**Author details**

Ricardo U. Sorensen, and *Department of Pediatrics, New Orleans, Louisiana, USA*

Tammy Harvey *Jeffrey Modell Center for Immunodeficiencies, New Orleans, Louisiana, USA*

Lily E. Leiva *Louisiana State University Health Science Center, New Orleans, Louisiana, USA*

### **12. References**

[1] Centers of Disease Control and Prevention. Prevention of pneumococcal disease: Recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR Morb Mortal Wkly Rep 1997;46:1-24.

Selective Antibody Deficiency with Normal Immunoglobulins 203

[11] Bonilla FA, Bernstein IL, Khan DA, Ballas ZK, Chinen J, Frank MM, et al. Practice parameter for the diagnosis and management of primary immunodeficiency. Ann

[12] Bossuyt X, Borgers H, Moens L, et al. Age- and serotype-dependent antibody response

[13] Boyle R, Le C, Balloch A, et al. The clinical syndrome of specific antibody deficiency in

[14] Burton R, Nahm M. Development and validation of a fourfold multiplexed opsonization assay (MOPA4) for pneumococcal antibodies. Clin Vaccine Immunol

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Ricardo U. Sorensen, and

*Department of Pediatrics, New Orleans, Louisiana, USA*

MMWR Morb Mortal Wkly Rep 1997;46:1-24.

*Jeffrey Modell Center for Immunodeficiencies, New Orleans, Louisiana, USA*

*Louisiana State University Health Science Center, New Orleans, Louisiana, USA*

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**Chapter 9**

© 2012 Tassone et al., licensee InTech. This is an open access chapter 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.

© 2012 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,

**Emerging Role of MicroRNAs in**

Marco Rossi, Pierosandro Tagliaferri and

Additional information is available at the end of the chapter

Pierfrancesco Tassone

http://dx.doi.org/10.5772/51543

**1.1. Overview of MiRNA biology**

30% of all protein-encoding genes [3].

called passenger strand, is degraded.

have shown that miRNA can increase translation [8].

**1. Introduction**

**the Pathophysiology of Immune System**

MicroRNAs are small (22 nucleotides), noncoding, double-stranded RNA molecules, that can regulate gene expression primarily by reducing the abilities of specific mRNAs to be transduced to their encoded proteins. The first recognized miRNA found in 1993 is lin-4, that controls the cell fate at larval stages in Caenorhabditis elegans [1, 2]. Bioinformatic approaches suggested that the mammalian miRNA repertoire is involved in regulation of

Human miRNAs are encoded within introns of coding genes and introns and exons of noncoding transcripts [4]. Generation of mature miRNAs is due to a series of endonucleolytic steps starting from long primary transcripts (pri-miRNAs). The primiRNAs are cleaved in the nucleus to a\_70 nt intermediate with the typical stem-loop hairpin structure, precursor miRNAs (pre-miRNAs) by the Drosha- DGCR8 microprocessor complex [5, 6]. The pre-miRNAs are further processed into\_22 nt double-stranded miRNA duplex by the cytoplasmic RNase III enzyme Dicer [7]. One strand of this miRNA duplex (the guide strand) incorporates into a large protein complex, RNA-induced silencing complex (RISC), formed by Dicer, TRBP (a dsRNA-binding domain protein) and Ago2 (the Argonaute protein 2), and finally becomes the mature miRNA. The other strand, the so-

Each mature miRNA interacts with a specific mRNA in the mRNA's 3´-untranslated region (3´UTR), leading to translational repression or mRNA degradation. Besides, some evidences

and reproduction in any medium, provided the original work is properly cited.

**Chapter 9**


**2. Cases**

118 Neuromuscular Disorders

**Congenital myopathies**: characterized by developmental delays in motor skills; skeletal

**Muscular dystrophies**: caused by progressive weakness in voluntary muscles; sometimes

**Mitochondrial myopathies**: caused by genetic abnormalities in mitochondria, cellular

**Glycogen storage diseases of muscle**: caused by mutations in genes controlling enzymes that metabolize glycogen and glucose (blood sugar); include Pompe's, Anderson's and

**Myoglobulinurias**: caused by disorders in the metabolisum of a fuel (myoglobulin)

**Common muscular cramps and stiffness and tetany**: characterized by prologed spasms

biopsy

ALD 5.8 myopathy ANA 20

6. F 55 DM SLD 5.7 nd nv 3.11 y no remission 7. F 60 DM CK 364 nd nv 2.0 y no remission

Table 2. Myopathy and myositis in primary Sjögren's syndrome (pSjS) and dermatomyositis

