**1. Introduction**

40 Urinary Incontinence

Wilson, P.D.; Herbison, R.M. & Herbison, G.P. (1996). Obstetric practice and the prevalence

Yang X, Zhang HX, Yu HY, Gao XL, Yang HX & Dong Y. (2010). The prevalence of fecal

1996), pp. 154-161, ISSN 0306-5456

of urinary incontinence three months after delivery. *BJOG,* Vol.103, No.2 (Feb

incontinence and urinary incontinence in primiparous postpartum Chinese women. *Eur J Obstet Gynecol Reprod Biol,* Vol.152, No.2 (June 2010), pp. 214-217, ISSN1872-7654

> Urinary incontinence (UI) has a multifactorial etiology, and as a rule lasts for many years (Abrams et al., 2002; Milart & Gulanowska-Gędek, 2002; Rechberger & Skorupski, 2005). It is most commonly seen in women (Foldspang & Mommsen, 1997; Rechberger, 2004; Rechberger & Skorupski, 2005; Garstka-Namysł, 2006, 2009). According to a recent epidemiological study (Minnasian, 2003), in a population of 230,000 people, the frequency of occurrence of UI was approximately 27.6% among women, as opposed to 10% among men. It is estimated that about 3% of men between the ages of 15 and 64 have UI-related problems, as compared to 7-10% of men over 64 (Evans, 2005). The prevalence of UI in women is likewise dependent on age: it affects nearly one-third of all women before menopause and over 45% after (Rogers et al., 2001a, 2001b). A detailed analysis of the occurrence of UI in various age brackets indicates that it affects 25% of women under 18, 18% under 29, and 37% between 35 and 54, whereas 39% of women with UI are over 55 years old (Bø, 2004). However, approximately 70-80% of women over 65 have UI (Milsom et al., 1993, 2001; Hannestad et al., 2000; Nygaard et al., 2003, 2004). This has been confirmed by several studies (cf. Diokno et al., 2004; Hunskaar et al., 2004; Furelly et al., 2003; Kirby et al., 2006; Rechberger & Tomaszewski, 2007).

> On the basis of separate studies, sponsored by the National Association for Incontinence in the United Sates, it can be inferred that, among all diseases of social significance, UI is the most common.  It should be emphasized in this context that the evaluation of HRQOL has become in recent times the most important diagnostic and prognostic index for the functioning of patients, regardless of the disease (Majkowicz et al., 1997; Brown et al., 1999a; Swithinbank & Abrams, 1999; Woodman et al., 2001), and is recommended by the

The analyses performed to date have dealt with particular types and degrees of clinical intensity of UI, as well as particular symptoms (e.g. UI during intercourse) before and after the application of various treatment methods (Sutherst, 1979; Lamm et al., 1986; Hilton, 1988; Lagro-Janssen et al.,1992b; Samuelsson et al., 1997; DuBeau et al., 1998; Robinson et al., 1998; Temml et al., 2000; Carcamo & Lledo, 2001; Fultz & Herzog, 2001; Hägglund et al., 2001; Scarpero et al., 2003; Abrams, 2003; Bidzan et al., 2006a, 2006b; Skrzypulec et al., 2006; Bidzan, Smutek & Bidzan, 2010). Most of this research has pointed to a connection between the variable assessed by the psychologist and those assessed by the physician, although there have been differing conclusions as to the strength of these dependencies. New possibilities are emerging for psychotherapeutic intervention, and the opportunity now exists to improve the outcome of complex, holistic treatment. It should be noted, however, that research on these connections among younger women (in the 18-34 age bracket) is relatively scant (Debus–Thiede & Dimpfl, 1993; Dugan et al., 1998; Fultz & Herzog, 2001; Hägglund et al., 2001; Shaw et al., 2001; Papanicolau et al., 2005). Attention has been drawn to the fact that the consequences of UI in younger patients are more visible than in older persons (Debus–Thiede & Dimpfl, 1993; Dugan et al., 1998; Fultz & Herzog, 2001; Hägglund et al., 2001; Shaw et al., 2001; Papanicolau et al., 2005). This may be associated with the fact that difficulties in retaining urine are generally ascribed to aging, and are perceived as a typical complaint in the elderly (Dowd, 1991; Bush et al., 2001; Dugan et al., 2001; Davey 1993). A state that is perceived as being normal is less often treated, either by public opinion or by the persons involved, as exerting a negative impact on emotional and social functioning, than when the same state is perceived as a deviation from the norm. Moreover, both the lifestyle and the range of obligations (family duties, vocational responsibilities) change with age, a fact which may support greater adaptation to UI in older persons than in

In this chapter, based on the results of earlier research, we would like to draw attention to the particular nature of the conditions surrounding UI in young women, and to advocate an multi-disciplinary approach (gynecology, urology, psychology, psychiatry, and physiotherapy) at every stage of the diagnosis and treatment of persons with UI. Based on our own experience with functional research on the lower urinary tract and the pelvic floor in a group of female patients reporting problems with urination, we shall present the most common discrepancies in the evaluation of the causes and the indications for treatment of UI. All these discrepancies may occur even in patients with correctly performed standard diagnostic tests, because the latter are not always enough to reveal disturbances of functional origin. We discuss three diagnostic problems that in our opinion are most often

International Continence Society (Abrams et al., 2002; Williams, 2004).

