Deterministic and Stochastic Modeling of Human Papillomavirus Gene Regulatory Network

In this thesis a novel stochastic and deterministic mathematical model of Human papillomavirus (HPV) gene regulatory network was developed. The novelty of this project is both on methodological and biological /clinical site. The former is in line with the current challenge in recent years to have a holistic view of the basics regulatory mechanisms interconnected to form a complex machinery, where complex patterns can arise, only form the interconnection of basics modules. In fact, HPV offers a case of study of great interest in molecular systems biology. It involves a number of relevant regulatory mechanisms (e.g. transcription, translation, promoter modulation, polyadenylation regulation, splicing,…) connected together to form a complex network, albeit its genome is relatively simple, thus suitable for an accurate deterministic and even stochastic modeling. HPVs cause a series of diseases of the cutaneous and mucosal epithelium, ranging from minor lesions to precancerous cervical lesions and cervical cancer, which is considered one of the most common cancer in the women worldwide. Therefore, on the biological/clinical aspect the development of a mathematical model of HPV gene expression, is of great interest in order to dispose of an in silico simulator useful to achieve a better comprehension of the complex gene regulatory network, and capable to predict different scenarios from the first stages of viral infection up to a cervical cancer condition. As far as we know, there is no model of HPV gene regulation available in literature. A new synthesis of the HPV molecular biology with especial regard to gather/infer from literature the parameters useful for designing a dynamical model, and to shed light in what is still lacking in the biological literature, was preformed. The biological knowledge was translated into a stochastic model in terms of biochemical reactions. In particular, we modeled the HPV early and late promoters that account for the transcripts and proteins evolution during the entire viral life cycle. Even the post-transcriptional and post-translational modifications were modeled in order to properly capture the complex viral regulation known from literature. As far as we know, it is the first time a stochastic model accounts for the complex post-transcriptional control, modeling the splicing and polyadenylation sites regulation, and connect this latter to the transcriptional control layer, mediated by the promoters activities, in order to explore complex patterns that can arise only from the interconnection of different control layers. The Master Equation (ME) of the system was considered in order to predict and investigate its stochastic behavior. Because of the complex system structure it wasn't possible to solve the whole ME analytically, hence numerical exact simulations were performed by means of the Gillespie's algorithm. A quasi-equilibrium approximation of the ME was developed in order to get a deterministic approximation of the model. The model structure together with the fixed parameters we have gathered/inferred from literature was able to fit a dataset consistent of the early promoter activity and to qualitatively reproduce the main dynamical behavior of two of the most important regulatory transcripts during viral late phase. Different in silico experiments were designed to opportunely explore both the capability of the stochastic model to follows the deterministic predictions, when in fast fluctuations regimen, and to discover complex stochastic patterns, that can arise through the interconnection of the transcriptional and post-transcriptional control layers. In general, both the stochastic and deterministic formulation of the model showed the capability to reproduce the HPV gene expression dynamics, during the entire viral life cycle, in good agreement with the current biological knowledge

Uurimus signaalide töötlemisest S. cerevisiae tsükliinsõltuva kinaasi inhibiitori Sic1 mitmikfosforüülitavatel radedel

