Topological stability through tame retractions

A smooth map is said to be stable if small perturbations of the map only differ from the original one by a smooth change of coordinates. Smoothly stable maps are generic among the proper maps between given source and target manifolds when the source and target dimensions belong to the so-called nice dimensions, but outside this range of dimensions, smooth maps cannot generally be approximated by stable maps. This leads to the definition of topologically stable maps, where the smooth coordinate changes are replaced with homeomorphisms. The topologically stable maps are generic among proper maps for any dimensions of source and target. The purpose of this thesis is to investigate methods for proving topological stability by constructing extremely tame (E-tame) retractions onto the map in question from one of its smoothly stable unfoldings. In particular, we investigate how to use E-tame retractions from stable unfoldings to find topologically ministable unfoldings for certain weighted homogeneous maps or germs. Our first results are concerned with the construction of E-tame retractions and their relation to topological stability. We study how to construct the E-tame retractions from partial or local information, and these results form our toolbox for the main constructions. In the next chapter we study the group of right-left equivalences leaving a given multigerm f invariant, and show that when the multigerm is finitely determined, the group has a maximal compact subgroup and that the corresponding quotient is contractible. This means, essentially, that the group can be replaced with a compact Lie group of symmetries without much loss of information. We also show how to split the group into a product whose components only depend on the monogerm components of f. In the final chapter we investigate representatives of the E- and Z-series of singularities, discuss their instability and use our tools to construct E-tame retractions for some of them. The construction is based on describing the geometry of the set of points where the map is not smoothly stable, discovering that by using induction and our constructional tools, we already know how to construct local E-tame retractions along the set. The local solutions can then be glued together using our knowledge about the symmetry group of the local germs. We also discuss how to generalize our method to the whole E- and Z- series.

Bayesian statistical analysis of bacterial diversity

Bacteria play an important role in many ecological systems. The molecular characterization of bacteria using either cultivation-dependent or cultivation-independent methods reveals the large scale of bacterial diversity in natural communities, and the vastness of subpopulations within a species or genus. Understanding how bacterial diversity varies across different environments and also within populations should provide insights into many important questions of bacterial evolution and population dynamics. This thesis presents novel statistical methods for analyzing bacterial diversity using widely employed molecular fingerprinting techniques. The first objective of this thesis was to develop Bayesian clustering models to identify bacterial population structures. Bacterial isolates were identified using multilous sequence typing (MLST), and Bayesian clustering models were used to explore the evolutionary relationships among isolates. Our method involves the inference of genetic population structures via an unsupervised clustering framework where the dependence between loci is represented using graphical models. The population dynamics that generate such a population stratification were investigated using a stochastic model, in which homologous recombination between subpopulations can be quantified within a gene flow network. The second part of the thesis focuses on cluster analysis of community compositional data produced by two different cultivation-independent analyses: terminal restriction fragment length polymorphism (T-RFLP) analysis, and fatty acid methyl ester (FAME) analysis. The cluster analysis aims to group bacterial communities that are similar in composition, which is an important step for understanding the overall influences of environmental and ecological perturbations on bacterial diversity. A common feature of T-RFLP and FAME data is zero-inflation, which indicates that the observation of a zero value is much more frequent than would be expected, for example, from a Poisson distribution in the discrete case, or a Gaussian distribution in the continuous case. We provided two strategies for modeling zero-inflation in the clustering framework, which were validated by both synthetic and empirical complex data sets. We show in the thesis that our model that takes into account dependencies between loci in MLST data can produce better clustering results than those methods which assume independent loci. Furthermore, computer algorithms that are efficient in analyzing large scale data were adopted for meeting the increasing computational need. Our method that detects homologous recombination in subpopulations may provide a theoretical criterion for defining bacterial species. The clustering of bacterial community data include T-RFLP and FAME provides an initial effort for discovering the evolutionary dynamics that structure and maintain bacterial diversity in the natural environment.

Endoplasmic reticulum stress and cell death mechanisms in the cell model of Huntington’s disease

