To enable monitoring and quantification of proteasome activity in vivo, a photoconvertible reporter system was developed to live image proteasome activity in C. elegans. By using this system, it was uncovered that proteasome activity varies between different tissues, as GABAergic and dopaminergic neurons showed faster reporter degradation than muscle cells, and that aging affects proteasome activity in a tissue-dependent manner. Subsequently, it was investigated whether signaling pathways regulating aging also affect proteasome activity. Insulin/IGF-1 signaling (IIS) is an evolutionarily conserved signaling pathway regulating lifespan in worms, flies, rodents and possibly humans. By using C. elegans as a model organism, this thesis shows that IIS regulates proteasome activity through the FOXO/DAF-16 and Nrf/SKN-1 transcription factors. Moreover, it was demonstrated that DAF-16, which is activated by reduced IIS, inhibits the expression of the proteasome-associated deubiquitinating enzyme UBH-4, which was shown to function as a tissue-specific proteasome inhibitor. The role of UBH-4 appears to be well-conserved, as downregulation of its mammalian ortholog, UCHL-5, increases proteasome activity and degradation of proteotoxic proteins in mammalian cells.

Taken together, this thesis provides tools to study proteasome activity in vivo, and establishes a molecular mechanism linking IIS to efficiency of proteasomal degradation. These results can be utilized when designing new therapies for proteasome-associated diseases, especially for disorders that may be alleviated with tissue-specific modulation of proteasome activity.

The systemic levels of matrix metalloproteinases (MMPs) -7, -8 and -9 and the tissue inhibitor of metalloproteinases-1 (TIMP-1) were investigated in 877 critically ill patients. In Study I, 15 intensive care unit-(ICU) treated adult patients with secondary peritonitis were prospectively included. Peritoneal fluid, blood and urine samples were collected at the ICU after surgery. The serum and urine levels of MMP-8 were compared with those obtained from ten healthy volunteers, and were found to be significantly higher in patients. In peritoneal fluid, MMP-8 levels were significantly higher than in serum and urine. This study was the first to identify MMP-8 in the peritoneal fluid of peritonitis patients.

Study II was a sub-study of the FINNSEPSIS study where patients with severe sepsis or septic shock were prospectively included in 24 Finnish ICUs. Serum samples of 248 patients were analysed for MMP-8, MMP-9 and TIMP-1 levels, that were found to be higher than those of healthy controls. MMP-8, -9 and TIMP-1 levels were compared between ICU survivors and non-survivors. Median MMP-8 (p<0.01) and TIMP-1 (p PIENEMPI 0.001) were higher and median MMP-9 levels lower (p=0.047) in ICU non-survivors than in ICU survivors.

Study III investigated MMP-7, MMP-8, MMP-9, and TIMP-1 levels on 51 patients resuscitated from cardiac arrest (CA). The patients were a subgroup of the Hypothermia After Cardiac Arrest study. Thirty patients had received mild therapeutic hypothermia treatment (MTH) and 21 non-hypothermia treatment (non-MTH). Serum samples taken at 24 and 48 hours from restoration of spontaneous circulation were analysed and compared between patients and healthy volunteers. Serum MMP and TIMP-1 levels of MTH-treated patients were compared during and after MTH with the levels of non-MTH-treated patients. MMP-8 and MMP-9 were higher in CA patients than controls. Patients receiving MTH treatment had lower median MMP-9 levels during MTH than non-MTH-treated patients (p PIENEMPI 0.001). This is one novel potential mechanism of how MTH treatment improves outcome of CA patients.

Study IV was a 563-patient sub-study of the FINNALI study that included acute respiratory failure patients at 25 Finnish ICUs. MMP-8 and TIMP-1 were analysed from blood samples taken on study admission and 48 hours thereafter. Association of MMP-8 and TIMP-1 with 90-day mortality was examined. Serum MMP-8 predicted 90-day mortality of acute respiratory failure patients poorly. Admission TIMP-1 levels were higher in non-survivors than in survivors (p PIENEMPI 0.001).TIMP-1 was an independent predictor of 90-day mortality, with a moderate discriminative power (AUC 0.633, 95% CI 0.580- 0.686). TIMP-1 was also associated with the severity of oxygenation disturbance. Thus, TIMP-1 is a potentially useful biomarker for predicting outcome in acute respiratory failure patients.

