Liquid biopsy and next-generation sequencing of cell-free DNA (cfDNA) in cancer patients’ plasma offer a minimally-invasive solution to detect tumor cell genomic information to aid real-time clinical decision-making. Reliability and sensitivity in the detection of genomic alterations is crucial for patient management and it is particularly relevant in the context of targeted therapies. However, biological and technical factors, such as low cfDNA tumor fraction and sequencing errors, affect the correct interpretation of genomic data limiting the utility of non-invasive cfDNA-based tumor profiling. To address these issues, we designed a prostate cancer bespoke assay, PCF_SELECT, that includes an innovative sequencing panel covering ∼25 000 high minor allele frequency SNPs and tailored analytical solutions to enable allele-informed evaluation of patients’ tumor. The framework also implements ABEMUS, an ad-hoc computational procedure we specifically designed for cfDNA samples to accurately detect somatic point mutations that could emerge under treatment pressure and as drug resistance mechanism. When applied on serial plasma samples from three patients receiving PARP inhibition harboring DNA repair gene aberrations, PCF_SELECT demonstrated high sensitivity in detecting BRCA2 allelic imbalance with decreasing tumor fractions resultant from treatment and identified complex ATM genomic states that may be incongruent with protein losses. As a step towards a more sensitive detection of tumor features in circulation of cancer patients, we next hypothesized that recent WGS-based approaches exploiting cfDNA fragments characteristics could be extrapolated for targeted sequencing data and that gene-region specific measures could improve detection metrics. Preliminary results suggest an increased sensitivity compared to copy-number-based methods supporting the integration at no extra cost in our targeted assay. Overall, this work is relevant to the context of precision oncology. It provides an innovative platform for the management of cancer patients, delivering detailed patient-specific molecular information which could be applied to guide treatment and improve clinical outcomes.

Infertility is an issue for approximately 12% of couples attempting to have a child. Of this group, 50% of the cases are due to male factor infertility. There are many reasons for decreased fecundity in men, but there remains 10% to 15% of infertile men that are diagnosed with the most severe form of infertility, non-obstructive azoospermia (NOA). A diagnosis of NOA implies the lack of sperm cells in the ejaculate with no physiological reason. The current diagnostic test and treatment consist of microscopic examination of seminal fluid and a biopsy to extract any viable sperm from the testis. This treatment is known to be problematic because of the destructive nature of surgery as well as expense. A non-invasive diagnostic test that could identify the presence of sperm in the testis at the beginning of fertility treatment would inform the patient and the physician about the functionality of the testis and thus lead to more informed decisions about treatment and potentially a decrease in cost. The ability to identify the tissue source of DNA present in the reproductive tract could facilitate a fertility diagnostic tool. Tissue specific epigenetic mechanisms are known to play a role in an organism's development. The identification of an epigenetic signature unique to sperm DNA would allow for the identification of sperm DNA in a heterologous mixture. Our lab has been able to identify a methylation signature that can consistently differentiate between sperm DNA and somatic DNA. We compared the sperm DNA signature with that of blood and testicular tissue and found that there was no overlap in epigenetic markers. To create an assay that could evaluate the presence of sperm DNA we used an Oxford Nanopore next-generation sequencing platform. Sequencing bisulfite converted DNA; we were able to retrieve the methylation status at locations of interest. A bioinformatic tool was created to analyze the thousands of reads obtained and analyze the individual methylation points within single molecules of DNA. To create a more accessible fertility test, we used the sperm DNA analysis tool to evaluate seminal cell-free DNA (cfDNA). The presence of sperm cfDNA in a patient's seminal fluid may indicate that there is sperm somewhere in the male reproductive tract even if the cells are not intact. A clinician could use this information to better advise the patient about treatment and potentially decrease cost of care.

