The plasma or serum assay of 17a-hydroxyprogesterone is a more sensitive and accurate index of this enzyme deficiency than measurement of pregnanetriol treatment vitiligo 250 mg lariam with visa. Normally treatment 1 degree av block order genuine lariam online, the blood level of 17a-hydroxyprogesterone is less than 100 ng/dL medicine 1920s buy cheap lariam online, although after ovulation and during the luteal phase of a normal menstrual cycle medicine 801 buy cheap lariam on line, a peak of 200 ng/dL can be reached treatment 1st 2nd degree burns purchase genuine lariam online. With excessive accumulation of stromal tissue or in the presence of an androgen-producing tumor medicine 013 discount 250 mg lariam with mastercard, testosterone becomes a significant secretory product medicine reactions 250mg lariam for sale. Occasionally treatment emergent adverse event cheap lariam 250 mg line, a nonfunctioning tumor can induce stromal proliferation and increased androgen production. The normal accumulation of stromal tissue at midcycle results in a rise in circulating levels of androstenedione and testosterone at the time of ovulation. The adrenal cortex produces 3 groups of steroid hormones, the glucocorticoids, the mineralocorticoids, and the sex steroids. The adrenal sex steroids represent intermediate byproducts in the synthesis of glucocorticoids and mineralocorticoids, and excessive secretion of the sex steroids occurs only with neoplastic cells or in association with enzyme deficiencies. Under normal circumstances, adrenal gland production of the sex steroids is less significant than gonadal production of androgens and estrogens. However, short-term variations in the blood levels due to episodic secretion require multiple sampling for absolutely accurate assessment. Although frequent sampling is necessary for a high degree of accuracy, a random sample is sufficient for clinical purposes to determine whether a level is within a normal range. The testosterone-binding capacity is decreased by androgens; hence, the binding capacity in men is lower than that in normal women. The binding globulin level in women with increased androgen production is also depressed. Androgenic effects are dependent on the unbound fraction that can move freely from the vascular compartment into the target cells. Thus, a total testosterone concentration can be in the normal range in a woman who is hirsute or even virilized, but because the binding globulin level is depressed by the androgen effects, the percent free and active testosterone is elevated. The need for a specific assay for the free portion of testosterone can be questioned because the very presence of hirsutism or virilism indicates increased androgen effects. In the face of hirsutism, one can reliably interpret a normal testosterone level as compatible with decreased binding capacity and increased active free testosterone. Both total and unbound testosterone are normal in only a few women with hirsutism. In these cases, the hirsutism, heretofore regarded as idiopathic, most likely results from excessive intracellular androgen effects (specifically increased intracellular conversion of testosterone to dihydrotestosterone). The 5b-derivatives are not androgenic, and this is not an important pathway; however, the 5a-derivative (a very active pathway) is extremely potent. Testosterone is also aromatized to a significant extent in the brain, liver, and breast; and in some circumstances. Excretion of Steroids Active steroids and metabolites are excreted as sulfo and glucuro conjugates. Conjugation of a steroid converts a hydrophoboic compound into a hydrophilic one and generally reduces or eliminates the activity of a steroid. This is not completely true, however, because hydrolysis of the ester linkage can occur in target tissues and restore the active form. Furthermore, estrogen conjugates can have biologic activity, and it is known that sulfated conjugates are actively secreted and may serve as precursors, present in the circulation in relatively high concentrations because of binding to serum proteins. Ordinarily, however, conjugation by liver and intestinal mucosa is a step in deactivation preliminary to , and essential for, excretion into urine and bile. Cellular Mechanism of Action Hormones circulate in extremely low concentrations and, in order to respond with specific and effective actions, target cells require the presence of special mechanisms. One mediates the action of tropic hormones (peptide and glycoprotein hormones) with receptors at the cell membrane level. In contrast, the smaller steroid hormones enter cells readily, and the basic mechanism of action involves specific receptor molecules within the cells. It is the affinity, specificity, and activity of the receptors, together with the large concentration of receptors in cells, that allow a small amount of hormone to produce a biologic response. The many different types of receptors can be organized into the following basic categories. Intracellular Receptors Receptors in the nucleus lead to transcription activation. G Protein Receptors these receptors are composed of a single polypeptide chain that spans the cell membrane. Binding to a specific hormone leads to interaction with G proteins that, in turn, activate second messengers. The second messengers include the adenylate cyclase enzyme, the phospholipase system, and calcium ion changes. Ion Gate Channels these cell surface receptors are composed of multiple units, that after binding, open ion channels. Receptors With Intrinsic Enzyme Activity these transmembrane receptors have an intracellular component with tyrosine or serine kinase activity. Examples include the receptors for insulin and growth factors (tyrosine kinase) and the receptors for activin and inhibin (serine kinase). Mechanism of Action for Steroid Hormones the specificity of the reaction of tissues to sex steroid hormones is due to the presence of intracellular receptor proteins. Different types of tissues, such as liver, kidney, and uterus, respond in a similar manner. The steroid hormone receptors primarily affect gene transcription, but also regulate post transcriptional events and nongenomic events. Each of the major classes of the sex steroid hormones, including estrogens, progestins, and androgens, has been demonstrated to act according to this general mechanism. Glucocorticoid, mineralocorticoid, and probably androgen receptors, when in the unbound state, reside in the cytoplasm and move into the nucleus after hormone-receptor binding. Estrogens and progestins are transferred across the nuclear membrane and bind to their receptors within the nucleus. Steroid hormones are rapidly transported across the cell membrane by simple diffusion. The factors responsible for this transfer are unknown, but the concentration of free (unbound) hormone in the bloodstream seems to be an important and influential determinant of cellular function. Once in the cell, the sex steroid hormones bind 37, 38 and 39 to their individual receptors. Transformation refers to a conformational change of the hormone-receptor complex revealing or producing a binding site that is necessary in order for the complex to bind to the chromatin. Activation of the receptor is driven by hormone binding that causes a dissociation of the receptor-heat shock protein complex. The principal action of steroid hormones is the regulation of intracellular protein synthesis by means of the receptor mechanism. Biologic activity is maintained only while the nuclear site is occupied with the hormone-receptor complex. The dissociation rate of the hormone and its receptor as well as the half-life of the nuclear chromatin-bound complex are factors in the biologic response because the hormone response elements are abundant and, under normal 40 conditions, are occupied only to a small extent. Thus, an important clinical principle is the following: duration of exposure to a hormone is as important as dose. One reason only small amounts of estrogen need be present in the circulation is the long half-life of the estrogen hormone-receptor complex. Indeed, a major factor in the potency differences among the various estrogens (estradiol, estrone, estriol) is the length of time the estrogen-receptor complex occupies the nucleus. Cortisol and progesterone must circulate in large concentrations because their receptor complexes have short half-lives in the nucleus. An important action of estrogen