Broome, 2003). As mentioned earlier, it is estimated that the statistics are similar for European countries (Hunskaar et al., 2002). Since UI affects much more than 5% of the population, and has a significant impact on personal, family, and vocational functioning, it is considered a disease of social significance (Cioskowska-Paluch, 2000; Adamiak & Rechberger, 2005; Bidzan, 2008).

The spectrum of psychological problems associated with the symptoms of UI is particularly broad when the disease is at an advanced stage (Lagro-Janssen et al., 1992a, 1992b). Lalos et al. (2001) found that the life of persons with UI undergoes a diametrical change, which affects many aspects of life:

For many patients UI continues to be shameful and stigmatizing; it may well be the "last taboo of the 21st century" (Roe & May, 1999; Thom, 2000; Bidzan, Smutek & Bidzan, 2005b). Treatment outcomes in UI, though systematically improving, are still unsatisfactory: there is too high a percentage of relapse, an insufficient understanding of the reasons for treatment failure, and non-compliance with recommended therapy. This has led to increasing interest in an multi-disciplinary approach to UI, in which the process of diagnosing and treating UI involves the joint efforts of urologists and uro-gynecologists with clinical psychologists, psychiatrists, neurologists and physiotherapists.

 In recent years many researchers have focused on the evaluation of Health Related Quality of Life (HRQOL) in persons with UI, including their psychosocial functioning, broadly understood

Broome, 2003). As mentioned earlier, it is estimated that the statistics are similar for European countries (Hunskaar et al., 2002). Since UI affects much more than 5% of the population, and has a significant impact on personal, family, and vocational functioning, it is considered a disease of social significance (Cioskowska-Paluch, 2000; Adamiak &

The spectrum of psychological problems associated with the symptoms of UI is particularly broad when the disease is at an advanced stage (Lagro-Janssen et al., 1992a, 1992b). Lalos et al. (2001) found that the life of persons with UI undergoes a diametrical change, which

1. The manner and style of family life is changed, as is sexual activity (cf. Norton et al., 1988). As many as 25% of female patients experience some urine emission during sexual intercourse, while 35% have difficulties achieving orgasm (Veerecken, 1989). Moreover, UI can be a drain on the family budget, due to expenses entailed in treating and

2. Career plans are changed, vocational opportunities are limited, and sometimes it is

3. Social life (including quality of life, QOL) is impaired, with the loss of good social functioning and limitations on social contacts (cf. Brown et al., 1998a; Wein & Rovner, 1999; Anders, 2000; Thom, 2000; Tołłoczko, 2002; Smutek et al., 2004a; Bidzan et al., 2005a,b; Garstka-Namysł et al., 2007, 2008). It is estimated that ca. 35% of persons with UI are on disability pension, where one of the main reasons for an adjudication of disability is the significant degree of incontinence and the concomitant inability to maintain a job. This can cause a feeling of reduced self-worth, a loss of social position, a deterioration of mood, and social isolation, all of which serve to reduce subjective QOL

For many patients UI continues to be shameful and stigmatizing; it may well be the "last taboo of the 21st century" (Roe & May, 1999; Thom, 2000; Bidzan, Smutek & Bidzan, 2005b). Treatment outcomes in UI, though systematically improving, are still unsatisfactory: there is too high a percentage of relapse, an insufficient understanding of the reasons for treatment failure, and non-compliance with recommended therapy. This has led to increasing interest in an multi-disciplinary approach to UI, in which the process of diagnosing and treating UI involves the joint efforts of urologists and uro-gynecologists with clinical psychologists,

