No presente estudo investigou-se amostras de plasma com DNA livre circulante (DNA LC) por meio da técnica Varredura Z. Esta é uma técnica eficiente na determinação de parâmetros de diferentes materiais, tais como cristais líquidos, ferrofluidos e compostos biológicos. Esta experiência é realizada através da focalização de um feixe laser de perfil gaussiano numa amostra. Na medida em que a amostra se aproxima do foco da lente, a intensidade do feixe aumenta e alcança seu valor máximo no ponto focal, então diminui para pontos distantes do foco. Na região próxima ao ponto focal se amplificam os fenômenos não-lineares. Recentemente foi demonstrado que níveis elevados de DNA LC no plasma ocorrem com frequência em pacientes com vários tipos de câncer, podendo ser utilizados para discriminar pacientes com malignidade de pessoas saudáveis. As amostras de DNA LC, submetidas ao experimento Varredura Z, forneceram respostas ópticas devido ao fenômeno de lente térmica. Os resultados revelaram que a amplitude de lente térmica das amostras extraídas do plasma de pacientes com malignidade difere daquela de doadores sãos. A técnica Varredura Z se mostrou mais vantajosa em relação a outras biológicas porque revelou uma maior diferença entre os grupos estudados e tem o caráter de detectar mudanças estruturais no DNA LC. / In the present study plasma samples with cell-free DNA were investigated by means of the Z-Scan technique. This is a powerfull technique in determining parameters of different materials, such as liquid crystals, ferrofluids and biological compounds. This experiment is performed by focusing a Gaussian profile laser beam on a sample. As the sample approaches the focus of the lens, the intensity of the beam increases and reaches its maximum value at the focal point, then decreases to points distant from the focus. In the region near the focal point non-linear phenomena are amplified. It has recently been demonstrated that high levels of plasma cell-free DNA occur frequently in patients with various cancers and can be used to discriminate patients with malignancy from healthy donors. The cell-free DNA samples, submitted to the Z-Scan experiment, provided optical responses due to the thermal lens phenomenon. The results revealed that the thermal lens amplitude of samples extracted from the plasma of patients with malignancy differs from that of healthy donors. The Z-Scan technique was more advantageous than other biological ones because it revealed a greater difference between the studied groups and has the character of detecting structural changes in cell-free DNA.

The primary purposes of this dissertation were to explore relationship between cell free DNA (cf-DNA), creatine kinase (CK), C-reactive protein (CRP), vertical jump testing delayed onset muscle soreness (DOMS) in response to a high-volume resistance training protocol, and to assess the sensitivity of cf-DNA to different resistance training volume loads. The secondary purpose was to examine the relationship between cf-DNA and relative strength. Study 1 was an exploratory attempt to discover relationships between cf-DNA, CK, CRP, delayed onset muscle soreness, and performance variables. Seventeen resistance trained males were recruited, 9 were randomly assigned to receive BCAAs while 8 received a placebo. Participants performed a high-volume resistance training session consisting of the back squat and bench press. Blood was drawn to measure serum cf-DNA, CK, and CRP levels prior to the training session, with cf-DNA collected immediately post, and CK and CRP at 24hr and 48hrs post. Self-reported DOMS on a scale of 1 to 10 was collected prior to training on day 2, day 3, and day 4. SJH, CMJH, and BOSCO were collected on day 1, day 3, and day 4. Fifty-seven correlations were run to explore the relationships between variables. Only the correlation between %Δ DOMS 48hr and %Δ CRP 48hr in the non-supplement group was significant (p = 0.02). The second study, designed to assess the sensitivity of cf-DNA to different resistance training volume loads, consisted of a high-volume resistance training protocol. Blood was drawn immediately before the resistance training session (T1), immediately after the third lifting set (T2), and immediately after the sixth lifting set (T3). cf-DNA increased significantly from T1 to T2 (p < 0.01) and T1 to T3 (p < 0.01). The linear regression model used to examine the capabilities of relative strength to predict %Δ cf-DNA from T1 to T3 was significant (p = 0.04). The results of this study demonstrate the short response time of cf-DNA in relation to variations in resistance training volume-load, suggesting it may be a valuable marker in monitoring the immune response to volume-load. Results also demonstrated the positive relationship between relative strength and %Δ cf-DNA.

