-β fibrils. GSK-3β is a kinase that controls various cellular processes, such as inflammation by regulating the activity of abundant transcription factors that are valuable for cytokine production. The aim of this thesis project was to investigate if GSK-3 Inhibitor IV, SB-216763, in a concentration-dependent manner had an effect on production of IL-1β in LPS- and Nigericin-stimulated THP-1 ASC-GFP-macrophages. In addition to the gene expression analysis of IL-1β, the amount of secreted IL-1β, and the possible correlation between treated THP-1 cells with and without GSK-3 inhibitor evaluated. The gene expression analysis was performed by using qPCR and the amount of secreted IL-1β was done using sandwich enzyme-linked immunosorbent assay. The results from this study showed no significant difference in gene expression and amount secreted of IL-1β in THP-1 cells when treated with the GSK-3 Inhibitor IV, SB-216763.
When exposed to pathogenic stress, cellular processes and survival are dependent on cytoskeletal proteins for structure and organisation of the cell to adapt and maintain homeostasis during inflammation. Vimentin is type III cytoskeletal protein, with an extensive cytoplasmic meshwork, across the cell and regulate the cell structure and cellular space and expressed strongly under tumorigenic events. GSK-3, a regulatory component of inflammation expressed in abundance of cell together with reactive oxygen species (ROS), a group of key complex signalling molecules that are oxygen metabolites which are partially reduced, with robust oxidising abilities, are believed to influence inflammasome formation and specifically vimentin expression upon inflammation. This project investigated the potential modulation vimentin mRNA expression utilising the two signal NLRP3 inflammasome activation theory, by inhibiting GSK-3 and ROS in signal I and or signal II in LPS and nigericin stimulated THP-1 cells, compared to non-inhibited LPS and nigericin THP-1 cells. Inhibition of GSK-3 in signal II downregulated vimentin expression, reflecting repressed phosphorylation of GSK-3 hence also the components required for vimentin; whilst upregulation of vimentin in signal I, reflects possible alternative pathways phosphorylating vimentin components. Overall upregulation of vimentin upon inhibiting ROS in both signal I and II, further proved that inflammasome activation is independent of ROS in the priming step. More research is required integrating vimentin activity and either GSK-3 or ROS, as the potential of these prominent inflammatory markers and their major regulatory presence across an abundance of cell may contribute to the future of drug development for inflammatory diseases.
Inflammation is a part of the innate immune system. It protects the body against foreign invaders such as bacteria and viruses. Inflammation helps to restore the body by removing harmful stimuli and starting the healing process. Inflammation is produced in response to damage-associated molecular patterns (DAMPS) or pathogen-associated molecular patterns (PAMPS). Glycogen synthase kinase 3 (GSK-3) is a key regulator of a variety of pathways, making it a promising therapeutic target. Therefore, this experiment aims to see how inhibiting GSK-3 affects the generation of IL-8 in THP-1 ASC GFP macrophage cells. For this study qPCR was used to measure IL-8 expression, while ELISA was used for protein secretion. An ANOVA test was utilized for the statistical analysis. Obtained results from this study showed that there is a significant difference between stimulated cells with LPS and nigericin against unstimulated samples both in protein and mRNA levels. When it comes to the stimulated cells against inhibited cells, the ANOVA test showed there is no significant difference between the samples both in protein and mRNA levels. This might suggest that GSK-3 does not influence the development of inflammasomes in THP-1 macrophage cells. Another possible reason is that other pathways such as the MAPK and JAK-STAT may mask potential inhibitory effects on the NLRP3 inflammasome pathway by producing even more IL-8, which interfered with qPCR and ELISA results. In conclusion, additional research is needed to confirm the involvement of GSK-3 in NLRP3 inflammation.