The high-throughput measurement technologies enable the measurement of molecular determinants in cells. However, measurement technologies produce remarkable large data sets and the research questions have become increasingly complex. Thus computational methods are needed to discover molecular mechanisms behind the phenotypes. In many cases, analysis of molecular determinants that contribute to the phenotype proceeds by first identifying putative candidates by using a priori information and high-throughput measurements. Then further analysis can focus on most promising molecules. In many cases, the aim is to identify relevant markers or targets from a set of candidate molecules.

Often biomedical studies result in a long list of candidate genes, and to interpret these candidates, information on their context in cell functions is needed. This context information can give insight to synergistic effects of molecular machinery in cells when functions of individual molecules do not explain the observed phenotype. In addition, the context information can be used to generate candidates. One of the methods in this thesis provides a computational data integration method that provides a link in between candidate genes from molecular pathways and genetic variants. It uses publicly available biological knowledge bases to systematically create functional context of candidate genes. This approach is especially important when studying cancer, that is dependent of complex molecular signaling.

Genotypes associated with inherited disease predispositions have been studied successfully in the past, however, traditional methods are not applicable in wide variety of analysis conditions. Thus, this thesis introduces a method that uses haplotype sharing to identify genetic loci inherited by multiple distantly related individuals. It is flexible and can be used in various settings, also with very limited number of samples.

Increasing the number of biological replicates in gene expression analysis increases the reliability of the results. In many cases, however, the number of samples is limited. Therefore, pooling gene expression data from multiple published studies can increase the understanding of the molecular background behind cell types. This is shown in this thesis by an analysis that identifies gene expression differences in two cell types using publicly available gene expression samples from previous studies.

Finally, when candidate molecules are available to characterize phenotypes, they can be compiled into biomarkers. In many cases, a combination of multiple molecules serves as a better biomarker than a single molecule. This thesis also includes a machine learning approach that is used to discover a classifier that predicts the phenotype.

Materials and methods: The localization of signaling molecules (c-Jun N-terminal kinase (JNK), p38, extracellular signal-regulated kinase (ERK), Akt, mammalian target of rapamycin (mTOR), cyclic AMP response element binding protein (CREB), and Bcl-associated death protein (Bad)) was studied using immunofluorescence staining in ruptured and unruptured IA samples, collected during microneurosurgical clipping. The levels of phosphorylated and total forms of signaling molecules were determined semi-quantitatively from IA tissue homogenates using Western blotting (WB). Altogether, 40 ruptured and 37 unruptured IA samples were investigated. IA size and shape indices were determined using 3D remodeling, and the associations between the levels of signaling molecules and IA rupture status and morphology were analyzed. The JNK signaling pathway was characterized further with c-Jun and pro-MMP-9 (MMP, matrix metalloproteinase) WB. Cell death in the IA wall was studied using terminal deoxynucleotidyl transferase (dUTP) nick end labeling (TUNEL) and cleaved caspase-3 immunostaining and caspase-3 WB. The activation of caspase cascades (caspase-9, intrinsic apoptosis; caspase-8, extrinsic apoptosis) was investigated using WB. The role of oxidative stress in IA wall degeneration was studied using heme oxygenase-1 (HO-1). Potential sources of oxidative stress were revealed using endogenous peroxidase stainings.

Results: All signaling molecules (JNK, p38, ERK, Akt, mTOR, CREB, and Bad) were found in the IA wall in immunofluorescence staining. Stress-activated kinases (phospho-p38 and phospho-JNK) were associated with size and rupture, probably indicating that stress signaling contributes to IA growth and rupture. Pro-MMP-9 levels were increased in ruptured IAs. Of well-known growth-promoting factors, phospho-Akt was associated with size, but not with rupture, indicating active cell proliferation in large IAs. On the other hand, phospho-mTOR levels were downregulated in ruptured IAs suggesting that cell proliferation signaling is decreased in ruptured IAs. Phospho-CREB levels were associated with the non-sphericity and ellipticity indices, whereas a negative association between total CREB levels and the undulation index was observed. On the other hand, phosphorylated levels of p38 and Akt correlated negatively with the undulation index, and total levels of CREB correlated positively with size indices, suggesting a complex role of signaling molecules in IA remodeling. Furthermore, TUNEL staining was associated with rupture. A few cleaved caspase-3-positive cells were also detected. Intrinsic activation of apoptosis (cleaved caspase-9) was observed in WB, mainly in ruptured samples, but cleaved caspase-8 was not detected. Unphosphorylated Bad levels, a regulator of the intrinsic apoptotic pathway, were also associated with rupture. HO-1 was associated with wall degeneration and rupture, and endogenous peroxidase staining localized mainly in polymorphonuclear cells of the intraluminal thrombus.

