From neural stem cells to precursors : Molecular regulation of self-renewal and differentiation

Stem cells are responsible for tissue turnover throughout lifespan. Only highly controlled specific environment, the stem cell niche , can sustain undifferentiated stem cell-pool. The balance between maintenance and differentiation is crucial for individual s health: uncontrolled stem cell self-renewal or proliferation can lead to hyperplasia and mutations that further provoke malignant transformation of the cells. On the other hand, uninhibited differentiation may result in diminished stem cell population, which is unable to maintain tissue turnover. The mechanisms that control the switch from maintenance to differentiation in stem cells are not well known. The same mechanisms that direct the self-renewal and proliferation in normal stem cells are likely to be also involved in maintenance of cancer stem cell . Cancer stem cells exhibit stem cell like properties such as self-renewal- and differentiation capacity and they can also regenerate the tumor tissue. In this thesis, I have investigated the effect of classical oncogenes E6/E7 and c-Myc, tumor suppressors p53 and retinoblastoma (pRb) family, and vascular endothelial growth factor (VEGF) subfamily and glial cell line-derived neurothropic factor (GDNF) family ligands on behavior of embryonic neural stem cells (NSCs) and progenitors. The study includes also the characterization of cytoskeletal tumor suppressor neurofibromatosis 2 (NF2) protein merlin and ezrin-radixin-moesin (ERM) protein ezrin expression in neural progenitors cells and their progeny. This study reveals some potential mechanisms regarding to NSCs maintenance. In summary, the studied molecules are able to shift the balance either towards stem cell maintenance or differentiation; tumor suppressor p53 represses whereas E6/E7 oncogenes and c-Myc increase the proportion of self-renewing and proliferating NSCs or progenitors. The data suggests that active MEK-ERK signaling is critical for self-renewal of normal and oncogene expressing NSCs. In addition, the results indicate that expression of cytoskeletal tumor suppressor merlin and ERM protein ezrin in central nervous system (CNS) tissue and progenitors indicates their role in cell differentiation. Furthermore, the data suggests that VEGF-C a factor involved in lymphatic system development, angiogenesis, neovascularization and metastasis but also in maintenance of some neural populations in brain is a novel thropic factor for progenitors in early sympathetic nervous system (SNS). It seems that VEGF-C dose dependently through ERK-pathway supports the proliferation and survival of early sympathetic progenitor cells, and the effect is comparable to that of GDNF family ligands.

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.

Differentiation of neural stem cells in fragile X syndrome

Multipotent stem cells can self-renew and give rise to multiple cell types. One type of mammalian multipotent stem cells are neural stem cells (NSC)s, which can generate neurons, astrocytes and oligodendrocytes. NSCs are likely involved in learning and memory, but their exact role in cognitive function in the developing and adult brain is unclear. We have studied properties of NSCs in fragile X syndrome (FXS), which is the most common form of inherited mental retardation. FXS is caused by the lack of functional fragile X mental retardation protein (FMRP). FMRP is involved in the regulation of postsynaptic protein synthesis in a group I metabotropic glutamate receptor 5 (mGluR5)-dependent manner. In the absence of functional FMRP, the formation of functional synapses is impaired in the forebrain which results in alterations in synaptic plasticity. In our studies, we found that FMRP-deficient NSCs generated more neurons and less glia than control NSCs. The newborn neurons derived from FMRP-deficient NSCs showed an abnormally immature morphology. Furthermore, FMRP-deficient NSCs exhibited aberrant oscillatory Ca2+ responses to glutamate, which were specifically abolished by an antagonist of the mGluR5 receptor. The data suggested alterations in glutamatergic differentiation of FMRP-deficient NSCs and were further supported by an accumulation of cells committed to glutamatergic lineage in the subventricular zone of the embryonic Fmr1-knockout (Fmr1-KO) neocortex. Postnatally, the aberrant cells likely contributed to abnormal formation of the neocortex. The findings suggested a defect in the differentiation of distinct glutamatergic mGluR5 responsive cells in the absence of functional FMRP. Furthermore, we found that in the early postnatal Fmr1-KO mouse brain, the expression of mRNA for regulator of G-protein signalling-4 (RGS4) was decreased which was in line with disturbed G-protein signalling in NSCs lacking FMRP. Brain derived neurotrophic factor (BDNF) promotes neuronal differentiation of NSCs as the absence of FMRP was shown to do. This led us to study the effect of impaired BDNF/TrkB receptor signaling on NSCs by overexpression of TrkB.T1 receptor isoform. We showed that changes in the relative expression levels of the full-length and truncated TrkB isoforms influenced the replication capacity of NSCs. After the differentiation, the overexpression of TrkB.T1 increased neuronal turnover. To summarize, FMRP and TrkB signaling are involved in normal differentiation of NSCs in the developing brain. Since NSCs might have potential for therapeutic interventions in a variety of neurological disorders, our findings may be useful in the design of pharmacological interventions in neurological disorders of learning and memory.