Autoantibody

ANA nv SSA 61.4 SSB 21.6

KL-6 2485 nd nv 4.3 y intestinal

ALD 11.2 myositis nv 2.6 y no remission

ALD 8.4 myositis nv 3.8 y no remission

myositis ANA 20 2.0 y pneumonia

Observation term

Compli-

3.0 y no remission

SSB 15.7 8 m no remission

cation Outcome

peumonia remission

and cancer death

**Familiar periodic paralysis**: characterized by episodes of weakness in arms and legs **Polymyositis inclusion body myositis and related myopathies**: inflammatory

**Neuromyotonia**: characterized by alternating episodes of twiching and stiffness **Stiff-man syndrome**: characterized by episodes of rigidity and reflex spasms

(National Institute of Neurological Disorders and Stroke, National Institutes of Health, USA1)

abnormatities myopathy

and facial abnormalities are occationally evident at birth

structures that control energy; include Kearns-Sayre syndrome,

**Dermatomyositis**: an inflammatory myopathy and skin lesions **Myositis ossificans**: characterized by bone growing in muscle tissue

years Disease Enzyme Muscle

CK 1706 ALD 8.2 KL-6 1876 myoglobin 29.7

ALD: aldolase, ANA: anti-nuclear antibody, CK: creatine kinase, DM: dermatomyositis, F: female, M: male, nd: not done, Nv: negative, pSjS: primary Sjögren's syndrome,

Myopathy: non-inflammatory myopathy, Myositis: inflammatory myopathy

necessary for muscle work; include McArdle, Tarui and DiMauro diseases

evident at birth

Cori's disease

myopathies of skeletal muscles

Table 1. Classification of myopathy

1. F 37 pSjS No

2. M 32 pSjS CK 147

3. F 29 DM CK 883

4. F 42 DM CK 212

5. M 45 DM CK 302

SSA: anti-SjS A antibody, SSB: anti-SjS B antibody

8. M 66 DM

(DM)

of the arms and legs

Patient Gender Age

**pSjS**: A 37-year-old woman (Case 1 in Table 2) was suffered from fever around 37.5℃, fatigue, proximal muscle pain and weakness in her limbs and arthralgia since a week before her visiting our hospital. She presented herself with swelling and helio-trope-like colored erythema on the eye-lids (Fig. 1a,b), purpurish erythema-spots on the elbows, thin-reddish erythema patches on the legs (Fig.1c) and red palms. On the dorsa of the hand-fingers, the eruption looked like Gottron's papules was seen and thin purpuric spots were also noted on the paronychial areas. The electromyography showed low amplitude motor units (less than 1 mV) from muscles of the upper extremities that were suggestive myopathy. We suspected the patient had DM and the skin biopsy was taken from the erythematous patches on the left leg. The histology revealed so-called "lymphocytic vasculitis" which showed swollen

Myopathy in Autoimmune Diseases – Primary Sjögren's Syndrome and Dermatomyositis 121

vascular walls surrounded by mainly monocytes and a few of neutrophils in the middle and deep dermis (Fig. 2a,b). No deposit of IgG, IgA, IgM and C3 was seen at the dermoepidermal (D-E) junction and vascular walls in the dermis by immunofluorescent microscopy. The immunochemical histology revealed CD4+>CD8+>CD56+ cells distributed around vessels in the deep dermis. A muscle biopsy was performed from the biceps muscle of the left-upper arm where the patient was complaining of pain. No features of inflammation associated the "muscle fiber degeneration" could be found though slight

a) b) Fig. 2. a) A skin biopsy from the left side of the lower leg revealed, a. vascular infiltration with lymphoid cells and a few neutrophils in the middle and deep dermis (H&E stain, x40). b) The magnified view showed swelling of the vascular endothelial cells with slight degeneration infiltrated by lymphoid cells, suggesting so-called "lymphocytic vasculitis" (x 200)

