Components of fractionated stallion seminal plasma and the effects of seminal plasma on sperm longevity

Seminal plasma (SP) is the fluid portion of semen, secreted by the epididymides and the accessory glands before and during ejaculation. The stallion s ejaculate is a series of jets that differ in sperm concentration, semen volume and biochemical composition. Before the actual ejaculation, a clear and watery pre-sperm fluid is secreted. The first three jets form the sperm-rich fractions, and contain ¾ of the total number of sperm. The semen volume and sperm concentration in each of the jets decrease towards the end of the ejaculation, and the last jets are sperm-poor fractions with a low sperm concentration. The aims of these studies were to examine the effects of the different SP fractions, and the presence of SP, on sperm survival during storage. Pre-sperm fluid, and semen fractions with a high (sperm-rich) and low (sperm-poor) sperm concentration were collected in five experiments. The levels of selected enzymes, electrolytes and proteins in different SP fractions were determined. These studies also aimed at assessing the individual variation in the levels of the selected SP components and in the effects of SP on spermatozoa. The association between the components of SP and semen quality, sperm longevity, and fertility was examined with a stepwise linear regression analysis. Compared to samples containing SP during storage, centrifugation and the subsequent removal of SP reduced sperm motility parameters during 24 h of cooled storage in all SP fractions, but sperm membrane integrity was not affected. Some of the measured post-thaw motility parameters were also higher in samples containing SP compared to samples stored without SP. In contrast, the proportion of DNA-damaged spermatozoa was greater in the samples stored with SP than those without SP, and this effect was seen in both sperm-rich and sperm-poor fractions. There were no differences in DNA integrity between fractions stored with SP, but the sperm-rich fraction showed less DNA damage than the sperm-poor fraction after SP removal. The differences between fractions in sperm motility after cooled storage were non-significant. The levels of alkaline phosphatase, acid phosphatase and β-glucuronidase were higher in the sperm-rich fractions compared to the sperm-poor fractions, while the concentrations of calcium and magnesium were higher in sperm-poor fractions than in sperm-rich fractions. The concentrations of sodium and chloride were highest in pre-sperm fluid. In the sperm-poor fraction, the level of potassium was associated with the maintenance of sperm motility during storage. The levels of alkaline and acid phosphatase were associated with sperm concentration and the total number of spermatozoa in the ejaculates. None of the measured SP components were correlated to the first cycle pregnancy rate. In summary, the removal of SP improved DNA integrity after cooled storage compared with samples containing SP. There were no differences in the maintenance of sperm motility between the sperm-rich and sperm-poor fractions and whole ejaculates during cooled storage, irrespective of the presence of SP. The lowest rate of DNA damage was found in the sperm-rich fractions stored without SP. In practice, the results presented in this thesis support the use of individual modifications of semen processing techniques for cooled transported semen from subfertile stallions.

Perception of surface brightness

The human visual system has adapted to function in different lighting environments and responds to contrast instead of the amount of light as such. On the one hand, this ensures constancy of perception, for example, white paper looks white both in bright sunlight and in dim moonlight, because contrast is invariant to changes in overall light level. On the other hand, the brightness of the surfaces has to be reconstructed from the contrast signal because no signal from surfaces as such is conveyed to the visual cortex. In the visual cortex, the visual image is decomposed to local features by spatial filters that are selective for spatial frequency, orientation, and phase. Currently it is not known, however, how these features are subsequently integrated to form objects and object surfaces. In this thesis the integration mechanisms of achromatic surfaces were studied by psychophysically measuring the spatial frequency and orientation tuning of brightness perception. In addition, the effect of textures on the spread of brightness and the effect of phase of the inducing stimulus on brightness were measured. The novel findings of the thesis are that (1) a narrow spatial frequency band, independent of stimulus size and complexity, mediates brightness information (2) figure-ground brightness illusions are narrowly tuned for orientation (3) texture borders, without any luminance difference, are able to block the spread of brightness, and (4) edges and even- and odd-symmetric Gabors have a similar antagonistic effect on brightness. The narrow spatial frequency tuning suggests that only a subpopulation of neurons in V1 is involved in brightness perception. The independence of stimulus size and complexity indicates that the narrow tuning reflects hard-wired processing in the visual system. Further, it seems that figure-ground segregation and mechanisms integrating contrast polarities are closely related to the low level mechanisms of brightness perception. In conclusion, the results of the thesis suggest that a subpopulation of neurons in visual cortex selectively integrates information from different contrast polarities to reconstruct surface brightness.

