Medical Biology - Theses
Now showing items 1-12 of 259
A CRISPR/Cas9-based investigation of inflammasomes in infectious disease and autoinflammation
Inflammasomes are a family of innate immune signalling platforms that are activated in response to tissue damage or infection. Inflammasome stimulation results in activation of the inflammatory protease caspase-1, which induces a lytic cell death program known as pyroptosis, and maturation and release of the pro-inflammatory cytokines Interleukin-1β (IL-1β) and IL-18. The potent inflammatory cascade triggered through activation of the inflammasomes is protective against many bacterial pathogens that either invade host cells or produce toxins that deregulate key homeostatic mechanisms within innate immune cells such as monocytes and macrophages. De-regulation of inflammasome signalling, such as gain-of-function mutations in inflammasome components, can result in autoinflammatory pathology. In order to investigate the function and regulation of inflammasomes, Clustered, Regularly Interspersed, Short, Palindromic Repeats (CRISPR)/Cas9 gene editing technology has been utilised to delete various inflammasome components from human myeloid cell lines or from mice. The alternative inflammatory caspases, caspase-11 in mice and caspases-4 and -5 in humans are activated directly by cytoplasmic lipopolysaccharide (LPS), a key component of the cell wall of gram-negative bacteria. These caspases are able to induce pyroptosis independently of caspase-1, but are only able to trigger IL-1β and IL-18 release in a caspase-1-dependent manner. In this thesis, the roles of caspase-4 and caspase-5 in the response to cytoplasmic lipopolysaccharide (LPS) and invasive gram-negative bacteria have been investigated in a human monocytic cell line. While both caspases responded to infection with live gram-negative bacteria, free LPS that was transfected into the cytoplasm activated only caspase-4. This suggests that caspases-4 and -5 may be activated by distinct stimuli or through different mechanisms. This work also interrogates the role of the inflammasome-forming receptor pyrin, in both autoinflammatory disease and the anti-bacterial immune response. A serine to arginine mutation in pyrin at amino acid position 242 results in a newly described autoinflammatory condition known as Pyrin-Associated Autoinflammation with Neutrophilic Dermatosis (PAAND). A monocytic cell line expressing the S242R mutant of pyrin has been created and it was demonstrated that this mutation results in spontaneous inflammasome activity. Under homeostatic conditions, serine 242 is phosphorylated and interacts with the 14-3-3 family of adapter proteins to keep pyrin inactive. Deletion of specific 14-3-3 isoforms also resulted in spontaneous production of mature IL-1β. Finally, the expression of pyrin in various myeloid compartments and its role in in vivo models of bacterial infection have been investigated using a pyrin-deficient mouse line. Two isoforms of pyrin were detected that were differentially expressed among myeloid populations. Additionally, no role for the pyrin inflammasome was observed in a Dextran Sodium Sulfate (DSS)-induced colitis model, or Citrobacter rodentium, Salmonella Typhimurium or Mycobacterium tuberculosis infection models.