Conclusions: This study revealed associations of several intracellular signaling molecules with IA morphology and rupture and also suggested oxidative stress as an important factor in the degeneration of an IA wall. Characterization of signaling pathways in the human IA wall may aid in finding biomarkers differentiating rupture-prone IAs and in developing less invasive treatment methods for IAs. However, the role of signaling pathways in IA growth and rupture must first be evaluated with appropriate experimental models.

Neovessel formation is a prerequisite for tumor growth. Pericellular modulation of the ECM by MT1-MMP releases matrix-associated growth factors and bioactive peptides, which further affect angiogenesis and tumor cell biology. We found that MT1-MMP modulated subendothelial extracellular matrix, and cleaved latent TGF-β binding protein-1 (LTPB-1), with subsequent release of latent TGF-β complexes from the ECM. Thus, MT1-MMP-mediated LTBP-1 cleavage provides a mechanism for the tightly controlled release of matrix-associated TGF-β at the sites of neovessel formation.

To elucidate the functions of MT-MMPs in melanoma cell invasion, we analyzed the expression of MT1-MMP and MT3-MMP from biopsies of normal human skin, benign nevi, and melanoma metastases. MT3-MMP was upregulated in lymph node metastases of human melanoma, while MT1-MMP expression was comparable in all biopsies. By culturing melanoma cells in 3D collagen and fibrin matrices, we found that MT3-MMP was associated with expansive melanoma growth in 3D collagen, but promoted their sprouting growth in 3D fibrin. In in vivo xenograft experiments, MT3-MMP expressing melanoma xenografts grew slowly, while MT3-MMP silencing enhanced tumor growth rate by over twofold. Interestingly, high MT3-MMP expression in murine xenografts and a human melanoma tumor was associated with prominent lymphatic vessel invasion but negligible blood vessel invasion of melanoma cells. Silencing of MT3-MMP reduced lymphatic invasion but facilitated blood vessel invasion of melanoma cells over ~10-fold. MT3-MMP reduced cell surface MT1-MMP in vitro and in vivo, resulting in limited collagen invasion in vitro and collagen accumulation in vivo. This suggested that low collagenolytic ability of MT3-MMP-expressing melanoma cells resulted in decreased blood vascular invasion. These cells invaded instead into more permissive lymphatic vessels. Since lymphatic vessel invasion is associated with metastatic spread in melanoma, MT3-MMP expression may serve as a new prognostic factor in this disease.

To study the development, we analysed the trends in case fatality (CF) of the first ACS and first ischaemic stroke, the incidence of lower-extremity amputations and population attributable-fractions (PAFs) of ACS and ischaemic stroke due to diabetes.

We executed a register study combining information from the national reimbursement registers of medicines, Hospital Discharge Register and Causes-of-Death Register to identify all persons that have been treated with hypoglycaemic medication or registered with a diabetes diagnosis, and their macrovascular complications from 1992 to 2002. Comparisons were made in both genders and various age groups in the nondiabetic population. Data for comparison were available through the National Cardiovascular Disease Register.

Our main findings were that the diabetic population has benefited from the favourable development to the same extent as the nondiabetic population (I, II). Prognoses after the first ACS and the first ischaemic stroke are improving. The incidence of lower-extremity amputation is decreasing (III). The prognoses of patients with diabetes are still, however, far from the prognoses of the nondiabetic population. Patients with type 2 diabetes (T2DM) had a total 1-year CF of 65.2% compared with 56.5% among nondiabetic men after their first ACS (I). The corresponding figures for women were 61.0% and 48.3%. The risk ratio (RR) for 28-day CF after the first ACS was 1.34 (95% CI (confidence interval) 1.24–1.45) among T2DM men 35–64 years of age compared with nondiabetic men in the same age group, and 1.69 (95% CI 1.44–1.97) among T2DM women with nondiabetic women, again in the same age group. The difference was greatest in the younger age groups and became less clear in the older age groups. The risk of fatal outcome within a year for 28-day survivors was considerable in patients with T2DM, RR 2.87 (95% CI 2.19–3.75) among men and 5.84 (3.70–9.23) among women 35–64 years of age. There was a statistically significant downward trend in 28-day CF and in 1-year CF of 28-day survivors both among patients with T2DM and nondiabetic patients. The trends did not differ between patients with T2DM and nondiabetic persons.