Stem Cell Markers in Cancer: Do They Have Clinical Significance?

In cancer, a subpopulation of malignant cells expresses markers of normal stem cells. These cells have the potential of initiating tumor growth and therefore also tumor recurrence. Thus, these cells are called cancer stem cells. A myriad of markers have been applied to identify these cells, but no single marker can be found exclusively in cancer stem cells. In many types of cancer, clinical recurrence and tumor progression are the main causes of mortality, despite intense oncological treatment. It has been proposed that the presence of cancer stem cells causes this resistance to therapy. The scope of this thesis is to investigate the role of stem cell markers and genes in the clinical setting. Especially, the aim was to elucidate the clinical significance of stem cell markers as novel prognostic and diagnostic tools in cancer. Tumor biopsy material from central nervous system tumors (oligodendroglioma, astrocytoma and glioblatoma), neural crest derived tumors (pheochromocytomas) and oral carcinoma was screened for stem cell markers. Initially, 15 stem cell markers were screened in a test series of gliomas. The markers applied for expanded tumor analyses (in 305 cases of glioma, 42 cases of pheochromocytoma, and 73 cases of oral carcinoma) were BMI-1, Snail, p16, mdm2, and c-Myc. Data on marker expression was compared with clinical and pathological parameters. In gliomas, BMI-1 expression was found in nearly all tumors analyzed, but the frequency of BMI-1 expressing cells was highly variable, ranging from 1 to 100%. In oligodendroglioma, BMI-1 expression was identified as a prognostic marker independent of tumor grade and clinical parameters. In pheochromocytoma, Snail expression was shown to distinguish between the metastatic and non-metastatic forms of the tumor. Snail expression was seen only in metastatic tumors, whereas non-metastatic tumors did not commonly express Snail. Finally, in oral carcinoma, BMI-1 expression was seen in roughly 80% of tumors, and Snail expression was high or very high in all cases. The lack of BMI-1 expression was associated with early relapse in oral carcinoma.

In vitro development of islets from human adult pancreatic tissues

Transplantation of isolated islets from cadaver pancreas is a promising possibility for the optimal treatment of type 1 diabetes. The lack of islets is a major problem. Here we have investigated the possibility of generating islets in tissue culture of human pancreatic cells. We first reproduced a previously reported method of in vitro generation of endocrine cells from human adult pancreatic tissue. By tracing the bromodeoxyuridine-labeled cells in differentiated islet buds, we found that the pancreatic progenitor cells represented a subpopulation of cytokeratin 19 (CK19)-positive ductal cells. Serum-free medium and Matrigel overlay were essential for the endocrine differentiation. We then examined the involvement of preexisting islet cells in islet neogenesis. About 6-10% of endocrine cells dedifferentiated and acquired a transitional phenotype by coexpressing CK19. Significant cell proliferation was only observed in CK19-positive cells, but not in chromogranin A-positive endocrine cells. The in vitro-derived human islets were morphologically and functionally immature when compared with normal islets. Their insulin mRNA levels were only 4-5% of that found in fresh human islets, and glucose-stimulated insulin release was 3 times lower than that of control islets. Moreover, some immature endocrine cells coexpressed insulin and glucagon. After transplantation in nude mice, the in vitro-generated islets became mature with one type of hormone per endocrine cell. In addition, we also found that also in both fresh islet transplants many cells coexpressed endocrine markers and ductal marker CK19 as a sign of ductal to endocrine cell transition. Finally, we studied the effects of clinically used immunosuppressive drugs on precursor cell proliferation and differentiation. Mycophenolate mofetil (MMF) severely hampered duct-cell proliferation, and significantly reduced the total DNA content indicating its antiproliferative effect on the precursors. Tacrolimus mainly affected differentiated beta cells by decreasing the insulin content per DNA as well as the proportion of insulin-positive cells. Sirolimus and daclizumab did not show any individual or synergistic side effects suggesting that these drugs are amenable for use in clinical islet transplantation. In summary, we confirm the capacity of endocrine differentiation from progenitors present in the adult human pancreas. The plasticity of differentiated cell types of human pancreas may be a potential mechanism of human pancreas regeneration. Ductal cell differentiation into endocrine cells in transplanted islets may be an important factor in sustaining the long-term function of islet transplants. The immunosuppressive protocol is likely to be an important determinant of long-term clinical islet graft function. Moreover, these results provide new information on the mechanisms of pancreatic islet regeneration and provide the basis for the development of new strategies for the treatment of insulin deficient diabetes mellitus.