is the modification of its own and other steroid hormone activity by affecting receptor concentrations. Estrogen increases target tissue responsiveness to itself and to progestins and androgens by increasing the concentration of its own receptor and that of the intracellular progestin and androgen receptors. Progesterone and clomiphene, on the other hand, limit tissue response to estrogen by blocking the replenishment mechanism, thus decreasing over time the concentration of estrogen receptors. Replenishment is very responsive to the available amount of steroid and receptors. Small amounts of receptor depletion and small amounts of steroid in the blood activate the mechanism. Replenishment, the synthesis of the sex steroid receptors, obviously takes place in the cytoplasm, but with estrogen and progestin receptors, synthesis must be 41 quickly followed by transportation into the nucleus. If the cell is growing rapidly, about 3 newly assembled ribosomes will be transported every minute in the other direction. In the case of steroid hormone receptor proteins, the signal sequences are in the hinge region. Estrogen and progestin receptors exit continuously from the nucleus to the cytoplasm and are actively transported back to the nucleus. This is a constant shuttle; constant diffusion into the cytoplasm is balanced by the active transport into the nucleus. The fate of the hormone-receptor complex after gene activation is referred to as hormone-receptor processing. In the case of estrogen receptors, processing involves the conversion of high-affinity estrogen receptor sites to a rapidly dissociating form followed by loss of binding capacity, which is completed in about 6 hours. The continuous presence of estrogen is an important factor for continuing response. The best example of the importance of these factors is the difference between estradiol and estriol. But if the effective concentration is kept equivalent to that of estradiol, it can produce a similar 42 biologic response. The depletion of estrogen receptors in target tissues by progestational agents is the fundamental reason for adding progestins to estrogen treatment programs. The progestins accelerate the turnover of pre-existing receptors, and this is followed by inhibition of estrogen-induced receptor synthesis. Using monoclonal antibody immunocytochemistry, this action has been pinpointed to the interruption of transcription in estrogen-regulated genes. Steroid hormone receptors share a 44 common structure with the receptors for thyroid hormone, 1, 25-dihydroxy vitamin D3, and retinoic acid; thus, these receptors are called a superfamily. Each receptor contains characteristic domains that are similar and interchangeable. Therefore, it is not surprising that the specific hormones can interact with more than one receptor in this family. Analysis of these receptors suggests a complex evolutionary history during which gene duplication and swapping between domains of different 45 origins occurred. This family now includes about 150 proteins, present in practically all species, from worms to insects to humans. Many are called orphan receptors because specific ligands for these proteins have not been identified. The estrogen receptor-alpha was 48, 49 and 50 discovered about 1960, and the amino acid sequence was reported in 1986. Different genetic messages can result not only because of differences in binding affinity, but through variations in the mechanisms to be discussed, notably differences in conformational shape and cellular contexts. A/B Region, the Regulatory Domain the amino acid terminal is the most variable in the superfamily of receptors, ranging in size from 20 amino acids in the Vitamin D receptor, to 600 amino acids in the mineralocorticoid receptor. Hormone binding induces a conformational change that allows binding to the hormone-responsive elements in the target gene. This domain is very similar for each member of the steroid and thyroid receptor superfamily; however, the genetic message is specific for the hormone that binds to the hormone-binding domain. The specificity of receptor binding to its hormone responsive element is determined by the zinc finger region, especially the first finger. The specific message can be changed by changing the amino acids in the base of the fingers. Functional specificity is localized to the second zinc finger in an area designated the d (distal) box. This nuclear localization signal must be present for the estrogen receptor to remain within the nucleus in the absence of hormone. This region is also a site of rotation (hence the hinge designation) in achieving conformational change. The hormone-binding domain of the steroid receptors contains a characteristic 54 structure, containing helices that form a pocket (also referred to as a sandwich fold). After binding with a hormone, this pocket undergoes a conformational change that creates new surfaces with the potential to interact with co-activator and co-repressor proteins. This region modulates gene transcription by estrogen and antiestrogens, having a role that influences 55 antiestrogen efficacy in suppressing estrogen-stimulated transcription. The conformation of the receptor-ligand complex is different with estrogen and antiestrogens, and this conformation is different with and without the F region. The F region is not required for transcriptional response to estrogen; however, it affects the magnitude of ligand-bound receptor activity. It is speculated that this region affects conformation in such a way that protein interactions are influenced. Thus, it is appropriate that the effects of the F domain vary according to cell type and protein context. Mechanism of Action the steroid family receptors are predominantly in the nucleus even when not bound to a ligand, except for the androgen, mineralocorticoid, and glucocorticoid receptors where nuclear uptake depends on hormone binding. But the estrogen receptor does undergo what is called nucleocytoplasmic shuttling. The estrogen receptor constantly diffuses out of the nucleus and is rapidly transported back in. When this shuttling is impaired, receptors are more rapidly degraded in the cytoplasm. Prior to binding, the estrogen receptor is an inactive complex that includes a variety of proteins, including the heat shock proteins. Heat shock protein 90 appears to be a critical protein, and many of the others are associated with it. This heat shock protein is not only important for maintaining an inactive state, but also for causing 57 proper folding for transport across membranes. Imagine the unoccupied steroid receptor as a loosely packed, mobile protein complexed with heat shock proteins. The conformational change induced by hormone binding involves a dissociating process to form a tighter packing of the receptor. The hormone-binding domain contains helices that form a pocket (also 54 referred to as a sandwich fold). Conformational shape is an important factor in determining the exact message transmitted to the gene. Conformational shape is slightly but significantly different with each ligand; estradiol, tamoxifen, and raloxifene each induce a distinct conformation that contributes to 58, 59 the ultimate message of agonism or antagonism. The weak estrogen activity of estriol is because of its altered conformation shape when combined with the 60 estrogen receptor in comparison with estradiol. The hormone-binding domain of the estrogen receptors contains a cavity surrounded by a wedge-shaped structure, and it is the fit into this cavity that is so influential in influencing the genetic message. The size of this cavity on the estrogen receptor is relatively large, larger than the volume of an estradiol molecule, explaining the acceptance of a large variety of ligands. Thus, estradiol, tamoxifen, and raloxifene each bind at the same site within the hormone binding domain, but the conformational shape with each is not identical. Conformational shape is a major factor in determining the ability of a ligand and its receptor to interact with coactivators and corepressors. Members of the thyroid and retinoic acid receptor subfamily do not exist in inactive complexes with heat shock proteins. These mutants can form dimers with natural estrogen receptor (wild type), and then bind 61 to the estrogen response element, but they cannot activate transcription. This indicates that transcription is dependent on the result after estradiol binding to the estrogen receptor, an estrogen-dependent structural change. Molecular modeling and physical energy calculations indicate that binding of estrogen with its receptor is not a simple key and lock mechanism. It involves conversion of the estrogen-receptor complex to a preferred geometry dictated to a major degree by the specific binding site of the receptor. The estrogenic response depends on the final bound conformation and the electronic properties of functional groups that contribute energy. Estrogen, progesterone, androgen, and glucocorticoid receptors bind to their response elements as dimers, one molecule of hormone to each of the two units in the dimer. The estrogen receptor-alpha can form dimers with other alpha receptors (homodimers) or with an estrogen receptor-beta (heterodimer). Similarly, the estrogen receptor-beta can form homodimers or heterodimers with the alpha receptor. This creates the potential for many pathways for estrogen signaling, alternatives that are further increased by the possibility of utilizing various response elements in target genes. Cells that express only one of the estrogen receptors would respond to the homodimers; cells that express both could express to a homodimer and a heterodimer.