This thesis clarifies important molecular pathways that are activated during the cell death observed in Huntington’s disease. Huntington’s disease is one of the most common inherited neurodegenerative diseases, which is primarily inherited in an autosomal dominant manner. HD is caused by an expansion of CAG repeats in the first exon of the IT15 gene. IT15 encodes the production of a Huntington’s disease protein huntingtin. Mutation of the IT15 gene results in a long stretch of polyQ residues close to the amino-terminal region of huntingtin. Huntington’s disease is a fatal autosomal neurodegenerative disorder. Despite the current knowledge of HD, the precise mechanism behind the selective neuronal death, and how the disease propagates, still remains an enigma. The studies mainly focused on the control of endoplasmic reticulum (ER) stress triggered by the mutant huntingtin proteins. The ER is a delicate organelle having essential roles in protein folding and calcium regulation. Even the slightest perturbations on ER homeostasis are effective enough to trigger ER stress and its adaptation pathways, called unfolded protein response (UPR). UPR is essential for cellular homeostasis and it adapts ER to the changing environment and decreases ER stress. If adaptation processes fail and stress is excessive and prolonged; irreversible cell death pathways are engaged. The results showed that inhibition of ER stress with chemical agents are able to decrease cell death and formation of toxic cell aggregates caused by mutant huntingtin proteins. The study concentrated also to the NF-κB (nuclear factor-kappaB) pathway, which is activated during ER stress. NF-κB pathway is capable to regulate the levels of important cellular antioxidants. Cellular antioxidants provide a first line of defence against excess reactive oxygen species. Excess accumulation of reactive oxygen species and subsequent activation of oxidative stress damages motley of vital cellular processes and induce cell degeneration. Data showed that mutant huntingtin proteins downregulate the expression levels of NF-κB and vital antioxidants, which was followed by increased oxidative stress and cell death. Treatment with antioxidants and inhibition of oxidative stress were able to counteract these adverse effects. In addition, thesis connects ER stress caused by mutant huntingtin to the cytoprotective autophagy. Autophagy sustains cellular balance by degrading potentially toxic cell proteins and components observed in Huntington’s disease. The results revealed that cytoprotective autophagy is active at the early points (24h) of ER stress after expression of mutant huntingtin proteins. GADD34 (growth arrest and DNA damage-inducible gene 34), which is previously connected to the regulation of translation during cell stress, was shown to control the stimulation of autophagy. However, GADD34 and autophagy were downregulated at later time points (48h) during mutant huntingtin proteins induced ER stress, and subsequently cell survival decreased. Overexpression GADD34 enhanced autophagy and decreased cell death, indicating that GADD34 plays a critical role in cell protection. The thesis reveales new interesting data about the neuronal cell death pathways seen in Huntington’s disease, and how cell degeneration is partly counteracted by various therapeutic agents. Expression of mutant huntingtin proteins is shown to alter signaling events that control ER stress, oxidative stress and autophagy. Despite that Huntington’s disease is mainly an untreatable disorder; these findings offer potential targets and neuroprotective strategies in designing novel therapies for Huntington’s disease.

Surface Proteins of Lactobacillus crispatus: Adhesive Properties and Cell Wall Anchoring

Bacterial surface-associated proteins are important in communication with the environment and bacteria-host interactions. In this thesis work, surface molecules of Lactobacillus crispatus important in host interaction were studied. The L. crispatus strains of the study were known from previous studies to be efficient in adhesion to intestinal tract and ECM. L. crispatus JCM 5810 possess an adhesive surface layer (S-layer) protein, whose functions and domain structure was characterized. We cloned two S-layer protein genes (cbsA; collagen-binding S-layer protein A and silent cbsB) and identified the protein region in CbsA important for adhesion to host tissues, for polymerization into a periodic layer as well as for attachment to the bacterial cell surface. The analysis was done by extensive mutation analysis and by testing His6-tagged fusion proteins from recombinant Escherichia coli as well as by expressing truncated CbsA peptides on the surface of Lactobacillus casei. The N-terminal region (31-274) of CbsA showed efficient and specific binding to collagens, laminin and extracellular matrix on tissue sections of chicken intestine. The N-terminal region also contained the information for formation of periodic S-layer polymer. This region is bordered at both ends by a conserved short region rich in valines, whose substitution to leucines drastically affected the periodic polymer structure. The mutated CbsA proteins that failed to form a periodic polymer, did not bind collagens, which indicates that the polymerized structure of CbsA is needed for collagen-binding ability. The C-terminal region, which is highly identical in S-layer proteins of L. crispatus, Lactobacillus acidophilus and Lactobacillus helveticus, was shown to anchor the protein to the bacterial cell wall. The C-terminal CbsA peptide specifically bound to bacterial teichoic acid and lipoteichoic acids. In conclusion, the N-terminal domain of the S-layer protein of L. crispatus is important for polymerization and adhesion to host tissues, whereas the C-terminal domain anchors the protein to bacterial cell-wall teichoic acids. Lactobacilli are fermentative organisms that effectively lower the surrounding pH. While this study was in progress, plasminogen-binding proteins enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were identified in the extracellular proteome of L. crispatus ST1. In this work, the cell-wall association of enolase and GAPDH were shown to rely on pH-reversible binding to the cell-wall lipoteichoic acids. Enolase from L. crispatus was functionally compared with enolase from L. johnsonii as well as from pathogenic streptococci (Streptococcus pneumoniae, Streptococcus pyogenes) and Staphylococcus aureus. His6-enolases from commensal lactobacilli bound human plasminogen and enhanced its activation by human plasminogen activators similarly to, or even better than, the enolases from pathogens. Similarly, the His6-enolases from lactobacilli exhibited adhesive characteristics previously assigned to pathogens. The results call for more detailed analyses of the role of the host plasminogen system in bacterial pathogenesis and commensalism as well of the biological role and potential health risk of the extracellular proteome in lactobacilli.