Laboratory examinations revealed within normal-limit (WNL) ranges of white blood cells (WBC), red blood cells (RBC) and serum AST (aspartate aminotransferase), ALT (alanine aminotransferase), and CK (creatine kinase) (28 IU/L, WNL: 48~259) and aldolase (ALD) (2.2 IU/L, WNL: 2.5~5.6) were rather lower than WNL. The serum levels of complement were high in CH50 (58.4 u/ml, WNL: 31.0~48.0) but levels of C3 and C4 were WNL. Although auto-antibodies (auto-Abs) including anti-nuclear Ab (ANA), anti-Jo-1, anti-DNA and anti-acetylcholine receptor Abs and RA factor were negative, anti-Sjögren's syndrome A (anti-SSA) and anti-SSB Abs showed the titers of 61.4 and 21.6 (WNL: less than 1.0 ), respectively, which were highly positive in detection by ELISA. Serum carcinoembryonic antigen (CEA) was not detected. Urinalysis revealed no abnormalities except detection of acetone body. The chest X-ray and positron emission tomography (PET) with 18FDG (2 deoxy-2-fluoro-D-glucose) were performed but no abnormal uptakes were detected except for an enlarged lymphnode at the right-side neck and slight enlargement of the liver and spleen. Ophthalmological examinations revealed positive Schirmer's test (10mm/6mm) and fluorescein test which showed dry-eyes, although the amount of saliva was 3.5 ml/10 min which seemed to be low But WNL. The pain of visual analog scale (VAS) score was 75 mm

The patient was diagnosed as having pSjS with myalgia mimicking DM, which may be classified as the extraglandular type. She was treated with prednisolone (PSL) 20mg/day and non-steroidal anti-inflammatory drugs (NSAIDs). The symptoms including the

vascular infiltration was seen in the interstitial tissue.

when she initially visited our clinic.

b)

c)

Fig. 1. a,b) Close-up view of the right upper eye-lid showing slightly swollen and heliotrope-like colored erythema (Case 1 in Table 2). c) Gottron's nodule-like eruption on the dorsa of the fingers and thin-reddish erythematous patches on the lower legs.

b)

c)

Fig. 1. a,b) Close-up view of the right upper eye-lid showing slightly swollen and heliotrope-like colored erythema (Case 1 in Table 2). c) Gottron's nodule-like eruption on the

dorsa of the fingers and thin-reddish erythematous patches on the lower legs.

vascular walls surrounded by mainly monocytes and a few of neutrophils in the middle and deep dermis (Fig. 2a,b). No deposit of IgG, IgA, IgM and C3 was seen at the dermoepidermal (D-E) junction and vascular walls in the dermis by immunofluorescent microscopy. The immunochemical histology revealed CD4+>CD8+>CD56+ cells distributed around vessels in the deep dermis. A muscle biopsy was performed from the biceps muscle of the left-upper arm where the patient was complaining of pain. No features of inflammation associated the "muscle fiber degeneration" could be found though slight vascular infiltration was seen in the interstitial tissue.

Fig. 2. a) A skin biopsy from the left side of the lower leg revealed, a. vascular infiltration with lymphoid cells and a few neutrophils in the middle and deep dermis (H&E stain, x40). b) The magnified view showed swelling of the vascular endothelial cells with slight degeneration infiltrated by lymphoid cells, suggesting so-called "lymphocytic vasculitis" (x 200)

Laboratory examinations revealed within normal-limit (WNL) ranges of white blood cells (WBC), red blood cells (RBC) and serum AST (aspartate aminotransferase), ALT (alanine aminotransferase), and CK (creatine kinase) (28 IU/L, WNL: 48~259) and aldolase (ALD) (2.2 IU/L, WNL: 2.5~5.6) were rather lower than WNL. The serum levels of complement were high in CH50 (58.4 u/ml, WNL: 31.0~48.0) but levels of C3 and C4 were WNL. Although auto-antibodies (auto-Abs) including anti-nuclear Ab (ANA), anti-Jo-1, anti-DNA and anti-acetylcholine receptor Abs and RA factor were negative, anti-Sjögren's syndrome A (anti-SSA) and anti-SSB Abs showed the titers of 61.4 and 21.6 (WNL: less than 1.0 ), respectively, which were highly positive in detection by ELISA. Serum carcinoembryonic antigen (CEA) was not detected. Urinalysis revealed no abnormalities except detection of acetone body. The chest X-ray and positron emission tomography (PET) with 18FDG (2 deoxy-2-fluoro-D-glucose) were performed but no abnormal uptakes were detected except for an enlarged lymphnode at the right-side neck and slight enlargement of the liver and spleen. Ophthalmological examinations revealed positive Schirmer's test (10mm/6mm) and fluorescein test which showed dry-eyes, although the amount of saliva was 3.5 ml/10 min which seemed to be low But WNL. The pain of visual analog scale (VAS) score was 75 mm when she initially visited our clinic.