The total 1-year cardiovascular disease (CVD) CF after the first ischaemic stroke was 1.2–1.6 times higher among patients with T2DM compared with nondiabetic persons, depending on gender and age group (II). The 28-day CF was 1.1–1.3 times higher among patients with T2DM. The difference was biggest in the younger age group of men. The 1-year CVD CF of 28-day survivors was 1.4–2.2 times higher in patients with T2DM and this difference was biggest in the younger age group of women. There was a statistically significant downward trend during the study period in CF of the first ischaemic stroke at 0–27 days and 28–364 days after the stroke. The trends did not differ between patients with T2DM and nondiabetic persons.

The crude rate of first amputations decreased from 924 to 387 per 100 000 diabetic persons (III). The decrease was similar in men and women. There were major differences in the pace of development among the hospital districts. The ratio of minor and major amputations had a positive development during the study period and the rate of first major amputations among diabetic patients decreased in the Finnish population.

The PAF of the first CVD event due to diabetes showed an upward trend among men from 11.4% (95% CI 10.8–12.0%) to 13.8% (95% CI 13.2–14.5%), p for trend less than 0.0001, and a downward trend among women from 20.1% (95% CI 19.2–21.0%) to 16.9% (95% CI 15.9–17.8%), p for trend 0.0005 (IV). The upward trend was statistically significant in the first ACS event among men and the downward trend was statistically significant in the first ischaemic stroke in women.

In conclusion, our results showed that the contribution of diabetes to the burden of first CVD events remained considerable. However, despite the increase in prevalence of diabetes, the PAF of the first CVD event decreased in women and increased only slightly in men.

Strong actions should be taken to improve comprehensive treatment of risk factors after diagnosis of diabetes mellitus and efficiency of secondary prevention after a cardiovascular event. Special attention should be focused on better secondary prevention, because the largest difference between T2DM and nondiabetic persons was in 1-year prognosis of 28-day survivors after the first ACS or ischaemic stroke.

We also studied the possibility to exploit neuron-specific miRNA expression patterns in the modification of tissue tropism of an alphavirus Semliki Forest virus (SFV). We engineered SFV genome to contain sequences complementary to the neuron-specific miRNA miR124. In vitro characterization of this novel virus showed that the modification of the SFV genome per se did not affect polyprotein processing or oncolytic potency. Intraperitoneally administered miR124-targeted SFV displayed an attenuated spread into the central nervous system (CNS) and increased survival of infected mice. Also, mice pre-infected with miR124-targeted SFV elicited strong protective immunity against otherwise lethal challenge with a highly virulent wild-type SFV strain.

In conclusion, these results show that miRNA-targeting is a potent new strategy to engineer viral tropism in development of safer and more efficient reagents for virotherapy applications.

This thesis aims to shed light on these newly emerging vulnerable groups in terms of assessing their voices through questionnaires, perceptual and acoustic voice assessment, and videolaryngoscopic examination. The thesis includes four studies. Nine subjects with suspected occupational rhinitis or asthma participated in Studies I and II. They had single blinded exposures to organic dust and placebo substances. Subjective and perceptual voice assessment was done in addition to acoustic analysis of 180 samples using glottal inverse filtering. In Study III, children’s voices were perceptually assessed as well as their health- and voice-related quality of life. In Study IV, 119 female kindergarten teachers responded to a questionnaire on voice habits, voice symptoms, and impact of various working conditions on voice. In addition, videolaryngoscopy examinations took place in these teachers’ workplaces.

Studies I and II showed that some self-assessed voice and throat symptoms changed significantly after organic dust exposure, although perceptual assessment failed to record these changes. However, inverse filtering analysis revealed changes that represent the ones reported by the subjects. In

Study III, voice-related quality of life and perceptual assessment of the study group showed lower scores than the controls’.