Stress and developmental responses in Arabidopsis thaliana: regulation through the transcription factor-interacting protein RCD1

Plants constantly face adverse environmental conditions, such as drought or extreme temperatures that threaten their survival. They demonstrate astonishing metabolic flexibility in overcoming these challenges and one of the key responses to stresses is changes in gene expression leading to alterations in cellular functions. This is brought about by an intricate network of transcription factors and associated regulatory proteins. Protein-protein interactions and post-translational modifications are important steps in this control system along with carefully regulated degradation of signaling proteins. This work concentrates on the RADICAL-INDUCED CELL DEATH1 (RCD1) protein which is an important regulator of abiotic stress-related and developmental responses in Arabidopsis thaliana. Plants lacking this protein function display pleiotropic phenotypes including sensitivity to apoplastic reactive oxygen species (ROS) and salt, ultraviolet B (UV-B) and paraquat tolerance, early flowering and senescence. Additionally, the mutant plants overproduce nitric oxide, have alterations in their responses to several plant hormones and perturbations in gene expression profiles. The RCD1 gene is transcriptionally unresponsive to environmental signals and the regulation of the protein function is likely to happen post-translationally. RCD1 belongs to a small protein family and, together with its closest homolog SRO1, contains three distinguishable domains: In the N-terminus, there is a WWE domain followed by a poly(ADP-ribose) polymerase-like domain which, despite sequence conservation, does not seem to be functional. The C-terminus of RCD1 contains a novel domain called RST. It is present in RCD1-like proteins throughout the plant kingdom and is able to mediate physical interactions with multiple transcription factors. In conclusion, RCD1 is a key point of signal integration that links ROS-mediated cues to transcriptional regulation by yet unidentified means, which are likely to include post-translational mechanisms. The identification of RCD1-interacting transcription factors, most of whose functions are still unknown, opens new avenues for studies on plant stress as well as developmental responses.

Ion-regulatory proreins in neuronal development and communication

Brain function is critically dependent on the ionic homeostasis in both the extra- and intracellular compartment. The regulation of brain extracellular ionic composition mainly relies on active transport at blood brain and at blood cerebrospinal fluid interfaces whereas intracellular ion regulation is based on plasmalemmal transporters of neurons and glia. In addition, the latter mechanisms can generate physiologically as well as pathophysiologically significant extracellular ion transients. In this work I have studied molecular mechanisms and development of ion regulation and how these factors alter neuronal excitability and affect synaptic and non-synaptic transmission with a particular emphasis on intracellular pH and chloride (Cl-) regulation. Why is the regulation of acid-base equivalents (H+ and HCO3-) and Cl- of such interest and importance? First of all, GABAA-receptors are permeable to both HCO3- and Cl-. In the adult mammalian central nervous system (CNS) fast postsynaptic inhibition relies on GABAA-receptor mediated transmission. Today, excitatory effects of GABAA-receptors, both in mature neurons and during the early development, have been recognized and the significance of the dual actions of GABA on neuronal communication has become an interesting field of research. The transmembrane gradients of Cl- and HCO3- determine the reversal potential of GABAA-receptor mediated postsynaptic potentials and hence, the function of pH and Cl- regulatory proteins have profound consequences on GABAergic signaling and neuronal excitability. Secondly, perturbations in pH can cause a variety of changes in cellular function, many of them resulting from the interaction of protons with ionizable side chains of proteins. pH-mediated alterations of protein conformation in e.g. ion channels, transporters, and enzymes can powerfully modulate neurotransmission. In the context of pH homeostasis, the enzyme carbonic anhydrase (CA) needs to be taken into account in parallel with ion transporters: for CO2/HCO3- buffering to act in a fast manner, CO2 (de)hydration must be catalyzed by this enzyme. The acid-base equivalents that serve as substrates in the CO2 dehydration-hydration reaction are also engaged in many carrier and channel mediated ion movements. In such processes, CA activity is in key position to modulate transmembrane solute fluxes and their consequences. The bicarbonate transporters (BTs; SLC4) and the electroneutral cation-chloride cotransporters (CCCs; SLC12) belong the to large gene family of solute carriers (SLCs). In my work I have studied the physiological roles of the K+-Cl- cotransporter KCC2 (Slc12a5) and the Na+-driven Cl--HCO3- exchanger NCBE (Slc4a10) and the roles of these two ion transporters in the modualtion of neuronal communication and excitability in the rodent hippocampus. I have also examined the cellular localization and molecular basis of intracellular CA that has been shown to be essential for the generation of prolonged GABAergic excitation in the mature hippocampus. The results in my Thesis provide direct evidence for the view that the postnatal up-regulation of KCC2 accounts for the developmental shift from depolarizing to hyperpolarizing postsynaptic EGABA-A responses in rat hippocampal pyramidal neurons. The results also indicate that after KCC2 expression the developmental onset of excitatory GABAergic transmission upon intense GABAA-receptor stimulation depend on the expression of intrapyramidal CA, identified as the CA isoform VII. Studies on mice with targeted Slc4a10 gene disruption revealed an important role for NCBE in neuronal pH regulation and in pH-dependent modulation of neuronal excitability. Furthermore, this ion transporter is involved in the basolateral Na+ and HCO3- uptake in choroid plexus epithelial cells, and is thus likely to contribute to cerebrospinal fluid production.