Lymphoma is a common hematopoietic malignancy in canines. Current diagnostic techniques to diagnose lymphoma are often invasive and expensive. Additionally, tumor heterogeneity complicates the accurate classification and diagnosis of specific subtypes, hindering the development of targeted therapy and prognostic assessments. We propose a minimally invasive liquid biopsy technique involving blood collection to detect cell-free DNA from tumors using Next-generation sequencing. We hypothesize that identical tumor aberrations can be found in matching plasma and tumor DNA. Five dogs diagnosed with B-cell lymphoma through flow cytometry or PAAR were enrolled in the study. Samples collected included: (1) blood for plasma (cfDNA), (2) tumor tissue fine-needle aspirates (tumor DNA), and (3) buccal swabs (genomic DNA, germline control). Whole Genome Sequencing was performed using Illumina NovaSeq 6000, and the sequenced output was analyzed with bioinformatics tools to detect somatic variants in plasma and tumor samples. Our results revealed many shared somatic variants between matched cfDNA and tumor DNA samples, with 1.7-49% of tumor variants also found in corresponding plasma samples. Shared variants constituted only 0.5-9% of all plasma somatic variants. Specific B-cell lymphoma mutations were identified in cfDNA, including MYC, POT1, and TRAF3, alongside other cancer-related genes. Tumor samples showed mutations in genes associated with canine and human B-cell lymphoma. This study suggests that tumor-specific genomic mutations can be detected in plasma, supporting the potential of liquid biopsy as a less invasive diagnostic tool. However, cfDNA may not capture the full genetic heterogeneity of tumors due to low tumor-derived DNA content in limited plasma volumes. / Master of Science / Lymphoma is a type of blood cancer affecting white blood cells. Canine lymphoma is a common neoplasia, with an incidence rate of 20 to 100 cases per 100,000 dogs, making it a significant research focus. Current diagnostic methods are invasive and costly. Additionally, the wide variety of tumor types in lymphoma makes it challenging to determine the exact subtypes, which is crucial for selecting the best treatment approach. To overcome these challenges, we proposed a less invasive method known as "liquid biopsy". This technique involves taking a blood sample of a dog to find cell-free DNA from tumor cells using Next-Generation Sequencing technologies. We aimed to see if blood DNA could provide the same information as tumor DNA. In our study, we worked with five dogs diagnosed with B-cell lymphoma through traditional methods. We collected blood, tissue from needle biopsies, and buccal swabs from each dog. We then performed DNA extraction and sequencing on these samples. Our findings showed that 1.7-50% of the mutations in tumor DNA were also detected in matched blood DNA, though these represented only a small fraction of all changes found in blood samples. Additionally, the blood samples also revealed mutations related to canine B-cell lymphoma in genes like MYC, POT1, and TRAF3. In conclusion, our study supports the use of liquid biopsy as a feasible and less invasive method to diagnose lymphoma in dogs. However, they might not show all genetic variations of the tumor due to limited tumor DNA content.

Ubiquitination is an important biochemical reaction found in all eukaryotic organisms and is involved in a wide range of cellular processes. Conventional ubiquitination requires the formation of polyubiquitin chains linked through Lys48 of the ubiquitin, which targets proteins for degradation, while the noncanonical Lys63-linked polyubiquitination of the proliferating cell nuclear antigen is required for error-free DNA damage tolerance (DDT or postreplication repair) in yeast. The ubiquitin-conjugating enzyme <i>Ubc13</i> and a cognate Ubc enzyme variant (Uev or Mms2) are involved in this process. Because there is less information available on either Lys63-linked ubiquitination or error-free DDT in plants, the goal of my research was to study the functions of <i>Ubc13</i> and Uev in plants using <i>Arabidopsis thaliana</i> as the model organism.<p> Four <i>UEV1</i> genes from <i>Arabidopsis thaliana</i> were isolated and characterized. All four <i>Uev1</i> proteins can form a stable complex with AtUbc13 and can promote <i>Ubc13</i> mediated Lys63 polyubiquitination. All four <i>UEV1</i> genes can replace yeast MMS2 in DDT function in vivo. Although these genes are ubiquitously expressed in most tissues, <i>UEV1D</i> appears to be expressed at a much higher level in germinating seeds and pollen. We obtained and characterized two <i>uev1d</i> null mutant T-DNA insertion lines. Compared with wild-type plants, seeds from uev1d null plants germinated poorly when treated with a DNA-damaging agent. Seeds that germinated grew slow and the majority ceased growth within 2 weeks. Pollen from uev1d plants also displayed a moderate but significant decrease in germination in the presence of DNA damage agent. These results indicate that <i>Ubc13-Uev</i> complex functions in DNA damage response in <i>Arabidopsis thaliana.</i> <i>Arabidopsis thaliana</i> contains two <i>UBC13</i> genes, AtUBC13A and AtUBC13B, that are highly conserved with respect to DNA sequence, protein sequence and genomic organization, suggesting that they are derived from a recent gene duplication event. Both <i>AtUbc13</i> proteins are able to physically interact with human and yeast Mms2, implying that plants also employ a Lys63-linked polyubiquitination reaction. Furthermore, Both <i>AtUBC13</i> genes were able to functionally complement the yeast ubc13 null mutants, suggesting the existence of an error-free DNA damage tolerance pathway in plants. The <i>AtUBC13</i> genes appear to be expressed ubiquitously and were not induced by various conditions tested.<p> The <i>ubc13a/b</i> double mutant lines were created and displayed strong phenotypic changes. The double mutant plants were delayed in seed germination as well as cotyledon and true leaf development. When seedlings were grown vertically on plates, the roots of the double mutant were shorter and grew in a zig-zag manner, compared to the straight growth of wild type roots. Root length and number of lateral roots on wild type and <i>ubc13a</i> and <i>ubc13b</i> single mutant plants were about 3 times longer than those of double mutant plants after 9 and 12 days of growth. When double mutant seeds were sown directly into soil, many did not germinate and those that germinated grew much slower than wild type. At 35 days, double mutant plants were smaller with thinner, flatter, and lighter coloured rosette leaves compared to wild type plants. These phenotypes indicate that <i>AtUbc13</i> not only plays a role in DDT to protect genome integrity but also is involved in plant development. Hence, this study set a cornerstone for future investigations into the roles of <i>Ubc13</i> and <i>Uev1</i> in plant development.