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Head and neck squamous cell carcinoma is the fifth most common cancer worldwide. They are associated with high morbidity and mortality. Despite considerable advances in surgical and oncological treatment over the past two decades, overall treatment outcome has only slightly improved. In my thesis I focused on serum gene expression analysis of head and neck cancer patients, which followed the tissue gene expression analysis in same patients. Further we investigated gene expression analysis in tumour stroma, which is now considered as significant factor in cancer initiation and progression. We revealed several candidate genes, which are involved in signalling pathways connected with cell differentiation and proliferation and are involved in apoptotic pathway (BCl-2, BCl-XL a MAX). As well we detected down-regulation of the main tumour suppressor p 53 protein. In peritumoural tissue we detected overexpression of cytokines typical for embryonal development and ectoderm differentiation - IGF-2 and BMP-4, which significantly influence the phenotype of normal keratinocytes. Further we identified several candidate genes relating with overexpression of Gal-1 in stromal myofibroblasts rich tissue (SPIN1, FUSIP1, TRIM23, SLC25A40, PTPLAD1, MP3K2). HNSCC is a heterogeneous disease despite the presence of...

Proteins—polymers of amino acids—are a major class of biomolecules whose myriad functions facilitate many crucial biological processes. Accordingly, human control over these biological processes depends upon the ability to study, produce, and modify proteins. One innovative tool for accomplishing these aims is cell-free protein synthesis (CFPS). This technique, rather than using living cells to make protein, simply extracts the cells' natural protein-making machinery and then uses it to produce protein in vitro. Because living cells are no longer involved, scientists can freely adapt the protein production environment in ways not otherwise possible. However, improved versatility and yield of CFPS protein production is still the subject of considerable research. This work focuses on two ideas for furthering that research.The first idea is the adaptation of CFPS to make proteins containing unnatural amino acids. Unnatural amino acids are not found in natural biological proteins; they are synthesized artificially to possess useful properties which are then conferred upon any protein made with them. However, current methods for incorporating unnatural amino acids do not allow incorporation of more than one type of unnatural amino acid into a single protein. This work helps lay the groundwork for the incorporation of different unnatural amino acid types into proteins. It does this by using modified aminoacyl-tRNA synthetases (aaRSs), which are key components in CFPS, to be compatible with unnatural amino acids. The second idea is the preservation of DNA templates from enzyme degradation in CFPS. Among the advantages of CFPS is the option of using linear expression templates (LETs) in place of plasmids as the DNA template for protein production. Because LETs can be produced more quickly than plasmids can, using LETs greatly reduces the time required to obtain a DNA template for protein production. This renders CFPS a better candidate for high-throughput testing of proteins. However, LETs are more susceptible to enzyme-mediated degradation than plasmids are, which means that LET-based CFPS protein yields are lower than plasmid-based CFPS yields. This work explores the possibility of increasing the protein yield of LET-based CFPS by addition of sacrificial DNA, DNA which is not used as a protein-making template but which is degraded by the enzymes in place of the LETs.

Sepsis is a clinical syndrome characterized by the systemic activation of inflammatory and coagulation pathways in response to microbial infection of normally sterile parts of the body. Despite considerable advances in our understanding of sepsis pathophysiology, sepsis remains the leading cause of death in non-coronary intensive care units (ICU) with a global disease burden between 15 and 19 million cases per year (Dellinger et al., 2008). Severe sepsis, defined as sepsis associated with organ dysfunction is associated with mortality rates of 33% to 45%. The incidence of severe sepsis continues to increase by 1.5% per annum due to the aging population, a rise in the prevalence of comorbidities, and the wider use of immunosuppressive agents and invasive procedures (Angus et al., 2001). Over the past several decades, many potential treatments for sepsis have shown early promise, yet have failed to improve survival in over 100 Phase II and Phase III clinical trials (Marshall, 2014) suggesting that some fundamental knowledge is lacking in our understanding of sepsis pathophysiology. Emerging studies on cell-free DNA (cfDNA), DNA released extracellularly into the circulation, demonstrate that cfDNA is a crucial link between inflammation and coagulation . In various conditions characterized by excessive inflammatory responses or aberrant prothrombotic responses, cfDNA has been implicated in exacerbating disease pathology (Atamaniuk, Kopecky, Skoupy, Säemann, & Weichhart, 2012; Fuchs, Brill, & Wagner, 2012; Swystun, Mukherjee, & Liaw, 2011). In clinical sepsis, levels of cfDNA upon admission into the ICU have strong prognostic value in predicting mortality (Dwivedi et al., 2012; Saukkonen et al., 2008). However, it is unclear whether these increases in cfDNA are an epiphenomenon during sepsis progression, or whether cfDNA actively plays a role in sepsis pathophysiology. In this work, in vivo studies were conducted to characterize the role of cfDNA in sepsis, the effects of DNase administration, and the potential mechanism by which cfDNA is released during experimental sepsis. In addition, mortality studies were conducted to identify surrogate markers of death to promote the design of humane and ethical animal studies in conducting sepsis research. Polymicrobial sepsis was induced via a surgical procedure whereby the cecum is exteriorized, ligated and punctured twice to introduce a continuous source of microorganisms, a model termed cecal ligation and puncture (CLP). In our CLP sepsis model, levels of cfDNA increased in a time-dependent manner. These increases accompanied an early pro-inflammatory response marked by increased pro-inflammatory IL-6, a transient increase in anti-inflammatory IL-10, and elevated lung myeloperoxidase (MPO) activity. Septic mice with elevated cfDNA levels also had high bacterial loads in the lungs, blood, and peritoneal cavity fluid. Organ damage was also observed in mice following CLP surgery versus mice subjected to the non-septic sham control surgery marked by increased levels of creatinine and alanine aminotransferase (ALT) indicative of kidney and liver injury, respectively. Histological analyses further confirmed lung and kidney damage following CLP surgery. Changes in coagulation were also observed in septic mice as mice subjected to CLP had sustained increases in thrombin-antithrombin (TAT) complexes. In addition, plasma from CLP-operated mice had increased thrombin generation (i.e. increased endogenous thromin potential, increased peak thrombin, decreased time to peak, and decreased lag time) mediated by FXIIa and enhanced by platelets. Following CLP-induced sepsis, elevations in cfDNA levels accompanied pro-inflammatory and pro-coagulant responses. The effects of in vivo DNase treatment in septic mice were time-dependent. Early DNase treatment when cfDNA levels were low resulted in an exaggerated pro-inflammatory response marked by increased plasma IL-6 levels and increased lung damage. In contrast, delayed DNase treatment at time-points when cfDNA levels were elevated suppressed inflammation characterized by an increase in anti-inflammatory IL-10 and reductions in cfDNA, IL-6, lung MPO, and ALT activity. Furthermore, delayed DNase administration resulted in decreased bacterial load in the lungs, blood, and peritoneal cavity fluid. Delayed DNase treatment also resulted in blunted pro-coagulant responses as levels of TAT complexes were suppressed and thrombin generation from septic mouse plasma was normalized. Moreover, DNase treatment when cfDNA levels were elevated increased survival in CLP-operated mice by 80% and reduced lung and liver damage. These findings suggest that administration of DNase when cfDNA levels are elevated may reduce pro-inflammatory and pro-coagulant responses and that delayed DNase treatment may infer protection in the CLP model of sepsis. One mechanism by which cfDNA is released is via the formation of neutrophil extracellular traps (NETs). Upon inflammatory stimulation, some neutrophils release chromatin material and antimicrobial proteins (i.e. neutrophil elastase, MPO, and histones) in an active process termed NETosis. Although NETs ensnare bacteria and exert antimicrobial properties, NETs may also exert harmful effects on the host by activating inflammation and coagulation. While some in vitro evidence suggest that neutrophils are the main source of cfDNA released following inflammatory stimulation, others have reported that neutrophils are not the main source of circulating cfDNA following septic challenge. To determine whether NETs contribute to cfDNA released during CLP sepsis, genetically modified mice that are incapable of forming NETs, PAD4-/- mice, were used. Levels of cfDNA in PAD-/- mice were significantly lower than cfDNA levels in C57Bl/6 mice following CLP surgery, suggesting that NETs were a source of cfDNA in our model. Levels of IL-6, MPO, and bacterial load in the lungs, blood, and peritoneal cavity were significantly reduced, indicating that NETs exert pro-inflammatory effects in CLP sepsis. Thrombin generation was also suppressed in PAD4-/- mice which suggests that NETs contribute to thrombin generation following CLP sepsis. NETs contribute to increases in circulating cfDNA and may exacerbate pathology by driving pro-inflammatory and pro-coagulant responses in CLP-induced sepsis. Appreciating the implications of conducting research using animals, it is pertinent that researchers ensure the highest ethical standards and design animal studies in the most humane, yet scientifically rigorous manner. Using mortality studies, we validated the utility of physiological and phenotypic markers to assess disease