Modern techniques in detection, identification and quantification of bacteria and peptides from foods

Standards have been placed to regulate the microbial and preservative contents to assure that foods are safe to the consumer. In a case of a food-related disease outbreak, it is crucial to be able to detect and identify quickly and accurately the cause of the disease. In addition, for every day control of food microbial and preservative contents, the detection methods must be easily performed for numerous food samples. In this present study, quicker alternative methods were studied for identification of bacteria by DNA fingerprinting. A flow cytometry method was developed as an alternative to pulsed-field gel electrophoresis, the golden method . DNA fragment sizing by an ultrasensitive flow cytometer was able to discriminate species and strains in a reproducible and comparable manner to pulsed-field gel electrophoresis. This new method was hundreds times faster and 200,000 times more sensitive. Additionally, another DNA fingerprinting identification method was developed based on single-enzyme amplified fragment length polymorphism (SE-AFLP). This method allowed the differentiation of genera, species, and strains of pathogenic bacteria of Bacilli, Staphylococci, Yersinia, and Escherichia coli. These fingerprinting patterns obtained by SE-AFLP were simpler and easier to analyze than those by the traditional amplified fragment length polymorphism by double enzyme digestion. Nisin (E234) is added as a preservative to different types of foods, especially dairy products, around the world. Various detection methods exist for nisin, but they lack in sensitivity, speed or specificity. In this present study, a sensitive nisin-induced green fluorescent protein (GFPuv) bioassay was developed using the Lactococcus lactis two-component signal system NisRK and the nisin-inducible nisA promoter. The bioassay was extremely sensitive with detection limit of 10 pg/ml in culture supernatant. In addition, it was compatible for quantification from various food matrices, such as milk, salad dressings, processed cheese, liquid eggs, and canned tomatoes. Wine has good antimicrobial properties due to its alcohol concentration, low pH, and organic content and therefore often assumed to be microbially safe to consume. Another aim of this thesis was to study the microbiota of wines returned by customers complaining of food-poisoning symptoms. By partial 16S rRNA gene sequence analysis, ribotyping, and boar spermatozoa motility assay, it was identified that one of the wines contained a Bacillus simplex BAC91, which produced a heat-stable substance toxic to the mitochondria of sperm cells. The antibacterial activity of wine was tested on the vegetative cells and spores of B. simplex BAC91, B. cereus type strain ATCC 14579 and cereulide-producing B. cereus F4810/72. Although the vegetative cells and spores of B. simplex BAC91 were sensitive to the antimicrobial effects of wine, the spores of B. cereus strains ATCC 14579 and F4810/72 stayed viable for at least 4 months. According to these results, Bacillus spp., more specifically spores, can be a possible risk to the wine consumer.

