Evaluación de la involución, salud uterina y retorno de la actividad ovárica con la adición de zeolita 2% en la dieta basal de vacas lecheras

El objetivo de esta investigación fue evaluar el efecto de zeolita (Clinoptilolita) adicionado en la dieta basal de vacas lecheras durante 105 días, 60 días pre-parto (dap) y 45 días post-parto (dpp) y su influencia en la involución (IU) y salud uterina (SU), el retorno de la actividad ovárica (AO) y la condición corporal (CC). El proyecto se realizó en tres ganaderías de la comunidad de Soldados del Cantón Cuenca – Azuay, en 50 vacas secas, con una CC ≥ 3,5, entre 2 a 5 partos, clínicamente sanas y todas en las mismas condiciones sanitarias y de manejo; fueron divididas en un grupo control (n1=25), alimentadas con dieta basal y un experimental (n2=25), con dieta basal + 2% de Zeolita del consumo de materia (CMS). Se evaluó retorno de AO considerando folículos ≥ 10 mm de diámetro a los 15, 22, 35 y 45 dpp por Ultrasonografía transrectal, SU (>10 PMN) a los 35 dpp por cytobrush, IU considerando posición del útero con respecto a la pelvis (PU), simetría de los cuernos (SCU), y diámetro del cérvix (DC), a los 22 y 45 dpp por palpación rectal, y finalmente la CC a los 15 dap, parto y 45 dpp. Se usó un diseño completamente al azar (DCA) y los resultados fueron analizados con el programa estadístico SPSS versión 22.0, posteriormente fueron aplicados los estadísticos de “U de Mann Withney y Kruscall Wallis”. Se obtuvo eficacia en el GE con mayor porcentaje de vacas que retornaron su AO (respectivamente, para GC y GE fueron: 35 dpp 29,6% vs. 70,4%; 45 dpp 44,4% vs. 55,6%; p<0,05). La IU fue a los 45 dpp, los valores obtenido para GC y GE fueron: respectivamente, PU 40,0% vs. 76,0%, SCU 32,0% vs. 76,0% y DC 28,0% vs. 68,0%, (p<0,05). Y en SU también mostró eficacia en el GE obteniendo un 3,4% vs. 22,2% de PMN en comparación con el GC, (p<0,05). Finalmente, se comprobó diferencias significativas (p<0,05) en la CC al parto y 45 dpp con eficacia atribuida al GE. En conclusión, la adición de zeolita al 2% en la dieta basal mostró eficacia en la involución y salud uterina, retorno de la actividad ovárica y la condición corporal en vacas en transición, recomendando su uso en las ganaderías lecheras

Association of follicle stimulating hormone receptor promoter with ovarian response in IVF-ET patients

Background: Poor ovarian response phenomenon has been observed in some of the in vitro fertilization-embryo transfer patients. Some investigations found that follicle stimulating hormone receptor (FSHR) gene plays a role in the process, but no direct evidence shows the correlation between genotypes of FSHR and ovarian response. Objective: Exploring the molecular mechanism behind the mutation of FSHR promoter association with ovarian granulosa cells and poor ovarian response. Materials and Methods: This cross sectional study was performed using 158 women undergoing the controlled short program ovarian stimulation for IVF treatment. The 263 bp DNA fragments before the follicle stimulating hormone (FSH) receptor 5' initiation site were sequenced in the patients under IVF cycle, 70 of which had poor ovarian response and 88 showed normal ovarian responses. Results: With a mutation rate of 40%, 63 in 158 cases showed a 29th site G→A point mutation; among the mutated cases, the mutation rate of the poor ovarian responders was significantly higher than the normal group (60% versus 23.9%; χ2=21.450, p<0.001). Besides, the variability was also obvious in antral follicle count, and ovum pick-ups. The estradiol peak values and the number of mature eggs between the two groups had significant difference. However, there was no obvious variability (t=0.457, p=0.324) in the basic FSH values between the two groups (normal group, 7.2±2.3 U/L; mutation group, 7.1±2.0 U/L). Conclusion: The activity of FSHR promoter is significantly affected by the 29th site G→A mutation that will weaken promoter activity and result in poor response to FSH.