Study IV showed that 71.5% of the teachers examined reported frequent strain on the voice. Organic findings were observed in 10.9% of the subjects and did not correlate with subjective voice symptoms. The thesis added new information on these high-risk groups, identifying an occupational voice-disorder risk group related to laryngeal reactions rather than voice abuse. It also added information on the long-term effects of surgery for subglottic stenosis in early infancy. Nevertheless, field videolaryngoscopy was quite accurate in determining the percentage of organic findings among nursery teachers.

In this study, 127 follicular thyroid neoplasia patients (83 FTAs and 44 FTCs) treated at Helsinki University Central Hospital (HUCH) in Finland between 1990 and 2009 were examined to find methods for differential diagnosis between follicular thyroid lesions. Tissue markers were investigated by immunohistochemistry in follicular neoplasms and non-neoplastic control tissues, and were correlated with clinical parameters, such as with metastatic disease and survival. Additionally, cancer registry data were gathered, concerning the diminishing incidence of FTC accompanied by an increase in the incidence of papillary thyroid carcinoma.

Carcinomas were reanalysed according to the new World Health Organization classification of endocrine tumours, in which a new tumour entity, poorly differentiated carcinoma of the thyroid, was introduced. Markers with possible clinical utility were found; e.g. in an attempt to differentiate between non-neoplastic and neoplastic follicular lesions of the thyroid (HES5) as well as between FTA and FTC (MIB-1, Cyclin D1, TLR-2, ERβ), a marker with prognostic value in carcinomas (ERβ), as well as a marker correlating with the presence of metastatic disease (TLR-4).

These results aid in the challenging field of diagnostics in follicular thyroid lesions. Measuring the expression of HES5 may help in differentiating between neoplastic and non-neoplastic follicular thyroid lesions. Markers, such as MIB-1 and ERβ, are partly able to differentiate between benign and malignant follicular thyroid neoplasias, whereas ERβ and TLR-4 have prognostic value in FTC.

Four prospective randomized control trials consisting of 293 ERCP patients were performed. PCS was compared with anesthesiologist administered sedation (AAS) using manually adjusted propofol infusion (I) and target-controlled infusion (TCI) (III). Remifentanil and alfentanil were compared in sedative mixture for PCS (II) and dexmedetomidine was evaluated for sedation of patients with chronic alcohol-ism (IV). Self-administration device was adjusted to deliver 1ml single bolus-dose of propofol or propo-fol-opioid mixture. Loading dose of 1 mcg • kg-1 of dexmedetomidine was infused in 10 minutes before ERCP start thereafter maintenance infusion at the constant rate of 0.7 mcg • kg-1 • h-1 was continued un-til the end of procedure (IV). In control groups sedation was administered by anesthesiologist. Hy-poxemia, respiratory depression, hypotension, arrhythmia, and pulmonary aspiration were considered as sedation related adverse events (SRAE). Consumption of propofol was the main objective. Secondary objectives were success rate of PCS, SRAE, patient satisfaction with sedation, easiness of ERCP per-formance, and rapidity of the recovery.

With the use of PCS propofol consumption was significantly lesser than with AAS. The success rate of PCS was 88 -100 %. Patients received PCS recovered faster than received AAS. Combined with propo-fol, remifentanil depressed spontaneous respiration and produced nausea more frequently than alfen-tanil. Increase of alfentanil concentration in sedative mixture from 0.04 mg • ml-1 to 0.08 mg • ml-1 did not provide any demonstrable benefit. The studied regimen of dexmedetomidine administration showed poor suitability for sedation of alcoholics during ERCP. Instead, PCS might be successful in such pa-tients.

In conclusion, PCS with combination of propofol and alfentanil is recommended as a primary method of sedation during ERCP.

A group of 283 DAVF patients admitted in Helsinki and Kuopio Departments of Neurosurgery in Finland, between 1944 and 2010, is one of the largest reported series in the world. The overall long-term clinical outcome of patients admitted between 1944-2006 was assessed. Long-term excess mortality was estimated using the relative survival ratio (RSR), which provides a measure of the excess mortality experienced by the patients compared with the general Finnish population matched by age, sex and calendar time. The results of microsurgical treatment of DAVFs were analyzed in patients treated between 1980 and 2010.