The patient was diagnosed as having pSjS with myalgia mimicking DM, which may be classified as the extraglandular type. She was treated with prednisolone (PSL) 20mg/day and non-steroidal anti-inflammatory drugs (NSAIDs). The symptoms including the

Myopathy in Autoimmune Diseases – Primary Sjögren's Syndrome and Dermatomyositis 123

a) b)

A 66 years old man (Case 8) was referred to our clinic for sudden episode of helio-tropelike colored erythematous eruption on the sun-exposed areas including the face, V-neck area, upper back and upper extremities associated with "myopathy" exhibiting muscle weakness and myalgia (Fig. 5). The patient had heavily smoked cigarettes a pack or more a day. He was initially suspected to have photosensitive dermatitis due to some photosensitizer and/ or DM and the examinations including skin and muscle biopsy from left-upper arm were performed. Laboratory examinations revealed WBC 11,070 /μl, RBC 327 x 104 /μl , CRP 14.28 mg/dL (WNL: 0.30), WNL of serum transaminases (AST and ALT), high levels of CK 1,706 U/L (WNL: 259-2.5) , ALD 8.2 IU/L (WNL: 5.6-2.5), myoglobulin-U 29.7 ng/mg (WNL: 10~0) and KL-6 1876 U/mL (WNL: 499-0). The titers of auto-Abs showed WNL as to anti-DNA, anti-Jo-1, anti-RNP, Sm, and anti-SSA and anti-SSB Abs except for ANA 20x (speckled type). Although CEA, CA15-3, AFP-L3, -L2,-L2, 3 and CA602 were negative, we suspected the patient might have an association with lung carcinoma after the chest X-ray and CT examination. The skin histology of the helio-tropelike erythema revealed as SLE-like findings exhibiting liquefaction degeneration of the D-E junction and edema of the upper dermis with a little lymphoid cell infiltration (Fig. 6) and immunohistologically, IgG, IgM and complement C3 were linearly deposited at the D-E junction. The muscle biopsy from the biceps of the left arm exhibiting myalgia shows a tiny interstitial perivascular infiltration between the muscle bundles, suggesting "myositis", although obvious muscular-degeneration was not found. The symptoms of the skin rash and myalgia of the extemities were improved temporary after treatment with oral PSL and NSAIDs. However, the patient died by lung cancer 2 years after his first

Fig. 4. a) A biopsy specimen of muscle tissue from the left biceps muscle of patient with dermatomyositis (DM) (Case 3). Lymphoid cell infiltration around the vessels was found in the interstitial tissue (HE, 200x). b) Immunohistology of CD4+T cells in the interstitial perivascular infiltration of the biceps muscle (Avidin-biotin stain, 20x). The infiltrated cells

are CD4+>CD8+>CD56+ mononuclear cells.

visit to our clinic.

cutaneous manifestations completely disappeared and pain VAS score also gradually decreased to 55 mm in a week. The patient was quickly recovered from fatigue, subfebrile state, myalgia and arthralgia two weeks after treatment by oral steroid. However, PSL administration of within 10 mg/day was needed to keep her well condition, although more than two years have been passed since her first visit.

Although the other 32 year-old male patient (Case 2) complained of dry eyes, mild fever, myalgia and muscle weakness of the upper extremities for more than one year, we initially suspected DM and examined for possibility of his having SjS regardless of absence of a DMlike eruption. Ophthalmologically he was suggested to have SjS. The blood examination revealed ALT 40 U/L (WNL: 6-36), ALD 5.8 IU/L, titers of ANA 20x (speckled type) and anti-SSA Ab 15.7 which were relatively high. However, a biopsy from the biceps muscle of the left upper arm was free from inflammation, and no internal malignancy was associated through examinations including PET with 18FDG. He has been followed as pSjS similarly to the Case 1 for the duration of more than half a year.