No relationship between most polymorphisms of steroidogenic acute regulatory (StAR) gene with polycystic ovarian syndrome

Background: Polycystic ovary syndrome (PCOS) is one of the most common endocrine women’s disorders in reproductive age. Hyperandrogenism has a critical role in the etiology of PCOS and it can cause fault in Steroidogenesis process. During steroidogenesis, steroidogenic acute regulatory protein (StAR) seems to increase the delivery of cholesterol through mitochondrial membrane. Therefore, polymorphisms of StAR might effect on this protein and play a role in the etiology of PCOS. Objective: The aim of this study was to investigate the association between StAR SNPs with PCOS. Thus, seven polymorphisms in this gene: rs104894086, rs104894089, rs104894090, rs137852689, rs10489487, rs104894085 were detected. Materials and Methods: In this case control study, 45 PCOS women, 40 male factor/unexplained infertile women, and 40 fertile women as two control groups were participated from 2008-2012. Polymorphisms were detected using restriction fragment length polymorphism (PCR-RFLP) method. Results: Heterozygote genotyping for rs137852689 SNP (amino acid 218 C > T) was only seen in seven PCOS patients, one in normal ovulatory women, and five in male factor/unexplained infertile women (15.5%, 2.5%, 12.5%, respectively) (p= 0.12). While, it has shown no association between other SNPS with PCOs. Conclusion: The RFLP results for seven chosen SNPs, which located in exon 5 and 7 showed normal status in three groups, it means no heterozygous or homozygous forms of selected SNPs were observed. So, it seems evaluation of the active amino acid sites should be investigated and also the study population should be increased.

Reproductive aspects of the oceanic whitetip shark, Carcharhinus longimanus (Elasmobranchii: Carcharhinidae), in the equatorial and southwestern Atlantic Ocean

The present study sought to study the reproductive biology of the oceanic whitetip shark, Carcharhinus longimanus, in the equatorial and southwestern Atlantic Ocean. A total of 234 specimens were collected as bycatch during pelagic longline fisheries targeting tunas and swordfish, between December 2003 and December 2010. The fishing area was located between latitudes 10N and 35S and longitudes 3E and 40W. Of the 234 individuals sampled, 118 were females (with sizes ranging from 81 to 227 cm TL, total length) and 116 males (ranging from 80 to 242 cm TL). The reproductive stages of the females were classed as immature, mature, preovulatory and pregnant, while males were divided into immature, maturing and mature. The size at maturity for females was estimated at 170.0 cm TL, while that for males was between 170.0 and 190.0 cm TL. Ovarian fecundity ranged from 1 to 10 follicles and uterine fecundity from 1 to 10 embryos. The reproductive cycle of this species is most likely biennial, with parturition occurring once every two years.

Pretreatment malnutrition and quality of life - association with prolonged length of hospital stay among patients with gynecological cancer: A cohort study

Background Length of hospital stay (LOS) is a surrogate marker for patients' well-being during hospital treatment and is associated with health care costs. Identifying pretreatment factors associated with LOS in surgical patients may enable early intervention in order to reduce postoperative LOS. Methods This cohort study enrolled 157 patients with suspected or proven gynecological cancer at a tertiary cancer centre (2004-2006). Before commencing treatment, the scored Patient Generated - Subjective Global Assessment (PG-SGA) measuring nutritional status and the Functional Assessment of Cancer Therapy-General (FACT-G) scale measuring quality of life (QOL) were completed. Clinical and demographic patient characteristics were prospectively obtained. Patients were grouped into those with prolonged LOS if their hospital stay was greater than the median LOS and those with average or below average LOS. Results Patients' mean age was 58 years (SD 14 years). Preoperatively, 81 (52%) patients presented with suspected benign disease/pelvic mass, 23 (15%) with suspected advanced ovarian cancer, 36 (23%) patients with suspected endometrial and 17 (11%) with cervical cancer, respectively. In univariate models prolonged LOS was associated with low serum albumin or hemoglobin, malnutrition (PG-SGA score and PG-SGA group B or C), low pretreatment FACT-G score, and suspected diagnosis of cancer. In multivariable models, PG-SGA group B or C, FACT-G score and suspected diagnosis of advanced ovarian cancer independently predicted LOS. Conclusions Malnutrition, low quality of life scores and being diagnosed with advanced ovarian cancer are the major determinants of prolonged LOS amongst gynecological cancer patients. Interventions addressing malnutrition and poor QOL may decrease LOS in gynecological cancer patients.