Bacillus cereus spores and cereulide in food-borne illness

B. cereus is a gram-positive bacterium that possesses two different forms of life:the large, rod-shaped cells (ca. 0.002 mm by 0.004 mm) that are able to propagate and the small (0.001 mm), oval shaped spores. The spores can survive in almost any environment for up to centuries without nourishment or water. They are insensitive towards most agents that normally kill bacteria: heating up to several hours at 90 ºC, radiation, disinfectants and extreme alkaline (≥ pH 13) and acid (≤ pH 1) environment. The spores are highly hydrophobic and therefore make them tend to stick to all kinds of surfaces, steel, plastics and live cells. In favorable conditions the spores of B. cereus may germinate into vegetative cells capable of producing food poisoning toxins. The toxins can be heat-labile protein formed after ingestion of the contaminated food, inside the gastrointestinal tract (diarrhoeal toxins), or heat stable peptides formed in the food (emesis causing toxin, cereulide). Cereulide cannot be inactivated in foods by cooking or any other procedure applicable on food. Cereulide in consumed food causes serious illness in human, even fatalities. In this thesis, B. cereus strains originating from different kinds of foods and environments and 8 different countries were inspected for their capability of forming cereulide. Of the 1041 isolates from soil, animal feed, water, air, used bedding, grass, dung and equipment only 1.2 % were capable of producing cereulide, whereas of the 144 isolates originating from foods 24 % were cereulide producers. Cereulide was detected by two methods: by its toxicity towards mammalian cells (sperm assay) and by its peculiar chemical structure using liquid-chromatograph-mass spectrometry equipment. B. cereus is known as one of the most frequent bacteria occurring in food. Most foods contain more than one kind of B. cereus. When randomly selected 100 isolates of B. cereus from commercial infant foods (dry formulas) were tested, 11% of these produced cereulide. Considering a frequent content of 103 to 104 cfu (colony forming units) of B. cereus per gram of infant food formula (dry), it appears likely that most servings (200 ml, 30 g of the powder reconstituted with water) may contain cereulide producers. When a reconstituted infant formula was inoculated with >105 cfu of cereulide producing B. cereus per ml and left at room temperature, cereulide accumulated to food poisoning levels (> 0.1 mg of cereulide per serving) within 24 hours. Paradoxically, the amount of cereulide (per g of food) increased 10 to 50 fold when the food was diluted 4 - 15 fold with water. The amount of the produced cereulide strongly depended on the composition of the formula: most toxin was formed in formulas with cereals mixed with milk, and least toxin in formulas based on milk only. In spite of the aggressive cleaning practices executed by the modern dairy industry, certain genotypes of B. cereus appear to colonise the silos tanks. In this thesis four strategies to explain their survival of their spores in dairy silos were identified. First, high survival (log 15 min kill ≤ 1.5) in the hot alkaline (pH >13) wash liquid, used at the dairies for cleaning-in-place. Second, efficient adherence of the spores to stainless steel from cold water. Third, a cereulide producing group with spores characterized by slow germination in rich medium and well preserved viability when exposed to heating at 90 ºC. Fourth, spores capable of germinating at 8 ºC and possessing the psychrotolerance gene, cspA. There were indications that spores highly resistant to hot 1% sodium hydroxide may be effectively inactivated by hot 0.9% nitric acid. Eight out of the 14 dairy silo tank isolates possessing hot alkali resistant spores were capable of germinating and forming biofilm in whole milk, not previously reported for B. cereus. In this thesis it was shown that cereulide producing B. cereus was capable of inhibiting the growth of cereulide non-producing B. cereus occurring in the same food. This phenomenon, called antagonism, has long been known to exist between B. cereus and other microbial species, e.g. various species of Bacillus, gram-negative bacteria and plant pathogenic fungi. In this thesis intra-species antagonism of B. cereus was shown for the first time. This brother-killing did not depend on the cereulide molecule, also some of the cereulide non-producers were potent antagonists. Interestingly, the antagonistic clades were most frequently found in isolates from food implicated with human illness. The antagonistic property was therefore proposed in this thesis as a novel virulence factor that increases the human morbidity of the species B. cereus, in particular of the cereulide producers.

Pre- and post-copulatory sexual selection in the least killifish, Heterandria formosa

It has been only recently realized that sexual selection does not end at copulation but that post-copulatory processes are often important in determining the fitness of individuals. In this thesis, I experimentally studied both pre- and post-copulatory sexual selection in the least killifish, Heterandria formosa. I found that this species suffers from severe inbreeding depression in male reproductive behaviour, offspring viability and offspring maturation times. Neither sex showed pre-copulatory inbreeding avoidance but when females mated with their brothers, less sperm were retrieved from their reproductive system compared to the situation when females mated with unrelated males. Whether the difference in sperm numbers is due to female or male effect could not be resolved. Based on theory, females should be more eager to avoid inbreeding than males in this species, because females invest more in their offspring than males do. Inbreeding seems to be an important part of this species biology and the severe inbreeding depression has most likely selected for the evolution of the post-copulatory inbreeding avoidance mechanism that I found. In addition, I studied the effects of polyandry on female reproductive success. When females mated with more than one male, they were more likely to get pregnant. However, I also found a cost of polyandry. The offspring of females mated to four males took longer to reach sexual maturity compared to the offspring of monandrous females. This cost may be explained by parent-offspring conflict over maternal resource allocation. In another experiment, in which within-brood relatedness was manipulated, offspring sizes decreased over time when within-brood relatedness was low. This result is partly in accordance with the kinship theory of genomic imprinting. When relatedness decreases, offspring are expected to be less co-operative and demand fewer resources from their mother, which leads to impaired development. In the last chapter of my thesis, I show that H. formosa males do not prefer large females as in other Poeciliidae species. I suggest that males view smaller females as more profitable mates because those are more likely virgin. In conclusion, I found both pre- and post-copulatory sexual selection to be important factors in determining reproductive success in H. formosa.