**DM**: A 29 year-old female (Case 3) visited our clinic for helio-trope-like eruptions on the sun-exposed areas including upper eye-lids, cheeks and V-neck area (Fig. 3a), swollen fingers with periungual hemorrhage of the hands (Fig. 3b) and myalgia of the upper extremities. The examination revealed a rise of AST and ALT (65 and 46 U/L; WNL: 35-11 and 39-6, respectively), CK 883 U/L and ADL 11.2 IU/L, but no auto-Abs including ANA, anti- SSA and SSB Abs were found. No internal malignancies were found. However, a biopsy from left biceps muscle revealed the typical myositis with intersititial vascular infiltration (Fig. 4a). The immunochemistry of infiltrated cells around the interstitial vessels of the muscle tissue revealed CD4+> CD8+> CD68+ cells and little of CD20+ cells as similarly seen in the cutaneous findings of Case 1 (Fig.4b). We made a diagnosis of early stage of DM.

Fig. 3. a) A 29 year-old female with dermatomyositis (DM) (Case 3). Swollen helio-trope-like erthema on the upper eye-lids and cheeks. b) Swollen erythema on the dorsa of the hands and periungual hamorrhage of the fingers.

cutaneous manifestations completely disappeared and pain VAS score also gradually decreased to 55 mm in a week. The patient was quickly recovered from fatigue, subfebrile state, myalgia and arthralgia two weeks after treatment by oral steroid. However, PSL administration of within 10 mg/day was needed to keep her well condition, although more

Although the other 32 year-old male patient (Case 2) complained of dry eyes, mild fever, myalgia and muscle weakness of the upper extremities for more than one year, we initially suspected DM and examined for possibility of his having SjS regardless of absence of a DMlike eruption. Ophthalmologically he was suggested to have SjS. The blood examination revealed ALT 40 U/L (WNL: 6-36), ALD 5.8 IU/L, titers of ANA 20x (speckled type) and anti-SSA Ab 15.7 which were relatively high. However, a biopsy from the biceps muscle of the left upper arm was free from inflammation, and no internal malignancy was associated through examinations including PET with 18FDG. He has been followed as pSjS similarly to

**DM**: A 29 year-old female (Case 3) visited our clinic for helio-trope-like eruptions on the sun-exposed areas including upper eye-lids, cheeks and V-neck area (Fig. 3a), swollen fingers with periungual hemorrhage of the hands (Fig. 3b) and myalgia of the upper extremities. The examination revealed a rise of AST and ALT (65 and 46 U/L; WNL: 35-11 and 39-6, respectively), CK 883 U/L and ADL 11.2 IU/L, but no auto-Abs including ANA, anti- SSA and SSB Abs were found. No internal malignancies were found. However, a biopsy from left biceps muscle revealed the typical myositis with intersititial vascular infiltration (Fig. 4a). The immunochemistry of infiltrated cells around the interstitial vessels of the muscle tissue revealed CD4+> CD8+> CD68+ cells and little of CD20+ cells as similarly seen in the cutaneous findings of Case 1 (Fig.4b). We made a diagnosis of early stage of DM.

a) b)

Fig. 3. a) A 29 year-old female with dermatomyositis (DM) (Case 3). Swollen helio-trope-like erthema on the upper eye-lids and cheeks. b) Swollen erythema on the dorsa of the hands

than two years have been passed since her first visit.

the Case 1 for the duration of more than half a year.

and periungual hamorrhage of the fingers.

Fig. 4. a) A biopsy specimen of muscle tissue from the left biceps muscle of patient with dermatomyositis (DM) (Case 3). Lymphoid cell infiltration around the vessels was found in the interstitial tissue (HE, 200x). b) Immunohistology of CD4+T cells in the interstitial perivascular infiltration of the biceps muscle (Avidin-biotin stain, 20x). The infiltrated cells are CD4+>CD8+>CD56+ mononuclear cells.