Prevalence and determinants of the health promotion and risk reduction practices of younger female survivors of breast cancer

This thesis describes a discrete component of a larger mixed-method (survey and interview) study that explored the health-promotion and risk-reduction practices of younger premenopausal survivors of ovarian, breast and haematological cancers. This thesis outlines my distinct contribution to the larger study, which was to: (1) Produce a literature review that thoroughly explored all longer-term breast cancer treatment outcomes, and which outlined the health risks to survivors associated with these; (2) Describe and analyse the health-promotion and risk-reduction behaviours of nine younger female survivors of breast cancer as articulated in the qualitative interview dataset; and (3) Test the explanatory power of the Precede-Proceed theoretical framework underpinning the study in relation to the qualitative data from the breast cancer cohort. The thesis reveals that breast cancer survivors experienced many adverse outcomes as a result of treatment. While they generally engaged in healthy lifestyle practices, a lack of knowledge about many recommended health behaviours emerged throughout the interviews. The participants also described significant internal and external pressures to behave in certain ways because of the social norms surrounding the disease. This thesis also reports that the Precede-Proceed model is a generally robust approach to data collection, analysis and interpretation in the context of breast cancer survivorship. It provided plausible explanations for much of the data in this study. However, profound sociological and psychological implications arose during the analysis that were not effectively captured or explained by the theories underpinning the model. A sociological filter—such as Turner’s explanation of the meaning of the body and embodiment in the social sphere (Turner, 2008)—and the psychological concerns teased out in Mishel’s (1990) Uncertainty in Illness Theory, provided a useful dimension to the findings generated through the Precede-Proceed model. The thesis concludes with several recommendations for future research, clinical practice and education in this context.

Extracellular matrix of the human cyclic corpus luteum

Extracellular matrix regulates many cellular processes likely to be important for development and regression of corpora lutea. Therefore, we identified the types and components of the extracellular matrix of the human corpus luteum at different stages of the menstrual cycle. Two different types of extracellular matrix were identified by electron microscopy; subendothelial basal laminas and an interstitial matrix located as aggregates at irregular intervals between the non-vascular cells. No basal laminas were associated with luteal cells. At all stages, collagen type IV α1 and laminins α5, β2 and γ1 were localized by immunohistochemistry to subendothelial basal laminas, and collagen type IV α1 and laminins α2, α5, β1 and β2 localized in the interstitial matrix. Laminin α4 and β1 chains occurred in the subendothelial basal lamina from mid-luteal stage to regression; at earlier stages, a punctate pattern of staining was observed. Therefore, human luteal subendothelial basal laminas potentially contain laminin 11 during early luteal development and, additionally, laminins 8, 9 and 10 at the mid-luteal phase. Laminin α1 and α3 chains were not detected in corpora lutea. Versican localized to the connective tissue extremities of the corpus luteum. Thus, during the formation of the human corpus luteum, remodelling of extracellular matrix does not result in basal laminas as present in the adrenal cortex or ovarian follicle. Instead, novel aggregates of interstitial matrix of collagen and laminin are deposited within the luteal parenchyma, and it remains to be seen whether this matrix is important for maintaining the luteal cell phenotype.