A quantitative analysis of B cell responses to specific antigen
Humoral immune responses arise when B lymphocytes respond to activation signals, enter mitosis and proliferate rapidly. Concurrent differentiation to antibody secreting and isotype switched effector cells is tightly linked to cell division, such that the degree of proliferation strongly influences the nature of the response that is mounted. Previous versions of a quantitative model of lymphocyte proliferation based on inherent variation in the time cells take to divide or die were able to accurately describe the entry of naïve, resting cells into division and subsequent population expansion. In the work described here, the model was tested and extended by investigating the proliferation cessation and population contraction phases of in vitro B cell responses. Experiments designed to assess the distribution of times to die of cells that had ceased proliferating revealed that the number of divisions achieved by individual cells is stochastically distributed in the population and varied in response to different stimuli. Both the concentration and duration of stimulation regulate the number of divisions undergone. A cell that stops dividing is described as having reached its division destiny. Further investigation revealed that cells reach a maximum division destiny even during repeated high-dose stimulation. This limit is dictated by cellular progression through divisions, and is not dependent on the survival capacity of the cells or time. Incorporation of division destiny in the quantitative model allows proliferation cessation to be described and the distribution of times to die after this point to be assessed. This extended model can describe the full course of in vitro lymphocyte proliferative responses to various different stimuli. (For complete abstract open document)
A quantitative analysis of T cell responses to stimulatory inputs
Upon activation, CD8+ T cells undergo rapid proliferation followed by division cessation and eventual cell death. How T cells integrate a range of alternative signal combinations to make decisions affecting tolerance and response strength still poses a considerable theoretical challenge. In this thesis the number of times a cell divides before reverting to a quiescent state, termed its division destiny, was identified as a key proliferation parameter regulated by multiple T cell stimuli. T cell receptor, costimulation and cytokine signals summed together using a linear addition calculus to regulate division destiny. This simple integration mechanism converted the linear additive components into a geometric increase in peak population expansion. As a consequence the strength of the T cell response was profoundly sensitive to combinations of inputs and could be predicted from the sum of the underlying components. A new method to efficiently measure the regulation of T cell division destiny at a clonal level was developed. Using this method it was demonstrated that the progeny cells of an individual CD8+ T cell clone exhibited highly concordant division destiny outcomes. Furthermore, the linear addition of division destiny observed at the population level also applied at the level of the clonal family tree. The regulation of division destiny by the classic T cell growth factor, interleukin-2 (IL-2), was dissected further. Positive feedback between IL-2 and IL-2 receptor alpha chain (IL-2Rα) expression was examined and the relationship with division destiny found to follow a set of simple deterministic rules. This enabled the onset of T cell division destiny to be predicted from the initial IL-2Rα expression and IL-2 concentration in culture. Collectively, these findings demonstrate that a diverse array of signal inputs following a simple set of quantitative principles can sensitively regulate T cell activation and tolerising decisions to generate complex but predictable response outcomes.
A quantitative framework for lymphocyte fate decisions
During an adaptive immune response activated B and T lymphocytes undergo rapid clonal expansion and generate extensive cellular heterogeneity. How lymphocytes guarantee the emergence of functional diversity amongst responding cells is not fully understood. In this thesis, the strategies utilised by the adaptive immune system for the diversification of B and T cells is investigated at the cellular, molecular and clonal levels in a quantitative manner. Activated B cell heterogeneity is predominantly driven by two critical programs. Firstly, the differentiation of antibody-secreting cells (ASCs) and secondly, the diversification of antibody isotype by class switch recombination (CSR). The regulation of these two processes was investigated through combined clonal and molecular analysis using a high-throughput proliferative lineage tracing approach to study ASC differentiation and CSR across thousands of clones. Two distinct fate programs emerged. Firstly, the timing of ASC differentiation within clones was strongly correlated. Diversity in commitment to the ASC lineage is established early and could be traced to the naive founder cell, from where it is transmitted to all progeny during clonal expansion. In striking contrast, isotype switching was highly variable across related cells irrespective of common ancestry, revealing a highly stochastic, cell-autonomous process regulated late within activated single cells. Further analysis demonstrated that single cells faced with a choice of two heavy chain isotypes solve the conflict using stochastic selection that is independent of their clonal lineage. As the principle molecular drivers of CSR are well known, their variation amongst single cell within clonal families was measured. Extensive variation was demonstrated in the expression of both activation-induced cytidine deaminase (AID) and the transcription of the germline noncoding RNAs. Furthermore, there was no correlation between AID expression and germline transcription, nor was the expression of distinct germline transcripts correlated. Thus, the net effect of stochastic influences over these two components can account for the single cell autonomy governing CSR. This stochastic molecular mechanism of CSR was developed into a quantitative model that accurately described and predicted B cell fate decisions across cell division and under varying experimental conditions. Quantitative analysis was applied to multi-parameter data of CD8 T cell heterogeneity, generated in response to diverse external stimulation. Using a combination of novel and established analytical techniques, the influence of time and division progression on T cell diversification, and their control by external signals, was accurately measured. The results of this investigation was subsequently used to construct a kinetic model of time- and division-dependent expression patterning for the molecule CD69 under varying external conditions. This model accurately described the expression dynamics of CD69 over time and division and highlighted the utility of a quantitative modelling approach to understanding CD8 T cell heterogeneity. Collectively, the work presented in this thesis represents a set of quantitative principles that describe lymphocyte fate decisions.