Ubiquitination is an important biochemical reaction found in all eukaryotic organisms and is involved in a wide range of cellular processes. Conventional ubiquitination requires the formation of polyubiquitin chains linked through Lys48 of the ubiquitin, which targets proteins for degradation, while the noncanonical Lys63-linked polyubiquitination of the proliferating cell nuclear antigen is required for error-free DNA damage tolerance (DDT or postreplication repair) in yeast. The ubiquitin-conjugating enzyme <i>Ubc13</i> and a cognate Ubc enzyme variant (Uev or Mms2) are involved in this process. Because there is less information available on either Lys63-linked ubiquitination or error-free DDT in plants, the goal of my research was to study the functions of <i>Ubc13</i> and Uev in plants using <i>Arabidopsis thaliana</i> as the model organism.<p> Four <i>UEV1</i> genes from <i>Arabidopsis thaliana</i> were isolated and characterized. All four <i>Uev1</i> proteins can form a stable complex with AtUbc13 and can promote <i>Ubc13</i> mediated Lys63 polyubiquitination. All four <i>UEV1</i> genes can replace yeast MMS2 in DDT function in vivo. Although these genes are ubiquitously expressed in most tissues, <i>UEV1D</i> appears to be expressed at a much higher level in germinating seeds and pollen. We obtained and characterized two <i>uev1d</i> null mutant T-DNA insertion lines. Compared with wild-type plants, seeds from uev1d null plants germinated poorly when treated with a DNA-damaging agent. Seeds that germinated grew slow and the majority ceased growth within 2 weeks. Pollen from uev1d plants also displayed a moderate but significant decrease in germination in the presence of DNA damage agent. These results indicate that <i>Ubc13-Uev</i> complex functions in DNA damage response in <i>Arabidopsis thaliana.</i> <i>Arabidopsis thaliana</i> contains two <i>UBC13</i> genes, AtUBC13A and AtUBC13B, that are highly conserved with respect to DNA sequence, protein sequence and genomic organization, suggesting that they are derived from a recent gene duplication event. Both <i>AtUbc13</i> proteins are able to physically interact with human and yeast Mms2, implying that plants also employ a Lys63-linked polyubiquitination reaction. Furthermore, Both <i>AtUBC13</i> genes were able to functionally complement the yeast ubc13 null mutants, suggesting the existence of an error-free DNA damage tolerance pathway in plants. The <i>AtUBC13</i> genes appear to be expressed ubiquitously and were not induced by various conditions tested.<p> The <i>ubc13a/b</i> double mutant lines were created and displayed strong phenotypic changes. The double mutant plants were delayed in seed germination as well as cotyledon and true leaf development. When seedlings were grown vertically on plates, the roots of the double mutant were shorter and grew in a zig-zag manner, compared to the straight growth of wild type roots. Root length and number of lateral roots on wild type and <i>ubc13a</i> and <i>ubc13b</i> single mutant plants were about 3 times longer than those of double mutant plants after 9 and 12 days of growth. When double mutant seeds were sown directly into soil, many did not germinate and those that germinated grew much slower than wild type. At 35 days, double mutant plants were smaller with thinner, flatter, and lighter coloured rosette leaves compared to wild type plants. These phenotypes indicate that <i>AtUbc13</i> not only plays a role in DDT to protect genome integrity but also is involved in plant development. Hence, this study set a cornerstone for future investigations into the roles of <i>Ubc13</i> and <i>Uev1</i> in plant development.