Väitekirja elektrooniline versioon ei sisalda publikatsiooneRakkude jagunemine käivitatakse DNA-sünteesi algatamisega. See toimub aga alles siis, kui rakud on valmis kogu järgneva raku jagunemise tsükli peatumata läbi tegema. Rakkude jagunemise käivitavad ja kogu edasist protsessi juhivad tsükliinsõltuvad kinaasid (Tsk). Need on valgud, mis kontrollivad teiste valkude asukohta, eluiga või aktiivsust neile foforhappejääke lisades. Rakutsükli etapid käivitatakse erinevate kindla spetsiifikaga Tsk komplekside poolt. Rakkude jagunemist kontrolliva mehhanismi uurimiseks kasutatakse laialdaselt mudelorganismina pagaripärmi S. cerevisiae, mille jagunemise tsükli üldine ülesehitus meenutab lihtsustatult inimese rakkudes toimuvat. Pärmirakkude kasvufaasi (G1) käigus rakud akumuleerivad mitmeid Tsk´se. Osad TSK´d on koheselt aktiivsed ning asuvad oma sihtmärke fosforüülima, valmistades rakku S-faasiks ette. Osad Tsk´d, mille aktiivust läheb tarvis alles S-faasi käivitamiseks ja läbiviimiseks, toodetakse inhibeeritud olekus. Nende aktiivsus on blokeeritud valgu Sic1 poolt. Kogu S-faasi käivitamiseks vajalik Tsk toodetakse kasvufaasis tihedalt kompleksis Sic1 valguga, et vältida juhuslikku S-faasi käivitava aktiivsuse lekkimist enne õiget aega. S-faasi käivitamiseks tuleb Sic1 lagundada, mis vabastab S-faasi spetsiifilise Tsk aktiivsuse. Sic1 ise on Tsk´de sihtmärk ja sisaldab palju fosforüülimiseks sobivaid aminohappeid. Nende fosforüülimine suunab Sic1 lagundamisele, mis määrab S-faasi käivitamise ajastuse ja dünaamika ning on üheks keskseks osaks rakutsükli reguleerimise mehhanismis. Varasemalt ei olnud teada, millised kinaasid millise dünaamikaga Sic1 fosforüülivad ning seetõttu puudus detailne mudel, kuidas seeläbi saavutatakse kontroll raku jagunemise käivitamise üle. Käesoleva uurimistöö käigus selgitati välja, mis kinaasid ja millise dünaamikaga Sic1 valku fosforüülivad. Sellega avastastasid uurimistöö autorid kvantitatiivse S-faasi käivitamise kontrollmehhanismi, mis reguleerib minutilise täpsusega rakkude jagunemise algust ja tagab selle käivitamiseks piisava koguse Tsk aktiivsust. Selle mehhanismi keskne osa – mitmiksfosforüülitav valk Sic1 – toimib mikrokiibina, mis salvestab kasvufaasis olevas rakus jagunemist toetavad ja takistavad signaalid. Neid signaale tõlgendatakse S-faasi käivitamise ajaks ja dünaamikaks S-faasi käivitava Tsk poolt Sic1 sidumise käigus.The cell division cycle is initiated with the onset of S-phase where cells replicate their DNA. The DNA-replication is triggered only when cells are sufficiently prepared to conduct following events of cellular division without stopping. The cell division is triggered and following events conducted by cyclin-dependent kinase holoenzymes (Cdk). These multi-component proteins that phosphorylate others to control the localization, lifespan or activity of target proteins. For proper initiation of each cell cycle phase specific Cdk holoenzyme complex is activated. To study the control mechanism of the cell cycle the S. cerevisiae is widely used as a model due to its simplicity and similarity to human cells. During the growth phase (G1) of yeast cells they accumulate different Cdk´s. Some of them are instantly active and begin to phosphorylate their targets preparing cell for the S-phase. Others Cdks necessary to trigger and conduct upcoming S-phase are accumulated in inhibited form. The activity is inhibited by Sic1 protein. All the Cdk necessary for S-phase initiation is accumulated in tight complex with Sic1 protein to avoid early leakage of S-phase kinase activity. To initiate S-phase the Sic1 protein must be degraded that in turn release S-phase specific Cdk activity. Sic1 itself is a Cdk target and contains multiple phosphorylation sites. For Sic1 destruction its phosphorylation from multiple sites is necessary. The phosphorylation of Sic1 determines the timing and dynamics of S-phase onset and has therefore central importance in cell cycle regulating mechanism. Limited knowledge about phosphorylation dynamics of Sic1 shielded quantitative regulatory mechanism of S-phase initiation and left the question of how is the onset of S-phase controlled unsolved. This study unveiled missing details of Sic1 phosphorylation inputs and their quantitative dynamics. Authors discovered a regulatory mechanism, how yeast cells regulate the timing and dynamics of S-phase onset. The central part of this discovery is that Sic1 works as a microprocessor that records growth phase signals and enables these to be transformed to S-phase initiation parameters via S-phase Cdks