Genetic aspects of outcome in kidney transplantation: cytokine and thrombosis associated candidate genes and gene expression biomarkers

Kidney transplantation (Tx) is the treatment of choice for end stage renal disease. Immunosuppressive medications are given to prevent an immunological rejection of the transplant. However, immunosuppressive drugs increase e.g. the risk of infection, cancer or nephrotoxicity. A major genetic contributors to immunological acceptance of the graft are human leukocyte antigen (HLA) genes. Also other non-HLA gene polymorphisms may predict the future risk of complications before Tx, possibly enabling individualised immunotherapy. Graft function after Tx is monitored using non-specific clinical symptoms and laboratory markers. The definitive diagnosis of graft rejection however relies on a biopsy of the graft. In the acute rejection (AR) diagnostics there is a need for an alternative to biopsy that would be an easily repeatable and simple method for regular use. Frequent surveillance of acute or subclinical rejection (SCR) may improve long-term function. In this thesis, associations between cytokine and thrombosis associated candidate genes and the outcome of kidney Tx were studied. Cytotoxic and co-stimulatory T lymphocyte molecule gene expression biomarkers for the diagnosis of the AR and the SCR were also investigated. We found that polymorphisms in the cytokine genes tumor necrosis factor and interleukin 10 (IL10) of the recipients were associated with AR. In addition, certain IL10 gene polymorphisms of the donors were associated with the incidence of cytomegalovirus infection and occurrence of later infection in a subpopulation of recipients. Further, polymorphisms in genes related to the risk of thrombosis and those of certain cytokines were not associated with the occurrence of thrombosis, infarction, AR or graft survival. In the study of biomarkers for AR, whole blood samples were prospectively collected from adult kidney Tx patients. With real-time quantitative PCR (RT-QPCR) gene expression quantities of CD154 and ICOS differentiated the patients with AR from those without, but not from the patients with other causes of graft dysfunction. Biomarkers for SCR were studied in paediatric kidney Tx patients. We used RT-QPCR to quantify the gene expression of immunological candidate genes in a low-density array format. In addition, we used RT-QPCR to validate the results of the microarray analysis. No gene marker differentiated patients with SCR from those without SCR. This research demonstrates the lack of robust markers among polymorphisms or biomarkers in investigated genes that could be included in routine analysis in a clinical laboratory. In genetic studies, kidney Tx can be regarded as a complex trait, i.e. several environmental and genetic factors may determine its outcome. A number of currently unknown genetic factors probably influence the results of Tx.

Death pathways activated in the neurotrophic factor-deprived neurons

Programed cell death (PCD) is a fundamental biological process that is as essential for the development and tissue homeostasis as cell proliferation, differentiation and adaptation. The main mode of PCD - apoptosis - occurs via specifi c pathways, such as mitochondrial or death receptor pathway. In the developing nervous system, programed death broadly occurs, mainly triggered by the defi ciency of different survival-promoting neurotrophic factors, but the respective death pathways are poorly studied. In one of the best-characterized models, sympathetic neurons deprived of nerve growth factor (NGF) die via the classical mitochondrial apoptotic pathway. The main aim of this study was to describe the death programs activated in these and other neuronal populations by using neuronal cultures deprived of other neurotrophic factors. First, this study showed that the cultured sympathetic neurons deprived of glial cell line-derived neurotrophic factor (GDNF) die via a novel non-classical death pathway, in which mitochondria and death receptors are not involved. Indeed, cytochrome c was not released into the cytosol, Bax, caspase-9, and caspase-3 were not involved, and Bcl-xL overexpression did not prevent the death. This pathway involved activation of mixed lineage kinases and c-jun, and crucially requires caspase-2 and -7. Second, it was shown that deprivation of neurotrophin-3 (NT-3) from cultured sensory neurons of the dorsal root ganglia kills them via a dependence receptor pathway, including cleavage of the NT- 3 receptor TrkC and liberation of a pro-apoptotic dependence domain. Indeed, death of NT-3-deprived neurons was blocked by a dominant-negative construct interfering with TrkC cleavage. Also, the uncleavable mutant of TrkC, replacing the siRNA-silenced endogeneous TrkC, was not able to trigger death upon NT-3 removal. Such a pathway was not activated in another subpopulation of sensory neurons deprived of NGF. Third, it was shown that cultured midbrain dopaminergic neurons deprived of GDNF or brainderived neurotrophic factor (BDNF) kills them by still a different pathway, in which death receptors and caspases, but not mitochondria, are activated. Indeed, cytochrome c was not released into the cytosol, Bax was not activated, and Bcl-xL did not block the death, but caspases were necessary for the death of these neurons. Blocking the components of the death receptor pathway - caspase-8, FADD, or Fas - blocked the death, whereas activation of Fas accelerated it. The activity of Fas in the dopaminergic neurons could be controlled by the apoptosis inhibitory molecule FAIML. For these studies we developed a novel assay to study apoptosis in the transfected dopaminergic neurons. Thus, a novel death pathway, characteristic for the dopaminergic neurons was described. The study suggests death receptors as possible targets for the treatment of Parkinson s disease, which is caused by the degeneration of dopaminergic neurons.

