Topoisomerase 1 (Top1)-associated Genome Instability in Yeast: Effects of Persistent Cleavage Complexes or Increased Top1 Levels

Topoisomerase 1 (Top1), a Type IB topoisomerase, functions to relieve transcription- and replication-associated torsional stress in DNA. Top1 cleaves one strand of DNA, covalently associates with the 3’ end of the nick to form a Top1-cleavage complex (Top1cc), passes the intact strand through the nick and finally re-ligates the broken strand. The chemotherapeutic drug, Camptothecin, intercalates at a Top1cc and prevents the crucial re-ligation reaction that is mediated by Top1, resulting in the conversion of a nick to a toxic double-strand break during DNA replication or the accumulation of Top1cc. This mechanism of action preferentially targets rapidly dividing tumor cells, but can also affect non-tumor cells when patients undergo treatment. Additionally, Top1 is found to be elevated in numerous tumor tissues making it an attractive target for anticancer therapies. We investigated the effects of Top1 on genome stability, effects of persistent Top1-cleavage complexes and elevated Top1 levels, in Saccharomyces cerevisiae. We found that increased levels of the Top1cc resulted in a five- to ten-fold increase in reciprocal crossovers, three- to fifteen fold increase in mutagenesis and greatly increased instability within the rDNA and CUP1 tandem arrays. Increased Top1 levels resulted in a fifteen- to twenty-two fold increase in mutagenesis and increased instability in rDNA locus. These results have important implications for understanding the effects of CPT and elevated Top1 levels as a chemotherapeutic agent.

Dynamic Compartmentalization of Persistent UPEC in the Superficial Bladder Epithelium

Urinary tract infections (UTIs) are typically caused by bacteria that colonize different regions of the urinary tract, mainly the bladder and the kidney. Approximately 25% of women that suffer from UTIs experience a recurrent infection within 6 months of the initial bout, making UTIs a serious economic burden resulting in more than 10 million hospital visits and $3.5 billion in healthcare costs in the United States alone. Type-1 fimbriated Uropathogenic E. coli (UPEC) is the major causative agent of UTIs, accounting for almost 90 % of bacterial UTIs. The unique ability of UPEC to bind and invade the superficial bladder epithelium allows the bacteria to persist inside epithelial niches and survive antibiotic treatment. Persistent, intracellular UPEC are retained in the bladder epithelium for long periods, making them a source of recurrent UTIs. Hence, the ability of UPEC to persist in the bladder is a matter of major health and economic concern, making studies exploring the underlying mechanism of UPEC persistence highly relevant.

In my thesis, I will describe how intracellular Uropathogenic E.coli (UPEC) evade host defense mechanisms in the superficial bladder epithelium. I will also describe some of the unique traits of persistent UPEC and explore strategies to induce their clearance from the bladder. I have discovered that the UPEC virulence factor Alpha-hemolysin (HlyA) plays a key role in the survival and persistence of UPEC in the superficial bladder epithelium. In-vitro and in-vivo studies comparing intracellular survival of wild type (WT) and hemolysin deficient UPEC suggested that HlyA is vital for UPEC persistence in the superficial bladder epithelium. Further in-vitro studies revealed that hemolysin helped UPEC persist intracellularly by evading the bacterial expulsion actions of the bladder cells and remarkably, this virulence factor also helped bacteria avoid t degradation in lysosomes.

To elucidate the mechanistic basis for how hemolysin promotes UPEC persistence in the urothelium, we initially focused on how hemolysin facilitates the evasion of UPEC expulsion from bladder cells. We found that upon entry, UPEC were encased in “exocytic vesicles” but as a result of HlyA expression these bacteria escaped these vesicles and entered the cytosol. Consequently, these bacteria were able to avoid expulsion by the cellular export machinery.