KSHV Rta Promoter Specification and Viral Reactivation

Viruses are obligate intracellular pathogens whose biological success depends upon replication and packaging of viral genomes, and transmission of progeny viruses to new hosts. The biological success of herpesviruses is enhanced by their ability to reproduce their genomes without producing progeny viruses or killing the host cells, a process called latency. Latency permits a herpesvirus to remain undetected in its animal host for decades while maintaining the potential to reactivate, or switch, to a productive life cycle when host conditions are conducive to generating viral progeny. Direct interactions between many host and viral molecules are implicated in controlling herpesviral reactivation, suggesting complex biological networks that control the decision. One viral protein that is necessary and sufficient to switch latent Kaposi’s sarcoma-associated herpesvirus (KSHV) into the lytic infection cycle is called K-Rta. K-Rta is a transcriptional activator that specifies promoters by binding DNA directly and interacting with cellular proteins. Among these cellular proteins, binding of K-Rta to RBP-Jk is essential for viral reactivation. In contrast to the canonical model for Notch signaling, RBP-Jk is not uniformly and constitutively bound to the latent KSHV genome, but rather is recruited to DNA by interactions with K-Rta. Stimulation of RBP-Jk DNA binding requires high affinity binding of Rta to repetitive and palindromic “CANT DNA repeats” in promoters, and formation of ternary complexes with RBP-Jk. However, while K-Rta expression is necessary for initiating KSHV reactivation, K-Rta’s role as the switch is inefficient. Many factors modulate K-Rta’s function, suggesting that KSHV reactivation can be significantly regulated post-Rta expression and challenging the notion that herpesviral reactivation is bistable. This review analyzes rapidly evolving research on KSHV K-Rta to consider the role of K-Rta promoter specification in regulating the progression of KSHV reactivation

Rewiring of signaling pathways by HCMV-encoded GPCRs

Smit, M.J. [Promotor]Siderius, M.H. [Copromotor

Bacteriophage 186 - Investigating the role of transcriptional regulators CI, Apl, CII and Tum at the lytic/lysogenic switch during 186 prophage induction

I am not a living entity, but I am very much alive. I am of tiny proportions and yet endowed with immense power. I am constantly waging war across the prokaryotic kingdom and have proven to be an aggressive, formidable and exceptionally deadly enemy of bacteria and archaea. Who am I? I am bacteriophage. Coliphage 186 is a UV-inducible, non-lambdoid temperate phage of the family Myoviridae (genus P2-likevirus). As a temperate phage, 186 has the ability to undergo two alternative modes of development - lytic development is the active, developmental default state and lysogeny is the alternate, dormant state, where the phage DNA integrates into its host’s genome. The lysogenic state is reversible and thus the lytic pathway can be resumed upon activation of the host SOS response, a phenomenon termed prophage induction. To control the entry into, and the transition between these states, 186 employs both a lytic/lysogenic transcriptional switch and an SOS inducible operon, each existing as independent modules in the 186 genome. Whilst extensive studies of 186 have provided significant insights into how the lytic and lysogenic cycles are regulated and into the process of prophage induction, there are a number of unique aspects for which our understanding remained incomplete. To progress our understanding of 186 prophage induction and how this phage makes its developmental decisions, four separate studies we undertaken to investigate the role(s) of four key transcriptional regulatory proteins (CI, Apl, CII and Tum) at the 186 switch. This knowledge was then used to re-wire the 186 modules to design and build a simple bistable memory circuit, capable of switching between alternate states in response to a chemical signal. In Chapter 2, to investigate the role of the CI immunity repressor in prophage induction, we asked, does disruption of CI negative autoregulation reduce prophage induction efficiency? Using the goa8 mutation (a 5bp deletion between the two promoters CI regulates, pR and pL) we demonstrated that when CI negative autoregulation is disrupted, this has a negative impact on prophage induction efficiency. This outcome underlined the importance of 186 being able to establish the correct lysogenic level of CI, so as to not only maintain stable lysogeny, but to remain optimally primed for prophage induction. To investigate the role of the Apl protein, we asked, why does Apl act as a weak transcriptional repressor at pR and pL during prophage induction? A series of hypothesises were framed on the idea that Apl binding at pR.pL is required to control cII, cI and/or int gene expression during prophage induction. With the experimental outcomes resulting in the rejection of all hypotheses however, this investigation contributed only to our understanding of what Apl does not do at the 186 switch. In the context of 186 prophage induction, the role of the Tum antirepressor and the host SOS response were investigated in Chapter 3. Using a series of minimal 186-like UV- and chemically-inducible, chromosomally-integrated reporter systems and a cumic acid-inducible 186 phage, we confirmed that Tum is essential and sufficient in single-copy for stable 186 lysogenic to lytic switching and that the fundamental role of host SOS activation is to induce expression of the tum gene. In Chapter 4, we asked, what is the significance of having a short-lived/protease sensitive CII protein? By replacing the short-lived, wildtype CII with a stabilised variant (CII145), we demonstrated that not only was there a significant bias towards lysogeny, but also that prophage induction efficiency was very strongly inhibited. The outcomes of this study suggested that the key purpose of having a highly active, and rapidly degraded CII is to quickly equilibrate CI levels in a lysogen to ensure the lysogen is established and ready for induction as soon after infection as possible. Lastly, in Chapter 5 we used the data collected throughout this thesis, combined with existing knowledge on 186 to engineer a bacterial whole-cell biosensor that can establish impressively stable cellular memory, with two distinct alternate, stable states. Specific features of the 186 lytic/lysogenic switch and SOS operon were isolated, remodelled and progressively optimised to engineer such a system. We are confident that with the appropriate modifications this system could potentially serve as an environmental sensor or one that can detect and diagnose (e. g. cancer) with high sensitivity and specificity.Thesis (Ph.D.) -- University of Adelaide, School of Biological Sciences, 202