In recent decades, the study of the gut microbiota has gained attention as a crucial element in human health and the onset of diseases. Technological advancements, such as high-throughput sequencing, have enabled a deeper study of intestinal microbiota composition and functions. Among the environmental factors modulating the gut microbial configuration, the impact of nutrients is of particular significance. Nonetheless, research on dietary lipids, particularly omega-3 fatty acids, remains underexplored. This study aimed to contribute to the metagenomic analysis of the 16 subunit of the ribosomal RNA gene in faecal samples. A nutritional intervention was carried out focused on fish consumption rich in omega-3. Successful DNA extraction from faecal samples yielded notable, with defined genomic DNA bands in the gel electrophoresis and an adequate concentration. Polymerase chain reactions, essential for library preparation, revealed distinct products in the gel electrophoresis, confirming the successful amplification of the V3-V4 16 ribosomal RNA region with proper adapter addition for DNA sequencing. Comprehensive quality control assessment of sequenced samples confirmed the effectiveness of the refined laboratory procedures. The steps undertaken in this project were critical for subsequent bioinformatics and biostatistics analyses, providing insights into the impact of omega-3-rich fish consumption on the relative abundance and diversity of studied microbial taxa. Integrating metagenomics analysis with other omics and non-omics disciplines, alongside standardising methods across different laboratories, will pave the way for the future application and democratisation of precision nutrition to enhance overall wellbeing.
Sclerotinia sclerotiorum is a notorious phytopathogenic fungus and is the causal agent of the disease Sclerotinia stem rot (SSR) of rapeseed (Brassica napus). SSR is one of the main diseases affecting the yield and oil quality of rapeseed crops worldwide. This disease is very hard to predict and control due to all the different factors that are involved in the development of the disease. Successful disease management depends on accurate identification and early detection of plant pathogens. qPCR is a fast, specific, reproducible, and reliable technique for plant pathogen diagnostics. However, one limitation of qPCR is that it is unsuitable to identify and study unknown species, other than those intended, making the detection of unknown pathogens very difficult. An alternative solution is to apply single molecule sequencing, which can provide information at species and strain level. In this study, a total sample of 15 rapeseed leaves coming from three different fields in Sweden with known incidence of SSR disease were analyzed using qPCR and other 15 leaves, coming from the same fields, were analyzed using Oxford Nanopore sequencing to attempt to identify pathogens, S. sclerotiorum being the main target. S. sclerotiorum was not identified with none of the previous mentioned techniques in any of the samples. Perhaps, S. sclerotiorum was not present on the samples at the time of the collection, due to the unfavorable weather conditions for the release of the spores. However, some issues were present during the development of the qPCR assays that also could have affected the results. Regarding Oxford Nanopore sequencing, other fungal species were identified instead.
Chronic kidney disease greatly raises cardiovascular disease risk. Heart disease and death risk grow proportionately with renal disease progression. Investigate the link between cardiovascular disease prevalence and chronic renal disease severity and mortality using meta-analysis. In this study, 155 publications were found after searching several databases (including PubMed and Google Scholar). 48 studies that matched the inclusion criteria were included in the literature review, however, only 20 were included in the meta-analysis. 17101 people had CKD, while 8883 had CVD or non-CVD. Using the R programming language, a meta-analysis was performed to get a pooled impact of the influence of CVD on the severity of CKD (odds ratio OR), and a funnel plot was also generated to check for publication bias. The outcomes of the meta-analysis indicate that cardiovascular disease has a moderate impact on the severity of chronic kidney disease (OR=2.28, 95% CI, 1.90-2.73). All data will give essential insights into the epidemiology of the cardiovascular disease in chronic kidney disease (CKD), disclose the influence of individual risk variables on bad outcomes, and serve as the platform for future interventional research. Further investigation of the particular (non-traditional) risk factors associated with the renal illness that contribute to accelerated atherosclerosis in this population is necessary to improve the efficacy of cardiovascular treatments for patients with CKD. The purpose of this research is to determine whether and how these variables affect the development of CKD.