From a "Student" to a Lifelong "Consumer" : Constructions of Educability in Adult Students' Narrative Life Histories

The focus of this study was to examine the constructions of the educable subject of the lifelong learning (LLL) narrative in the narrative life histories of adult students at general upper secondary school for adults (GUSSA). In this study lifelong learning has been defined as a cultural narrative on education, “a system of political thinking” that is not internally consistent, but has contradictory themes embedded within it (Billig et al., 1988). As earlier research has shown and this study also confirms, the LLL narrative creates differences between those who are included and those who fall behind and are excluded from the learning society ideal. Educability expresses socially constructed interpretations on who benefit from education and who should be educated and how. The presupposition in this study has been that contradictions between the LLL narrative and the so-called traditional constructions of educability are likely to be constructed as the former relies on the all-inclusive interpretation of educability and the latter on the meritocratic model of educating individuals based on their innate abilities. The school system continues to uphold the institutionalized ethos of educability that ranks students into the categories “bright”, “mediocre”, and “poor” (Räty & Snellman, 1998) on the basis of their abilities, including gender-related differences as well as differences based on social class. Traditional age-related norms also persist, for example general upper secondary education is normatively completed in youth and not in adulthood, and the formal learning context continues to outweigh both non-formal and informal learning. Moreover, in this study the construction of social differences in relation to educability and, thereafter unequal access to education has been examined in relation to age, social class, and gender. The biographical work of the research participants forms a peephole that permits the examination of the dilemmatic nature of the constructions of educability in this study. Formal general upper secondary education in adulthood is situated on the border between the traditional and the LLL narratives on educability: participation in GUSSA inevitably means that one’s ability and competence as a student and learner becomes reassessed through the assessment criteria maintained by schools, whereas according to the principles of LLL everyone is educable; everyone is encouraged to learn throughout their lives regardless of age, social class, or gender. This study is situated in the field of adult education, sociology of education, and social psychological research on educability, having also been informed by feminist studies. Moreover, this study contributes to narrative life history research combining the structural analysis of narratives (Labov & Waletzky, 1997), i.e. mini-stories within life history, with the analysis of the life histories as structural and thematic wholes and the creation of coherence in them; thus, permitting both micro and macro analyses. On accounting for the discontinuity created by participation in general upper secondary school study in adulthood and not normatively in youth, the GUSSA students construct coherence in relation to their ability and competence as students and learners. The seven case studies illuminate the social differences constructed in relation to educability, i.e. social class, gender, age, and the “new category of student and learner”. In the data of this study, i.e. 20 general upper secondary school adult graduates’ narrative life histories primarily generated through interviews, two main coherence patterns of the adult educable subject emerge. The first performance-oriented pattern displays qualities that are closely related to the principles of LLL. Contrary to the principles of lifewide learning, however, the documentation of one’s competence through formal qualifications outweighs non-formal and informal learning in preparation for future change and the competition for further education, professional careers, and higher social positions. The second flexible learning pattern calls into question the status of formal, especially theoretical and academically oriented education; inner development is seen as more important than such external signs of development — grades and certificates. Studying and learning is constructed as a hobby and as a means to a more satisfactory life as opposed to a socially and culturally valued serious occupation leading to further education and career development. Consequently, as a curious, active, and independent learner, this educable but not readily employable subject is pushed into the periphery of lifelong learning. These two coherence patterns of the adult educable subject illuminate who is to be educated and how. The educable and readily employable LLL subject is to participate in formal education in order to achieve qualifications for working life, whereas the educable but not employable subject may utilize lifewide learning for her/his own pleasure. Key words: adult education, general upper secondary school for adults, educability, lifelong learning, narrative life history