severity and predict death in murine sepsis. Temperature via a rectal probe monitor and sepsis scoring systems which assess components such as orbital tightening, level of consciousness, and activity were effective surrogate markers of death. These tools offer a non-invasive assessment of disease progression which do not artificially exacerbate sepsis pathology and immediate information regarding any changes in the health status. Surrogate markers of death also provide reliable monitoring to meet increasing standards of ethical, humane animal research and a feasible and cost-efficient means to obtain vital signs in small rodents. We have proposed a scoring system which can be used for assessing disease severity, endpoint monitoring, and predicting death to obviate inhumane methods of using death as an endpoint in sepsis studies. In summary, cfDNA levels are elevated in CLP-induced sepsis and these elevations accompany pro-inflammatory and pro-coagulant responses. NETosis may be a mechanism by which cfDNA is released and NETs may drive inflammation and coagulation in CLP sepsis. Delayed DNase administration may suppress inflammation and coagulation and may be protective in polymicrobial sepsis. In future animal sepsis studies, surrogate markers of death and a sepsis scoring system can be used in place of death as an endpoint to raise the standards in conducting ethical, humane sepsis research. / Thesis / Doctor of Philosophy (PhD)

In breast cancer, the genetic profiling of circulating cell-free DNA (cfDNA) from blood plasma was shown to have good potential for clinical use. In contrast, only a few studies were performed investigating urinary cfDNA. In this pilot study, we analyzed plasma-derived and matching urinary cfDNA samples obtained from 15 presurgical triple-negative breast cancer patients. We used a targeted next-generation sequencing approach to identify and compare genetic alterations in both body fluids. The cfDNA concentration was higher in urine compared to plasma, but there was no significant correlation between matched samples. Bioinformatical analysis revealed a total of 3339 somatic breast-cancer-related variants (VAF ≥ 3%), whereof 1222 vs. 2117 variants were found in plasma-derived vs. urinary cfDNA, respectively. Further, 431 shared variants were found in both body fluids. Throughout the cohort, the recovery rate of plasma-derived mutations in matching urinary cfDNA was 47% and even 63% for pathogenic variants only. The most frequently occurring pathogenic and likely pathogenic mutated genes were NF1, CHEK2, KMT2C and PTEN in both body fluids. Notably, a pathogenic CHEK2 (T519M) variant was found in all 30 samples. Taken together, our results indicated that body fluids appear to be valuable sources bearing complementary information regarding the genetic tumor profile.

Joint Communication and Sensing (JCAS) technology enables the sharing of infrastructure, resources, and signals between communication and sensing. However, studying the performance and algorithms using appropriate target reflectivity models for detection poses a significant challenge. Moreover, the increasing demand for efficient sensing systems in large-scale environments necessitates the study of distributed sensing for handling extensive data collection and processing. This study investigates the impact of target mobility on the choice between the Swerling-I and Swerling-II models for target reflectivity and proposes a brief method for reflectivity models in multi-static sensing. This method constructed a dedicated decorrelation area for a single radar detector using its decorrelation angle. Multiple radar system constructs an intersection of these areas. For targets expected to remain in this area, the Swerling-I model is preferred, while for targets likely to move to the outside intersection, the Swerling-II model is more suitable. Additionally, this thesis proposes and derives the test statistics for the distributed sensing in JCAS cell-free massive MIMO (multiple-input multiple-output) systems, where only the statistical distribution of transmitted signals is known at the receiver access points for the sensing purpose. This thesis compares the sensing performance of the proposed distributed processing with the centralized processing. Moreover, the results of a power allocation algorithm that maximizes sensing performance are compared against a baseline algorithm that minimizes total power consumption. In terms of sensing performance via guaranteeing the quality of service of the communication, the results indicate that the sensing algorithm consistently outperforms the power-minimizing algorithm, regardless of the selected reflectivity model. Furthermore, the Swerling-II model performs relatively worse when the reflectivity of the target is low, but exhibits improved performance on a relatively high reflectivity target. Regarding distributed sensing, its implementation may lead to a deterioration in sensing performance. However, the results show that distributed sensing can approach the performance of centralized sensing when the target has high reflectivity. The major advantage of distributed sensing is the reduced fronthaul signaling load in a