The advances in diagnostic methods have increased the detection rate of DAVFs. The incidence of DAVFs in a defined Southern Finnish population was 0.51 per 100,000 per year, and they represented 32% of all the brain arteriovenous malformations (AVMs), which is twice as much as in previous studies. The results of treatment and outcome of patients have markedly improved with the introduction of endovascular techniques and stereotactic radiosurgery (SRS). Major hemorrhagic or ischemic complications were seen especially after surgical treatment of DAVFs. Currently, microsurgery is of limited use in those DAVFs where endovascular treatment and radiosurgery have failed to reduce the high risk of hemorrhage or relieve intolerable symptoms.

During the first 12 months, there was excess mortality among the patients, mainly due to treatment complications. Thereafter, their overall long-term survival became similar to that of matched general population, except for those that were located elswhere than transverse and sigmoid sinuses or presented with cortical venous drainage. The patients had more cardiovascular and cerebrovascular deaths than would be expected in the general Finnish population, for an unknown reason.

Data on women who participated in two Finnish opulation-based studies (Health 2000 and FINRISK 1997, 2002 and 2007) were analyzed. Data were collected through face-to-face interviews, self-administered questionnaires and health examinations. Different structured (BDI-21, BDI-13, GHQ-12, CIDI) and non-structured tools were used to assess mental health and psychological well-being. Study I focused on the association between mental health and miscarriage, by history and number; Studies II and III focused on the relationship with use of hormonal contraception (either oral or intrauterine) and its duration; Study IV concentrated on the associations between mental health and hormone therapy in perimenopausal and postmenopausal women.

Study I showed that a miscarriage as a pregnancy outcome was related to a high prevalence of depressive disorders, and to more severe depressive or anxiety symptoms compared with other pregnancy outcomes. Moreover, the higher the number of miscarriages was, the worse the current state of mood was and the higher the frequency of a psychiatric diagnosis. Studies II and III revealed that the use of hormonal contraception was not associated with adverse psychological status or depressive symptoms/disorders. Additionally, no effect of different hormonal compounds was detected. The main finding in Study IV was the high prevalence of depressive and anxiety disorders among women in connection with the menopausal transition. Moreover, in this group, an association between current use of hormone therapy and worse psychological well-being or mental health was detected.

The results of this study support the hypothesis of an association between psychological well-being and reproductive features in women. The importance of considering reproductive health and events when assessing psychological status and mental health in women is discussed.

Diabetic retinopathy is the leading cause of acquired visual disability among people of working age in all industrialised countries. Established risk factors for diabetic retinopathy include the duration of diabetes, glycaemic control, blood pressure and dyslipidaemia. However, these risk factors explain less than half of the risk for diabetic retinopathy. It is, thus, obvious that a large proportion of the risk remains to be explored.

Aim

The aim of the present study was to investigate potential risk factors that could affect the development of severe forms of retinopathy in type 1 diabetes.

Patients and Methods

The study patients were drawn from the large FinnDiane (Finnish Diabetic Nephropathy study) database. The FinnDiane study is an observational cohort study which since 1997 has collected comprehensive data on patients with type 1 diabetes at 92 centers throughout Finland with the aim of identifying genetic and environmental risk factors for diabetic complications. The patients’ retinopathy status were verified from ophthalmic and medical files and fundus photographs when available and graded with the ETDRS-scale. All patients underwent a thorough clinical characterisation of their clinical diabetes status by the attending physician at the participating study centers.

Results

Proliferative diabetic retinopathy showed significant familial clustering in siblings with type 1 diabetes and the heritability h2 adjusted for conventional risk factors suggested a significant genetic contribution to the risk. The siblings first affected by type 1 diabetes had a lower risk of proliferative retinopathy as compared to the siblings later affected by type 1 diabetes. The risk of both proliferative retinopathy and clinically significant macular edema were modified by the patient’s age at onset of type 1 diabetes. The patients with higher age at onset of type 1 diabetes had a lower risk of proliferative retinopathy but conversely, a higher risk of clinically significant macular edema. The HbA1c variability was lower in those patients with higher age at onset of type 1 diabetes and the patients with lower HbA1c variability had a lower cumulative incidence and risk of laser treatment and proliferative retinopathy.

Conclusion

In addition to the conventional risk factors, such as diabetes duration, glycaemic control and blood pressure, familial factors, age at onset of type 1 diabetes and glycaemic profile may explain a significant proportion of the risk of severe forms of diabetic retinopathy.