Quality of life and genetics in men with prostate cancer

As family history has been established as a risk factor for prostate cancer, attempts have been made to isolate predisposing genetic variants that are related to hereditary prostate cancer. With many genetic variants still to be identified and investigated, it is not yet possible to fully understand the impact of genetic variants on prostate cancer development. The high survival rates among men with prostate cancer have meant that other issues, such as quality of life (QoL), have also become important. Through their effect on a person’s health, a range of inherited genetic variants may potentially influence QoL in men with prostate cancer, even prior to treatment. Until now, limited research has been conducted on the relationship between genetics and QoL. Thus, this study contributes to an emerging field by aiming to identify certain genetic variants related to the QoL found in men with prostate cancer. It is hoped that this study may lead to future research that will identify men who have an increased risk of a poor QoL following prostate cancer treatment, which will aid in developing treatments that are individually tailored to support them. Previous studies have established that genetic variants of Vascular Endothelial Growth Factor (VEGF) and Insulin-like Growth Factor 1 (IGF-1) may play a role in prostate cancer development. VEGF and IGF-1 have also been reported to be associated with QoL in people with ovarian cancer and colorectal cancer, respectively. This study completed a series of secondary analyses using two major data-sets (from 850 men newly diagnosed with prostate cancer, and approximately 550 men from the general Queensland population), in which genetic variants of VEGF and IGF-1 were investigated for associations with prostate cancer susceptibility and QoL. The first aim of this research was to investigate genetic variants in the VEGF and IGF-I gene for an association with the risk of prostate cancer. It was found that one IGF-1 genetic variant (rs35765) had a statistically significant association with prostate cancer (p = 0.04), and one VEGF genetic variant (rs2146323) had a statistically significant association with advanced prostate cancer (p = 0.02). The estimates suggest that carriers of the CA and AA genotype for rs35765 may have a reduced risk of developing prostate cancer (Odds Ratio (OR) = 0.72, 95% Confidence Interval (CI) = 0.55, 0.95, OR = 0.60, 95% CI = 0.26, 1.39, respectively). Meanwhile, carriers of the CA and AA genotype for rs2146323 may be at increased risk of advanced prostate cancer, which was determined by a Gleason score of above 7 (OR = 1.72, 95% CI = 1.12, 2.63, OR = 1.90, 95% CI = 1.08, 3.34, respectively). Utilising the widely used short-form health survey, the SF-36v2, the second aim of this study was to investigate the relationship between prostate cancer and QoL prior to treatment. Assessing QoL at this time-point was important as little research has been conducted to evaluate if prostate cancer affects QoL regardless of treatment. The analyses found that mean SF-36v2 scale scores related to physical health were higher by at least 0.3 Standard Deviations (SD) among men with prostate cancer than the general population comparison group. This difference was considered clinically significant (defined by group differences in mean SF-36v2 scores by at least 0.3 SD). These differences were also statistically significant (p<0.05). Mean QoL scale scores related to mental health were similar between men with prostate cancer and those from the general population comparison group. The third aim of this study was to investigate genetic variants in the VEGF and IGF-1 gene for an association with QoL in prostate cancer patients prior to their treatment. It was essential to evaluate these relationships prior to treatment, before the involvement of these genes was potentially interrupted by treatment. The analyses found that some genetic variants had a small clinically significant association (0.3 SD) to some QoL domains experienced by these men. However, most relationships were not statistically significant (p>0.05). Most of the associations found identified that a small sub-group of men with prostate cancer (approximately 2%) reported, on average, a slightly better QoL than the majority of the prostate cancer patients. The fourth aim of this research was to investigate whether associations between genetic variants in VEGF and IGF-1 and QoL were specific to men with prostate cancer, or were also applicable to the general male population. It was found that twenty out of one-hundred relationships between the genetic variants of VEGF and IGF-1 and QoL health-measures and scales examined differed between these groups. In the majority of the relationships involving VEGF SNPs that differed, a clinically significant difference (0.3 or more SD) between mean scores among the genotype groups in prostate cancer patients was found, while mean scores among men from the general-population comparison group were similar. For example, prostate cancer participants who carried at least one T allele (CT or TT genotype) for rs3024994 had a clinically significant higher (0.3 SD) mean QoL score in terms of the role-physical scale, than participants who carried the CC genotype. This was not seen among men from the general population sample, as the mean score was similar between genotype groups. The opposite was seen in regards to the IGF-1 SNPs examined. Overall, these relationships were not considered to directly impact on the clinical options for men with prostate cancer. As this study utilised secondary data from two separate studies, there are a number of important limitations that should be acknowledged including issues of multiple comparisons, power, and missing or unavailable data. It is recommended that this study be replicated as a better-designed study that takes greater consideration of the many factors involved in prostate cancer and QoL. Investigation into other genetic variants of VEGF or IGF-1 is also warranted, as is consideration of other genes and their relationship with QoL. Through identifying certain genetic variants that have a modest association to prostate cancer, this project adds to the knowledge surrounding VEGF and IGF-1 and their role in prostate cancer susceptibility. Importantly, this project has also introduced the potential role genetics plays in QoL, through investigating the relationships between genetic variants of VEGF and IGF-1 and QoL.