Outcome of singleton pregnancy after assisted reproductive treatment

Singleton pregnancies achieved by means of assisted reproductive treatment (ART) are associated with increased obstetric and neonatal risks in comparison with spontaneously conceived singleton pregnancies. The impact of infertility- and treatment-related factors on these risks is not properly understood. In addition, the psychological effects of infertility and its treatment on the experience of pregnancy have scarcely been studied. Thus, the aim of the present study was to evaluate the importance of infertility- and treatment-related factors on prediction of pregnancy outcome, obstetric and neonatal risks, fear-of-childbirth and pregnancy-related anxiety. The subjects consisted of infertile women who achieved a singleton pregnancy by means of in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI). The control groups comprised spontaneously conceiving women with singleton gestations. Early pregnancy outcome was assessed by means of assay of serum human chorionic gonadoptrophin (hCG) in single samples. Other outcome data were collected from patient records, national Health Registers and via prospective questionnaire surveys. Viable pregnancies were associated with significantly higher serum hCG levels 12 days after embryo transfer than non-viable pregnancies. Among singleton pregnancies, aetiological subgroup, treatment type or the number of transferred embryos did not impair the predictive value of single hCG assessment. According to the register-based data, age-, parity- and socioeconomic status- adjusted risks of gestational hypertension, preterm contractions and placenta praevia were more frequent in the ART pregnancies than in the control pregnancies. Significantly higher rates of induction of delivery and Caesarean section occurred in the ART group than in the control group. The risks of preterm birth and low birth weight (LBW) were increased after ART pregnancy. Duration or aetiology of infertility, treatment type (fresh or frozen IVF or ICSI) or rank of treatment did not contribute to the risks of preterm birth or LBW. In addition, the risks of preterm birth and LBW remained elevated in spite of of the number of transferred embryos. Although mean duration of pregnancy was shorter and mean birth weight lower in the ART pregnancies than in the control pregnancies, these differences were hardly of clinical significance. Fear-of-childbirth and pregnancy-related anxiety were equally common to women conceiving by means of ART, or spontaneously. Partnership of five to ten years appeared to be protective as regards severe fear-of-childbirth, whereas long preceding infertility (≥ seven years) had the opposite effect. In conclusion, an early hCG assessment maintained its good predictive value regardless of infertility- or patient-related factors. Further, we did not recognise any infertility- or patient-related factors that would expose infertile women to increased obstetric or neonatal risks. However, a long period of infertility was associated with severe fear-of-childbirth.

Carotid Stenosis, Endarterectomy, and the Brain : Brain microcirculation, diffusion, and cognitive function before and after carotid endarterectomy in patients with a high-grade carotid stenosis

Carotid atherosclerotic disease is a major cause of stroke, but it may remain clinically asymptomatic. The factors that turn the asymptomatic plaque into a symptomatic one are not fully understood, neither are the subtle effects that a high-grade carotid stenosis may have on the brain. The purpose of this study was to evaluate brain microcirculation, diffusion, and cognitive performance in patients with a high-grade stenosis in carotid artery, clinically either symptomatic or asymptomatic, undergoing carotid endarterectomy (CEA). We wanted to find out whether the stenoses are associated with diffusion or perfusion abnormalities of the brain or variation in the cognitive functioning of the patients, and to what extent the potential findings are affected by CEA, and compare the clinically symptomatic and asymptomatic subjects as well as strictly healthy controls. Coagulation and fibrinolytic parameters were compared with the rate microembolic signals (MES) in transcranial Doppler (TCD) and the macroscopic appearance of stenosing plaques in surgery. Patients (n=92) underwent CEA within the study. Blood samples pertaining to coagulation and fibrinolysis were collected before CEA, and the subjects underwent repeated TCD monitoring for MES. A subpopulation (n= 46) underwent MR imaging and repeated neuropsychological examination (preoperative, as well 4 and 100 days after CEA). In MRI, the average apparent diffusion coefficients were higher in the ipsilateral white matter (WM), and altough the interhemispheric difference was abolished by CEA, the levels remained higher than in controls. Symptomatic stenoses were associated with more sluggish perfusion especially in WM, and lower pulsatility of flow in TCD. All patients had poorer cognitive performance than healthy controls. Cognitive functions improved as expected by learning effect despite transient postoperative worsening in a few subjects. Improvement was greater in patients with deepest hypoperfusion, primarily in executive functions. Symptomatic stenoses were associated with higher hematocrit and tissue plasminogen activator antigen levels, as well as higher rate of MES and ulcerated plaques, and better postoperative improvement of vasoreactivity and pulsatility. In light of the findings, carotid stenosis is associated with differences in brain diffusion, perfusion, and cognition. The effect on diffusion in the ipsilateral WM, partially reversible by CEA, may be associated with WM degeneration. Asymptomatic and symptomatic subpopulations differ from each other in terms of hemodynamic adaptation and in their vascular physiological response to removal of stenosis. Although CEA may be associated with a transient cognitive decline, a true improvement of cognitive performance by CEA is possible in patients with the most pronounced perfusion deficits. Mediators of fibrinolysis and unfavourable hemorheology may contribute to the development of a symptomatic disease in patients with a high-grade stenosis.

Transition to parenthood after assisted reproductive treatment: Follow-up study of singleton pregnancies

Infertility treatments are relatively easily available in most Western countries today, but the psychological consequences of these high-tech treatments have scarcely been addressed. The purpose of this controlled longitudinal study was to explore the early environment of the infant born by assisted reproductive treatment (ART). We focused on the parents mental well-being, marital relations and experience of parenting. In addition to this, we assessed parent child interaction and parents mental representations of their child after long-standing infertility and several unsuccessful ART attempts. The subjects were infertile couples who achieved a singleton pregnancy by in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). The control group comprised of spontaneously conceiving couples with singleton pregnancies. ART women showed fewer depressive symptoms than controls during pregnancy and after delivery, but the difference vanished by the end of the child s first year. ART men consistently had lower levels of anxiety symptoms, sleeping difficulties, and social dysfunction than control men. Control women experienced a decrease in dyadic consensus during the child s first year, which did not happen among ART women. After the child was born, ART men reported a higher level of sexual affection compared with control men. Psychic symptoms and stressful life events were differently related to marital relations in ART and control groups. The parenting experiences of ART mothers were in general at a higher level, compared with controls, and they changed in a positive direction during the child s first year. Fathering experiences were at the same level in both groups, and they changed positively in both groups by the end of the child s first year. The parenting experiences of ART mothers and fathers were more resilient to certain child-related stressors than those of control group. Both mothers and fathers with long-term infertility showed more sensitive behaviour with their child in toddler-age than in infancy. Correspondingly, children s cooperation increased. Mothers often mentioned a fear of miscarriage and difficulty in creating representations of the child during pregnancy. Descriptions of the infants were mainly rich, vivid and loaded with positive features. In conclusion, ART parents in general seem to adapt well to the transition to parenthood. Former infertility and ART do not seem to constitute a risk for parents mental health, marital relations or experience of parenting. Even longstanding infertility with several unsuccessful treatment attempts did not create a risk as regards parenting behaviour or parents mental representations of their child. In this group, however, women were found to have fear for losing the child and difficulty in creating representations of the child during pregnancy, which in some cases may indicate need for psychosocial support. Even though our results are encouraging, infertility and infertility treatments are generally considered as a stressful experience. It is a challenge for health authorities to recognize those couples who need professional help to overcome the distressing experiences of infertility and ART.

Afrikkalaisen sikaruton mahdollisia maahantuloreittejä

Afrikkalainen sikarutto on lakisääteisesti vastustettava helposti leviävä sikojen ja villisikojen virustauti, jolla on myös vakavia sosioekonomisia vaikutuksia. Akuutissa tautimuodossa eläimillä esiintyy syömättömyyttä, korkeaa kuumetta, verenpurkaumia iholla, veristä ulostetta sekä mahdollisesti ripulia. Kuolleisuus on lähes 100 % ja tauti johtaa kuolemaan 7-10 vrk tartunnan jälkeen. Tauti ei tartu ihmisiin. Tautia esiintyy sekä kesy- että villisioissa suurimmassa osassa Saharan eteläpuolista Afrikkaa ja Sardiniassa. Vuodesta 2007 lähtien tautia on esiintynyt Kaukasuksen alueella ja vuonna 2011 lähellä Suomen rajaa: Leningradin alueella ja Kuolan niemimaalla. Tässä riskiprofiilissa kartoitetaan reitit ja tapahtumasarjat, jotka voivat johtaa siihen, että afrikkalainen sikarutto tulee Suomeen ensimmäisen kerran. Näistä oleellisimmat ovat: maahantulo infektioalueella matkustaneiden ihmisten mukana, infektoituneen lihan tai lihatuotteen mukana, elävien kesysikojen ja sperman mukana, kontaminoituneiden eläinkuljetusajoneuvojen mukana, kansainvälisen liikenteen ruokajätteen mukana ja Suomeen vaeltavan infektoituneen villisian mukana. Tilatason tautisuojaus sekä tehokas ja kohdennettu tiedottaminen taudin vaaroista ovat avainasemassa, kun halutaan suojata Suomen sikaelinkeinoa afrikkalaiselta sikarutolta.

Impaired spermatogenesis in Finnish boars and bulls

Maintenance of breeding efficiency and high semen quality is essential for reproductive success in farm animals. Early recognition of possible inheritable factors causing infertility requires constant attention. This thesis focuses on describing different manifestations of impaired spermatogenesis, their impact on fertility and partly also their incidence in populations. The reasons for spermatogenic failure are various. An interruption of germ cell differentiation, spermatogenic arrest, can lead to infertility. The incidence of azoospermia was investigated in the 1996 2005 survey of Finnish AI and farm breeding boars. We focused on the diagnosis, testicular morphometry and the possible reasons for the condition. The incidence of azoospermia was significantly higher in Yorkshire boars than in the Landrace breed. The most common diagnosis in Yorkshire boars was germ cell arrest at the primary spermatocyte level. The second most frequent diagnosis in Yorkshire boars was segmental aplasia of the Wolffian ducts with idiopathic epididymal obstruction. Other reasons for azoospermia were infrequent. In the second study we investigated the incidence of two relatively well-defined specific sperm defects in Finnish Yorkshire and Landrace boars during the same survey, the immotile short-tail sperm (ISTS) defect and the knobbed acrosome (KA) defect. In the Finnish Yorkshire boars the inherited ISTS defect, and the probably inherited KA defect, were important causes of infertility during 1996 2005. The ISTS defect was found in 7.6% and the KA defect in 0.8% of the Yorkshire boars. No Landrace boars were diagnosed with either of these two defects. In the third study we described a new sterilizing sperm defect in an oligoasthenoterazoospermic bull. Because of its morphological characteristics this defect was termed the multinuclear-multiflagellar sperm (MNMFS) defect. The number of Sertoli cells in the seminiferous tubuli was highly increased in the MNMFS bull compared with the number in normal bulls. In the following two studies we used a combined approach of fluorescence in situ hybridization (FISH), flow cytometry and morphometric studies to provide information on the cytogenetic background of macrocephalic bull spermatozoa. We described cellular features of diploid spermatozoa and compared the failures in the first and second meiotic divisions. In the last study we describe how the transplantation of testicular cells was used to determine whether spermatogonia derived from donor animals are able to colonize and produce motile spermatozoa in immune-competent unrelated boars suffering the ISTS defect. Transplantation resulted in complete focal spermatogenesis, indicated by the appearance of motile spermatozoa and confirmed by genotyping.