Theoretical and Experimental Examination of EGF Receptor Endocytosis

In der hier präsentierten Arbeit behandeln wir die Zusammenführung von prädiktiven mathematischen Modellen und experimentellen Daten zu EGF Rezeptor Endozytose. Dieser Rezeptor ist vor allem für die Weiterleitung von Wachstumssignalen verantwortlich. In der Arbeit wurde insbesondere die Internalisierung selbst, sowie die Sortierung in Clathrin-abhängige wie -unabhängige Pathways untersucht. Das genaue Verständnis der Mechanismen von EGF Rezeptor Endozytose ist aus mehrfacher Hinsicht erstrebenswert: Endozytose, d.h. die Verlagerung des aktivierten Rezeptors von der Zelloberfläche in intrazelluläre Kompartimente, dient dazu, die Signalweiterleitung (Spezifität, Stärke, Dauer) zu kontrollieren. Entsprechend können abnorme Veränderungen des Endozytoseapparats z.B. durch Mutationen oder virale Infektion schwerwiegende Folgen für den zellulären Haushalt (Krebs) haben. Im ersten Teil der Arbeit präsentieren wir eine Kombination aus mathematischer Modellierung, konfokaler Mikroskopie und Mehrfarben-Flow Cytometry, um die Internalisierung in einzelnen Zellen quantitativ zu erfassen. Das Modell wird benutzt, um die Wirkung von Rezeptorüberexpression auf den Internalisierungsvorgang zu untersuchen (H.S-G et al, zur Publikation akzeptiert in JBC). Im zweiten Teil der Arbeit legen wir ein mathematisches Modell vor, welches die Sortierung von aktivierten Rezeptoren in unterschiedliche Endozytosewege beschreibt. Dies ist nicht nur aus biologischmedizinischen Gründen, sondern auch aus Sicht der mathematischen Modellierung und Systembiologie interessant, da sie als Beispiel für einen allgemeinen zellulären Entscheidungsprozess aufgefasst werden kann. Das hier vorgelegte Modell beinhaltet den Mechanismus eines Schalterverhaltens, welches kontinuerliche Signale in ein 'Alles-oder-Nichts' Outputverhalten umwandelt (H.S-G et al, BMC Systems Biology, 2008). Die mathematische Struktur und die Relevanz für zellbiologische Prozesse dieser Art von Modellen wird in der Einleitung ausführlich besprochen. Schließlich, um das erarbeitete Modell experimentell zu testen, wurden quantitative Vorhersagen abgeleitet, die vor allem mit Hilfe von konfokaler Mikroskopie untersucht wurden. Um die Bilddaten auszuwerten, wurde im Rahmen dieser Arbeit eine Bildverarbeitungssoftware entwickelt, mit der sich Vesikelstrukturen automatisch detektieren und quantifizieren lassen (Manuskript zur Veröffentlichung eingereicht). Diese experimentellen Untersuchungen werden im dritten Ergebnisteil vorgestellt. Hierbei wurde insbesondere selektive Hemmung von Clathrin- oder Caveolin-abhangigen pathways auf das Endozytoseverhalten des EGF Rezeptors untersucht. Das Hauptergebnis dieses Teils der Arbeit ist, dass aktivierte Rezeptoren vorrangig über Clathrin-abhängige Mechanismen internalisieren. Caveolin hingegen ist für den intrazellulären Transport und die Inaktivierung des internalisierten Rezeptors wichtig (Manuskript in Vorbereitung). Insgesamt stellt die Arbeit einen biomathematischen Ansatz zur Beschreibung von Wachstumsfaktoren auf zelluläre Prozesse dar und liefert damit einen Beitrag zu dem sich im Entstehenden befindenden Gebiet der Systembiologie