Methods used in cross-cultural comparisons of psychological symptoms and their determinants

This paper is the second in a series of reviews of cross-cultural studies of menopausal symptoms. The goal of this review is to compare and contrast methods which have been previously utilized in Cross-Cultural Midlife Women's Health Studies with a view to (1) identifying the challenges in measurement across cultures in psychological symptoms and (2) suggesting a set of unified questions and tools that can be used in future research in this area. This review also aims to examine the determinants of psychological symptoms and how those determinants were measured. The review included eight studies that explicitly compared symptoms in different countries or different ethnic groups in the same country and included: Australian/Japanese Midlife Women's Health Study (AJMWHS), Decisions At Menopause Study (DAMeS), Four Major Ethnic Groups (FMEG), Hilo Women's Health Survey (HWHS), Penn Ovarian Aging Study (POAS), Study of Women's Health Across the Nation (SWAN), Women's Health in Midlife National Study (WHiMNS), and the Women's International Study of Health and Sexuality (WISHeS). This review concludes that mental morbidity does affect vasomotor symptom prevalence across cultures and therefore should be measured. Based on the review of these eight studies it is recommended that the following items be included when measuring psychological symptoms across cultures, feeling tense or nervous, sleeping difficulty, difficulty in concentrating, depressed and irritability along with the CES-D Scale, and the Perceived Stress Scale. The measurement of these symptoms will provide an evidence based approach when forming any future menopause symptom list and allow for comparisons across studies.

Methods used in cross-cultural comparisons of sexual symptoms and their determinants

This is the fourth in a series of reviews of cross-cultural studies of menopausal symptoms. The purpose of this review is to examine methods used in cross-cultural comparisons of sexual symptoms among women at midlife, and to examine the determinants of sexual symptoms and how those determinants were measured. The goal of this review is to make recommendations that will improve cross-cultural comparisons in the future. The review included nine studies that explicitly examined symptoms in different countries or different ethnic groups in the same country and included: Australian/Japanese Midlife Women's Health Study (AJMWHS), Decisions At Menopause Study (DAMeS), Four Major Ethnic Groups (FMEG), Hilo Women's Health Survey (HWHS), Mid-Aged Health in Women from the Indian Subcontinent (MAHWIS), Penn Ovarian Aging Study (POAS), Study of Women's Health Across the Nation (SWAN), Women's Health in Midlife National Study (WHiMNS), and Women's International Study of Health and Sexuality (WISHeS). Although methods used for assessing sexual symptoms across cultures differed between studies, statistically significant differences were reported. Cross-cultural differences in sexual symptoms exist, and should be measured by including the following symptoms: loss of interest in sex, vaginal dryness, and the Females Sexual Function Index which covers desire, arousal, lubrication, orgasm, satisfaction, and pain on intercourse. The measurement of these symptoms will provide an evidence-based approach when forming any future menopause symptom list and allow for comparisons across studies.

Methods used in cross-cultural comparisons of vasomotor symptoms and their determinants

Methodological differences among studies of vasomotor symptoms limit rigorous comparison or systematic review. Vasomotor symptoms generally include hot flushes and night sweats although other associated symptoms exist. Prevalence rates vary between and within populations, but different studies collect data on frequency, bothersomeness, and/or severity using different outcome measures and scales, making comparisons difficult. We reviewed only cross-cultural studies of menopausal symptoms that explicitly examined symptoms in general populations of women in different countries or different ethnic groups in the same country. This resulted in the inclusion of nine studies: Australian/Japanese Midlife Women's Health Study (AJMWHS), Decisions At Menopause Study (DAMeS), Four Major Ethnic Groups (FMEG), Hilo Women's Health Survey (HWHS), Mid-Aged Health in Women from the Indian Subcontinent (MAHWIS), Penn Ovarian Aging Study (POAS), Study of Women's Health Across the Nation (SWAN), Women's Health in Midlife National Study (WHiMNS), and Women's International Study of Health and Sexuality (WISHeS). These studies highlight the methodological challenges involved in conducting multi-population studies, particularly when languages differ, but also highlight the importance of performing multivariate and factor analyses. Significant cultural differences in one or more vasomotor symptoms were observed in 8 of 9 studies, and symptoms were influenced by the following determinants: menopausal status, hormones (and variance), age (or actually, the square of age, age2), BMI, depression, anxiety, poor physical health, perceived stress, lifestyle factors (hormone therapy use, smoking and exposure to passive smoke), and acculturation (in immigrant populations). Recommendations are made to improve methodological rigor and facilitate comparisons in future cross-cultural menopause studies.