Selfcare Mobility Social function Yes 32% 42% 23% No 58% 55% 61% Missing value 10% 3% 16% % of respondents that rated this answer medicine examples buy lariam 250mg lowest price. Selfcare Mobility Social function Yes 7% 16% 10% No 87% 74% 71% Missing value 7% 10% 19% % of respondents that rated this answer medicine to increase appetite buy lariam 250 mg lowest price. Lastly medicine expiration buy lariam online now, a majority of the respondents would not add items to the particular domains (table 2 medicine 8 pill purchase cheap lariam. With regard to the question whether all the important facets are represented (table 2 medications a to z cheap lariam 250 mg line. Written comments of some respondents suggested that there are too large steps between the subsequent items to measure clinically meaningful change in functional status symptoms 4 days post ovulation lariam 250 mg sale. General comments were reported concerning the presence of spelling-mistakes and inconsistent usage of language treatment example trusted lariam 250mg. One respondent missed qualitative aspects of performance medicine 0552 best lariam 250 mg, such as starting position and quality of movement. New items were added because all the members of the review committee agreed that these items would strengthen the construct of the scales. Bicycling can be considered as an important 29 functional skill in the Netherlands. Bicycling is the main means of transportation to go to school, friends or shopping centres in the Netherlands. Therefore, the item bicycling was added to the mobility domain of the functional skills scale. Based on the content validity study, no further items were added to the questionnaire. A lower percentage of the respondents, but still a majority, have the same opinion regarding the evaluative potentials, i. In both cases, the social function domain was markedly lower rated than the selfcare and mobility domain. It is supposed that changes in this more abstracted level of social skills, rendered as behaviour, are more difficult to measure over time. Despite this, the same number of respondents was not prepared to remove any of the items of the particular domains. Some respondents even suggested that there are too large steps between the subsequent items to measure clinically meaningful change in functional status. More (smaller) steps between item levels led to a larger number of items resulting in lengthening the instrument 31 Chapter 2 and thus the assessment. The suggestions of the respondents regarding spelling-mistakes and inconsistent usage of language were gratefully applied in the revised version. As equivalence is also necessary with regard to reliability, additional studies (inter-respondent, inter interviewer, and test-re-test reliability, as well as internal consistency) were started. Postma for their extensive participation in the review-committee during the adaptation process. Generic and disease-specific measures in assessing health status and quality of life. Children with Cerebral Palsy: A Functional Approach to Physical Therapy [dissertation]. Evaluating research in developmental disabilities: a conceptual framework for reviewing treatment outcomes. Correlates of disablement in polyarticular juvenile chronic arthritis A cross sectional study. Explanation: the child walks outside on most sorts of surfaces without needing support from a walking aid or the caregiver and carry a book bag, for example. Explanation: the child begins of his own accord a shared activity (such as playing hide and seek or marbles) with one or more other children and continues doing exclusively that for a specific time. While playing, the child is able to negotiate with and attune his play to the other child. For example: it is decided who gets to take turn first, who gets assigned a certain role, etcetera. Explanation: the child is able, without help, to get on and off a (specially adapted) three-wheeler and moved forward by turning the pedals. Explanation: the child is able, without help, to get on and off a bicycle and to ride it independently. Explanation: the child is able, without help, to get on and off a bicycle with no training-wheels and to ride it independently. Design: Inter-interviewer reliability was studied after scoring audiotaped interviews by a second researcher. For test-retest reliability the same parent was interviewed twice within three weeks; in inter-respondent reliability both parents of a child were interviewed independently within a few days. Subjects: Parents of 63 non-disabled and 53 disabled (various diagnoses) children aged between 7 and 88 months were interviewed. On item level for the Functional Skills Scale, the mean percentage identical scores varied from 89 to 99, and for the Caregiver Assistance Scale from 54 to 90. Different scores between interviewers resulted partially from ambiguous interpretation of the item and/or the explanation. This instrument, originally developed for the North-American population, has 7 recently been translated and cross-culturally adapted for use in the Netherlands. In the adaptation process four new items were added, while many other items were adapted. First, inter-interviewer reliability was studied to find out whether items and the accompanying explanation are interpreted in a uniform way. Second, internal consistency was established by studying the extent in which items within a scale are related. Third, test-retest reliability was examined to give an indication of stability of measures and last, inter-respondent reliability was studied to find out if there are differences in the judgements of both parents. Both the capability of the child and the amount of help they get from their parents as well as the equipment used in daily tasks are measured by a structured interview with parent(s). Functional status is determined in three domains: self-care, mobility and social function. Summed scores can be computed in every domain; the American version also gives standardized scores. A first group consisted of children visiting a primary health care centre for infants where growth and development of healthy children is recorded routinely. The health care centre sent a letter with an outline of the study and a request to participate to parents of all children aged two and living in a small town in the centre of the Netherlands (n= 260). A second group of non-disabled children were already participating in another study when they were asked to participate in this study: 20 of them agreed. They have neurometabolic disorders (n = 29), spina bifida (n = 7), osteogenesis imperfecta (n = 11) and infantile encephalopathy (n = 6). All children have stable or slowly progressive limitations in performing daily activities. Parents who agreed to participate were asked whether they also agreed into audiotaping the interview, whether it was possible to interview also the other parent, and/or to (partially) redo the interview within a few weeks. In sum, we audiotaped 31 interviews, we interviewed both parents of 32 children, while 20 parents were interviewed twice within a few weeks. Analysis Inter-respondent and inter interviewer reliability was established using scores from both disabled and non-disabled children. In all reliability