Doctor of Philosophy

dissertationZBTB33 is the first identified zinc finger containing methyl-CpG binding protein (MBP), the cellular function of which is still unclear in both normal and disease state. Gene regulation by ZBTB33 is mediated through recognition of both methylated as well as sequence specific nonmethylated deoxyribonucleic acid (DNA) sites. DNA recognition is followed by recruitment of corepressors resulting in transcription changes. While evidence suggests that transcriptional regulation by ZBTB33 plays a role in cancer, an extensive analysis of the gene targets and signaling pathways regulated by this protein have yet to be investigated. The goal of this research is to begin to determine how ZBTB33 directs disease relevant transcription. To investigate this, a combination of techniques to characterize in cell global gene occupation, DNA methylation status, and transcriptome changes were utilized. Further, the effect of bimodal DNA recognition exhibited by ZBTB33 on transcriptional regulation and biological pathways in cancer were investigated. Powerful techniques have emerged for investigating genome-wide protein-DNA interactions. One such technique incorporates chromatin immunoprecipitation followed by exonuclease digestion and next generation sequencing (ChIP-exo). While this technique maps global gene occupations of transcription factors at high resolution, it also harbors technique specific issues, including the significant generation of artifact peaks, limiting its application. Thus, a method for generating a background control that substantially reduces these artifacts and improves the confidence level in peak identification for ChIP-exo data sets was developed. Application of our improved ChIP-exo method to ZBTB33 revealed additional issues with ChIP-based techniques coupled with next generation sequencing that can affect a number of systems. Thus, efforts to identify and resolve these issues are discussed. Along with ChIP-based methods, whole genome shotgun bisulfite sequencing (WGSBS) and RNA sequencing (RNA-seq) are other powerful methodologies informing on the global DNA methylation status and transcriptome of a cell, respectively. By combining these techniques with ZBTB33 depletion/overexpression studies and phenotypic assays, we found mechanistic details for how ZBTB33 mediates cell-specific cell cycle regulation in a cancerous cell line. Specifically, we identified a mechanism by which ZBTB33 mediates the cyclin D1/cyclin E1/RB1/E2F pathway, controlling passage through the G1-restriction point and accelerating cancer cell proliferation

Temporal control principles in cell cycle regulation

Cell division is characterized by a sequence of events by which a cell gives rise to two daughter cells. In order to maintain the integrity of the genome, cell growth, chromosome duplication and chromosome segregation must be coordinated in a manner that ensures the faithful transmission of hereditary information from one generation to the next. Mis- segregation of chromosomes, chromosomal abnormalities and de-regulation of cell division timing are well known hallmarks of cancer. Even though the molecular machinery that drives cell division is the same in all tissues cell cycle duration in mammalian cells varies for different cell types: embryonic cells divide faster than differentiated cells, for example. In addition, classical experiments have highlighted a high level of variability in cell cycle duration even within the same cell type. Understanding temporal control of cell division and, in particular, how the same cell cycle regulators control differences in cell cycle length is a fundamental question in cell and developmental biology. To address this question, a multidisciplinary approach combining live cell imaging of human cells and mathematical modelling was used. It was found that duration of mitosis is strictly dependent on feedback regulation networks that control mitotic entry. Positive feedback within these networks ensures that mitosis is short and temporally insulated from upstream cell cycle events. Modulation of regulatory feedbacks was also found to allow fine-tuning of cell division frequency, showing once more the importance of feedback control in temporal regulation of cell division. The work presented in this thesis suggests that understanding cell cycle dynamics at a single cell level provides a powerful way to derive control principles in cell cycle regulation. Moreover, combining experimental approaches with computational analysis allows a quantitative understanding of temporal control in cell division at a single cell level. This is likely to lead to transformative insights into cell cycle in health and disease states.Open Acces