The present thesis will provide an overview of how the four humor styles, affiliative, self-enhancing, aggressive, and self-defeating humor, are connected to different brain areas. The thesis will also include an overview of how humor in general, and especially three factors of humor including, processing, appreciation, and comprehension is connected to different brain areas. The present study found a connection between these three factors of humor and activation in the prefrontal cortex (PFC) and inferior frontal gyrus (IFG). The four humor styles were all connected to activity in the midbrain and nucleus accumbens (NAc), though they were found to differ in other parts of the brain. Affiliative humor and self-enhancing humor are humor styles found to share activation of similar brain areas, whereas self-enhancing and aggressive humor was found to the least extent share activation of the same brain areas. No neural differences in relation to the four humor styles have been found between men and woman, or between cultures.
The in vitro cell culture models of human pluripotent stem cells (hPSC)-derived cardiomyocytes (CMs) have gained a predominant value in the field of drug discovery and is considered an attractive tool for cardiovascular disease modellings. However, despite several reports of different protocols for the hPSC-differentiation into CMs, the development of an efficient, controlled and reproducible 3D differentiation remains challenging. The main aim of this research study was to understand the changes in the gene expression as an impact of spatial orientation ofhPSC-derived CMs in 2D(two-dimensional) and 3D(three-dimensional) culture conditions and to identify the topologically important Hub and Hub-Bottleneck proteins using centrality measures to gain new knowledge for standardizing the pre-clinical models for the regeneration of CMs. The above-mentioned aim was achieved through an extensive bioinformatic analysis on the list of differentially expressed genes (DEGs) identified from RNA-sequencing (RNA-Seq). Functional annotation analysis of the DEGs from both 2D and 3D was performed using Cytoscape plug-in ClueGO. Followed by the topological analysis of the protein-protein interaction network (PPIN) using two centrality parameters; Degree and Betweeness in Cytoscape plug-in CenTiScaPe. The results obtained revealed that compared to 2D, DEGs in 3D are primarily associated with cell signalling suggesting the interaction between cells as an impact of the 3D microenvironment and topological analysis revealed 32 and 39 proteins as Hub and Hub-Bottleneck proteins, respectively in 3D indicating the possibility of utilizing those identified genes and their corresponding proteins as cardiac disease biomarkers in future by further research.
Uropathogenic Escherichia coli is a leading cause of urinary tract infections and bacterial prostatitis, the most common UTI complication in men. The initial stages of a successful infection involve bacterial adhesion to host cells through specialized adhesins. FimH, a protein located at the tip of type 1 pili, plays a crucial role as the main mediator for UPEC binding to bladder cells. While the host partners of FimH in the bladder are well-established, the interactions between FimH and prostate cells remain elusive. Consequently, the overarching goal is to enhance comprehension of the initial steps in prostate infection by investigating the interaction of FimH with prostate proteins. To achieve this, a recombinant FimH was constructed and expressed in an inducible expression vector, and an immunofluorescence staining assay was performed which demonstrated distinctive binding patterns in prostate cells compared to the bladder cell line. A Far Western overlay assay, revealed six distinct protein bands in human prostate cells and two in mouse prostate cells, indicating different potential protein partners. These interactions were examined under native conditions by establishing and optimizing a co-immunoprecipitation assay with cell proteins derived from both human and mouse prostates, with the 5637 cell line serving as a positive control. In summary, this study reveals striking differences between FimH binding to prostate and bladder cells, emphasizing the importance of FimH in adhesion and the need for further exploration of FimH interaction with prostate cells.
Cancer is still one amongst the leading death causes in the present world. Prostate, rectal, and gynecological cancers are prevalent types of cancers in the pelvic region. Approximately one million patients undergo pelvic radiotherapy worldwide every year. Unfortunately, the surrounding tissues can suffer from unintended radiation damage, leading to symptoms collectively known as pelvic radiation disease. In the project BIOSURV (BIOpsies in cancer SURVivors), 24 pelvic cancer survivors and four non-irradiated controls were previously biopsied in colorectal areas that had received no, high, or low dose of radiation. Even after 20 years, the biopsy samples that received high doses of radiation exhibited low-grade inflammation. In the current study, we hypothesize that some of the symptoms are a result of the inflammation, and that there is an overlap in the molecular pathways of Inflammatory Bowel Disease (IBD) and Pelvic Radiation Disease (PRD), which could potentially be exploited for therapeutic purposes in PRD patients. One of the common pathways identified between the PRD and IBD data sets is the TNF-alpha signaling pathway. TNF-α blockers are known to be used to treat IBD. In this thesis, the major laboratory work involved validating TNF-alpha by staining FFPE biopsy sections using immunohistochemistry (IHC) techniques to detect TNF-α at the protein level and using RNA scope® ISH to validate TNF-α at the mRNA level. Our findings from visual inspection of the graphs from RNA scope data indicates an increased expression of TNF-alpha mRNA in irradiated biopsies compared to non-irradiated biopsies in PRD.