Psychological research to date has concentrated on the psychopathology of persons with UI, especially depression (Vereecken, 1989; Lew-Starowicz, 2002; Chiverton et al., 1996; Valvanne et al., 1996; Kinn & Zaar, 1998; Bodden-Heidrich et al., 1999; Zorn et al., 1999; Dugan et al., 2000; Watson et al., 2000; Zajda et al., 2000; Fultz & Herzog, 2001; Meade-D'Alisera et al., 2001; Broome, 2003; Nuotio et al., 2003; Nygaard et al., 2003; Bodden-Heidrich,2 004; Perry et al., 2006), anxiety, and mental discomfort (Chiara et al., 1998; Watson et al., 2000; Libalová et al., 2001; Bogner et al., 2002; Bogner & Gallo, 2002; Perry et al., 2006), as well as the impact of psychosocial factors on the course of UI, the coping methods used by patients, and treatment outcomes (Sand et al., 1999; Thom, 2000; Janssen et al., 2001; Shaw, 2001; Miller & Hoffman, 2006; Sand & Appell, 2006; Bidzan, 2008). In recent years many researchers have focused on the evaluation of Health Related Quality of Life (HRQOL) in persons with UI, including their psychosocial functioning, broadly understood

mitigating the symptoms (sanitary pads, diaper-panties, etc.).

impossible to work outside the home.

psychiatrists, neurologists and physiotherapists.

Rechberger, 2005; Bidzan, 2008).

affects many aspects of life:

(Norton et al., 1988).

 It should be emphasized in this context that the evaluation of HRQOL has become in recent times the most important diagnostic and prognostic index for the functioning of patients, regardless of the disease (Majkowicz et al., 1997; Brown et al., 1999a; Swithinbank & Abrams, 1999; Woodman et al., 2001), and is recommended by the International Continence Society (Abrams et al., 2002; Williams, 2004).

The analyses performed to date have dealt with particular types and degrees of clinical intensity of UI, as well as particular symptoms (e.g. UI during intercourse) before and after the application of various treatment methods (Sutherst, 1979; Lamm et al., 1986; Hilton, 1988; Lagro-Janssen et al.,1992b; Samuelsson et al., 1997; DuBeau et al., 1998; Robinson et al., 1998; Temml et al., 2000; Carcamo & Lledo, 2001; Fultz & Herzog, 2001; Hägglund et al., 2001; Scarpero et al., 2003; Abrams, 2003; Bidzan et al., 2006a, 2006b; Skrzypulec et al., 2006; Bidzan, Smutek & Bidzan, 2010). Most of this research has pointed to a connection between the variable assessed by the psychologist and those assessed by the physician, although there have been differing conclusions as to the strength of these dependencies. New possibilities are emerging for psychotherapeutic intervention, and the opportunity now exists to improve the outcome of complex, holistic treatment. It should be noted, however, that research on these connections among younger women (in the 18-34 age bracket) is relatively scant (Debus–Thiede & Dimpfl, 1993; Dugan et al., 1998; Fultz & Herzog, 2001; Hägglund et al., 2001; Shaw et al., 2001; Papanicolau et al., 2005). Attention has been drawn to the fact that the consequences of UI in younger patients are more visible than in older persons (Debus–Thiede & Dimpfl, 1993; Dugan et al., 1998; Fultz & Herzog, 2001; Hägglund et al., 2001; Shaw et al., 2001; Papanicolau et al., 2005). This may be associated with the fact that difficulties in retaining urine are generally ascribed to aging, and are perceived as a typical complaint in the elderly (Dowd, 1991; Bush et al., 2001; Dugan et al., 2001; Davey 1993). A state that is perceived as being normal is less often treated, either by public opinion or by the persons involved, as exerting a negative impact on emotional and social functioning, than when the same state is perceived as a deviation from the norm. Moreover, both the lifestyle and the range of obligations (family duties, vocational responsibilities) change with age, a fact which may support greater adaptation to UI in older persons than in younger (Umlauf et al., 1996).

In this chapter, based on the results of earlier research, we would like to draw attention to the particular nature of the conditions surrounding UI in young women, and to advocate an multi-disciplinary approach (gynecology, urology, psychology, psychiatry, and physiotherapy) at every stage of the diagnosis and treatment of persons with UI. Based on our own experience with functional research on the lower urinary tract and the pelvic floor in a group of female patients reporting problems with urination, we shall present the most common discrepancies in the evaluation of the causes and the indications for treatment of UI.

All these discrepancies may occur even in patients with correctly performed standard diagnostic tests, because the latter are not always enough to reveal disturbances of functional origin. We discuss three diagnostic problems that in our opinion are most often encountered, and propose optimum solutions.

A Multi-Disciplinary Perspective on the Diagnosis

maintaining the pelvic floor muscles in constant tension.

and Treatment of Urinary Incontinence in Young Women 45

towards the end of the first visit, the women voided their bladders without abdominal pressing, with normal flow and without retention, or, in the case of abnormal urethra function, with a wavy curve but without urinary retention. Urodynamic testing, including profilometry (performed 2-3 weeks after the first visit), with relaxed pelvic floor muscles, confirmed the suspicion of low urethral closure pressure (P clos max oscillated from 25 – 35 cm H2O), or abnormal urethra function. We were able to discover in the same patients during the first visit, that the P clos max could reach 80 or 120 cm H2O, if the patient were

Fig. 1 a,b. EMG recordings of perineal muscle resting tone made in supine position with Veriprobe® vaginal probe and MyoPlus® EMG unit (Verity Medical Ltd) from different

patients with increased muscle resting tone. Source: authors' own examinations.