Actin regulation in Plasmodium falciparum: towards understanding the elusive nature of malarial actin filaments
Malaria disease, caused by the unicellular parasites from the genus Plasmodium, is a major cause of morbidity and mortality in many developing countries throughout the world. While there have been many improvements in intervention strategies in recent years, parasite resistance to front-line therapeutics is on the rise, highlighting the need for new and improved treatments and vaccines. To this end, a greater understanding of the biological mechanisms underpinning the disease will be crucial in the push towards malaria eradication. Across the malaria life cycle the parasite must traverse tissues and invade host cells in order to establish an infection and replicate. A conserved acto-myosin motor, anchored at the parasite periphery, generates the requisite force to drive the parasite forward, facilitating both invasion and motility. The actin at the heart of this motor is extremely divergent, forming filaments that are highly dynamic and unstable. Tightly controlled regulation of malaria actin is therefore necessary to direct the formation and disassembly of filaments in an appropriate spatio-temporal manner. However, malaria parasites possess a markedly reduced repertoire of actin regulators, of which coronin is one of the only predicted filament regulators. Much of the current literature surrounding Plasmodium actin biology relies on the production of actin from recombinant sources. In this study I investigate the various published methods for purifying recombinant malaria actin, and determine that the unusual characteristics previously reported for this actin are likely artifacts driven by incomplete protein folding in heterologous expression systems. This finding lead to the identification of the key actin folding chaperonin CCT in the Plasmodium genome, an essential protein complex required for producing native, functional actin in the cell. In parallel, characterization of the filament regulator, coronin, revealed its critical role in the organization of actin filaments. Using in vitro observations from recombinant Plasmodium falciparum coronin (PfCoronin), I have demonstrated that PfCoronin binds to actin filaments and bundles them together in parallel arrays. Furthermore, in vivo observations revealed PfCoronin to be located at the periphery of the parasite, consistent with the pellicular space in which the actin-myosin motor is housed. This localization is likely mediated by peripheral interactions with PI(4,5)P2 at the plasma membrane. These data identify PfCoronin as a potentially key regulator of actin filament recruitment and bundling at the cell cortex of motile Plasmodium parasites. Taken together, the identification of Plasmodium CCT and the characterization of PfCoronin have opened up new avenues for further development of these as potential drug targets, with the eventual aim of potentially crippling the motile malaria parasite and halting the progression of disease.
Advancing a functional cure for HIV by identifying therapeutics that promote the death of latently infected cells
The persistence of a replication-competent HIV reservoir necessitates life-long antiretroviral adherence and precludes the possibility of a HIV cure via conventional therapy alone. Furthermore, recent clinical studies have made it increasingly clear that the predominant strategy for reservoir elimination, enforced transcriptional reactivation, does not diminish the size of the latent reservoir or reduce the time to viral rebound following treatment interruption. A novel approach seeks to purge the HIV reservoir by activating apoptotic pathways in latently infected cells and shifting the balance away from survival and towards cell death. Several lines of evidence implicate Bcl-2 family proteins in the long-term survival of memory CD4+ T cells – the major reservoir for HIV. Bcl-2 antagonism thus represents a viable strategy for sensitizing latent cells to death and delaying viral rebound. The development and clinical progression of BH3-mimetics, which induce apoptosis by binding pro-survival Bcl-2 homologs, has resulted in a well- characterised class of inhibitors with relatively few unknowns regarding toxicity, side effects and dosage. In this thesis, I hypothesise that there are apoptotic blocks in place, specifically a greater dependence on pro-survival Bcl-2 proteins, which prevent a minority of infected CD4+ T cells from dying during active infection. I hypothesise that latently infected cells are distinct from other infected or healthy cells, and that this pro-survival phenotype allows them to persist in such a way that renders them susceptible to pro- apoptotic therapeutics which target the intrinsic pathway, such as BH3-mimetics. In Chapter 3, I infect primary human CD4+ T cells with HIV in vitro to assess the ability of BH3-mimetics to kill actively infected