Targeting the Ubiquitin System in Glioblastoma

Glioblastoma is the most common primary brain tumor in adults with poor overall outcome and 5-year survival of less than 5%. Treatment has not changed much in the last decade or so, with surgical resection and radio/chemotherapy being the main options. Glioblastoma is highly heterogeneous and frequently becomes treatment-resistant due to the ability of glioblastoma cells to adopt stem cell states facilitating tumor recurrence. Therefore, there is an urgent need for novel therapeutic strategies. The ubiquitin system, in particular E3 ubiquitin ligases and deubiquitinating enzymes, have emerged as a promising source of novel drug targets. In addition to conventional small molecule drug discovery approaches aimed at modulating enzyme activity, several new and exciting strategies are also being explored. Among these, PROteolysis TArgeting Chimeras (PROTACs) aim to harness the endogenous protein turnover machinery to direct therapeutically relevant targets, including previously considered “undruggable” ones, for proteasomal degradation. PROTAC and other strategies targeting the ubiquitin proteasome system offer new therapeutic avenues which will expand the drug development toolboxes for glioblastoma. This review will provide a comprehensive overview of E3 ubiquitin ligases and deubiquitinating enzymes in the context of glioblastoma and their involvement in core signaling pathways including EGFR, TGF-β, p53 and stemness-related pathways. Finally, we offer new insights into how these ubiquitin-dependent mechanisms could be exploited therapeutically for glioblastoma

CHARACTERIZING pRB’S ROLE IN CELL CYCLE CONTROL, MAMMARY GLAND DEVELOPMENT, AND TUMORIGENESIS: A UNIQUE CONNECTION TO TGF-p SIGNALLING

The ability to respond to anti-growth signals is critical to maintain tissue homeostasis and loss of this proliferative control mechanism is considered a hallmark of cancer. Negative growth regulation generally occurs during the G0/G1 phase of the cell cycle, yet the redundancy and complexity among components of this regulatory network have made it difficult to discern how negative growth cues protect cells from aberrant proliferation. . Transforming growth factor (3(TGF-P) is a crucial mediator of mammary epithelial morphogenesis and can negatively regulate cell cycle progression. TGF-P has been shown to inhibit cyclin dependent kinase activity, which leads to activation of the retinoblastoma protein (pRB) and growth arrest. However, unlike other components of TGF-P cytostatic signalling, pRB is thought to be dispensable for mammary development. Using gene-targeted mice where the LXCXE binding cleft on pRB has been disrupted (R b l^ and RblNF), we have discovered that pRB plays a crucial role in mammary gland development. In particular, Rbl and RblNFmutant female mice have hyperplastic mammary epithelium due to insensitivity to TGF-P growth inhibition. In contrast with previous studies that highlight the inhibition of cyclin/CDK activity by TGF-P signalling, these experiments reveal that active transcriptional repression of E2F target genes by pRB is also a key component of TGF-p cytostatic signalling. However, loss of pRB-LXCXE interactions does not cause overt defects in other TGF-P signalling pathways such as apoptosis and differentiation. Taken together, this work demonstrates a unique functional connection between pRB and TGF-p in growth control and mammary development. iii These findings were extended to explore the importance of the pRB anti­ proliferative response during tumour formation and progression. Cytostatic control is considered a key tumour suppressive mechanism in the mammary gland. Here I show that LXCXE-dependent growth control by pRB blocks formation of mammary tumours in Wap-p53R172Htransgenic mice. In contrast, the same growth control mechanism is unnecessary to protect against Neu or 7,72-dimethylbenz[a]anthracene-induced mammary tumorigenesis. Taken together, this work demonstrates that anti-proliferative control by pRB can act as a barrier against oncogenic transformation. Strikingly, these data also reveals that this tumour suppressive effect is context-dependent