#### **2. Low urethral pressure or abnormal urethra function**

Our clinical observations suggest that as early as the preliminary patient history an awareness of the dependencies between the mental sphere and UI can enable the physician to avoid committing diagnostic errors and applying ineffective treatment methods. Particular striking were the discrepancies regarding young women (18-34 years old in our patients) treated with a diagnosis of high urethral pressure, with concomitant UI of greater or lesser intensity and disturbances of micturition. In our urodynamic clinic, we see a large number of patients previously treated surgically by urethral dilatation due to a diagnosis of high urethral pressure, and urodynamically normal detrusor profile. In many cases, however, the initial psychological interview, including a psychosexual biography (Bidzan, Smutek & Bidzan, 2010), suggested that the micturition disturbances might have existed since childhood. A questionnaire we have developed helped us to discover that these patients had frequently repressed from consciousness their memories of UI and bedwetting in childhood and youth, and in filling out the standard questionnaires had often denied that any such thing had occurred (or they had never been asked). They most often associated the onset of their complaints with childbirth or a bladder infection in childhood or adolescence. A detailed patient history (lack of neurological disorders), palpation of perineal muscles, an evaluation of muscle contraction in the pelvic floor and the state of conscious control of these muscles by surface electromyography, and a functional evaluation of the pelvic floor muscles using a transperineal ultrasonogram, along with data from the psychosexual biography, pointed to a diametrically different diagnosis even before urodynamic testing.

The majority of the symptoms reported by these patients (recurring urinary infections, episodic urinary retention, urinary incontinence, assisting urination by abdominal pressing, with a urodynamic image typical for a sub-vesical obstruction or disorders of sphincterdetrusor coordination) were caused by a state of permanent contraction (possible since childhood) of the pelvic floor muscles (Figures 1a,b). This mechanism, used sub-consciously for years to defend against UI and the feeling of the urge to urinate, makes it impossible for the pelvic floor muscles to relax normally, which is essential for unhampered urination. It should be emphasized that the inflow of urine to the upper urethra with low or dysfunctional pressure at moments of reduced urethral pressure can evoke the urinary reflex, which our patients (with urodynamically normal detrusor function) refer to as "sudden urges." The constant maintenance of increased muscle tone can cause not only difficulties with initiating voiding, but also overfilling, which gives a clinical picture of bladder outlet obstruction. The lack of normal relaxation of the pelvic floor muscles in virtually the entire group of patients with chronic low urethral pressure, and in those with abnormal changes in urethral closure pressure, definitely hampered sexual initiation, the maintenance of sexual activity satisfying for both partners, and the establishment of a lasting relationship, and for many patients caused constant discomfort in the urogenital region. These patients assessed their HRQOL much lower. During the first visit, after a patient history has been taken and the questionnaire developed by our team has been filled out, the patients are informed about the possible genesis of the problem, and they are taught to relax the pelvic floor muscles during palpation and a transperineal ultrasonogram in standing position, with visualization of muscle activity on the screen. Those patients who had trouble with mastering conscious muscle relaxation additionally benefited from EMG biofeedback exercises with a vaginal electrode. As a rule, during the uroflow testing