cells. I demonstrate that ABT-737 and Venetoclax, but not the Mcl-1 inhibitor S63845, preferentially kill activated, HIV infected CD4+ T cells in the setting of productive viral replication. These results shed light on the pro-survival role of Bcl-2 proteins during active HIV infection, and inform our progression into a preclinical model of HIV latency. Chapter 4 uses a humanized mouse model of HIV latency to further interrogate the importance of Bcl-2 pro-survival proteins in reservoir survival. I investigate the ability of Venetoclax, a clinically-approved Bcl-2 antagonist, as well as S63845, a preclinical Mcl-1 inhibitor, to delay viral rebound following analytical treatment interruption. This work provides the first compelling evidence that BH3-mimetics, either as monotherapy or in combination, can eliminate latently infected cells in vivo. In Chapter 5 I perform a tat/rev Induced Limiting Dilution Assay (TILDA) on CD4+ T cells from latently infected mice in order to quantify the impact of Venetoclax on the magnitude of the latent HIV reservoir. I confirm the existence of an inducible reservoir in our mouse latency model, although I do not observe a significant effect of Venetoclax treatment as measured by TILDA. I also use single-cell RNA sequencing to characterize peripheral CD4+ T cells from ART-suppressed human donors following Venetoclax treatment ex vivo, arriving at the suggestion that Venetoclax may target CD4+ T cells that are enriched for a gene signature associated with activation and cell metabolism. This work lays the foundation for furthering our understanding of which cells may contribute to HIV persistence and which may be susceptible to death- inducing compounds. Overall, this thesis represents a comprehensive assessment of the ability of BH3-mimetics to kill HIV active and latently infected cells, offering a strong justification for the translation of pro-apoptotic therapeutics such as Venetoclax into a clinical setting where reservoir eradication is the goal.
Advancing BH3 mimetics to treat cancers
The evasion of apoptosis, one of the hallmarks of cancer, is observed in many cancers. This can also impair the efficacy of many conventional chemotherapies. The BCL2 protein family is the central regulator of the intrinsic apoptotic pathway and plays a vital role during tumor development. In particular, the levels of the pro survival family members are often elevated in some cancers. Venetoclax, a BH3 mimetic inhibitor that mimics the BH3-only proteins, natural inhibitors of the pro-survival BCL2 proteins, has proven to be effective for treating hematological cancers by selectively targeting BCL2. This has translated into regulatory approvals of venetoclax for treating a subset of chronic lymphocytic leukemia and acute myeloid leukemia. In addition to targeting BCL2, potent and specific BH3 mimetic inhibitors of its relatives, BCLxL and MCL1, are now also available. However, their full clinical utility is poorly defined. This thesis focuses on advancing the utility of the BH3 mimetic compounds as anti-cancer agents. Previous studies have suggested roles for BCLxL and MCL1 in many solid cancers (e.g. colon, breast, lung). In particular, colorectal cancers have elevated levels of the pro survival protein, one usually associated with chemo resistance. Furthermore, colorectal cancer patients with advanced disease or those who carry poor prognostic markers do not respond well to the current stand of care therapies such as surgery and adjuvant chemo/radiotherapy. Given the pressing need to find better treatments for these patients, we first utilized a panel of validated BH3 mimetics to assess the feasibility of using them for treating colorectal cancer. By using cancer cell lines and patient-derived organoids, we identified and validated BCLxL and MCL1 as the most important survival factors for colorectal cancer. We then validated them as potential targets by pharmacological inhibition in a mouse model in vivo. Moreover, we found that even those tumors that harbor poor prognostic factors respond as avidly as those do not, further highlighting the potential of this approach for treating patients with colorectal cancer. Even though the targeting BCLxL might be a possible approach to kill cancers that depend on it, the clinical use of BCLxL selective inhibitors is limited due to the toxicity of BCLxL inhibition on platelets. I have screened for novel regulators of BCLxL using the CRISPR/Cas9 technology, which might offer potential approaches to target BCLxL safely. The final goal of this thesis is to identify biomarkers that predict response to BH3 mimetics, given that there are few reliable tools to stratify patients that might respond well to these novel anti-cancer agents. By using large scale transcriptomic datasets from publicly available RNA sequencing studies, I was able to identify a few candidate genes and achieve reasonable prediction performance.