Developmental, functional brain imaging and electrophysiological evidence of visual and auditory working memory

Intact function of working memory (WM) is essential for children and adults to cope with every day life. Children with deficits in WM mechanisms have learning difficulties that are often accompanied by behavioral problems. The neural processes subserving WM, and brain structures underlying this system, continue to develop during childhood till adolescence and young adulthood. With functional magnetic resonance imaging (fMRI) it is possible to investigate the organization and development of WM. The present thesis aimed to investigate, using behavioral and neuroimaging methods, whether mnemonic processing of spatial and nonspatial visual information is segregated in the developing and mature human brain. A further aim in this research was to investigate the organization and development of audiospatial and visuospatial information processing in WM. The behavioral results showed that spatial and nonspatial visual WM processing is segregated in the adult brain. The fMRI result in children suggested that memory load related processing of spatial and nonspatial visual information engages common cortical networks, whereas selective attention to either type of stimuli recruits partially segregated areas in the frontal, parietal and occipital cortices. Deactivation mechanisms that are important in the performance of WM tasks in adults are already operational in healthy school-aged children. Electrophysiological evidence suggested segregated mnemonic processing of visual and auditory location information. The results of the development of audiospatial and visuospatial WM demonstrate that WM performance improves with age, suggesting functional maturation of underlying cognitive processes and brain areas. The development of the performance of spatial WM tasks follows a different time course in boys and girls indicating a larger degree of immaturity in the male than female WM systems. Furthermore, the differences in mastering auditory and visual WM tasks may indicate that visual WM reaches functional maturity earlier than the corresponding auditory system. Spatial WM deficits may underlie some learning difficulties and behavioral problems related to impulsivity, difficulties in concentration, and hyperactivity. Alternatively, anxiety or depressive symptoms may affect WM function and the ability to concentrate, being thus the primary cause of poor academic achievement in children.

Neural stem cell characteristics affected by oncogenic pathways and in a human motoneuron disease

Neural stem cell characteristics affected by oncogenic pathways and in a human motoneuron disease Stem cells provide the self-renewing cell pool for developing or regenerating organs. The mechanisms underlying the decisions of a stem or progenitor cell to either self-renew and maintain multipotentiality or alternatively to differentiate are incompletely understood. In this thesis work, I have approached this question by investigating the role of the proto-oncogene Myc in the regulatory functions of neural progenitor cell (NPC) self-renewal, proliferation and differentiation. By using a retroviral transduction technique to create overexpression models in embryonic NPCs cultured as neurospheres, I show that activated levels of Myc increase NPC self-renewal. Furthermore, several mechanisms that regulate the activity of Myc were identified. Myc induced self-renewal is signalled through binding to the transcription factor Miz-1 as shown by the inhibited capacity of a Myc mutant (MycV394D), deficient in binding to Miz-1, to increase self-renewal in NPCs. Furthermore, overexpression of the newly identified proto-oncogene CIP2A recapitulates the effects of Myc overexpression in NPCs. Also the expression levels and in vivo expression patterns of Myc and CIP2A were linked together. CIP2A stabilizes Myc protein levels in several cancer types by inhibiting its degradation and our results suggest the same function for CIP2A in NPCs. Our results also support the conception of self-renewal and proliferation being two separately regulated cellular functions. Finally, I suggest that Myc regulates NPC self-renewal by influencing the way stem and progenitor cells react to the environmental cues that normally dictate the cellular identity of tissues containing self-renewing cells. Neurosphere cultures were also utilised in order to characterise functional defects in a human disease. Neural stem cell cultures obtained post-mortem from foetuses of lethal congenital contracture syndrome (LCCS) were used to reveal possible cell autonomous differentiation defects of patient NPCs. However, LCCS derived NPCs were able to differentiate normally in vitro although several transcriptional differences were identified by using microarray analysis. Proliferation rate of the patient NPCs was also increased as compared to NPCs of age-matched control foetuses.