Rational design of serine protease inhibitors

Proteases regulate a spectrum of diverse physiological processes, and dysregulation of proteolytic activity drives a plethora of pathological conditions. Understanding protease function is essential to appreciating many aspects of normal physiology and progression of disease. Consequently, development of potent and specific inhibitors of proteolytic enzymes is vital to provide tools for the dissection of protease function in biological systems and for the treatment of diseases linked to aberrant proteolytic activity. The studies in this thesis describe the rational design of potent inhibitors of three proteases that are implicated in disease development. Additionally, key features of the interaction of proteases and their cognate inhibitors or substrates are analysed and a series of rational inhibitor design principles are expounded and tested. Rational design of protease inhibitors relies on a comprehensive understanding of protease structure and biochemistry. Analysis of known protease cleavage sites in proteins and peptides is a commonly used source of such information. However, model peptide substrate and protein sequences have widely differing levels of backbone constraint and hence can adopt highly divergent structures when binding to a protease’s active site. This may result in identical sequences in peptides and proteins having different conformations and diverse spatial distribution of amino acid functionalities. Regardless of this, protein and peptide cleavage sites are often regarded as being equivalent. One of the key findings in the following studies is a definitive demonstration of the lack of equivalence between these two classes of substrate and invalidation of the common practice of using the sequences of model peptide substrates to predict cleavage of proteins in vivo. Another important feature for protease substrate recognition is subsite cooperativity. This type of cooperativity is commonly referred to as protease or substrate binding subsite cooperativity and is distinct from allosteric cooperativity, where binding of a molecule distant from the protease active site affects the binding affinity of a substrate. Subsite cooperativity may be intramolecular where neighbouring residues in substrates are interacting, affecting the scissile bond’s susceptibility to protease cleavage. Subsite cooperativity can also be intermolecular where a particular residue’s contribution to binding affinity changes depending on the identity of neighbouring amino acids. Although numerous studies have identified subsite cooperativity effects, these findings are frequently ignored in investigations probing subsite selectivity by screening against diverse combinatorial libraries of peptides (positional scanning synthetic combinatorial library; PS-SCL). This strategy for determining cleavage specificity relies on the averaged rates of hydrolysis for an uncharacterised ensemble of peptide sequences, as opposed to the defined rate of hydrolysis of a known specific substrate. Further, since PS-SCL screens probe the preference of the various protease subsites independently, this method is inherently unable to detect subsite cooperativity. However, mean hydrolysis rates from PS-SCL screens are often interpreted as being comparable to those produced by single peptide cleavages. Before this study no large systematic evaluation had been made to determine the level of correlation between protease selectivity as predicted by screening against a library of combinatorial peptides and cleavage of individual peptides. This subject is specifically explored in the studies described here. In order to establish whether PS-SCL screens could accurately determine the substrate preferences of proteases, a systematic comparison of data from PS-SCLs with libraries containing individually synthesised peptides (sparse matrix library; SML) was carried out. These SML libraries were designed to include all possible sequence combinations of the residues that were suggested to be preferred by a protease using the PS-SCL method. SML screening against the three serine proteases kallikrein 4 (KLK4), kallikrein 14 (KLK14) and plasmin revealed highly preferred peptide substrates that could not have been deduced by PS-SCL screening alone. Comparing protease subsite preference profiles from screens of the two types of peptide libraries showed that the most preferred substrates were not detected by PS SCL screening as a consequence of intermolecular cooperativity being negated by the very nature of PS SCL screening. Sequences that are highly favoured as result of intermolecular cooperativity achieve optimal protease subsite occupancy, and thereby interact with very specific determinants of the protease. Identifying these substrate sequences is important since they may be used to produce potent and selective inhibitors of protolytic enzymes. This study found that highly favoured substrate sequences that relied on intermolecular cooperativity allowed for the production of potent inhibitors of KLK4, KLK14 and plasmin. Peptide aldehydes based on preferred plasmin sequences produced high affinity transition state analogue inhibitors for this protease. The most potent of these maintained specificity over plasma kallikrein (known to have a very similar substrate preference to plasmin). Furthermore, the efficiency of this inhibitor in blocking fibrinolysis in vitro was comparable to aprotinin, which previously saw clinical use to reduce perioperative bleeding. One substrate sequence particularly favoured by KLK4 was substituted into the 14 amino acid, circular sunflower trypsin inhibitor (SFTI). This resulted in a highly potent and selective inhibitor (SFTI-FCQR) which attenuated protease activated receptor signalling by KLK4 in vitro. Moreover, SFTI-FCQR and paclitaxel synergistically reduced growth of ovarian cancer cells in vitro, making this inhibitor a lead compound for further therapeutic development. Similar incorporation of a preferred KLK14 amino acid sequence into the SFTI scaffold produced a potent inhibitor for this protease. However, the conformationally constrained SFTI backbone enforced a different intramolecular cooperativity, which masked a KLK14 specific determinant. As a consequence, the level of selectivity achievable was lower than that found for the KLK4 inhibitor. Standard mechanism inhibitors such as SFTI rely on a stable acyl-enzyme intermediate for high affinity binding. This is achieved by a conformationally constrained canonical binding loop that allows for reformation of the scissile peptide bond after cleavage. Amino acid substitutions within the inhibitor to target a particular protease may compromise structural determinants that support the rigidity of the binding loop and thereby prevent the engineered inhibitor reaching its full potential. An in silico analysis was carried out to examine the potential for further improvements to the potency and selectivity of the SFTI-based KLK4 and KLK14 inhibitors. Molecular dynamics simulations suggested that the substitutions within SFTI required to target KLK4 and KLK14 had compromised the intramolecular hydrogen bond network of the inhibitor and caused a concomitant loss of binding loop stability. Furthermore in silico amino acid substitution revealed a consistent correlation between a higher frequency of formation and the number of internal hydrogen bonds of SFTI-variants and lower inhibition constants. These predictions allowed for the production of second generation inhibitors with enhanced binding affinity toward both targets and highlight the importance of considering intramolecular cooperativity effects when engineering proteins or circular peptides to target proteases. The findings from this study show that although PS-SCLs are a useful tool for high throughput screening of approximate protease preference, later refinement by SML screening is needed to reveal optimal subsite occupancy due to cooperativity in substrate recognition. This investigation has also demonstrated the importance of maintaining structural determinants of backbone constraint and conformation when engineering standard mechanism inhibitors for new targets. Combined these results show that backbone conformation and amino acid cooperativity have more prominent roles than previously appreciated in determining substrate/inhibitor specificity and binding affinity. The three key inhibitors designed during this investigation are now being developed as lead compounds for cancer chemotherapy, control of fibrinolysis and cosmeceutical applications. These compounds form the basis of a portfolio of intellectual property which will be further developed in the coming years.