Analysing the impact of the absence of CARD containing caspases on different forms of cell death
Cell death is an important process during embryogenesis as well as tissue homeostasis in the adult. Apoptosis, pyroptosis and necroptosis are three of the major programmed cell death pathways. Dysregulation of either of these cell death pathways can promote the development of a variety of diseases, such as cancer or autoimmune pathologies. Cysteine-dependent aspartate-specific proteases, known as caspases, exert key functions in all of these cell death pathways. Of note, certain caspases have been shown to play a role in more than one cell death pathway. This thesis presents the functional analysis of different caspases, in particular caspase activation and recruitment domain (CARD) containing caspases and their contributions to the pyroptotic, apoptotic and other cell death pathways. We have generated a novel triple knockout mouse strain deficient for the CARD containing caspases-1, -11 and -12. We initially used this strain to improve our understanding on the contributions of caspases-1, -11 and-12 to sepsis and different forms of cell death. Previous studies have suggested a role for caspase-12 in endoplasmic reticulum (ER) stress-induced cell death. However, we were not able to attribute a role of caspase-12 to sepsis or ER stress-induced apoptosis in vitro and in vivo. In Chapter 4 we present a study on the roles of different caspases as well as RipK3 during Salmonella infection in vitro and in vivo. There is evidence for a substantial functional overlap between different cell death pathways in the cellular response to pathogens, such as Salmonella. We examined this functional overlap of different cell death processes in the organismal and cellular response to infection by generating mice deficient for multiple caspases and also RipK3, an essential mediator of necroptotic cell death. Upon infection with S. Typhimurium SL1344 strain, primary myeloid cells from caspase-1/11/12/8 RipK3-/- mice showed marked resistance to cell death and survived even at high bacterial loads for up to 24 hours. When infecting the caspase-1/11/12/8 RipK3-/- mice with the vaccine Salmonella Typhimurium strain, they were not able to clear the bacteria from primary organs. Collectively, these findings provide evidence that there is substantial functional overlap between the different cell death pathways and hence the caspases involved in these processes in the cellular as well as organismal response to infection with S. Typhimurium and possibly other pathogens. Lastly, I generated mice lacking all murine CARD containing caspases, i.e. caspase-1, -11, -12, -2 and -9. These preliminary analyses revealed no major defects when comparing the embryonic development of mice lacking caspases-1, -11, -12, -2 and -9 to wildtype. Furthermore, we isolated haematopoietic stem and progenitor cells (HSPCs) from foetal livers derived from caspase-1/11/12/2/9 deficient mice and reconstituted lethally irradiated wildtype mice. Surprisingly, we did not find notable defects in the lymphoid and myeloid compartments in the caspase-1/11/12/2/9 deficient mice at steady state. In thymocyte cell death assays, cells from the quintuple caspase knockout mice still could undergo cell death, induced by the cytotoxic agent ionomycin, albeit at a delayed rate.
Analysis of 6-cys proteins and calcium fluxes during erythrocyte invasion by Plasmodium falciparum parasites
Plasmodium parasites amplify their population within the human host by invading, growing and replicating within the body’s erythrocytes. When the population becomes high enough, the damage caused produces symptomatic malaria disease. To develop new drugs and vaccines against malaria it is therefore important to know as much possible about how parasites grow within the human host and particularly about how the extracellular merozoite stage invades erythrocytes, since this short-lived stage is highly vulnerable. This thesis provides new information from the most deadly human malaria pathogen P. falciparum, on the biochemical characteristics of a little known family of merozoite surface proteins which were thought to facilitate erythrocyte invasion as well revealing with unprecedented resolution, new details about how merozoites enter erythrocytes. P12, P38, P41, and P92 comprise a group of blood-stage merozoite surface proteins that belong to the 6-cys family and all except P41 are predicted to have membrane anchors. To functionally characterize the proteins, specific antibodies were made and were then employed to block merozoite invasion by interfering with the binding of 6-cys to erythrocytes. The effect of the antibodies was very weak and therefore not indicative of a major role for 6-cys in invasion. The antibodies were then used as localization probes and indicated that P12 and P41 were at the merozoite periphery with some concentrated towards the apex. In addition, the non-anchored P41 was held on the merozoite surface through heterodimerization with the membrane anchored P12. Despite the P12/P41 heterodimer being in prime position to bind erythrocytes during invasion no evidence for binding could be established. Characterisation of P92 was next conducted and revealed that like the P12/P41 heterodimer, it was tightly associated with the parasite membrane and later cleaved off possibly during invasion. On the other hand, P38 did not shed from the merozoite surface, and it was carried into the erythrocyte. P92 was strictly localised to the apical end of the merozoite while P38 displayed both apical and surface localisation. Similar to the P12/P41 heterodimer, P92 does not appear to bind erythrocytes. In a final attempt to derive a function for the blood stage 6-cys, their genes were individually knocked out but none of the mutants produced any defective growth or invasion phenotypes suggestive of function. To further study invasion, the morphology and kinetics of this process in P. falciparum merozoites was examined with high-speed live-cell microscopy. With greater temporal resolution, novel cellular actions of the merozoites were observed. For example, during the 7.5 s pre-invasion phase the merozoite deforms the erythrocyte plasma membrane multiple times whilst re-orientating. After a brief rest, the merozoite invaded over a ~17 s period forming a vacuole mainly from wrapping the erythrocyte’s membrane around itself. About 18.5 s after entry, the merozoite began spinning in a clockwise direction to possibly to help disconnect itself from the erythrocyte membrane. After spinning had commenced the host erythrocyte began to develop a spiculated appearance called echinocytosis. Suspecting that calcium influx into the erythrocyte during invasion might be responsible for the echinocytosis, the appearance of these fluxes was monitored during invasion by live cell imaging. These observations confirmed for the first time, that a calcium flux originated as an intense spot emanating from the area of contact between the merozoite and erythrocyte suggestive of pore formation between the cells. Further experiments with modified levels of calcium indicated the ion is required for efficient invasion and may play role in causing echinocytosis. Other work using the calcium flux as a visual marker indicated that pore formation coincided with the deployment of tight adhesive proteins from the merozoite that commit it to invasion. The live cell imaging work presented therefore sheds considerable light on many details of merozoite invasion that could inform future drug and vaccine development. Supplementary Videos: Video 1. High-speed time-lapse acquisition of 3D7 merozoite invading the erythrocyte (40 fps). Video 2. The 3D7 merozoite invading the BODIPY FL C12-sphingomyelin labelled erythrocyte (2 fps, 2× real speed). Video 3. The 3D7 merozoite invading the erythrocyte in the presence of Fluo-4 AM showing the punctate apical calcium and calcium influx in the infected erythrocyte (3 fps, 8× real speed) Video 4. Fluo-4-stained 3D7 parasite culture showing merozoites attempting to invade in the presence of R1 peptide (3 fps, 8× real speed). The punctate apical calcium and influx in the attached erythrocyte were detectable. Video 5. Fluo-4-stained 3D7 parasite culture showing merozoites attempting to invade in the presence of R1 peptide (variable speed). The echinocytotic erythrocyte had not recovered after ~20 min of recording. Video 6. CytD-treated 3D7 merozoite attempting to invade the Fluo-4 labelled erythrocyte (variable speed). The punctate apical calcium was visible but the calcium influx was difficult to observe. The echinocytotic erythrocyte had not recovered after ~20 min of recording.
Analysis of whole-genome sequencing data from cell-free DNA in maternal plasma to detect fetal aneuploidy
Since its discovery in 1948 there has been immense interest in circulating cell-free DNA as a source of genetic material that can be non-invasively obtained in a routine phlebotomy. With the rapid development of next-generation sequencing and the decreasing cost of associated reagents and platforms, the potential of cell-free DNA as a biomarker has been explored in diverse research areas such as in scanning the genetic landscape of tumors and the monitoring of organ transplant rejection. However, non-invasive prenatal testing has been the field that has experienced the greatest research reward including a subsequent translation to the clinic. A complex mixture of maternal and fetal DNA fragments circulates in the plasma of pregnant women and next-generation sequencing allows the high-resolution interrogation of this mixture to detect fetal chromosomal abnormalities. Prenatal screening based on cell-free DNA sequencing has been one of the most rapidly adopted genomic tests and is now widely available around the world. Current tests focus on detecting common autosomal trisomies such as Down Syndrome (trisomy 21) and aneuploidy conditions of sex chromosomes. Although these tests report high accuracy estimates, both false positive and false-negative results occur. Biological factors such as a low fetal DNA concentration and technical factors such as bias in the next-generation sequencing data have been implicated as the cause of some discordant results. However, recent reports show that the sequencing-based tests have facilitated a significant reduction in the number of invasive diagnostic procedures that have been performed. This thesis describes various statistical and bioinformatics analyses carried out on whole-genome sequencing data from cell-free DNA in maternal plasma. Most published studies focus on the diagnostic applications of cell-free DNA with only a few investigating its biological characteristics. The thesis first aims to expand the understanding of cell-free DNA by examining characteristics related to the biological cleavage processes that produce the fragments circulating in blood. This work has successfully uncovered novel sequence signatures associated with the origin of cell-free DNA along the human genome, the distribution of fragment sizes both between and within chromosomes and the nucleotide motifs stemming from the enzymatic cleaving of DNA. The secondary focus of this thesis is the implementation of bioinformatics algorithms that reduce the bias in next-generation sequencing data due to technical and biological factors such as GC content, genomic repeats and non-random fragmentation of DNA. Reducing the noise in maternal plasma sequencing data would facilitate the detection of any signal due to copy number changes in the fetal genome. The biological characteristics investigated under the first aim, such as fragment lengths and cleavage motifs are used to tailor the bias correction methods to fit the intricacies of cell-free DNA sequencing data. The methods applied in the thesis show substantial improvement in the sensitivity of trisomy 21 detection compared to the conventional bias correction protocols used in the literature. The proportion of fetal DNA in maternal plasma is one of the most important factors that affect the accuracy of cell-free DNA based tests. Estimating this factor prior to testing can identify samples with a fetal fraction too low to ensure a reliable result. The third and final aim for this thesis is to investigate an approach that can quantify fetal DNA directly from low coverage or ‘low-pass’ whole-genome sequencing data. The developed methodology exploits uneven sequence coverage due to the aforementioned technical and biological biases and genotypes known SNPs at regions with excess reads. A mixture model framework incorporates this allelic information and a maximum likelihood based algorithm is implemented to accurately calculate the proportion of fetal DNA.
Apoptotic caspases: silencing the mitochondrial danger within
Mitochondrial apoptosis is mediated by BAK and BAX, two proteins that, upon activation, oligomerise in the mitochondrial outer-membrane to induce its permeabilisation (MOMP). This event allows cytochrome-c efflux from the mitochondria subsequently triggering a family of cysteine-dependent, aspartic-specific proteases, the caspases. These caspases, once initiated, cleave hundreds of substrates to bring about global cellular demolition. In doing so, they mediate many key characteristics of apoptosis, such as DNA laddering, membrane blebbing, and phosphatidylserine exposure. For many years caspases were thought to be essential for death, but it is increasingly apparent that they do not instigate the killer event, instead acting to accelerate cellular demise. Furthermore, there exists a burgeoning literature suggesting apoptotic caspases may have functions beyond cell death. One such study, reported apoptotic caspases also control hematopoietic stem cell (HSC) proliferation and function, and recent work from our lab suggested this phenotype was driven by increased levels of circulating Type I interferons (IFNs). Yet the molecular pathway responsible for the increased Type I IFN secretion, and how the apoptotic caspases fit into this response, was unknown. The work presented here, characterizes the mechanism by which caspase blockade drives an IFN response. It shows that during intrinsic apoptosis, BAK/BAX-mediated damage to the mitochondria not only triggered cytochrome-c release but also, the subsequent efflux of mitochondrial DNA (mtDNA) into the cytoplasm. In the absence of caspase activation, mtDNA activated the innate anti-viral cGAS/STING-signaling pathway to induce IFN production. To further this investigation, a live-cell imaging assay was developed, which utilized lattice light-sheet microscopy, to document mitochondrial morphology and behavior during apoptosis. The resulting images showed mtDNA was delivered to the cytoplasm via an orchestrated process involving mitochondrial fragmentation and inner membrane herniation through large BAK/BAX pores. This event was downstream of BAK/BAX activation, occurred independently of caspases, and was assisted by, but not reliant on, DRP1-mediated fission. Thus, mtDNA release is a common consequence of BAK/BAX-mediated MOMP, however subsequent caspase activation prevents mtDNA-triggered IFN production from dying cells, thereby maintaining the immunological silence of apoptosis. Despite the extensive literature implicating mtDNA in disease, the images presented in this thesis represent the very first demonstration of mtDNA release in real-time, in any setting. Thus, this live-cell imaging assay presents an exciting opportunity to further our understanding of mtDNA release in a wide range of human pathologies. Furthermore, this thesis also presents preliminary data demonstrating that pharmacological caspase inhibition is capable of driving apoptotic-IFN production in vivo, and suggests that caspase-inhibitors may have thus-far unappreciated potential as anti-viral and anti-cancer therapies.
Aspartic proteases and their potential for transmission blocking strategies
Sexual stage development in Plasmodium spp. is essential for transmission through the mosquito and to the human host. It represents objects to study a broad range of biological processes, including stage conversion and parasite/host co-adaptation. After the bloodmeal, male and female gametes emerge from intracellular gametocytes and zygote formation follows fertilization. Ookinetes develop from the zygote and traverse through the midgut epithelial cell layer to the basal lamina side of outer wall and develop into oocysts, the only parasite developmental stage that grows extracellularly and this growth and development creates thousands of sporozoites. Once fully developed and egressed, these sporozoites are released into the mosquito hemocoel and they migrate to the salivary gland ready to infect next mammalian host and continue their life cycle. This sexual stage also represents a major bottleneck during the life cycle of Plasmodium as, in mosquito midgut, parasites have to persevere for up to 24 hours outside host cell, exposed themselves to various risk factors such as components of human immune system included within bloodmeal, natural midgut microbial flora in mosquito midgut, and mosquito innate immune system. This exposure can lead up to an approximate 300-fold decrease in parasite survivability during the transmission to mosquito. Due to this unique feature, sexual stage is prime target for transmission blocking intervention strategies aimed to inhibit spread of the disease by the mosquito. Protease enzymes are essential during many steps of malaria parasite development in the blood and transmission stages and an important group of these enzymes are the plasmepsins, of which there are 10 in Plasmodium acting at various points through the life cycle. So far, only 4 plasmepsins are identified to be involved in critical processes and required for transmission. Firstly, plasmepsin VI is highly expressed during sexual stages and was previously shown to be involved in sporozoite development in P. berghei. Secondly, plasmepsin VIII is expressed in mature sporozoite and responsible for sporozoite motility in P. berghei. Finally, PMIX and X are found to be essential in both blood and mosquito stages, making them stand out as promising drug targets. In this study, we attempted to determine the biological functions of plasmepsin VI, IX, and X during transmission of malaria parasites. We found that plasmepsin VI is required for transmission of P. falciparum and might plays an important role in sporozoite egress process instead of sporozoite development as observed in P. berghei. We also found that our dual inhibitor that target both plasmepsin IX and X is able to block the transmission of P. falciparum to mosquito while another antimalaria compound that target only plasmepsin X is enough to block transmission of P. berghei from mouse to mosquito suggesting that both plasmepsin IX and X are essential for transmission. Taken together, our data has identified 3 plasmepsins that play important roles in sexual stage of malaria parasites and more works are needed in order to determine the mechanism of action of these 3 proteases.