Our clinical observations suggest that as early as the preliminary patient history an awareness of the dependencies between the mental sphere and UI can enable the physician to avoid committing diagnostic errors and applying ineffective treatment methods. Particular striking were the discrepancies regarding young women (18-34 years old in our patients) treated with a diagnosis of high urethral pressure, with concomitant UI of greater or lesser intensity and disturbances of micturition. In our urodynamic clinic, we see a large number of patients previously treated surgically by urethral dilatation due to a diagnosis of high urethral pressure, and urodynamically normal detrusor profile. In many cases, however, the initial psychological interview, including a psychosexual biography (Bidzan, Smutek & Bidzan, 2010), suggested that the micturition disturbances might have existed since childhood. A questionnaire we have developed helped us to discover that these patients had frequently repressed from consciousness their memories of UI and bedwetting in childhood and youth, and in filling out the standard questionnaires had often denied that any such thing had occurred (or they had never been asked). They most often associated the onset of their complaints with childbirth or a bladder infection in childhood or adolescence. A detailed patient history (lack of neurological disorders), palpation of perineal muscles, an evaluation of muscle contraction in the pelvic floor and the state of conscious control of these muscles by surface electromyography, and a functional evaluation of the pelvic floor muscles using a transperineal ultrasonogram, along with data from the psychosexual biography, pointed to a diametrically different diagnosis even before urodynamic testing. The majority of the symptoms reported by these patients (recurring urinary infections, episodic urinary retention, urinary incontinence, assisting urination by abdominal pressing, with a urodynamic image typical for a sub-vesical obstruction or disorders of sphincterdetrusor coordination) were caused by a state of permanent contraction (possible since childhood) of the pelvic floor muscles (Figures 1a,b). This mechanism, used sub-consciously for years to defend against UI and the feeling of the urge to urinate, makes it impossible for the pelvic floor muscles to relax normally, which is essential for unhampered urination. It should be emphasized that the inflow of urine to the upper urethra with low or dysfunctional pressure at moments of reduced urethral pressure can evoke the urinary reflex, which our patients (with urodynamically normal detrusor function) refer to as "sudden urges." The constant maintenance of increased muscle tone can cause not only difficulties with initiating voiding, but also overfilling, which gives a clinical picture of bladder outlet obstruction. The lack of normal relaxation of the pelvic floor muscles in virtually the entire group of patients with chronic low urethral pressure, and in those with abnormal changes in urethral closure pressure, definitely hampered sexual initiation, the maintenance of sexual activity satisfying for both partners, and the establishment of a lasting relationship, and for many patients caused constant discomfort in the urogenital region. These patients assessed their HRQOL much lower. During the first visit, after a patient history has been taken and the questionnaire developed by our team has been filled out, the patients are informed about the possible genesis of the problem, and they are taught to relax the pelvic floor muscles during palpation and a transperineal ultrasonogram in standing position, with visualization of muscle activity on the screen. Those patients who had trouble with mastering conscious muscle relaxation additionally benefited from EMG biofeedback exercises with a vaginal electrode. As a rule, during the uroflow testing

**2. Low urethral pressure or abnormal urethra function**

towards the end of the first visit, the women voided their bladders without abdominal pressing, with normal flow and without retention, or, in the case of abnormal urethra function, with a wavy curve but without urinary retention. Urodynamic testing, including profilometry (performed 2-3 weeks after the first visit), with relaxed pelvic floor muscles, confirmed the suspicion of low urethral closure pressure (P clos max oscillated from 25 – 35 cm H2O), or abnormal urethra function. We were able to discover in the same patients during the first visit, that the P clos max could reach 80 or 120 cm H2O, if the patient were maintaining the pelvic floor muscles in constant tension.

Fig. 1 a,b. EMG recordings of perineal muscle resting tone made in supine position with Veriprobe® vaginal probe and MyoPlus® EMG unit (Verity Medical Ltd) from different patients with increased muscle resting tone. Source: authors' own examinations.

A Multi-Disciplinary Perspective on the Diagnosis

**underactive urination reflex**

rehabilitation (Figs. 2a,b).

**5. Discussion**

and Treatment of Urinary Incontinence in Young Women 47

over many years, give the patient a false sense of being in good health, and make it difficult to establish the cause-and-effect relationship of psychosomatic factors. Along with the appearance of static disturbances (e.g. due to childbirth or disturbances in the innervation of the pelvic floor muscles due to discopathy), the previous mechanism for voiding an overfilled bladder and reducing urine retention ceases to be effective, and difficulties with urination intensify. They are caused by the exacerbation of deformities (folding of the urethra), along with prolapse of the anterior vaginal wall when the effort is made to assist urination by abdominal pressing. In these patients, just as was the case in the group of patients with congenitally low or abnormal urethral pressure and with a normally sensitive and properly innervated bladder of the correct volume and an active urination reflex, there is an identical defense mechanism, i.e. permanently increased resting tone in the pelvic floor muscles. This mechanism is likewise the cause of psychosexual disturbances and a worse HRQOL. The lack of a diagnosis reached early enough using surface electromyography with vaginal electrode (or rectal electrode in children), produces a similar history of largely ineffective treatment. Unfortunately, some physicians still do not have the possibility to make an objective

evaluation of the rest tone in the pelvic floor muscles with surface electromyography.

**4. Underactive detrusor, bladder with increased cystometric capacity and**

The patients with long standing underactive detrusor and a weakened urination reflex were subjected to the same diagnostic procedures as the first two groups, and were taught to relax the pelvic floor muscles. The effect of the proposed treatment, education, and exercises was particularly spectacular in this group, since after we had taught them to urinate with relaxation of the pelvic floor muscles and referred them for rehabilitation of the pelvis in cases where the disturbances were severe, further treatment was no longer necessary, and the symptoms of urinary dysfunction did not recur. The EMG recordings of perineal muscles rsting tone done with vaginal probe were much lower than in patients without

Since the patients in this group had normal urethral closure pressure, there was no UI. If we found disturbances of statics and functional mechanisms producing a tendency to UI due to overfilling, e.g. kinking of the urethra, with reduction of hernia, we referred the patient for