studies we first looked at the proportion of identical answers in every item. In studying test-retest reliability the same parent of the same child was (partially) interviewed twice: mean time between the two interviews was 14. Self-care Mobility Social function (74 items) (61 items) (66 items) Inter-interviewer Researcher 1 35. Self-care Mobility Social function (74 items) (61 items) (66 items) Inter-interviewer % identical score 97. From the explanation it was not clear whether a child is capable only if they pronounce their name correctly, or also when they call themselves consequently in the same manner but not correct (which is often the case when a child has a name that is hard to pronounce). In the other two third of the differences, one of the researchers scored inaccurate. Both the researcher that took the interviews and the researcher who scored the audio taped interviews sometimes gave inaccurate scores. This means that the score in the first interview was just above or below the score in the second interview, the smallest possible difference. For example, an electric toothbrush is not a modification as meant, but it is when a parent chooses to use an electric toothbrush for their child because of its functional limitations. Differences found in retesting after a short period of time showed a decrease in independence in the self-care domain, while in the social function domain most differences showed an increase in independence and in the mobility domain both were found. In case of differences between two parents of the same child, mothers judged their child as more independent than fathers did, especially mothers of disabled children. Our results are not completely comparable: we controlled for development by computing alpha on a sample that was homogeneous for age, and we found lower alphas (. In our study inter-interviewer reliability was greater than test-retest reliability, 4, 5 and both were greater than inter-respondent reliability. Other researchers found the same results for test-retest and inter-respondent reliability, although their methods were slightly different from ours. We feel that differences between first and second measurement are due to a test-effect: parents mentioned that as a result of the first measurement they watched the performances of the child and their 8 assistance more consciously. In a Swedish study the Functional Skills Scale was administered as a written questionnaire and the other scales were administered as an interview: although they concluded that this did not reduce interview time sending a list with the subjects to be discussed may be a way to increase reliability by focusing attention on these subjects before the parents are interviewed. In all parts of this study, except for internal consistency, the mobility domain scored better than the self-care and social function domain. An explanation for this finding might be that items of the mobility domain, more than in other domains, are less subjected to choices parents make when raising their children. Both researchers scored inaccurately while they were interviewing and scoring; therefore, we do not think this is an interviewer effect. Mothers judged their children more capable and less dependent on their assistance than fathers did, especially when the child is disabled. We cannot conclude that the judgement of the mother is better than the judgement of the father, just because the mother spends more time with her child. It is a judgement-based structured interview for parents used by professionals in rehabilitation medicine and in health related outcome research. The children with disabilities scored significantly lower than the children without disabilities in the self-care and mobility domains. The results showed different outcome profiles, indicating possible inter-cultural differences. Examples of these adaptations are the conversion of weights and measures into the metric system and the addition of a shower to the items concerning tub transfers. Regarding the first group, we included children with a known psychomotor delay, spina bifida, or infantile encephalopathy. In addition, children with juvenile idiopathic arthritis, osteogenesis imperfecta, traumatic injury, and neuromuscular disorders represented the children with musculoskeletal involvement. In these children, it was assumed that functional limitations would be found mainly in the ambulation and self-care skills. Although it was not the main purpose of the study, we also looked at differences between the clinical groups. Methods Participants Between August 1999 and November 2000, 62 children without disabilities were recruited from a health care centre for infants and toddlers (table 4. Parents visited this outpatient clinic for routine health assessment of their child. A clinical sample was measured between January 1999 and October 2000, comprising 197 children with different kinds of disabilities (table 4. The children were previously diagnosed, with the exception of the children with symptoms of a neurometabolic disorder. These children, in whom there was not always a diagnosis at hand, presented different levels of psychomotor delay, sometimes associated with seizures, muscular conditions, failure to thrive, and sensory impairments. Children and parents were excluded if they were not able to actively use the Dutch language. This was determined at the introduction of the study when they were not able to iterate what they were told about the procedure. These scales contain a total of 201 questions organized within 41 subscales concerning 3 domains: self-care domain, mobility domain, and social function domain (table 4. These scales contain 20 questions concerning the same activities of the functional skills scales (table 4. Scores of 0 and 1 refer to the supportive participation of the caregiver for more than half of the activities, whereas scores of 2 to 5 refer to a progressive independence of the child. Parents of children with a traumatic injury were interviewed within 14 days after the incident, and they were asked to base their judgement on the actual functional status. All other interviews were performed during their visit to the outpatient department or within 1 month. Data Analysis 14 Based on an analysis of covariance, age-corrected scale scores were computed for each of the 6 outcome scales (3 functional skills scales and 3 caregiver-assistance scales). This was necessary to correct for age differences among the eight groups (table 4. Discriminant validity was examined by using discriminant analysis after we established the reliability and item-test correlations of each of the 6 outcome scales (which were around. Discussion the aim of discriminative measures is to distinguish between individuals or 3 groups on underlying dimensions. Discriminative measures in rehabilitation medicine are useful to determine the impact of a disorder with respect to functional status at a single point of time. However, we first performed an analysis of covariance and computed age-corrected scale scores, because discriminant analysis could not be conducted in this study with children of the same age because the sample sizes were too small. Our findings confirm a high degree of sensitivity (correct identification of children with disabilities within this population) and specificity (false prediction of children without disabilities who were identified as disabled). The sample was representative for current Dutch society with respect to age and gender. Therefore, the resulting normative standard scores can be used in Dutch pediatric rehabilitation practice to measure the deficit or delay in the development of functional status, and if so, the extent and content area of this deficit or delay. Carefully interpreting scores of originally non-Dutch children is recommended as these children were hardly represented in the Dutch normative sample. Instruments that proved to be reliable and valid in one country can also be used in other countries to save resources needed for the development of a new instrument. Of course, a translation is needed from the original language into the target language. However, when the aspect to be measured has a cultural component, a simple translation may not be sufficient: a cross-cultural adaptation and a subsequent re-standardization are than recommended.