Hepatitis C viral infection is a globally widespread blood-borne disease affecting the liver, causing cirrhosis and hepatocellular carcinoma. This type of liver cancer is mainly caused by chronic Hepatitis C and Hepatitis B. Specific detection with the following correct treatment is crucial to reduce the overall burden of the disease. This work focused on investigating whether the padlock probes and rolling circle amplification can detect Hepatitis C, determining the limit of detection, and if any blood components would inhibit the reactions. All oligonucleotides were tested for ligation functionality in 10% TBE-Ureal gel electrophoresis and used with rolling circle amplification and phi29 polymerase to determine if eye read-out was possible. The lowest concentration of detection was found to be 10 pM. To avoid inhibition in blood plasma, samples were pre-treated at 95 ˚C for five minutes. Eye read-out was possible after amplification, with 30% plasma at the highest and 5% plasma at the lowest in samples. In conclusion, this novel assay using padlock probes, a detection oligonucleotide, and rolling circle amplification holds promise in developing a simplified new detection technique for the diagnostics of Hepatitis C.
Apis mellifera is the most common honeybee species in the world. In recent years, there have been several reports of declines in wild, and domesticated populations. Central to honeybee health are the mutualistic relationships they have with their intestinal microbiome. The Lactobacillus species living in their digestive tract assist with nutrient digestion and pathogen protection. The aim of this study was to investigate which Lactobacillus spp. were present in the intestines of subspecies A. mellifera mellifera, and A. mellifera ligustica, and if they were able to inhibit growth of the pathogen Melissococcus plutonius. The 16S rRNA gene was amplified from gDNA extracted from two complete intestines per sample, in a PCR reaction with barcoded primers. Fragments were then analyzed with nanopore sequencing. In vitro assays of catalase-treated cell-free supernatants from Lactobacillus cultures were set up against living cultures of M. plutonius on KBHI agar plates and liquid broth media, in two experiments. The same seven Lactobacillus species previously found in honeybees were confirmed to be present in the bees of this study. The ratio of species was different between individual samples, which supports earlier findings suggesting the variation is dependent on factors such as individual health, food source, and sampling season. Liquid broth in vitro assay resulted in no inhibition of early growth phase, while the last cell count measure at24 h, recorded statistically significant difference in mean values between A. apinorum and negative control (p<0.001). Further research is needed to investigate optimum conditions for inhibition.
Honeybees are an important pollinator around the world and is used in agriculture, the bees also make products like honey, royal jelly and wax these products make up a market worth 15 billion dollars in the US. Honeybees are on the decline and a problem might be damage to the microbiota in the gut. The MinION is a next generation sequencing tool created by nanopore technologies; it’s often compared to Illumina sequencing. MinION is believed to be relatively inaccurate compared to other sequencing tools, MinION utilizes nanopores to measure electrical signals from the different nucleotides. The aim of this study was to analyze the MinIONs accuracy in identifying bacteria, and analyzing if the MinION is a tool appropriate to use in understanding the gut microbiota of bees. Bacteria was grown on LB plates and the genetic DNA was isolated and amplified using PCR. The 16S gene was sequenced using MinION and analyzed using the Epi2me desktop agent. The large number of analyzing shows that there are about 40% of the materials that never get approved by Epi2me. When identifying bacteria the program showed to be accurate in most cases even the contaminated samples were identified with high accuracy. However, Epi2me had some difficulties correctly identifying specific closely related bacteria. In conclusion the identifying bacterial species using MinION together with the Epi2me software was concluded to be accurate in the majority of cases even though a few blind spots were experienced.