The expression, regulation and function of kallikrein 4 in prostate cancer

Prostate cancer is a significant health problem faced by aging men. Currently, diagnostic strategies for the detection of prostate cancer are either unreliable, yielding high numbers of false positive results, or too invasive to be used widely as screening tests. Furthermore, the current therapeutic strategies for the treatment of the disease carry considerable side effects. Although organ confined prostate cancer can be curable, most detectable clinical symptoms occur in advanced disease when primary tumour cells have metastasised to distant sites - usually lymph nodes and bone. Many growth factors and steroids assist the continued growth and maintenance of prostatic tumour cells. Of these mitogens, androgens are important in the development of the normal prostate but are also required to sustain the growth of prostate cancer cells in the early stage of the disease. Not only are androgens required in the early stage of disease, but also many other growth factors and hormones interact to cause uncontrolled proliferation of malignant cells. The early, androgen sensitive phase of disease is followed by an androgen insensitive phase, whereby androgens are no longer required to stimulate the growth of the tumour cells. Growth factors such as transforming growth factor  and  (TGF/), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), insulin-like growth factors (IGFs), Vitamin D and thyroid hormone have been suggested to be important at this stage of disease. Interestingly, some of the kallikrein family of genes, including prostate specific antigen (PSA), the current serum diagnostic marker for prostate cancer, are regulated by androgens and many of the aforementioned growth factors. The kallikrein gene family is a group of serine proteases that are involved in a diverse range of physiological processes: regulation of local blood flow, angiogenesis, tissue invasion and mitogenesis. The earliest members of the kallikrein gene family (KLK1-KLK3) have been strongly associated with general disease states, such as hypertension, inflammation, pancreatitis and renal disease, but are also linked to various cancers. Recently, this family was extended to include 15 genes (KLK1-15). Several newer members of the kallikrein family have been implicated in the carcinogenesis and tumour metastasis of hormone-dependent cancers such as prostate, breast, endometrial and ovarian cancer. The aims of this project were to investigate the expression of the newly identified kallikrein, KLK4, in benign and malignant prostate tissues, and prostate cancer cell lines. This thesis has demonstrated the elevated expression of KLK4 mRNA transcripts in malignant prostate tissue compared to benign prostates. Additionally, expression of the full length KLK4 transcript was detected in the androgen dependent prostate cancer cell line, LNCaP. Based on the above finding, the LNCaP cell line was chosen to assess the potential regulation of full length KLK4 by androgen, thyroid hormone and epidermal growth factor. KLK4 mRNA and protein was found to be up-regulated by androgen and a combination of androgen and thyroid hormone. Thyroid hormone alone produced no significant change in KLK4 mRNA or protein over the control. Epidermal growth factor treatment also resulted in elevated expression levels of KLK4 mRNA and protein. To assess the potential functional role(s) of KLK4/hK4 in processes associated with tumour progression, full length KLK4 was transfected into PC-3 cells - a prostate cancer cell line originally derived from a secondary bone lesion. The KLK4/hK4 over-expressing cells were assessed for their proliferation, migration, invasion and attachment properties. The KLK4 over-expressing clones exhibited a marked change in morphology, indicative of a more aggressive phenotype. The KLK4 clones were irregularly shaped with compromised adhesion to the growth surface. In contrast, the control cell lines (parent PC-3 and empty vector clones) retained a rounded morphology with obvious cell to cell adhesion, as well as significant adhesion to their growth surface. The KLK4 clones exhibited significantly greater attachment to Collagen I and IV than native PC-3s and empty vector controls. Over a 12 hour period, in comparison to the control cells, the KLK4 clones displayed an increase in migration towards PC-3 native conditioned media, a 3 fold increase towards conditioned media from an osteoblastic cell line (Saos-2) and no change in migration towards conditioned media from neonatal foreskin fibroblast cells or 20% foetal bovine serum. Furthermore, the increase in migration exhibited by the KLK4 clones was partially blocked by the serine protease inhibitor, aprotinin. The data presented in this thesis suggests that KLK4/hK4 is important in prostate carcinogenesis due to its over-expression in malignant prostate tissues, its regulation by hormones and growth factors associated with prostate disease and the functional consequences of over-expression of KLK4/hK4 in the PC-3 cell line. These results indicate that KLK4/hK4 may play an important role in tumour invasion and bone metastasis via increased attachment to the bone matrix protein, Collagen I, and enhanced migration due to soluble factors produced by osteoblast cells. This suggestion is further supported by the morphological changes displayed by the KLK4 over-expressing cells. Overall, this data suggests that KLK4/hK4 should be further studied to more fully investigate the potential value of KLK4/hK4 as a diagnostic/prognostic biomarker or in therapeutic applications.