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Sometimes medicine 524 purchase 250mg lariam with amex, some parts of the conversation were not transcribed due to the irrelevance to the topic or question treatment ingrown hair order generic lariam from india. Then the codes were compared for similarities and differences medications prescribed for anxiety purchase generic lariam on-line, grouped into content areas and subsequently into preliminary categories and sub-categories treatment 4 hiv safe lariam 250mg. The concept of the study was given the moment the participant was contacted treatment 0f gout discount lariam 250mg fast delivery, and only the participants who voluntarily accepted to participate in this survey medicine 2015 song purchase lariam without prescription, were interviewed medicine 9 minutes buy lariam in india. During the interviews treatment 4s syndrome buy lariam 250mg with mastercard, no measurements were taken or made; we only asked questions and expected their opinion and experience on this topic. On one side, the participating mothers, doctors and midwives expressed great satisfaction with the protocol that is being followed nowadays and there is no need for more checkups; on the other side, they admitted that the protocol was and should evolve so that better care would be given to mothers and babies and that home monitoring could be a great help for mothers and doctors particularly during the last trimester and in risky pregnancies. The characteristics for the prototype the connection between these categories is pictured below, Figure 14, and the Table 4 describes these categories together with their sub-categories. Good experience, but need more checkups Most of the mothers considered the appointments during their pregnancy to be very good and that midwives and doctors checked everything. They recognized the role of the midwife as very important and having the same one throughout the pregnancy was felt as personal. However, there were mothers who were not as satisfied with the process as they were on their first pregnancy, during the summer or when they encountered miscarriage. Psychological and sociological examination and preparation for both parents is included in the protocol nowadays. Monitoring the baby was seen as really important, but routine blood pressure, blood sugar, hemoglobin (iron), weight, urine test for the mother were of the same importance. And I also talk about their social situation, how it is at home and what they need to prepare at home before the baby will come. However, they claimed that in some counties there might be some changes, and this was mostly due to resources, economy and/or people. They acknowledged the addition of the psycho-social preparation and counselling for parents as must and very crucial for having a good and successful pregnancy as a whole. I feel that women today have had a very protected life and that makes them more vulnerable. Some felt that having only one ultrasound was not enough and admitted the need for an th earlier ultrasound screening on 12 week, especially to catch risky or twin pregnancies as soon as possible. Some others requested that maybe a later ultrasound could be good too, st around 31 week, citing that many other countries had these screenings and maybe it was about time for Sweden to have those as well. They said the time should be flexible, in particular with women of special needs or different nationally. Many said they used official websites such as 1177 Health Care Guide, the official Swedish website and telephone service with information, counseling and services in the health and care, online forums or mobile applications that showed the pregnancy progress week by week. However, there were women that took the online search on the next level by reading conference or journal research articles. Family was the main source of comfort to all of them, followed by the husband and/or partner. Friends, especially the ones who had already been pregnant, were considered a good source of confirmation if they had any fear. Nevertheless, if any of them had any medical questions, the main contact was the medical staff, with the midwives being number one. Support groups were helpful as well, but they were only for first pregnancies and women were not very interactive with each other after those meetings. Two sides of technology Every participant was very content about new technology in general in medicine and in particular in prenatal care. Mothers were very open to such a device and expressed they would have used 18 also known as Pinard horn 48 it if they knew about it during their pregnancy. Some went on saying they would use it every day, but others were also concerned about the safety of using such device on the baby, the price of it and its aesthetics, having a normal everyday life while using the device/ wearable. Doctors argued that the use of such device on early stages of the pregnancy would not change much, neither treat any form of fetal abnormalities, but they suggested it could be very useful during the last trimester and specially with risky pregnancies, with pregnancies that were fine but the mother had miscarriage history, or when the mother was living far from the hospital, and in Sweden this was very often. The midwives showed their concerns by saying that one should be educated to interpret the information shown in these devices and home was not the right place to monitor the baby. And others were interested on trying if it could add value to the process, and the fact that it was a non-ultrasound device, was very much appreciated. In addition, they argued if this type of device could add any value to the process. If possible, all information Approximately every participant suggested monitoring the position and the heartbeat of fetus. They proposed to monitor such information as: blood pressure, blood sugar and some other metabolic diseases that need to be checked 2 to 3 times per week, fetal movements, uterine contractions and to check if it is a single or twin (or more) pregnancy. There were some who had not thought about such thing before, hence suggested measuring everything, if that was possible. Everyone, but in particular midwives, were very much in its favor, knowing that this way a person with medical experience was going to check the measurements and say if the pregnancy was going fine or not. At first all technologies, be it invasive or non-invasive, were seen as an option. Considering that an invasive solution would bring many ethical dilemmas, we chose a non-invasive solution, which would be as much accurate as an invasive one. Although a lot of progress has been done in this domain, a lot more needs to be done. One is still in the research stage, since 2011, and the other one is progressing but very slow. The interviews were broad, so we could get a better picture of the current situation in Sweden. The participants viewed the fetal health monitoring at home as a very good way to follow up risky pregnancies, mothers with miscarriage history in their late pregnancies and for mothers who lived far from the healthcare centers and clinics. And on the other side, as it could bring more anxiety and increase the number of checkups due to not appropriately wear the wearable or giving the full attention to the baby and ignoring the mother. Nowadays, more care is moving from the hospital to home because of wanting to improve care quality and reduce costs. The most challenging issues described by participants in this study, were the issues doctors and midwives raised concerning: 1. The use of the fetal monitoring devices as a medical measure for the benefit of fetal health when it was necessary 2. There are studies showing that ultrasound, and specifically Doppler ultrasound, can harm the fetus. The reason why the midwives are not as open as doctors is maybe because they are not as happy as they say about new technology, or they still might be for to it, but using new devices may feel like extra work and training. Home monitoring When participants were asked about what they thought of home monitoring, mothers and doctors were mostly excited about it, midwives not that much. It was interesting to hear the answers of the participants when asked about the measurements they would have wanted to monitor. It felt they were not prepared for such question, when they clearly answered the previous questions related to what is their main procedure or test during these pregnancy checkups. Almost all of the medical participants worried about the mothers not being able to