A 66 years old man (Case 8) was referred to our clinic for sudden episode of helio-tropelike colored erythematous eruption on the sun-exposed areas including the face, V-neck area, upper back and upper extremities associated with "myopathy" exhibiting muscle weakness and myalgia (Fig. 5). The patient had heavily smoked cigarettes a pack or more a day. He was initially suspected to have photosensitive dermatitis due to some photosensitizer and/ or DM and the examinations including skin and muscle biopsy from left-upper arm were performed. Laboratory examinations revealed WBC 11,070 /μl, RBC 327 x 104 /μl , CRP 14.28 mg/dL (WNL: 0.30), WNL of serum transaminases (AST and ALT), high levels of CK 1,706 U/L (WNL: 259-2.5) , ALD 8.2 IU/L (WNL: 5.6-2.5), myoglobulin-U 29.7 ng/mg (WNL: 10~0) and KL-6 1876 U/mL (WNL: 499-0). The titers of auto-Abs showed WNL as to anti-DNA, anti-Jo-1, anti-RNP, Sm, and anti-SSA and anti-SSB Abs except for ANA 20x (speckled type). Although CEA, CA15-3, AFP-L3, -L2,-L2, 3 and CA602 were negative, we suspected the patient might have an association with lung carcinoma after the chest X-ray and CT examination. The skin histology of the helio-tropelike erythema revealed as SLE-like findings exhibiting liquefaction degeneration of the D-E junction and edema of the upper dermis with a little lymphoid cell infiltration (Fig. 6) and immunohistologically, IgG, IgM and complement C3 were linearly deposited at the D-E junction. The muscle biopsy from the biceps of the left arm exhibiting myalgia shows a tiny interstitial perivascular infiltration between the muscle bundles, suggesting "myositis", although obvious muscular-degeneration was not found. The symptoms of the skin rash and myalgia of the extemities were improved temporary after treatment with oral PSL and NSAIDs. However, the patient died by lung cancer 2 years after his first visit to our clinic.

Myopathy in Autoimmune Diseases – Primary Sjögren's Syndrome and Dermatomyositis 125

It is rare to see the cases of pSjS with myalgia in Japan, but about 30 % of pSjS patients are reported to be associated with muscle involvement known as fibromyalgia in US and European countries (5, 6). The main cutaneous involvement is purpura, annular shaped erythema and/or macules, erythematous papules and ischemic ulcers due to microvasculitis in pSjS (7, 8). In this patient (Case 1 in Table 2), the cutaneous eruptions including the swollen and helio-trope-like colored erythema on the eye-lids, thin-reddish macules on the limbs, purpuric spots and Gottron's papule-like eruption on the dorsa of the fingers were recognized and quickly disappeared after administration with low dose of PSL. Although the clinical signs-like DM did not reappear by the treatment, the continuous treatment with PSL seems to be still needed. As reported that this disorder is a bothersome and slowly progressive disease (10-12), we should follow the clinical course whether the patient might develop lymphoproliferative disorders in a near future because the enlargement of the lymphnode and hepatosplenomegaly was noted initially. Regarding Case 2, the clinical symptoms and laboratory examination were suggestive of DM associated with SjS without the skin manifestations. We considered him as having pSjS associated with myopathy because dry-eye symptoms, positive anti-SSA Ab and no cutaneous symptom of DM were noted. Although these 2 cases of pSjS were associated with myalgia, the cause of their myopathy is not clear because no inflammation was found in the biopy specimens from their biceps muscle. However, it is reported that the myopathy might be due to small-vessel injury by auto-Abs or circulating immune complexes because electrondence deposits were noted (13). As to the infiltrated cells around the vessels in the cutaneous lesion, the CD4+ T

cells were dominant as similar to the findings in myositis of patients with DM.

DM is an idiopathic inflammatory disorder characterized by inflammatory myopathy, indicating myositis, and skin manifestations and it can be associated with the secondary SjS sometimes. The etiology is still unknown and the prevalence is estimated as 1-10 cases per million population, but in children 3.2 cases per million which are distributed in the whole world (3). The clinical types of DM are classified in Table 3 and the internal malignancies are frequently associated in the adult type of DM. The risk is reported to be a 6.5-fold higher than ordinary persons after 45 years of age (14). Regarding myopathy in DM patients it might be characterized by inflammatory myopathy progressing to myositis and degeneration of muscle fibers, and the helio-trope-like eruptions on the sun-exposed areas showed SLE-like changes histologically. On the other hand, there are the cases associated without myogenic symptoms in DM, which is called as "amyopathic DM" (DM sine myositis). However, these cases should be considered as "pre-myopathic DM" because they might Be rather early diagnosed (15). Though the direct cause of myopathy is still unknown, there are some pathogeneses reported, such as the presences of myositis- specific auto-Abs (15,16) and inflammatory cytokines from T cells including interleukin (IL)-17 and IL-23 in early stage of patients with DM (18). The study regarding Th1/Th2 balance showed that Th2 cell predominance was suggested in patients with active stage of DM (19). In plasma of patients with DM and/or polymyositis, microparticles derived from CD14+ mononuclear cells, CD3+ T cells and CD19+ B cells were found to be elevated by electron microscopic examination, which suggests these diseases were immunological disorders (20). It is reported that CD19+CD23+ cells are increased and that CD4+CD45RO+ cells are decreased in the peripheral blood of patients with DM, suggesting reduction of regulatory T cells (21). It

**3. Discussion**

Fig. 5.  Helio-trope-like erythema can be seen on the face and upper breast (so-called sun exposed areas).