Le cancer broncho-pulmonaire du non-fumeur est considéré comme une entité à part du fait de ses particularités épidémiologiques. Il est en outre un excellent modèle pour l'étude des facteurs de risque de cancer bronchique autres que le tabagisme actif. Il n'existe que très peu de données non-asiatiques concernant cette entité d'intérêt. Le bio-observatoire national des cancers bronchiques de non-fumeurs (BioCAST I IFCT-1002) est une étude épidémiologique multicentrique prospective. Son objectif principal est de décrire une population de patient strictement non-fumeur (moins de 100 cigarettes au cours de la vie) atteint de cancer bronchique, notamment sur le plan de leur profil moléculaire somatique et de leur exposition aux facteurs de risque. Les objectifs secondaires étaient d'étudier si l'exposition aux différents facteurs de risque pouvait influencer le profil moléculaire ; et d'utiliser cette cohorte particulière (grande fréquence et diversité de mutations somatiques attendue) afin de développer un test multiplex pour le diagnostic non-invasif du profil moléculaire somatique tumoral à partir d'ADN circulant. Au total, 384 patients non-fumeurs atteints de cancer broncho-pulmonaire ont été inclus dans cette cohorte. Deux-tiers d'entre eux étaient exposés au tabagisme passif, et il s'agissait essentiellement d'une exposition domestique touchant les femmes. Inversement, 35% des hommes étaient exposés de manière certaine à au moins un cancérogène professionnel, contre 8% des femmes. Au total, 72% des patients présentait une anomalie moléculaire, essentiellement au niveau de l'EGFR (51% de l'ensemble de la cohorte). Le genre, ou l'exposition à différents facteurs de risque (tabagisme passif, exposition professionnelle, exposition hormonale chez les femmes) n'affectait pas de manière significative et cliniquement pertinente le profil mutationnel, avec les limites liée à de faibles effectifs dans certains groupes et aux expositions multiples. Seule l'exposition professionnelle à l'amiante et / ou à la silice semble avoir pour effet de diminuer la fréquence des mutations de l'EGFR / Lung cancer in never smokers (LCINS) is considered as a separate entity given its epidemiological specificities. It is also a very interesting template to assess alternative risk factors for lung cancers than tobacco smoking. However, there is very little non-Asian data about this particular topic. The BioCAST / IFCT1002 study is a prospective, nationwide, and multi-centric epidemiological study. Its main objective was to describe a French population of lung cancers in lifelong never smokers (less than 100 cigarette during all lifetime); with a special focus on molecular somatic profile and risk-factors exposure. Secondary objectives were to assess the interaction between risk-factor exposure and molecular profile; and to use this particular cohort to develop a multiplex test for non-invasive diagnosis of tumor mutations in circulating free DNA. Overall, 384 patients were recruited in the cohort. Two-third were exposed to passive smoking (mainly women and in domestic setting). By contrast, 35% of men were definitely exposed to occupational carcinogens versus 8% of women. Finally, 72% were found with a somatic mutation, mainly in the EGFR gene (51% of the whole population). Gender or exposure to risk factors such as passive smoking, occupational exposure, or hormonal status in women, were not significantly associated with a specific and/or clinically meaningful molecular profile in tumor. These findings should be interpreted with caution given that some subgroups were small and/or with many simultaneous exposures. However, exposure to asbestos and/or silica was significantly associated to a decreased risk for EGFR mutation. On the pilot study (n=106), circulating free DNA was associated with tumor burden. The multiplex diagnosis (12 amplicons on 5 genes) by next-generation sequencing was feasible and gave encouraging results in stage 4 patients (67% sensitivity, 73% concordance rate). LCINS is an interesting entity for the study of non-tobacco-related cancer risk factors; or to optimize liquid biopsy strategy

The purpose of this investigation was to elucidate the changes in cf-DNA as it relates to fluctuations in resistance training workloads and intensities. The relationship between cell free DNA (cf-DNA), C-reactive protein (CRP), creatine kinase (CK), testosterone (T), cortisol (C), testosterone-cortisol ratio (T:C), body mass and body composition were also examined. Eight weightlifters (5 males and 3 females, age = 25 ± 3.5 yr, body mass = 88.3 ± 22.7 kg, height = 173.8 ±8.4 cm) volunteered to participate in this study. Venous blood samples, body mass and body composition were taken six times, each corresponding to the end of a training phase. CK (p = 0.018, η² = 0.409) and CK %Δ (p < 0.001, η² = 0.594) were the only biochemical variables to reach statistical significance at any point. A number of statistically significant correlations were found among variables. VLD4wk was related to CK %Δ (r = 0.86), VLD4wk %Δ was related CK %Δ (r = 0.86) and TID1wk was related to CRP (r = 0.83). cf-DNA %Δ was correlated with CRP and CRP %Δ (r = 0.83 and 0.86, respectively). CRP and CRP %Δ were correlated with BF % (r = 0.94 and 0.92, respectively). CK and CK %Δ were both related to T:C (r = 0.94 and 0.89, respectively) and T:C %Δ (r = 0.87 and 0.86, respectively). The correlation between cf-DNA and CRP suggests that cf-DNA may be a valuable indicator of inflammation in weightlifters.