understand the information being given to them. But how about the Doppler ultrasound or any other device that normal non-medical people use nowadays This feature, doctors being able to remotely monitor the baby, was very much appreciated by both groups. It was seen as important when following risk pregnancies and for mothers who lived far from the healthcare clinic or hospital. These waveform evaluations are generally inaccessible to researchers today due to lack of data. In the near future these questions will be addressed and answered, and the results will be incorporated into clinical algorithms that will improve the safety of obstetrics. Medical professionals, like it or not, are using technology on diagnosing, monitoring and treating patients on their everyday work routine, and technology is playing a significant role on managing the patients in general. The continuing advances in the potential of fetal monitoring at home to impact on pregnancy management will also increase the need for appropriate information and counselling. Together with parents, doctors and midwives therefore need to develop new ways of making the pregnancy duration and the health of the mother and the baby as safe as possible. Although there are some similar products in the market already, there is still a need for further discussions regarding the use of such devices. Parents on the other hand, trust the technology and they welcome it in any aspect of their life, especially if they could get closer to their unborn child. They want more checkups, and this wearable offers them what they want, and at the same time helps them feel safe knowing that the doctor is remotely checking them, and not relying only on their knowledge on any fetal health monitoring device. Alongside the medical aspects, sustainability is the other main concern of the thesis, as it has an impact on all three pillars of the sustainability, the people, the planet and the profit. Parents feel more comfortable and secure while staying home and still receiving constant care. Doctors and midwives still monitor the patients anywhere they are remotely, and most importantly they can follow up and have more time with the patients with risky pregnancies. Hospitals can reduce their expenses on hospital beds and still being able to take care of patients remotely. The prototype will be based on the findings of this thesis, in addition to taking a deeper look into the market and going further with the algorithms. Electronic fetal heart rate monitoring: retrospective reflections on a twentieth-century technology. You can create a new directory or go into the directory where you wish to install the Toolbox: 3. I checked with the hospital here in Skelleftea, and in a normal pregnancy there are 10 check-ups and one ultrasound (on week 18) during this period. Has any mother/parent express any feelings on using technology during the pregnancy Have you experience any certain fears about the health status of your baby in between check-ups Are you satisfied with the help support you have right now (or had) during the pregnancy) Rather than put a trademark symbol after every occurrence of a trademarked name, we use names in an editorial fashion only, and to the bene t of the trademark owner, with no intention of infringement of the trademark. McGraw-Hill Education eBooks are available at special quantity discounts to use as premiums and sales promotions, or for use in corporate training programs. This work represents his personal and professional views and not necessarily those of the U. Neither McGraw-Hill Education nor its licensors shall be liable to you or anyone else for any inaccuracy, error or omission, regardless of cause, in the work or for any damages resulting therefrom. McVary section i Pituitary, thyroid, and adrenal disorders section iii diabetes Mellitus, obesity, 2 Disorders of the Anterior Pituitary and liPoProtein MetabolisM Hypothalamus. Larry Jameson section iV disorders affecting MultiPle 8 Disorders of the Testes and Male Reproductive endocrine systeMs System. Bosl 23 Disorders Affecting Multiple Endocrine 10 the Female Reproductive System, Infertility, Systems. Byrne Chair in Clinical Oncology, Memorial Sloan-Kettering Cancer Center, New York, Robert F. Karl Professor of Endocrinology and Metabolism in Medicine, Washington University School of Medicine; J. Dunlop Professor of Medicine; Dean, University of Pennsylvania School of Medicine; Executive Vice President of the Stephen N. Woodward Professor and Chairman, Pennsylvania [1, 2, 4, 7, 8, 24] Department of Medicine, University of Maryland School of Medicine; Physician-in-Chief, University of Maryland Robert T. Pesce, PhD Mayo Clinic, Rochester, Minnesota [26] Professor Emeritus of Pathology and Cell Biology, Columbia University College of Physicians and Surgeons; Stephen M. Harrison Distinguished Professor of New York [Appendix] Medicine, Harvard Medical School; Massachusetts General Hospital, Boston, Massachusetts [25] John T. Davis Chair in Biomedical Science; Professor of Medicine, New York, New York [Appendix] Molecular Physiology, and Biophysics; Director, Vanderbilt Diabetes Center; Chief, Division of Diabetes, Endocrinology, and Metabolism, Henry M. Over time, ing Multiple Endocrine Systems; and (V) Disorders of the traditional textbook has evolved to meet the needs of Bone and Calcium Metabolism. In addition to the dramatic advances emanating ogy, now in its third edition, is a compilation of chapters from genetics and molecular biology, the introduction of related to the specialty of endocrinology. Numerous recent was to bring this information to readers in a more com clinical studies involving common diseases like diabetes, pact and usable form. Because the topic is more focused, obesity, hypothyroidism, and osteoporosis provide pow it was possible to increase the presentation of the mate erful evidence for medical decision making and treat rial by enlarging the text and the tables. These rapid changes in endocrinology are exciting included a Review and Self-Assessment section that in for new students of medicine and underscore the need cludes questions and answers to provoke refection and for practicing physicians to continuously update their to provide additional teaching points. The clinical manifestations of endocrine disorders Our access to information through web-based journals can usually be explained by considering the physiologic and databases is remarkably effcient. While these sources role of hormones, which are either defcient or exces of information are invaluable, the daunting body of data sive. Thus, a thorough understanding of hormone action creates an even greater need for synthesis and for high and principles of hormone feedback arms the clinician lighting important facts. Thus, the preparation of these with a logical diagnostic approach and a conceptual chapters is a special craft that requires the ability to distill framework for treatment approaches. The frst chapter core information from the ever-expanding knowledge of the book, Principles of Endocrinology, provides this base. Using numerous examples a group of internationally recognized authorities who are of translational research, this introduction links genet masters at providing a comprehensive overview while ics, cell biology, and physiology with pathophysiology being able to distill a topic into a concise and interesting and treatment. The book is divided into fve main We hope you fnd this book useful in your effort to sections that refect the physiologic roots of endocri achieve continuous learning on behalf of your patients. The global icons call greater attention to key epidemiologic and clinical differences in the practice of medicine throughout the world. The peripheral nervous system stimulates Accordingly, the practice of endocrinology is intimately the adrenal medulla. The immune and endocrine systems linked to a conceptual framework for understanding are also intimately intertwined. The adrenal hormone hormone secretion, hormone action, and principles of cortisol is a powerful immunosuppressant. For example, hormones play an important role in maintenance of blood pressure, intravascular volume, and peripheral resistance in the scope of eNdocriNology cardiovascular system. The term endocrine are involved in dynamic changes of vascular tone in was coined by Starling to contrast the actions of hormones addition to their multiple roles in other tissues. The secreted internally (endocrine) with those secreted externally heart is the principal source of atrial natriuretic peptide, (exocrine) or into a lumen, such as the gastrointestinal tract.