If we accept the criteria for the classification of dysfunctions of the lower urinary tract in the three groups of patients under discussion here, i.e. low urethral pressure/normal detrusor, abnormal urethral pressure/normal detrusor, and normal urethral pressure/underactive detrusor with increased capacity, the dysfunction was sufficiently mild and the defense mechanisms used sufficiently effective that the patients had not sought specialist medical care until additional factors had appeared, such as childbirth, reducing their ability to retain or pass urine. Ordinary medical care had been more oriented to symptomatic diagnosis and treatment. The complicated mechanisms underlying the appearance and development of UI and the broad background of psychosomatic conditions require adequate diagnostics and a thorough investigation of the etiopathology of pelvic floor muscle dysfunction, the absence of which retards properly targeted treatment and the choice of appropriate prevention methods.

appropriate surgical treatment, supplemented by rehabilitation process.

This explains why in some previous investigations these women had been diagnosed with high urethral pressure. Most likely, when the previous urodynamic testing had been done without prior training in muscles relaxation with visualization of muscle function, these women had not relaxed the pelvic floor muscles even for a moment (first of all, they were unaware of the problem). The increased resting tone in the pelvic floor muscles, or slight muscle tone changes during the various phases of urodynamic testing, could lead to an incorrect diagnosis, despite the EMG record from the skin electrodes, which results from the limitations of this method of registration.

The great majority of patients in this group reported, that after the first visit (complying with our recommendations), they had fewer disturbances of voiding and recurrent infections. Some of them, who had periodic episodes of UI, decided on rehabilitation with electrical muscle stimulation (EMS) and EMG biofeedback therapy, or surgical treatment with sling procedures for better urethral support, supplemented by post-operative rehabilitation.


**1. Introduction**

In cellular organisms, DNA replication is a crucial and highly regulated process that follows specific steps, which vary slightly between prokaryotes and eukaryotes. In general, the earliest step in DNA replication is the establishment of replication origins, i.e., the genomic loci where DNA synthesis begins [1]. The start of replication is preceded by the binding of an initiator at the replication origins, which recruits and activates the replisome in a process called origin firing. Each origin fired produces two replication forks in opposite directions (bidirectional movement), which are responsible for synthesizing DNA at a rate that varies according to the organism and cell type [1–3]. The replication time required for all chromosomes determines the S-phase duration. Although the S-phase length is referred to as a way of regulating the cell cycle progression [4,5], recent studies have suggested that it is extremely robust [6–8].

Studies indicated that bacteria [1,9] and some protozoan parasites, such as *Leishmania* spp. [10], typically have one single origin per chromosome. On the other hand, most other eukaryotes, such as *S. cerevisiae* and *S. pombe*, generally have multiple origins per chromosome [9,11–13]. The exact number of origins per chromosome can vary according to cell type and the cellular environment [14]. However, the minimum number of origins (MO) required to duplicate an entire chromosome within

a specific S-phase duration must show minimal variation because it depends on two very constant factors: average replication rate and chromosome size [8].

In trypanosomatids, single-celled eukaryotes that encompass human pathogens are of great medical importance, and the question about how many origins are needed to replicate an entire chromosome during the S-phase is totally open [8,10,15,16]. Even for the widely studied domain Bacteria and the model eukaryote *S. cerevisiae*, this discussion is not yet a closed subject [1,17–19].

Here, we developed a website that is able to determine the duration of each cell cycle phase—G1, S, G2, mitosis (M), and cytokinesis (C)—in any organism. After using this website to obtain the S-phase duration for the organisms analyzed, we applied a formula developed by our group [8] and showed a comparative analysis between the minimum number of origins (MO) in trypanosomatids (*T. cruzi*, *L. major*, and *T. brucei*) and yeasts (*S. cerevisiae* and *S. pombe*). In addition to contributing to a discussion of why some organisms use far more origins than the minimum required, this study provides a clue about the dynamic of replication during the S-phase, raising questions about the possible phenomena involved in this process.

## **2. Materials and Methods**

#### *2.1. Trypanosomatids Culture, Growth Curves, and Morphological Patterns*

Epimastigote forms of *T. cruzi* (CL Brener strain) were cultured at 28 ◦C in liver infusion tryptose (LIT) medium supplemented with 10% (*v*/*v*) fetal bovine serum and 1% (*v*/*v*) antibiotic/antimycotic solution. Promastigote forms of *L. major* (strain Friedlin) were cultured at 26 ◦C in an M199 medium supplemented with 10% (*v*/*v*) heat-inactivated fetal calf serum, 25 mM HEPES, and 1% (*v*/*v*) antibiotic/antimycotic solution.