Since bacteria found in the cytosol of host cells are typically recognized by the cellular autophagy pathway and transported to the lysosomes where they are degraded, we explored why this was not the case here. We observed that although cytosolic HlyA expressing UPEC were recognized and encased by the autophagy system and transported to lysosomes, the bacteria appeared to avoid degradation in these normally degradative compartments. A closer examination of the bacteria containing lysosomes revealed that they lacked V-ATPase. V-ATPase is a well-known proton pump essential for the acidification of mammalian intracellular degradative compartments, allowing for the proper functioning of degradative proteases. The absence of V-ATPase appeared to be due to hemolysin mediated alteration of the bladder cell F-actin network. From these studies, it is clear that UPEC hemolysin facilitates UPEC persistence in the superficial bladder epithelium by helping bacteria avoid expulsion by the exocytic machinery of the cell and at the same time enabling the bacteria avoid degradation when the bacteria are shuttled into the lysosomes.

Interestingly even though UPEC appear to avoid elimination from the bladder cell their ability to multiple in bladder cells seem limited.. Indeed, our in-vitro and in-vivo experiments reveal that UPEC survive in superficial bladder epithelium for extended periods of time without a significantly change in CFU numbers. Indeed, we observed these bacteria appeared quiescent in nature. This observation was supported by the observation that UPEC genetically unable to enter a quiescence phase exhibited limited ability to persist in bladder cells in vitro and in vivo, in the mouse bladder.

The studies elucidated in this thesis reveal how UPEC toxin, Alpha-hemolysin plays a significant role in promoting UPEC persistence via the modulation of the vesicular compartmentalization of UPEC at two different stages of the infection in the superficial bladder epithelium. These results highlight the importance of UPEC Alpha-hemolysin as an essential determinant of UPEC persistence in the urinary bladder.

Developmental exposure to a complex PAH mixture causes persistent behavioral effects in naive Fundulus heteroclitus (killifish) but not in a population of PAH-adapted killifish.

Acute exposures to some individual polycyclic aromatic hydrocarbons (PAHs) and complex PAH mixtures are known to cause cardiac malformations and edema in the developing fish embryo. However, the heart is not the only organ impacted by developmental PAH exposure. The developing brain is also affected, resulting in lasting behavioral dysfunction. While acute exposures to some PAHs are teratogenically lethal in fish, little is known about the later life consequences of early life, lower dose subteratogenic PAH exposures. We sought to determine and characterize the long-term behavioral consequences of subteratogenic developmental PAH mixture exposure in both naive killifish and PAH-adapted killifish using sediment pore water derived from the Atlantic Wood Industries Superfund Site. Killifish offspring were embryonically treated with two low-level PAH mixture dilutions of Elizabeth River sediment extract (ERSE) (TPAH 5.04 μg/L and 50.4 μg/L) at 24h post fertilization. Following exposure, killifish were raised to larval, juvenile, and adult life stages and subjected to a series of behavioral tests including: a locomotor activity test (4 days post-hatch), a sensorimotor response tap/habituation test (3 months post hatch), and a novel tank diving and exploration test (3months post hatch). Killifish were also monitored for survival at 1, 2, and 5 months over 5-month rearing period. Developmental PAH exposure caused short-term as well as persistent behavioral impairments in naive killifish. In contrast, the PAH-adapted killifish did not show behavioral alterations following PAH exposure. PAH mixture exposure caused increased mortality in reference killifish over time; yet, the PAH-adapted killifish, while demonstrating long-term rearing mortality, had no significant changes in mortality associated with ERSE exposure. This study demonstrated that early embryonic exposure to PAH-contaminated sediment pore water caused long-term locomotor and behavioral alterations in killifish, and that locomotor alterations could be observed in early larval stages. Additionally, our study highlights the resistance to behavioral alterations caused by low-level PAH mixture exposure in the adapted killifish population. Furthermore, this is the first longitudinal behavioral study to use killifish, an environmentally important estuarine teleost fish, and this testing framework can be used for future contaminant assessment.

Physiotherapy for persistent postnatal stress urinary incontinence : a randomized controlled trial

OBJECTIVE: The aim of this study was to compare the effectiveness of multimodal supervised physiotherapy programs with the absence of treatment among women with persistent postnatal stress urinary incontinence. METHODS: This was a single-blind randomized controlled trial. Sixty-four women with stress urinary incontinence were randomly assigned to 8 weeks of either multimodal pelvic floor rehabilitation (n = 21), multimodal pelvic floor rehabilitation with abdominal muscle training (n = 23), or control non–pelvic floor rehabilitation (n = 20). The primary outcome measure consisted of a modified 20-minute pad test. The secondary outcome measures included a Visual Analog Scale describing the perceived burden of incontinence, the Urogenital Distress Inventory, the Incontinence Impact Questionnaire, and pelvic floor muscle function measurements. RESULTS: Two patients dropped out, leaving 62 for analysis. At follow-up, more than 70% of the women in the treatment groups (14/20 in the pelvic floor and 17/23 in the pelvic floor plus abdominal group) were continent on pad testing compared with 0% of women in the control group. Scores on the pad test, Visual Analog Scale, Urogenital Distress Inventory, and Incontinence Impact Questionnaire improved significantly in both treatment groups (all P < .002), whereas no changes were observed in the control group. Pelvic floor muscle function, however, did not improve significantly in either active group. CONCLUSION: Multimodal supervised pelvic floor physiotherapy is an effective treatment for persistent postnatal stress urinary incontinence.

Psychosocial Functioning in Women with Symptoms of Persistent Genital Arousal

Persistent genital arousal disorder (PGAD) is characterized by physiological sexual arousal (vasocongestion, sensitivity of the genitals and nipples) that is described as distressing, and sometimes painful. Although awareness of PGAD is growing, there continues to be a lack of systematic research on this condition. The vast majority of published reports are case studies. Little is known about the symptom characteristics, biological factors, or psychosocial functioning associated with the experience of persistent genital arousal (PGA) symptoms. This study sought to characterize a sample of women with PGA (Study One); compare women with and without PGA symptoms on a series of biopsychosocial factors (Study Two); and undertake an exploratory comparison of women with PGA, painful PGA, and genital pain (Study Three)—all within a biopsychosocial framework. Symptom-free women, women with PGA symptoms, painful PGA, and genital pain, completed an online survey of biological factors (medical history, symptom profiles), psychological factors (depression, anxiety) and social factors (sexual function, relationship satisfaction). Study One found that women report diverse symptoms associated with PGA, with almost half reporting painful symptoms. In Study Two, women with symptoms of PGA reported significantly greater impairment in most domains of psychosocial functioning as compared to symptom-free women. In particular, catastrophizing of vulvar sensations was related to symptom ratings (i.e., greater severity, distress) and psychosocial outcomes (i.e., greater depression and anxiety). Finally, Study Three found that women with PGA symptoms reported some overlap in medical comorbidities and symptom expression as those with combined PGA and vulvodynia and those with vulvodynia symptoms alone; however, there were also a number of significant differences in their associated physical symptoms. These studies indicate that PGA symptoms have negative consequences for the psychosocial functioning of affected women. As such, future research and clinical care may benefit from a biopsychosocial approach to PGA symptoms. These studies highlight areas for more targeted research, including the role of catastrophizing in PGA symptom development and maintenance, and the potential conceptualization of both PGA and vulvodynia (and potentially other conditions) under a general umbrella of ‘genital paraesthesias’ (i.e., disorders characterized by abnormal sensations, such as tingling and burning).

Fatigue two months after myocardial infarction may indicate risk for persistent fatigue

Fatigue two months after myocardial infarction may indicate risk for persistent fatigue

Finite domination and Novikov homology over strongly Z-graded rings

Let L be a unital Z-graded ring, and let C be a bounded chain complex of finitely generated L-modules. We give a homological characterisation of when C is homotopy equivalent to a bounded complex of finitely generated projective L0-modules, generalising known results for twisted Laurent polynomial rings. The crucial hypothesis is that L is a strongly graded ring. 

Chronic inflammation in CF airways - a persistent issue for A20

Cystic Fibrosis (CF) is characterised by prolonged and exaggerated airways inflammation. Despite recent developments to overcome the underlying functional defect in CFTR (cystic fibrosis transmembrane conductance regulator), there is still an unmet need to reduce the inflammatory response. The NF-kB regulator A20 is a key target to normalise the inflammatory response and is reduced in CF. Here, we describe the plethora of functions of A20 as they apply to innate immune function within the airways. Pharmacological compounds can enhance A20 mRNA and protein expression, but we observed a blunted effect in CF primary epithelial cells. In CF cells pre-treatment with gibberellic acid (GA3) shows anti-inflammatory effects only in some patients. We show that cells with higher basal p38 expression respond with an increase in pro-inflammatory cytokines. Furthermore, all CF PNECs show increased p38 mRNA when stimulated in the presence of GA3. Our results suggest that those patients may benefit from therapeutics targeting p38.

Potential adverse health effects of persistent organic pollutants on sea turtles: Evidences from a cross-sectional study on Cape Verde loggerhead sea turtles

[EN] The Cape Verde nesting population of loggerhead sea turtles (Caretta caretta) is the third largest population of this species in the world. For conservation purposes, it is essential to determine how these reptiles respond to different types of anthropogenic contaminants. We evaluated the presence of organochlorine pesticides (OCPs), polychlorinated biphenyls (PCBs), and polycyclic aromatic hydrocarbons (PAHs) in the plasma of adult nesting loggerheads from Boa Vista Island, Cape Verde, and studied the effects of the contaminants on the health status of the turtles using hematological and biochemical parameters. All turtles had detectable levels of non-dioxin like PCBs, whereas dioxin-like congeners (DL-PCBs) were detected in only 30% of the turtles.

Database evolution on an orthogonal persistent programming system: A semi-transparent approach

In this paper the problem of the evolution of an object-oriented database in the context of orthogonal persistent programming systems is addressed. We have observed two characteristics in that type of systems that offer particular conditions to implement the evolution in a semi-transparent fashion. That transparency can further be enhanced with the obliviousness provided by the Aspect-Oriented Programming techniques. Was conceived a meta-model and developed a prototype to test the feasibility of our approach. The system allows programs, written to a schema, access semi-transparently to data in other versions of the schema.

Persistent identifier Studies

The studies have aimed to overcome the confusing variety of existing persistent identifier systems, by; analysing the current national URN:NBN and other identifier initiatives providing guidelines for an international harmonized persistent identifier framework that serves the long-term preservation needs of the research and cultural heritage communities advising these communities about a roadmap to gain the potential benefits. This roadmap also includes a blueprint for an organisation for the distribution and maintenance of the Persistent Identifier infrastructure. These studies are connected to the broader PersId project with DEFF, SURF, DANS, the national libraries of Germany, Finland and Sweden and CNR and FDR from Italy. A number of organisations have been involved in the process: Europeana, the British library, the Dutch Royal Library, the National library of Norway and the Ministry of Education, Flanders, Belgium. PersID - III: Current State and State of the Art (IIIa) & User Requirements (IIIb) (Persistent Identifier: urn:nbn:nl:ui:13-9g4-i1s) PersID - IV: Prototype for a Meta Resolver System/ Work on Standards (Persistent Identifier: urn:nbn:nl:ui:13-wt1-6n9) PersID - V: Sustainability (Persistent Identifier: urn:nbn:nl:ui:13-o4p-8py) Please note that there are also two broader reports on the project as a whole: PersID - I: Project report and II:Communication. For further information please visit the website of the Persistent Identifier project: www.persid.org

ANOSOV MAPS, POLYCYCLIC GROUPS AND HOMOLOGY

Many manifolds that do not admit Anosov diffeomorphisms are constructed. For example: the Cartesian product of the Klein bottle and a torus.