In contrast treatment 99213 discount lariam online amex, testis development in mice harbouring heterozygous Sox9 deletion progress normally despite perinatal lethality (Bi et al symptoms carbon monoxide poisoning best 250 mg lariam. These findings show that Sox9 can substitute Sry function in testis determination medicine 377 order lariam with visa. Fibroblast growth factor 9 (Fgf9) treatment 4 toilet infection discount lariam 250mg with amex, an extracellular signalling molecule medications overactive bladder discount lariam 250 mg otc, was proven essential to maintain Sox9 expression and therefore Sertoli cell differentiation (Reviewed by Kim and Capel atlas genius - symptoms lariam 250mg, 2006) medications with weight loss side effect cheap lariam 250mg visa. Fg/9 is one of the signalling molecules identified so far that can cause complete sex reversal in null mutant mice 5 medications that affect heart rate discount lariam 250mg on line. Studies on genetic interactions between Fg/9, Sox9 and Sry revealed the importance of Fg/9 during early stages of Sertoli cell specification. As a result, Sertoli cells undergo cell fate transition causing sex reversal (Kim and Capel, 2006). Studies on Wtl function after sex determination therefore have to rely on mutant mice generated through conditional deletion. Mice harbouring only one functional Wtl allele have lost the majority of Sertoli cells by E l5. Mini-summary on testis determination Sry is required to switch supporting cell fate from that of follicle cells to Sertoli cells, characteristic of the testis, and is sufficient to initiate the male pathway. Although Sry is normally required to do this, in certain circumstances it is, however, not essential for testis determination, because Sox9 has been shown able to substitute for Sry function during testis determination. More importantly, Sox9 is required for building up the Sertoli cell population, which is a crucial event during early stages of testis development. Without a threshold number of Sertoli cells, testis cords will not form and the testicular pathway will not be maintained. As Sry acts as the genetic switch towards testis development, its absence will allow the network of transcription factors and extracellular signalling in the bipotential gonad to proceed to female development. However, before this happens, genetic activities in somatic cells have already adopted the female pattern and cell fate decisions appear to have been taken. Several attempts have been made to identify genes that actively promote ovarian development, or at least act as anti-testis genes. This section focuses on several candidates that have proposed to be ovarian determinants. Daxl is expressed in the bipotential gonad at the same time as Sry, and becomes specific to ovarian somatic cells shortly after E l2. The function of Deal during testis/ovary differentiation will require further studies using conditional transgenesis. Mullerian ducts were not formed, but this is due to an additional role of Wnt4 common to both sexes. The gonads display some male specific characteristics, including coelomic vessels, and have Leydig-like cells that secrete testosterone. Although a very recent report suggests that there is transient up-regulation of Sox9 (Kim et al, 2006), testis cords do not form and there is no obvious differentiation of Sertoli cells, at least early on. The germ cells also do not follow the male pathway; instead oocytes are lost during mid-fetal stages (Vainio et al. This precedes the detection of Sertoli cell markers at perinatal stages, which was therefore attributed to a type of secondary sex reversal seen in a number of circumstances when germ cells are lost from ovaries after 25 they have begun to differentiate. These findings show that Sox9 upregulation in the gonad does not have to depend on Sry, as in this case it happened as a result of downregulation of Wnt4. It was proposed that the antagonism between Wnt4 and Fg/9 balances the bipotential gonad between two alternative fates. Foxl2 is specifically expressed in the ovary in mouse, chick, turtle and fish embryos at the time of sex determination (Loffler et al. After this stage, Foxl2 expression levels are maintained at a high level in follicle cells (Ottolenghi et al. In the adult ovary, Foxl2 expression was seen highest in the granulosa cells in early follicles, whereas expression levels decline during later stages of folliculogenesis (Schmidt et al, 2004). Foxl2 does not appear to affect initial ovary formation, but it is essential for ovarian maintenance. However, unlike Wnt4 null ovaries, Foxl2 null ovaries do not display massive loss of oocyte (Ottolenghi et al. In order to further address the function of Foxl2 during sex determination, transgenic mice misexpressing Foxl2 are needed for the investigation of possible sex reversal phenotypes. However, for most of these genes there is still relatively little understanding of underlying molecular mechanisms and more effort has to be made to uncover their place in the pathways and networks involved in cell fate decisions and morphogenesis of the ovary and testis. Genes that are not discussed here include in particular Dessert Hedgehog, a gene that is expressed in Sertoli cells shortly after Sry but plays roles in Leydig and germ cell survival; Dmrtl, which is expressed in Sertoli and germ cells in the testis, but whose function is not yet understood; Fst and Bmp2, expression of which is exclusive to somatic cells in the developing ovary and are important for germ cell survival, Vanin1, which may be involved in extracellular matrix remodelling specifically in the testis (Grimmond et al. After the initial establishment of supporting cell identity, sexually dimorphic gonad development proceeds to a further level, with other cell lineages being induced or permitted to follow the male or female pathway, while factors made specifically in the testis instruct the rest of the embryo to follow a male pathway. In the absence of such factors or their receptors, or in the absence of any gonads, the embryo will become female (Luo et al. This induces Mullerian duct regression, an early masculinisation event (Behringer et al. Amh eliminating the female reproductive tract Amh is responsible for Mullerian duct regression in the male, and is therefore an essential component of the male sexual developmental pathway in mammals (Josso et al. Other than Mullerian regression, Amh also appears to have a secondary role to eliminate any germ cells that have entered meiosis early in the testis (Josso, 1990). Sexual hormone production Sex hormone synthesis is a process downstream of sex determination. In the ovary, follicle-stimulating hormone is produced by granulosa cells, whereas estrogen is synthesized by theca cells during folliculogenesis. Six distinct forms of cytochrome p450 enzymes are involved in the synthetic pathway of steroid hormones from cholesterol. Biosynthesis of estrogens from androgens is catalyzed by P450 aromatase, which is important in follicular growth, especially towards ovulation. It has been postulated that estrodiol blocks expression of male-specific genes including Sox9 and Fgf9 in granulosa cells (Richards, 2001). Sfl is linked to these steroidogenic P450s by their coexpression pattern in various steroidogenic tissues (Honda et al. The expression of Sfl seems to correlate with the timing of hormone synthesis in gonads of both sexes. After sex determination, Sfl expression levels increase and are maintained in Leydig cells in the testis, but are decreased in the ovary. Puberty sees a dramatic change in the Sfl expression pattern: it becomes expressed at high levels in the theca and granulosa cells in the postnatal ovary around the first ovulation cycles (Hatano et al. This coincides to the expression of P450scc and aromatase in granulosa cells and reductase in theca cells (Reviewed by Richards, 2001), both of them synthesizing hormones including estrodial and progesterone. However, further studies will be required to address the function of Sfl in ovarian development. It has been shown that Fgf9~Amice failed to upregulate Cypllal, the gene encoding P450scc, resulting in failure in Leydig cell development (Colvin et al. During late folliculogenesis, aromatase is upregulated by