Sepsis is a serious condition characterized by a strong systematic inflammatory reaction that takes place because the immune system is not responding properly to an infection. This study focused on sepsis caused by bacterial infections. The most important factor in the diagnosis of sepsis is time and the current golden standard of blood culturing used can take several days and leaves room for improvement. This study aimed to evaluate the ability of nanopore sequencing of DNA extracted from whole human blood on the MinION device, to evaluate its efficiency and potential as a future tool for early sepsis diagnosis. Furthermore, the study aimed to examine if it is feasible to identify genes that encode antimicrobial resistance in the sequenced DNA. Three different DNA extraction kits were used to extract DNA from whole human blood spiked with microbial DNA. The obtained data from nanopore sequencing was analyzed using EPI2ME, and the FASTQ-files were further analyzed with What’s-In-My-Pot and BV-BRC taxonomic classification and metagenomic read mapping. Two of the DNA extraction kits yielded DNA extractions with higher quality, whereas DNA extractions performed with the third kit led to challenges with purity. The results from nanopore sequencing indicated a difference in reads depending on both the two flow cells as well as the DNA extraction kit used. The DNA from gram-positive bacterial species added to the whole blood was the focus of this project, and using taxonomic classification and metagenomic read mapping all DNA included in the microbial spike was detected, and genes that code for antimicrobial resistance were identified. Differences in the number of detected bacterial species could be seen depending on the algorithm used. However, the time aspect remains an issue. The results indicated that this technique could possibly be used in future studies, but there is a need for optimization.
Hepatocellular carcinoma is the most common type of primary liver cancer. Worldwide, metabolic dysfunction-associated steatotic liver disease and metabolic dysfunction-associated steatohepatitis are becoming the most common triggers of liver disease and are now major contributors to hepatocellular carcinoma development. Metabolic dysfunction-associated steatotic liver disease triggers hepatic lipid accumulation, inducing oxidative stress, inflammatory activation, and fibrosis, culminating in hepatocellular damage and proliferation, key drivers of metabolic dysfunction-associated steatohepatitis associated hepatocellular carcinoma. Translational studies in human cell lines and mouse models have identified serine/threonine kinase (STK)25 in regulating liver lipid homeostasis and the progression of metabolic dysfunction-associated steatotic liver disease/metabolic dysfunction-associated steatohepatitis. In this study, a nonclinical proof-of-principle investigation was performed to assess the benefits of Stk25- antisense oligonucleotides and N-acetylgalactosamine Stk25 antisense oligonucleotides, which were either designed to be hepatocyte-specific or broadly distributed to peripheral organs, in blocking the initiation and progression of metabolic dysfunction-associated steatohepatitis related hepatocellular carcinoma in mice. Results revealed reduced hepatic messenger RNA expression of genes controlling apoptosis, fibrosis, proliferation, oxidative/endoplasmic reticulum stress, and inflammation in mice treated with both Stk25- antisense oligonucleotides and N-acetylgalactosamine Stk25 antisense oligonucleotides, alongside decreased immunolabeling for proliferative and hepatocellular carcinoma diagnostic markers. Additionally, a 90% reduction in STK25 abundance was observed in Stk25- antisense oligonucleotides and N-acetylgalactosamine Stk25 antisense oligonucleotides-dosed mice. While STK25 antagonism effectively suppressed hepatocellular carcinoma development, it did not entirely prevent its occurrence. These findings suggest that targeting STK25 presents a promising therapeutic strategy for metabolic dysfunction-associated steatohepatitis-driven hepatocellular carcinoma.