Androgen receptor-dependent and independent functions of ARIP4

Androgen receptor (AR) is necessary for normal male phenotype development and essential for spermatogenesis. AR is a classical steroid receptor mediating actions of male sex steroids testosterone and 5-alpha-dihydrotestosterone. Numerous coregulators interact with the receptor and regulate AR activity on target genes. This study deals with the characterization of androgen receptor-interacting protein 4 (ARIP4). ARIP4 binds DNA, interacts with AR in vitro and in cultured yeast and mammalian cells, and modulates AR-dependent transactivation. ARIP4 is an active DNA-dependent ATPase, and this enzymatic activity is essential for the ability of ARIP4 to modulate AR function. On the basis of sequence homology in its ATPase domain, ARIP4 belongs to the SNF2 family of proteins involved in chromatin remodeling, DNA repair, and homologous recombination. Similar to its closest homologs ATRX and Rad54, ARIP4 does not seem to be a classical chromatin remodeling protein in that it does not appear to form large protein complexes in vivo or remodel mononucleosomes in vitro. However, ARIP4 is able to generate superhelical torsion on linear DNA fragments. ARIP4 is covalently modified by SUMO-1, and mutation of six potential SUMO attachment sites abolishes the ability of ARIP4 to bind DNA, hydrolyze ATP, and activate AR function. ARIP4 expression starts in early embryonic development. In mouse embryo ARIP4 is present mainly in the neural tube and limb buds. In adult mouse tissues ARIP4 expression is virtually ubiquitous. In mouse testis ARIP4 is expressed in the nuclei of Sertoli cells in a stage-dependent manner. ARIP4 is also present in the nuclei of Leydig cells, spermatogonia, pachytene and diplotene spermatocytes. Testicular expression pattern of ARIP4 does not differ significantly in wild-type, FSHRKO, and LuRKO mice. In the testis of hpg mice, ARIP4 is found mainly in interstitial cells and has very low, if any, expression in Sertoli and germ cells. Heterozygous Arip4+/ mice are fertile and appear normal; however, they are haploinsufficient with regard to androgen action in Sertoli cells. In contrast, Arip4 / embryos are not viable. They have significantly reduced body size at E9.5 and die by E11.5. Compared to wild-type littermates, Arip4 / embryos possess a higher percentage of apoptotic cells at E9.5 and E10.5. Fibroblasts derived from Arip4 / embryos cease growing after 2-3 passages and exhibit a significantly increased apoptosis and decreased proliferation rate than cells from wild-type embryos. Our findings demonstrate that ARIP4 plays an essential role in mouse embryonic development. In addition, testicular expression and AR coregulatory activity of ARIP4 suggest a role of ARIP4-AR interaction in the somatic cells of the testis.

Early detection of type 2 diabetes mellitus in Chinese and Indian adult population

Objectives of this study were to determine secular trends of diabetes prevalence in China and develop simple risk assessment algorithms for screening individuals with high-risk for diabetes or with undiagnosed diabetes in Chinese and Indian adults. Two consecutive population based surveys in Chinese and a prospective study in Mauritian Indians were involved in this study. The Chinese surveys were conducted in randomly selected populations aged 20-74 years in 2001-2002 (n=14 592) and 35-74 years in 2006 (n=4416). A two-step screening strategy using fasting capillary plasma glucose (FCG) as first-line screening test followed by standard 2-hour 75g oral glucose tolerance tests (OGTTs) was applied to 12 436 individuals in 2001, while OGTTs were administrated to all participants together with FCG in 2006 and to 2156 subjects in 2002. In Mauritius, two consecutive population based surveys were conducted in Mauritian Indians aged 20-65 years in 1987 and 1992; 3094 Indians (1141 men), who were not diagnosed as diabetes at baseline, were reexamined with OGTTs in 1992 and/or 1998. Diabetes and pre-diabetes was defined following 2006 World Health Organization/ International Diabetes Federation Criteria. Age-standardized, as well as age- and sex-specific, prevalence of diabetes and pre-diabetes in adult Chinese was significantly increased from 12.2% and 15.4% in 2001 to 16.0% and 21.2% in 2006, respectively. A simple Chinese diabetes risk score was developed based on the data of Chinese survey 2001-2002 and validated in the population of survey 2006. The risk scores based on β coefficients derived from the final Logistic regression model ranged from 3 – 32. When the score was applied to the population of survey 2006, the area under operating characteristic curve (AUC) of the score for screening undiagnosed diabetes was 0.67 (95% CI, 0.65-0.70), which was lower than the AUC of FCG (0.76 [0.74-0.79]), but similar to that of HbA1c (0.68 [0.65-0.71]). At a cut-off point of 14, the sensitivity and specificity of the risk score in screening undiagnosed diabetes was 0.84 (0.81-0.88) and 0.40 (0.38-0.41). In Mauritian Indian, body mass index (BMI), waist girth, family history of diabetes (FH), and glucose was confirmed to be independent risk predictors for developing diabetes. Predicted probabilities for developing diabetes derived from a simple Cox regression model fitted with sex, FH, BMI and waist girth ranged from 0.05 to 0.64 in men and 0.03 to 0.49 in women. To predict the onset of diabetes, the AUC of the predicted probabilities was 0.62 (95% CI, 0.56-0.68) in men and 0.64(0.59-0.69) in women. At a cut-off point of 0.12, the sensitivity and specificity was 0.72(0.71-0.74) and 0.47(0.45-0.49) in men; and 0.77(0.75-0.78) and 0.50(0.48-0.52) in women, respectively. In conclusion, there was a rapid increase in prevalence of diabetes in Chinese adults from 2001 to 2006. The simple risk assessment algorithms based on age, obesity and family history of diabetes showed a moderate discrimination of diabetes from non-diabetes, which may be used as first line screening tool for diabetes and pre-diabetes, and for health promotion purpose in Chinese and Indians.