Sepsis is life-threatening organ dysfunction produced by a dysregulated host response to infection in which neutrophils release neutrophil extracellular traps (NETs). NETs consist of DNA, histones, and antimicrobial peptides which kill pathogens. However, DNA and histones also exert damage by activating the intrinsic pathway of coagulation and inducing endothelial cell death, respectively. AADH, a 15kDa non-anticoagulant unfractionated heparin (UFH), prevents histone-mediated cytotoxicity in vitro and improves survival in septic mice. We explored the effectiveness of Vasoflux, a 5.5kDa low-molecular-weight-heparin as an anti-sepsis treatment as compared to enoxaparin and UFH. Vasoflux has reduced anticoagulant functions and hence reduces the risk of bleeding as compared to enoxaparin or UFH. We showed that UFH, enoxaparin, or Vasoflux at concentrations of up to 13.3uM, 40uM, or 40uM, neutralize histone-mediated cytotoxicity. These results suggest that these glycosaminoglycans (GAGs) are able to neutralize histone-mediated cytotoxicity independent of the AT-binding pentasaccharide. To quantitate the binding affinity between GAGs and histones, surface plasmon resonance was conducted. UFH is a more potent inhibitor of histone-mediated cytotoxicity compared to Vasoflux as UFH has a 10-fold greater binding affinity to histones compared to Vasoflux. To translate our in vitro findings to in vivo, Vasoflux, enoxaparin, and UFH were administered in a murine model of sepsis. Vasoflux at 8mg/kg - 50mg/kg reduced survival and exhibited damage in the lung, liver, and kidney in septic mice compared to 10 mg/kg of UFH or 8mg/kg of enoxaparin. This may be due to Vasoflux and UFH disrupting the DNA-histone complex, thereby releasing free procoagulant DNA. This is evident by our gel electrophoresis experiments, where addition of 1uM Vasoflux or 3.3uM UFH to DNA-histone complexes lead to histone dissociation from DNA. UFH bound to histones may be able to inhibit DNA-mediated thrombin generation, as it retains its anticoagulant properties, demonstrated by UFH-histone complexes attenuating DNA and TF-mediated thrombin generation. In contrast, Vasoflux may not neutralize the procoagulant DNA leading to a hypercoagulable state in the mice. Our study may have important clinical implications as there is an ongoing trial, HALO, which will administer intravenous UFH to patients suspected to have septic shock to reduce mortality. Based on our results, future clinical trials should consider the antithrombin-dependent anticoagulant activity of UFH being used as a sepsis treatment. / Thesis / Master of Science (MSc) / Sepsis is a life threatening condition caused by the body’s extreme response to microbial infection of the blood, whereby neutrophils release traps composed of cell-free DNA (cfDNA), histones, and antimicrobial proteins. In addition to fighting off infections, these traps also exert harmful effects like triggering clotting and killing host cells. Currently, no specific anti-septic drugs exist. Studies have shown that DNase1 (a recombinant protein that digests double stranded cfDNA) or a modified form of heparin (neutralizes histones) improves survival in septic mice. Our goal was to explore the protective effects of Vasoflux, (a non-anticoagulant heparin) and DNase1 in a mouse model of sepsis. We hypothesize that the combined therapy of DNase1 and Vasoflux will improve survival. We found that Vasoflux has minimal blood thinning activity and can prevent histones from killing cells. However, Vasoflux administered into septic mice worsened organ damage and decreased survival. We hypothesize that this damage may be due to Vasoflux’s ability to displace histones from histone-DNA complexes, thereby releasing free DNA, which promotes excessive blood clotting in sepsis.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23772号 / 医博第4818号 / 新制||医||1056(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 村川 泰裕, 教授 松田 文彦, 教授 篠原 隆司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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.