Sepsis is a very dangerous and life-threatening disease that develops when the body’s reaction to infection causes damage to the body’s tissues and organs. It is difficult to diagnose it and it develops fast leading to a high mortality rate. Current methods rely on blood culturing and multiple biomarkers, such as C-reactive protein and procalcitonin, that take too long to produce results. A possible solution to this problem lies in specific biomarkers such as microRNAs, which are small non-coding single stranded RNA molecules that contain around 22 nucleotides and have a big role in regulating gene expression. Being specific biomarkers for particular disease makes microRNAs promising biomarkers for sepsis. The aim of the project was to optimize the process from extraction to quantification of microRNAs using the miRNeasy Serum/Plasma Advanced Kit-Qiagen Kit (manual) and to see if the Two-tailed RT-qPCR (TATAA Biocenter) technique could quantify the samples. Blood plasma from healthy donors was used for microRNA extractions and was separated into two categories- spiked-in samples and non-spiked samples. Spiked-in samples were spiked with a synthetic microRNA- miR-223 and served as a positive control. All samples were quantified using the absolute quantification and the Two-tailed RT-qPCR method (TATAA Biocenter). Quantification was successful for all samples showing that the method was optimized, parameters for optimization were within the wanted range, and quantifiable. More research is needed, however, the method has potential in becoming a simple and quick novel tool in diagnosing sepsis in the early stages and thus saving lives.
Inflammasomes are large multiprotein complexes that are part of the innate immune response and assemble in response to microbial microorganisms. The function of the innate immune response is dependent upon the detection of pathogen-associated molecular patterns and Damage associated-molecular patterns by germline-encoded pattern recognition receptors. The NLRP3 is the most characterized member of the NOD-like receptors that are also capable of inflammasome activation, the activation leads to the cleavage of caspase-1 into its active form. Dysregulation of inflammasome activation has been linked to several autoimmune diseases such as type1 and 2 diabetes, and the mechanisms regulating the activation of the NLRP3 activation are not fully understood yet. This study aims to study the time dependence of processes related to the activated NLRP3 inflammasome. THP-1 cells were cultured and collected at set time points and the first objective was to perform a western blot analysis and the second was to do an RT-qPCR. The obtained results from the qPCR indicated that they were multiple products in the samples and therefore unspecific amplification during the run. Troubleshooting tests such as melting curve analysis was used to determine the melt profile of the amplicon and gel electrophoresis was used to determine if they were multiple products in the samples. The results from the western blot analysis showed unexpected bands and due to time constraints, further optimization for the antibodies could not be performed.
FBXL12 (UniProt_ID: Q9NXK8) is a largely uncharacterized F-box protein and bioinformatic analysis has revealed that its expression levels correlate with those of Cyclin-E and together play an unfavourable role in patient prognosis in breast cancer. This marks FBXL12 as a potential target for cancer therapy. In an MS-based screen, FANCD2 (Fanconi anaemia complementation group D2) was discovered as an interacting partner of FBXL12. FANCD2 is a protein of the Fanconi anaemia complementation group (FANC), a disorder of the bone marrow that leads to anaemia and other developmental defects. FBXL12 poly-ubiquitylates FANCD2, a modification that causes FANCD2 dissociation from the DNA and subsequent degradation. Presumably, FBXL12 inhibition results in FANCD2 accumulation on DNA that hinders the recruitment of DNA repair factors. The current study aims to focus on the implication of FBXL12-FANCD2 in cancer development and progression, particularly in triple-negative breast cancer cell lines. The role of FBXL12 during replication stress was assessed using stable FBXL12-KO cells, doxycycline regulated shRNAFBXL12 and siRNA-FBXL12-mediated depletion of FBXL12 treated with replication stressinducing drugs, including WEE 1 inhibitor AZD1775. Characterization of a putative phosphorylated FANCD2 site was analysed by Dot-blot and immunoblotting. The data suggest that depletion of FBXL12 sensitize cells to AZD1775-induced replication stress, presumably leading to lethal DNA damage and loss of cellular recovery. It can be concluded that FBXL12-FANCD2 interaction can be exploited as a potential target for cancer therapy to treat aggressive cancer types like triple-negative breast cancers that have poor patient outcomes and limited treatment options available.