Impacts of agriculture on amphibians at multiple scales

Agriculture-mediated habitat loss and degradation together with climate change are among the greatest global threats to species, communities, and ecosystem functioning. During the last century, more than 50% of the world's wetlands have been lost and agricultural activities have subjected wetland species to increased isolation and decreased quality of habitats. Likewise, as a part of agricultural intensification, the use of pesticides has increased notably, and pesticide residues occur frequently in wetlands making the exposure of wetland organisms to pesticides highly probable. In this thesis, a set of ecotoxicological and landscape ecological studies were carried out to investigate pesticide-effects on tadpoles, and species-habitat relationships of amphibians in agricultural landscapes. The results show that the fitness of R. temporaria tadpoles can be negatively affected by sublethal pesticide concentrations, and that pesticides may increase the costs of response to natural environmental stressors. However, tadpoles may also be able to compensate for some of the negative effects of pesticides. The results further demonstrate that both historic and current-day agricultural land use can negatively impact amphibians, but that in some cases the costs of living in agricultural habitats may only become apparent when amphibians face other environmental stressors, such as drought. Habitat heterogeneity may, however, increase the persistence of amphibians in agricultural landscapes. Hence, the results suggest that amphibians are likely to be affected by agricultural processes that operate at several spatial and temporal scales, and that it is probable that various processes related to current-day agriculture will affect both larval and adult amphibians. The results imply that maintaining dense wetland patterns could enhance persistence of amphibian populations in agricultural habitats, and indicate that heterogeneous landscapes may lower the risk of regional amphibian population declines under extreme weather perturbations.