Early growth and adult health: focus on blood pressure, glucose tolerance status and body composition

Theory of developmental origins of adult health and disease proposes that experiences during critical periods of early development may have consequences on health throughout a lifespan. Thesis studies aimed to characterize associations between early growth and some components of the metabolic syndrome cluster. Participants belong to two epidemiological cohorts with data on birth measurements and, for the younger cohort, on serial recordings of weight and height during childhood. They were born as singletons between 1924-33 and 1934-44 in the Helsinki University Central Hospital, and 500 and 2003 of them, respectively, attended clinical studies at the age of 65-75 and 56-70 years, respectively. In the 65-75 year old men and women, the well-known inverse relationship between birth weight and systolic blood pressure (SBP) was confined to people who had established hypertension. Among them a 1-kg increase in birth weight was associated with a 6.4-mmHg (95% CI: 1.0 to 11.9) decrease in SBP. This relationship was further confined to people with the prevailing Pro12Pro polymorphism of the peroxisome proliferator-activated receptor-γ2 (PPARγ2) gene. People with low birth weight were more likely to receive angiotensin-converting enzyme inhibitors/angiotensin-receptor blockers (ACEI/ARB, p=0.03), and, again, this relationship was confined to the carriers of the Pro12Pro (p=0.01 for interaction). These results suggest that the inverse association between birth weight and systolic BP becomes focused in hypertensive people because pathological features of BP regulation, associated with slow fetal growth, become self-perpetuating in adult life. Insulin resistance of the Pro12Pro carriers with low birth weight may interact with the renin-angiotensin system leading to raised BP levels. Habitual physical activity protected men and women who were small at birth, and thus at increased risk for the development of type 2 diabetes, against glucose intolerance more strongly. Among subjects with birth weight ≤3000 g, the odds ratio (OR) for glucose intolerance was 5.2 (95% CI: 2.1 to 13) in those who exercised less than 3 times per week compared to regular exercisers; in those who scored their exercise light compared with moderate exercisers (defined as comparable to brisk walking) the OR was 3.5 (1.5 to 8.2). In the 56-70 year old men a 1 kg increase in birth weight corresponded to a 4.1 kg (95% CI: 3.1 to 5.1) and in women to a 2.9 kg (2.1 to 3.6) increase in adult lean mass. Rapid gain in body mass index (BMI), i.e. crossing from an original BMI percentile to a higher one, before the age of 2 years increased adult lean mass index (LMI, lean mass/height squared) without excess fat accumulation whereas rapid gain in BMI during later childhood, despite the concurrent rise in LMI, resulted in a relatively higher increase in adult body fat mass. These findings illustrate how genes, the environment and their interactions, early growth patterns, and adult lifestyle modify adult health risks which originate from early life.