For the growth curves, each parasite culture was initiated with 1 × 106 cells.mL−1 . Each growth curve was harvested and counted daily until it reached the stationary phase. For the establishment of the morphological patterns, formaldehyde-fixed and DAPI-stained exponentially growing parasites (*T. cruzi* and *L. major*) were examined under an Olympus BX51 fluorescent microscope (Olympus, Tokyo, Japan) (100× oil objective) to observe the profile of organelles that contain DNA (nucleus and kinetoplast).

## *2.2. EdU Incorporation Assays and 'Click' Chemistry Reaction*

Exponentially growing parasites were incubated with 100 µM 5-ethynyl-2′ -deoxyuridine (EdU) (ThermoFisher Scientific, Waltham, MA, USA) for the time required according to each assay at species-specific temperatures (28 ◦C for *T. cruzi* and 26 ◦C for *L. major*). The parasites were then harvested by centrifugation at 2500 *g* for 5 min, washed three times in 1× PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 2 mM KH2PO4, pH 7.4), and the pellet was resuspended in 200 µL of the same buffer solution. Afterward, 100 µL of the cell suspension was loaded onto poly-L-lysine-pretreated microscope slides (Tekdon, Myakka, FL, USA), fixed for 20 min using 4% sterile paraformaldehyde (Merck, Darmstadt, Germany) diluted in 1× PBS, washed three times with 1× PBS, and then washed three times with 3% BSA (Sigma-Aldrich, Saint Louis, MO, USA) diluted in 1× PBS. Then, parasites were permeabilized for 10 min with 0.1% sterile Triton X-100 (Sigma Aldrich, Saint Louis, MO, USA) diluted in 1× PBS, washed three times with 1× PBS, and then washed three times with 3% BSA in 1× PBS. To detect incorporated EdU, we used the Click-iT EdU detection solution for 45 min protected from light. The Click-iT EdU detection mix solution consisted of 25 µL 500 mM ascorbic acid (C6H8O6), 5 µL 100 mM copper sulfate (CuSO4), 2.5 µL Alexa fluor azide 488 (ThermoFisher Scientific, Waltham, MA, USA), and 467.5 µL distilled water (for details about EdU procedure, see ref. [20]). Finally, the parasites were washed five times with 1× PBS. Vectashield Mounting Medium (Vector, Burlingame, CA, USA) containing 4′ ,6-diamidino-2-phenylindole dihydrochloride (DAPI) was used as an antifade mounting solution and to stain nuclear and kinetoplast DNA. Images were acquired using an Olympus Bx51 fluorescent microscope (100× oil objective) attached to an EXFO Xcite series 120Q lamp and a digital

Olympus XM10 camera with camera controller software Olympus Cell F (Olympus, Tokyo, Japan). Images were further analyzed using ImageJ software (National Institutes of Health, USA) to count the numbers of EdU-positive parasites, and the percentage of proliferating parasites was calculated for each sample relative to the total number of DAPI-positive parasites.

## *2.3. Development of the CeCyD Website and Analysis of the Cell Cycle*

The website CeCyD (Cell Cycle Duration estimator) was developed using the Python programming language plus the Django v.1.8 framework. CeCyD is available at the following address http: //cecyd.vital.butantan.gov.br/, and its source code is released under the GNU GPL-3 license at https: //github.com/bruno-fs/CeCyD.

To estimate the duration of mitosis (M) and cytokinesis (C), the CeCyD uses the Williams (1971) equation [21]:

$$\alpha = \frac{\ln(1 - y/2)}{-a} \tag{1}$$

where *x* is the cumulative time within the cell cycle necessary to reach the start of the phase in question, i.e., the difference between the doubling time and *x* will give the time of the remaining phase(s); *y* is the cumulative proportion of cells up the phase in question (expressed as a fraction of one unit), i.e., the difference between the total cells (1% or 100%) and the percentage of cells in C or M+C, will provide the *y* value for C and M, respectively. Finally, α is the specific growth rate.

To estimate the G2 phase, the CeCyD must receive from the user the value corresponding to the period required for a cell to pass through G2 and M phases. For this, the user must apply an EdU pulse (e.g., 15 min) and then collected parasites every 15 min until a single cell containing two EdU-labeled nuclei (2N2K in case of trypanosomatids) is observed. The difference between this value and the duration of mitosis previously calculated corresponds to the G2-phase duration.

The S-phase duration is estimated by the CeCyD according to the Stanners and Till (1960) equation [22]:

$$S = \frac{1}{\alpha} \ln[L + e^{\alpha(Z)}] - (Z + t) \tag{2}$$

where *L* is the proportion of cells exhibiting EdU-labeled nuclei, α = ln 2/T (T = doubling time expressed in hours), *Z* = *G*2 + *M* + *C*, and *t* is the duration of the EdU labeling period in hours. Finally, the G1-phase duration is estimated by the difference between the doubling time and the sum of the remaining phases.