Wnt4 and Foxl2, and is expressed in granulosa cells (Pannetier et al. However, the complexity of development demands further control of gene alteration in vivo to facilitate gene function analyses. In many cases, introducing genetic changes to the germ line also have severe developmental consequences, complicating or even precluding the desired experimental analysis. For example, the loss-of function of a gene might cause early embryonic lethality, rendering the study of its function 33 in other later-developed tissues impossible. Conditional mutagenesis is therefore required to overcome these undesired limitations. The establishment of several conditional technologies in mice has enabled considerable sophistication to be applied to gene function research. The most commonly applied effector involves a recombinase that rearranges the responder gene, by either activating or silencing it (for reviews see Lewandoski, 2001; Ryding et al. Both have become popular in conditional mutagenesis in the mouse, especially during embryogenesis (Figure 1. Recombinase-mediated changes range from excision, inversion, duplication, and chromosomal translocation. Cre recombinase has now evolved to become more suitable for in vivo usage in the mouse by changing a few codons in cis. In the classic set up, the Cre-recombinase would be driven by a regulatory element, which defines where and when homologous recombination takes place, i. This can either be a genomic sequence or a transgene driven by an exogenous regulatory element. This genetic set-up allows loss or gain-of-function studies based on the time and space specific Cre recombination profile, by conditional alteration of gene expression patterns in vivo. By doing this, although Cre expression is controlled by a tissue-specific promoter, its activities are prohibited until tamoxifen is introduced into the system. TetR specifically binds to both tetracycline and the 19bp operator sequences (tetO) of the tet operon in the target sequence, which results in its transcription (Gossen and Bujard, 1992). The TetR system is later on modified to enhance its efficiency and reduce basal transcription levels (Gossen et al. Also available now is a 36 doxycycline (Dox)-inducible system (Baron and Bujard, 2000; Hasan et al. This inducible TetR system is also applied in several cases to temporally control Cre activity (Saam and Gordon, 1999; Utomo et al. If region B is an essential region o f a gene, then the recom bination event results in gene inactivation. The other tw o products o f recom bination betw een loxP sites 2 and 3, Essential region and between 1 and 3 are also show n. The aim of my project is to manipulate the timing and cell-specificity of Sry and Sox9 expression, and to test the effect of this manipulation on sex determination. For this purpose I am using the tamoxifen-inducible Cre/loxP system, which allows gene expression in a highly controlled manner. In my experiments, gonad specific regulatory elements will be employed to control iCre expression, thereby controlling the conditional expression profiles of Sry or Sox9 (Figure 1. Following the next chapter, which outlines all materials and general techniques in use in my project, I describe how Cre-driver and responder elements were chosen and how these transgenes were made and tested (Chapters 3-5). I then describe the tools chosen for studies on the possible function of Sox9 (Chapter 6). Briefly, an inducible iCre driven by a ubiquitous promoter is used to study the effects of loss or gain-of-function of Sox9 in the developing mouse gonad. Experiments are performed in different systems, including cell cultures, in vivo or explant cultures. Using the new tools developed, we can explore the function of Sox9, as well as many other genes involved in gonadal development in the mouse. An example here is the conditional misexpression of Sry or Sox9 in the developing mouse gonad, which is mediated by a tamoxifen inducible Cre. Chemically transformation: the ligated mixture was mixed with 200pL freshly defrosted chemically competent bacteria on ice and incubated for 10 minutes. A list of all imported plasmid, and plasmids generated during my PhD in Lovell-Badge lab is listed in Appendix F. Plastics: Plasticwares, including culture dishes and plates, were purchased from either Costar Inc. Following tamoxifen administration, cells were cultured for a further 24-36 hours before harvest. During 45 licence transition, part of the protocols were carried out using licences 80/1761 and 80/1653. I declare that all procedures untaken in my project follows the regulations stated in the above licences, and are legal procedures listed in my personal licence (80/8481). For postnatal studies, the day when the pups were bom would be designated as postnatal day (P)0. With the exception to Z/Sox9 Line 10F, C and M, which were genotyped using X-gal staining (See section 2. R26R LacZ and ZlSox9 lines were genotyped using LacZ-specific primers, or X-gal staining. For Flox-Sox9, homozygous, heterozygous and knock-out alleles were genotyped using published primers (Akiyama et al. Administration of tamoxifen was either by oral gavage (carried out only under project licences 70/5042, 80/1949 and 80/1653) for embryonic stages, or intraperitoneal injection to adults. Postnatal and adult testes collected were either processed for sectioning (Section 2. Cords were then split into small portions and transferred onto SuperFrost Plus slides, and then 50 squashed with a thumb through a cover slip. Glutaraldehyde: For X-gal staining, samples were fixed with a solution that contains 0. For sectioning, samples were embedded in wax and cut at 6micron thickness with a 52 microtom (Leica). H&E staining was performed by the histology Service group after paraffin sectioning. Signals were visualized with fluorescent secondary antibodies, which were incubated in darkness at room temperature for 1 hour. Unless specified, fluorescent secondary antibodies were purchased from Molecular Probes. Washing and secondary antibodies incubation procedures were the same as for immunocytochemical staining. On paraffin sections: Sections were de-waxed by immersing in histoclear solution for 5 minutes, and then re-hydrated with washes in decrement in percentage of ethanol, 75%, 50%, 25%, 5 minutes each. Softwares used for texts for this thesis include Microsoft Word 2003 and Excel 2003. Previous members of the lab have generated several mouse lines for these purposes, however, none of them turned out to be appropriate to drive reporter gene expression in the gonads. For example, an Sry:Cre transgene made previously in the lab did not show evidence of functionality (Claire Canning, unpublished and data not shown), whereas Daxl:Cre was shown to be active in pre-implantation embryo (Amy Johnson, PhD thesis, 2002). Apart from gonad-specific sequences, a ubiquitous, strong promoter sequence was employed to drive the expression of Cre. In Daxl:LacZ transgenic mice, this region was consistently able to drive LacZ expression in a pattern identical to that of endogenous Daxl. The Daxl:Sry transgene resulted in female-to-male sex reversal at the same frequency as LacZ expression was seen in the Daxl:LacZ mice (Swain et al. Sfl is expressed in both the supporting and steroidogenic cell lineages at early stages, and while it is downregulated in the ovary, it is maintained in differentiated Sertoli cells and Leydig cells. In theory an appropriate regulatory region will be useful in gene alteration studies both cell lineages. For example, it could be used to ask if the expression of a Sertoli cell-specific gene in steroidogenic cells will lead to a change in cell fate. A 674bp Sfl promoter region was shown to be sufficient to activate LacZ reporter gene expression in El 1. In transgenic mice, this Sfl promoter driving Cre successfully deleted a conditional Sox9 allele in the gonads (Chacoissier et al. To enhance Cre recombination efficiency, the improved codon-Cre (iCre, Shimshek et al. This should allow control of Cre activation through the administration of the inducer tamoxifen at the desired embryonic stage. Expression was also seen in the Wolffian and Mullerian ducts of both sexes (Figure 3. The activity of this enhancer seems therefore to lose its Sertoli cell specificity (Figure 3. Moreover, it may be worth exploring the onset of Leydig cell expression within this line to see if it coincides with replacement of fetal by adult Leydig cells.