Purine Nucleoside Phosphorylase mediated molecular chemotherapy and conventional chemotherapy: A tangible union against chemoresistant cancer

Background Late stage Ovarian Cancer is essentially incurable primarily due to late diagnosis and its inherent heterogeneity. Single agent treatments are inadequate and generally lead to severe side effects at therapeutic doses. It is crucial to develop clinically relevant novel combination regimens involving synergistic modalities that target a wider repertoire of cells and lead to lowered individual doses. Stemming from this premise, this is the first report of two- and three-way synergies between Adenovirus-mediated Purine Nucleoside Phosphorylase based gene directed enzyme prodrug therapy (PNP-GDEPT), docetaxel and/or carboplatin in multidrug-resistant ovarian cancer cells. Methods The effects of PNP-GDEPT on different cellular processes were determined using Shotgun Proteomics analyses. The in vitro cell growth inhibition in differentially treated drug resistant human ovarian cancer cell lines was established using a cell-viability assay. The extent of synergy, additivity, or antagonism between treatments was evaluated using CalcuSyn statistical analyses. The involvement of apoptosis and implicated proteins in effects of different treatments was established using flow cytometry based detection of M30 (an early marker of apoptosis), cell cycle analyses and finally western blot based analyses. Results Efficacy of the trimodal treatment was significantly greater than that achieved with bimodal- or individual treatments with potential for 10-50 fold dose reduction compared to that required for individual treatments. Of note was the marked enhancement in apoptosis that specifically accompanied the combinations that included PNP-GDEPT and accordingly correlated with a shift in the expression of anti- and pro-apoptotic proteins. PNP-GDEPT mediated enhancement of apoptosis was reinforced by cell cycle analyses. Proteomic analyses of PNP-GDEPT treated cells indicated a dowregulation of proteins involved in oncogenesis or cancer drug resistance in treated cells with accompanying upregulation of apoptotic- and tumour- suppressor proteins. Conclusion Inclusion of PNP-GDEPT in regular chemotherapy regimens can lead to significant enhancement of the cancer cell susceptibility to the combined treatment. Overall, these data will underpin the development of regimens that can benefit patients with late stage ovarian cancer leading to significantly improved efficacy and increased quality of life.