JCAS cell-free massive MIMO system. / Joint Communication and Sensing (JCAS) teknologi möjliggör delning av infrastruktur, resurser och signaler mellan kommunikation och sensorik. Studier av prestanda och algoritmer med lämpliga modeller för detektering av målets reflektivitet utgör emellertid en betydande utmaning. Dessutom kräver den ökande efterfrågan på effektiva sensorsystem i storskaliga miljöer studier av distribuerad sensorik för att hantera omfattande datainsamling och -bearbetning. Detta studie undersöker påverkan av målets rörlighet på valet mellan SwerlingI och Swerling-II modellerna för målets reflektivitet och föreslår en kort metod för reflektivitetsmodeller i multi-statisk avkänning. Denna metod konstruerar ett dedikerat dekorrelationsområde för en enskild radardetektor med hjälp av dess dekorrelationsvinkel. Ett flertal radarsystem konstruerar en skärningspunkt av dessa områden. För mål som förväntas förbli i detta område föredras Swerling-I-modellen, medan för mål som troligen rör sig till den yttre skärningspunkten är Swerling-II-modellen mer lämplig. Dessutom föreslår och härleder denna avhandling teststatistik för distribuerad avkänning i JCAS cellfri massiv MIMO (multiple-input multiple-output) system, där endast den statistiska fördelningen av överförda signaler är känd vid mottagarens åtkomstpunkter för avkänningsändamål. Denna avhandling jämför avkänningsprestanda för föreslagen distribuerad bearbetning med centraliserad bearbetning. Dessutom jämförs resultaten av en effekttilldelningsalgoritm som maximerar avkänningsprestanda mot en baslinjealgoritm som minimerar total effektförbrukning. När det gäller avkänningsprestanda genom att garantera kommunikationens tjänstekvalitet indikerar resultaten att avkänningsalgoritmen konsekvent presterar bättre än effektminimeringsalgoritmen, oavsett vald reflektivitetsmodell. Dessutom presterar Swerling-II-modellen relativt sämre när målets reflektivitet är låg, men uppvisar förbättrad prestanda på ett relativt högreflekterande mål. När det gäller distribuerad avkänning kan dess implementering leda till försämrad avkänningsprestanda. Resultaten visar dock att distribuerad avkänning kan närma sig prestandan hos centraliserad avkänning när målet har hög reflektivitet. Den största fördelen med distribuerad avkänning är den minskade signalbelastningen i en JCAS cellfri massiv MIMO-system.

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Cyanobakterier är intressanta mikroorganismer för produktion av biobränslen från solljus, vatten och atmosfärisk koldioxid och anses därför vara potentiella mikrobiella cellfabriker. Men på grund av långsam tillväxt och låg produktion är genteknologi processen intensiv och tidskrävande för cyanobakterier. En alternativ metod till prototypteknik för metabola vägar är att använda cellfri metabolisk teknik där cellysat av överuttryckta enzymer används. I detta projekt försökte vi utveckla en metod för cellfri metabolisk ingenjörsteknik för cyanobakterien Synechocystis PCC 6803 med hjälp av den övre mevalonatvägen som exempelreaktionsväg. Vi började med att utveckla tre fluorescensbaserade metoder för att detektera proteinöveruttryck med hjälp av de tre enzymerna från mevalonatreaktionsvägen. Dessa metoder använde fusering av YFP-proteinet till målproteinet, en delad GFP-reporterprotein eller translationskoppling. Ett av de överuttryckta enzymerna verkade vara giftigt för Synechocystis-celler så flera inducerbara promotorer användes för att försöka uttrycka enzymet. Den högst uttryckande konstruktionen för varje gen valdes ut och proteiner extraherades och blandades i en cellfri metabolisk ingenjörsreaktion. Även om inget mevalonat kunde detekteras med hjälp av gaskromatografi i detta projekt, berodde detta sannolikt på otillräckligt högt proteinöveruttryck av mevalonatgenerna. / Cyanobacteria are desirable microorganisms for the production of biofuels from sunlight, water and atmospheric carbon dioxide, and are therefore considered potential microbial cell factories. But due to slow growth rate and low production rates, the engineering processes for bioproduction is labour intensive and time consuming. An alternative method to prototype metabolic pathway engineering is to use cell-free metabolic engineering, where cell lysates of enriched enzymes are used. In this project, we attempted to develop a method for cell-free metabolic engineering for the cyanobacterium Synechocystis PCC 6803 using the upper mevalonate pathway as an example pathway. We started by developing three fluorescence-based methods for detecting protein overexpression using the three enzymes from the mevalonate pathway. These methods used YFP fusion to target proteins, a split GFP reporter tag or translation coupling. One of the overexpressed enzymes appeared to be toxic to Synechocystis cells so several inducible promoters were used to try and express the enzyme. The highest expressing construct for each gene was selected and proteins were extracted and mixed in a cell free metabolic engineering reaction. Although no mevalonate could be detected using gas chromatography in this project, this was likely due to insufficiently high protein overexpression of the mevalonate pathway genes.