Adult-type hypolactasia: Genotype-phenotype correlation

Adult-type hypolactasia (primary lactose malabsorption, lactase non-persistence) is the most common enzyme deficiency worldwide, and manifests with symptoms of lactose intolerance such as abdominal pain, gas formation and diarrhea. In humans with adult-type hypolactasia, lactase activity is high at birth, but declines during childhood to about one-tenth of the activity at birth. In 2002, a one base polymorphism C/T-13910, located 14 kilobases from the starting codon of the lactase-phlorizin hydrolase (LPH) gene was observed to be associated with the persistence of lactase activity. The T-13910 allele (C/T-13910 and T/T-13910 genotypes) associates with persistence of lactase activity throughout life, whereas the C/C-13910 genotype associates with adult-type hypolactasia. In this thesis work, the timing and mechanism of decline of lactase enzyme activity during development was studied using the C/T-13910 polymorphism as a molecular marker. We observed an excellent correlation between low lactase activity and the C/C-13910 genotype in all subjects > 12 years of age, irrespective their ethnicity. In children of African origin, the lactase activity declined somewhat earlier than among Finnish children. Furthermore, we observed an increasing imbalance in the relative lactase mRNA expression from the C-13910 and T-13910 alleles in Finnish children beginning from five years of age. The genetic test for adult-type hypolactasia showed a sensitivity of 93% and a specificity of 100% in the Finnish children and adolescents > 12 years of age. The relation of milk consumption and the milk-related abdominal complaints to the C/T-13910 genotypes associated with lactase persistence/non-persistence was studied by a questionnaire-based approach in > 2100 Finns. Both Finnish children and adults with the C/C-13910 genotype consumed significantly less dairy products compared to those with the C/T-13910 and T/T-13910 genotypes. Flatulence was the only of the abdominal symptoms of lactose intolerance that subjects with the C/C-13910 genotype reported significantly more often than those with the C/T-13910 and T/T-13910 genotypes. A minor proportion (<10%) of subjects with the C/C-13910 genotype, nevertheless, reported drinking milk without any symptoms afterwards. There was no association between cow's milk allergy starting as a newborn and adult-type hypolactasia. In an association study an increased risk of colorectal cancer was observed among those with molecular diagnosis of adult-type hypolactasia. It warrants further studies to clarify whether the increased risk observed in the Finnish population is associated with lactose or decreased intake of dairy products in these subjects.

Automatic Lameness Detection in a Milking Robot : Instrumentation, measurement software, algorithms for data analysis and a neural network model

The aim of this thesis is to develop a fully automatic lameness detection system that operates in a milking robot. The instrumentation, measurement software, algorithms for data analysis and a neural network model for lameness detection were developed. Automatic milking has become a common practice in dairy husbandry, and in the year 2006 about 4000 farms worldwide used over 6000 milking robots. There is a worldwide movement with the objective of fully automating every process from feeding to milking. Increase in automation is a consequence of increasing farm sizes, the demand for more efficient production and the growth of labour costs. As the level of automation increases, the time that the cattle keeper uses for monitoring animals often decreases. This has created a need for systems for automatically monitoring the health of farm animals. The popularity of milking robots also offers a new and unique possibility to monitor animals in a single confined space up to four times daily. Lameness is a crucial welfare issue in the modern dairy industry. Limb disorders cause serious welfare, health and economic problems especially in loose housing of cattle. Lameness causes losses in milk production and leads to early culling of animals. These costs could be reduced with early identification and treatment. At present, only a few methods for automatically detecting lameness have been developed, and the most common methods used for lameness detection and assessment are various visual locomotion scoring systems. The problem with locomotion scoring is that it needs experience to be conducted properly, it is labour intensive as an on-farm method and the results are subjective. A four balance system for measuring the leg load distribution of dairy cows during milking in order to detect lameness was developed and set up in the University of Helsinki Research farm Suitia. The leg weights of 73 cows were successfully recorded during almost 10,000 robotic milkings over a period of 5 months. The cows were locomotion scored weekly, and the lame cows were inspected clinically for hoof lesions. Unsuccessful measurements, caused by cows standing outside the balances, were removed from the data with a special algorithm, and the mean leg loads and the number of kicks during milking was calculated. In order to develop an expert system to automatically detect lameness cases, a model was needed. A probabilistic neural network (PNN) classifier model was chosen for the task. The data was divided in two parts and 5,074 measurements from 37 cows were used to train the model. The operation of the model was evaluated for its ability to detect lameness in the validating dataset, which had 4,868 measurements from 36 cows. The model was able to classify 96% of the measurements correctly as sound or lame cows, and 100% of the lameness cases in the validation data were identified. The number of measurements causing false alarms was 1.1%. The developed model has the potential to be used for on-farm decision support and can be used in a real-time lameness monitoring system.