2.1.2. Anion Effect

For carbon steel and aluminium, the corrosivity of IL media strongly depends on the chemical structure of the cationic moiety and the nature of anion in the IL molecule. Specifically, the corrosion resistance of carbon steel in water-free ILs strongly depends on the IL anion. Depending on the type of IL, carbon steel may undergo severe corrosion in diluted IL media. Anions like tosylate and dimethyl phosphate generally trigger higher corrosivity specifically in water-diluted ILs. In this regard, Uerdingen et al. [39] investigated the behaviour of carbon steel, austenitic stainless steel, nickel-based alloy, copper, brass and aluminium in presence of various diluted ILs with different concentrations under flow conditions at temperatures up to 90 ◦C. The effect of the chemical structure of the IL cation and the nature of anion on the corrosivity of the metals has been studied. It is observed that diluted ILs (with water) could result in hydrolysis of IL's anions. As a result, they can produce acids (e.g., sulfuric acid, phosphoric acid) and, hence, cause a considerable increase of medium corrosivity, whereas in water-free ILs, most of the metals exhibited a high corrosion resistance.

Ashassi-Sorkhabi et al. [40] studied the effect of IL anion in corrosion inhibitor behaviour of two synthesized imidazolium ionic liquids with chlorides and hydrogen sulphate anion on mild steel in hydrochloric acid. In the structure of the imidazolium bases, the atoms of the imidazolium ring and the –C=N– group can form a big π bond. Then, in addition to the π electron of the imidazolium, bases enter unoccupied orbitals of iron. The π\* orbital can also accept the electrons of d orbitals of iron to form bonds, that produce more than one center of adsorption action. Moreover, the presence of the Cl and S on the imidazolium structure, which are electron donating groups, increases the electron density on the nitrogen of the –C=N– group, resulting in high inhibition efficiency. Therefore, in this study hydrogen sulphate counter-anion showed better inhibiting effect of mild steel in HCl compared with chloride anion.


2.4.2. Analysis of Tensile Strength in Flexion and Compression

For this test, guided by the ABNT NM 13279 [32] standard, it was necessary to prepare 3 prismatic parts for each mix, using Portland cement types CP II, CP III and CP V, with cures of 7, 14 and 28 days, in the different substitutions of sand by OSPW (10%, 30% and 60%).

After completing the curing days, the prismatic parts were subjected to flexural and compressive strength tests, using an EMIC 26–30 universal testing equipment with a maximum force of 30 kN. First, the flexural tensile strength test was performed, where the prismatic parts were positioned in the machine in order to avoid contact between the flat surface and the supporting and loading components.

Then, to run the compressive strength test, the halves of each part of the flexural test were used and also positioned in the machine in order to avoid contact between the flat surface and the supporting and loading components.


**Mechanics of Electric Rope Shovel Performance and Reliability in Formation Excavation Mechanics of Electric Rope Shovel Performance and Reliability in Formation Excavation**

Muhammad Azeem Raza and Samuel Frimpong Muhammad Azeem Raza and Samuel Frimpong

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/65333

#### **Abstract**

Large-capacity rope shovels are used as primary production equipment in many surface mining operations. Current rope shovels have payload capacities in excess of 100 tons per pass. The dynamic payload and formation resistive forces result in severe stress loading of the shovel front-end assembly. Material flaws, high stresses, and harsh excavation conditions can initiate cracks in the dipper-teeth assembly. These cracks, under high stresses, can propagate to critical lengths resulting in fatigue failure of frontend assembly. Dipper-related problems can significantly reduce shovel availability. There is no fundamental research for understanding dipper fatigue failure resulting from high stress intensity, crack initiation, and propagation, the subject matter of this study. The Newton-Euler algorithm is used to build kinematics and dynamic models of the cable shovel front-end assembly. The models incorporate the dynamic resistive forces on the dipper-teeth assembly. Numerical simulations are used to generate the dynamic payload force and its dynamic left. Virtual simulation, based on the P&H 4100XPC shovel prototype in ANSYS (R15), is run to generate stress loading of the dipper-teeth assembly and equivalent (von Mises) stresses. Stress intensity factors are computed for various crack lengths in the dipper-teeth assembly, and the crackpropagation lives are computed for these cracks. The results show that a 75-mm crack can propagate to the critical length in 16 days. This research study provides a pioneering effort toward understanding shovel dipper fatigue failure due to high stress intensity, crack initiation, and propagation for understanding shovel reliability and availability for production efficiency and bulk production economics.

**Keywords:** formation excavation, machine kinematics and dynamics, virtual simulation, stress profile intensity

Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons