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The obligate intracellular pathogen Chlamydia trachomatis is the causative agent of
trachoma related blindness and the sexually transmitted pelvic inflammatory disease.
Being an obligate intracellular pathogen, C. trachomatis has an intricate dependency
on the survival of the host cell. This relationship is indispensible owing to the fact that
the pathogen spends a considerable fraction of its biphasic lifecycle within a
cytoplasmic vacuole inside the host cell, the so-called chlamydial inclusion. The
cellular apoptotic-signalling network is governed by several finely tuned regulatory
cascades composed of pro- and anti-apoptotic proteins that respond to changes in
the cellular homeostasis. In order to facilitate its intracellular survival, Chlamydia has
been known to inhibit the premature apoptosis of the host cell via the stabilization of
several host anti-apoptotic proteins such as cIAP2 and Mcl-1. While the pro- and
anti-apoptotic proteins are the major regulators of the host apoptotic signalling
network, a class of the small non-coding RNAs called microRNAs (miRNAs) has
increasingly gained focus as a new level of regulatory control over apoptosis.
This work investigates the changes in the host miRNA expression profile post
Chlamydia infection using a high throughput miRNA deep sequencing approach.
Several miRNAs previously associated with the modulation for apoptotic signalling
were differentially expressed upon Chlamydia infection in human endothelial cells. Of
the differentially regulated miRNAs, miR-30c-5p was of particular interest since it had
been previously shown to target the tumor suppressor protein p53. Our lab and
others have previously demonstrated that Chlamydia can downregulate the levels of
p53 by promoting its proteasomal degradation. This work demonstrates that
Chlamydia infection promotes p53 downregulation by increasing the abundance of
miR-30c-5p and a successful infection cycle is hindered by a loss of miR-30c-5p.
Over the last decade, dedicated research aimed towards a better understanding of
apoptotic stimuli has greatly improved our grasp on the subject. While extrinsic
stress, deprivation of survival signals and DNA damage are regarded as major
proponents of apoptotic induction, a significant responsibility lies with the
mitochondrial network of the cell. Mitochondrial function and dynamics are crucial to
cell fate determination and dysregulation of either is decisive for cell survival and
pathogenesis of several diseases. The ability of the mitochondrial network to perform
its essential tasks that include ATP synthesis, anti-oxidant defense, and calcium
homeostasis amongst numerous other processes critical to cellular equilibrium is tied
closely to the fission and fusion of individual mitochondrial fragments. It is, thus,
8
unsurprising that mitochondrial dynamics is closely linked to apoptosis. In fact, many
of the proteins involved regulation of mitochondrial dynamics are also involved in
apoptotic signalling. The mitochondrial fission regulator, Drp1 has previously been
shown to be transcriptionally regulated by p53 and is negatively affected by a miR-
30c mediated inhibition of p53. Our investigation reveals a significant alteration in the
mitochondrial dynamics of Chlamydia infected cells affected by the loss of Drp1. We
show that loss of Drp1 upon chlamydial infection is mediated by the miR-30c-5p
induced depletion of p53 and results in a hyper-fused architecture of the
mitochondrial network.
While it is widely accepted that Chlamydia depends on the host cell metabolism for
its intracellular growth and development, the role of mitochondria in an infected cell,
particularly with respect to its dynamic nature, has not been thoroughly investigated.
This work attempts to illustrate the dependence of Chlamydia on miR-30c-5p induced
changes in the mitochondrial architecture and highlight the importance of these
modulations for chlamydial growth and development.
In mammals, anucleate blood platelets are constantly produced by their giant bone marrow (BM) progenitors, the megakaryocytes (MKs), which originate from hematopoietic stem cells. Megakaryopoiesis and thrombopoiesis have been studied intensively, but the exact mechanisms that control platelet generation from MKs remain poorly understood. Using multiphoton intravital microscopy (MP-IVM), thrombopoiesis and proplatelet formation were analyzed in the murine BM in real-time and in vivo, identifying an important role for several proteins, including Profilin1, TRPM7 and RhoA in thrombopoiesis. Currently, it is thought that blood cell precursors, such as MKs, migrate from the endosteal niche towards the vascular niche during maturation. In contrast to this paradigm, it was shown that MKs are homogeneously distributed within the dense BM blood vessel network, leaving no space for vessel-distant niches. By combining results from in vivo MP-IVM, in situ light-sheet fluorescence microscopy (LSFM) of the intact BM as well as computational simulations, surprisingly slow MK migration, limited intervascular space and a vessel-biased MK pool were revealed, contradicting the current concept of directed MK migration during thrombopoiesis.
Platelets play an essential role in hemostasis and thrombosis, but also in the pathogenesis of ischemic stroke. Ischemic stroke, which is mainly caused by thromboembolic occlusion of brain arteries, is among the leading causes of death and disability worldwide with limited treatment options. The platelet collagen receptor glycoprotein (GP) VI is a key player in arterial thrombosis and a critical determinant of stroke outcome, making its signaling pathway an attractive target for pharmacological intervention. The spleen tyrosine kinase (Syk) is an essential signaling mediator downstream of GPVI, but also of other platelet and immune cell receptors. In this thesis, it was demonstrated that mice lacking Syk specifically in platelets are protected from arterial thrombus formation and ischemic stroke, but display unaltered hemostasis. Furthermore, it was shown that mice treated with the novel, selective and orally bioavailable Syk inhibitor BI1002494 were protected in a model of arterial thrombosis and had smaller infarct sizes and a significantly better neurological outcome 24 h after transient middle cerebral artery occlusion (tMCAO), also when BI1002494 was administered therapeutically, i.e. after ischemia. These results provide direct evidence that pharmacological Syk inhibition might become a safe therapeutic strategy. The T cell receptor chain-associated protein kinase of 70 kDA (Zap-70) is also a spleen tyrosine kinase family member, but has a lower intrinsic activity compared to Syk and is expressed in T cells and natural killer (NK) cells, but not in platelets. Unexpectedly, arterial thrombus formation in vivo can occur independently of Syk kinase function as revealed by studies in Sykki mice, which express Zap-70 under the control of intrinsic Syk promoter elements.