Patients and methods The first study consisted of 60 consecutive patients treated by skin sparing mastectomy and immediate breast reconstruction for an ipsilateral breast tumour recurrence. Study II consisted of 40 consecutively operated patients who were treated with wide soft tissue resection and immediate flap reconstruction for an extensive chest wall recurrent breast cancer. In both studies I and II hospital records were analysed for surgical complications and oncological outcome. Study III was based on 522 consecutive patients who underwent mastectomy with or without breast reconstruction between January 2000 and December 2003. Data on cancer recurrence and survival were collected from hospital records and the files of the Finnish Cancer Registry. Study IV consisted of 10 patients treated surgically between 1999 and 2010 for a breast angiosarcoma following earlier radiotherapy for primary breast cancer. Surgical notes and pathology reports were reviewed for resection margins. Hospital records were analysed for oncological outcome.

Results Study I After a median follow-up of 84 months, 11 (18%) patients developed disease relapse (6 local re-recurrences (10%), 3 cases of regional lymph node metastasis and 2 cases of distant metastasis). 5 year overall survival (OS) was 94% and 5 year disease-free survival (DFS) was 83%.

Study II After a median follow-up of 31 months, the median DFS was 31 months and the median OS was 52 months. The 2 and 5 year OS rates were 71% and 32% respectively. Multivariate cox regression analysis revealed that resection size was an independent prognostic factor with a threefold increased risk of mortality for resections of more than 150 cm².

Study III After a median follow-up of 102 months, the 8-year LRR rate was 5.4% (n=21) in the mastectomy only group, 0.8% (n=1) in the delayed reconstruction group and 23.1% (n=3) in the immediate reconstruction group. Cox multivariate analysis revealed immediate reconstruction to be an independent risk factor for LRR. However Cox multivariate analysis revealed no reconstruction to be an independent risk factor for distant metastasis (DM), poor breast cancer specific survival (BCSS) and poor overall survival (OS).

Study IV Marked lateral macroscopic margins were at least 3 cm in 9/10 cases and clear histological margins were achieved in all cases. Defect coverage involved direct closure in 2 cases, reconstruction with a pedicled latissimus dorsi flap in 4 cases and skin grafting in 4 cases. After a median follow-up of 81 months, 3 patients suffered a recurrence and 5 year OS was 70%.

Conclusions Skin-sparing mastectomy and immediate breast reconstruction is a possible treatment option for selected patients who develop an ipsilateral breast tumour recurrence. In selected patients autologous flap reconstruction enables the resection of extensive chest wall recurrent breast tumours with acceptable morbidity and reasonable local control and survival. Palliative surgery can also be invaluable to improve the quality of life in patients with extensive symptomatic recurrences. In comparison to mastectomy only breast reconstruction does not appear to worsen the prognosis following treatment for primary breast cancer. Increased awareness of radiation-associated angiosarcoma, with no delay in diagnosis and ultimately excision with wide macroscopic margins, is vital in improving survival of this poorly known tumour.

The effects of dietary fat and obesity on gut barrier function were investigated in the diet induced obesity and ob/ob -mouse models. Fecal bile acids were quantified and profiles calculated from these mice. The effects of bile acids on intestinal permeability were studied in an in vivo feeding trial with deoxycholic acid and in vitro in an Ussing chamber. In vitro, tissue preparations were incubated with deoxycholic acid and/or ursodeoxycholic acid - two bile acids greatly different in their hydrophobicity.

Gut barrier function was impaired by a high-saturated fat diet in mice, but not in genetically obese mice that were fed normal low-fat chow. Barrier dysfunction by dietary fat was especially prominent in jejunum and colon - no significant difference was seen in the permeability of duodenum or ileum. Fecal bile acid hydrophobicity was increased only by dietary fat, not by genetic obesity, and was positively correlated with intestinal permeability.

Deoxycholic acid alone increased gut permeability both in vivo and in vitro. The effect was more evident in colonic than jejunal tissue preparations, and the mechanism seemed not to be inflammation-dependent. Barrier impairment was reduced by the hydrophilic ursodeoxycholic acid, which was also reflected as improved tissue morphology. Deoxycholic acid -induced barrier dysfunction seemed to be aggravated by translocated lipopolysaccharides.

The present results suggest that dietary fat, but not obesity itself, impairs gut barrier function. The data imply that luminal bile acids are one mechanism for barrier impairment, with hydrophobic bile acids initiating tissue disruption and lipopolysaccharides likely playing the role of a second hit.