Individualized immunosuppression after renal transplantation in childhood

Pediatric renal transplantation (TX) has evolved greatly during the past few decades, and today TX is considered the standard care for children with end-stage renal disease. In Finland, 191 children had received renal transplants by October 2007, and 42% of them have already reached adulthood. Improvements in treatment of end-stage renal disease, surgical techniques, intensive care medicine, and in immunosuppressive therapy have paved the way to the current highly successful outcomes of pediatric transplantation. In children, the transplanted graft should last for decades, and normal growth and development should be guaranteed. These objectives set considerable requirements in optimizing and fine-tuning the post-operative therapy. Careful optimization of immunosuppressive therapy is crucial in protecting the graft against rejection, but also in protecting the patient against adverse effects of the medication. In the present study, the results of a retrospective investigation into individualized dosing of immunosuppresive medication, based on pharmacokinetic profiles, therapeutic drug monitoring, graft function and histology studies, and glucocorticoid biological activity determinations, are reported. Subgroups of a total of 178 patients, who received renal transplants in 1988 2006 were included in the study. The mean age at TX was 6.5 years, and approximately 26% of the patients were <2 years of age. The most common diagnosis leading to renal TX was congenital nephrosis of the Finnish type (NPHS1). Pediatric patients in Finland receive standard triple immunosuppression consisting of cyclosporine A (CsA), methylprednisolone (MP) and azathioprine (AZA) after renal TX. Optimal dosing of these agents is important to prevent rejections and preserve graft function in one hand, and to avoid the potentially serious adverse effects on the other hand. CsA has a narrow therapeutic window and individually variable pharmacokinetics. Therapeutic monitoring of CsA is, therefore, mandatory. Traditionally, CsA monitoring has been based on pre-dose trough levels (C0), but recent pharmacokinetic and clinical studies have revealed that the immunosuppressive effect may be related to diurnal CsA exposure and blood CsA concentration 0-4 hours after dosing. The two-hour post-dose concentration (C2) has proved a reliable surrogate marker of CsA exposure. Individual starting doses of CsA were analyzed in 65 patients. A recommended dose based on a pre-TX pharmacokinetic study was calculated for each patient by the pre-TX protocol. The predicted dose was clearly higher in the youngest children than in the older ones (22.9±10.4 and 10.5±5.1 mg/kg/d in patients <2 and >8 years of age, respectively). The actually administered oral doses of CsA were collected for three weeks after TX and compared to the pharmacokinetically predicted dose. After the TX, dosing of CsA was adjusted according to clinical parameters and blood CsA trough concentration. The pharmacokinetically predicted dose and patient age were the two significant parameters explaining post-TX doses of CsA. Accordingly, young children received significantly higher oral doses of CsA than the older ones. The correlation to the actually administered doses after TX was best in those patients, who had a predicted dose clearly higher or lower (> ±25%) than the average in their age-group. Due to the great individual variation in pharmacokinetics standardized dosing of CsA (based on body mass or surface area) may not be adequate. Pre-Tx profiles are helpful in determining suitable initial CsA doses. CsA monitoring based on trough and C2 concentrations was analyzed in 47 patients, who received renal transplants in 2001 2006. C0, C2 and experienced acute rejections were collected during the post-TX hospitalization, and also three months after TX when the first protocol core biopsy was obtained. The patients who remained rejection free had slightly higher C2 concentrations, especially very early after TX. However, after the first two weeks also the trough level was higher in the rejection-free patients than in those with acute rejections. Three months after TX the trough level was higher in patients with normal histology than in those with rejection changes in the routine biopsy. Monitoring of both the trough level and C2 may thus be warranted to guarantee sufficient peak concentration and baseline immunosuppression on one hand and to avoid over-exposure on the other hand. Controlling of rejection in the early months after transplantation is crucial as it may contribute to the development of long-term allograft nephropathy. Recently, it has become evident that immunoactivation fulfilling the histological criteria of acute rejection is possible in a well functioning graft with no clinical sings or laboratory perturbations. The influence of treatment of subclinical rejection, diagnosed in 3-month protocol biopsy, to graft function and histology 18 months after TX was analyzed in 22 patients and compared to 35 historical control patients. The incidence of subclinical rejection at three months was 43%, and the patients received a standard rejection treatment (a course of increased MP) and/or increased baseline immunosuppression, depending on the severity of rejection and graft function. Glomerular filtration rate (GFR) at 18 months was significantly better in the patients who were screened and treated for subclinical rejection in comparison to the historical patients (86.7±22.5 vs. 67.9±31.9 ml/min/1.73m2, respectively). The improvement was most remarkable in the youngest (<2 years) age group (94.1±11.0 vs. 67.9±26.8 ml/min/1.73m2). Histological findings of chronic allograft nephropathy were also more common in the historical patients in the 18-month protocol biopsy. All pediatric renal TX patients receive MP as a part of the baseline immunosuppression. Although the maintenance dose of MP is very low in the majority of the patients, the well-known steroid-related adverse affects are not uncommon. It has been shown in a previous study in Finnish pediatric TX patients that steroid exposure, measured as area under concentration-time curve (AUC), rather than the dose correlates with the adverse effects. In the present study, MP AUC was measured in sixteen stable maintenance patients, and a correlation with excess weight gain during 12 months after TX as well as with height deficit was found. A novel bioassay measuring the activation of glucocorticoid receptor dependent transcription cascade was also employed to assess the biological effect of MP. Glucocorticoid bioactivity was found to be related to the adverse effects, although the relationship was not as apparent as that with serum MP concentration. The findings in this study support individualized monitoring and adjustment of immunosuppression based on pharmacokinetics, graft function and histology. Pharmacokinetic profiles are helpful in estimating drug exposure and thus identifying the patients who might be at risk for excessive or insufficient immunosuppression. Individualized doses and monitoring of blood concentrations should definitely be employed with CsA, but possibly also with steroids. As an alternative to complete steroid withdrawal, individualized dosing based on drug exposure monitoring might help in avoiding the adverse effects. Early screening and treatment of subclinical immunoactivation is beneficial as it improves the prospects of good long-term graft function.