Fig. 6. A biopsy specimen of the helio-trope-like erythema from the upper chest. Liquefaction degeneration can be seen at the D-E junction and edema and a few lymphoid cell infiltration are present in the upper dermis (HE, 200x).

#### **3. Discussion**

124 Neuromuscular Disorders

Fig. 5.  Helio-trope-like

erythema can be seen on the face and upper breast (so-called sun exposed areas).

Fig. 6. A biopsy specimen of the helio-trope-like erythema from the upper chest.

cell infiltration are present in the upper dermis (HE, 200x).

Liquefaction degeneration can be seen at the D-E junction and edema and a few lymphoid

It is rare to see the cases of pSjS with myalgia in Japan, but about 30 % of pSjS patients are reported to be associated with muscle involvement known as fibromyalgia in US and European countries (5, 6). The main cutaneous involvement is purpura, annular shaped erythema and/or macules, erythematous papules and ischemic ulcers due to microvasculitis in pSjS (7, 8). In this patient (Case 1 in Table 2), the cutaneous eruptions including the swollen and helio-trope-like colored erythema on the eye-lids, thin-reddish macules on the limbs, purpuric spots and Gottron's papule-like eruption on the dorsa of the fingers were recognized and quickly disappeared after administration with low dose of PSL. Although the clinical signs-like DM did not reappear by the treatment, the continuous treatment with PSL seems to be still needed. As reported that this disorder is a bothersome and slowly progressive disease (10-12), we should follow the clinical course whether the patient might develop lymphoproliferative disorders in a near future because the enlargement of the lymphnode and hepatosplenomegaly was noted initially. Regarding Case 2, the clinical symptoms and laboratory examination were suggestive of DM associated with SjS without the skin manifestations. We considered him as having pSjS associated with myopathy because dry-eye symptoms, positive anti-SSA Ab and no cutaneous symptom of DM were noted. Although these 2 cases of pSjS were associated with myalgia, the cause of their myopathy is not clear because no inflammation was found in the biopy specimens from their biceps muscle. However, it is reported that the myopathy might be due to small-vessel injury by auto-Abs or circulating immune complexes because electrondence deposits were noted (13). As to the infiltrated cells around the vessels in the cutaneous lesion, the CD4+ T cells were dominant as similar to the findings in myositis of patients with DM.

DM is an idiopathic inflammatory disorder characterized by inflammatory myopathy, indicating myositis, and skin manifestations and it can be associated with the secondary SjS sometimes. The etiology is still unknown and the prevalence is estimated as 1-10 cases per million population, but in children 3.2 cases per million which are distributed in the whole world (3). The clinical types of DM are classified in Table 3 and the internal malignancies are frequently associated in the adult type of DM. The risk is reported to be a 6.5-fold higher than ordinary persons after 45 years of age (14). Regarding myopathy in DM patients it might be characterized by inflammatory myopathy progressing to myositis and degeneration of muscle fibers, and the helio-trope-like eruptions on the sun-exposed areas showed SLE-like changes histologically. On the other hand, there are the cases associated without myogenic symptoms in DM, which is called as "amyopathic DM" (DM sine myositis). However, these cases should be considered as "pre-myopathic DM" because they might Be rather early diagnosed (15). Though the direct cause of myopathy is still unknown, there are some pathogeneses reported, such as the presences of myositis- specific auto-Abs (15,16) and inflammatory cytokines from T cells including interleukin (IL)-17 and IL-23 in early stage of patients with DM (18). The study regarding Th1/Th2 balance showed that Th2 cell predominance was suggested in patients with active stage of DM (19). In plasma of patients with DM and/or polymyositis, microparticles derived from CD14+ mononuclear cells, CD3+ T cells and CD19+ B cells were found to be elevated by electron microscopic examination, which suggests these diseases were immunological disorders (20). It is reported that CD19+CD23+ cells are increased and that CD4+CD45RO+ cells are decreased in the peripheral blood of patients with DM, suggesting reduction of regulatory T cells (21). It

Myopathy in Autoimmune Diseases – Primary Sjögren's Syndrome and Dermatomyositis 127

[6] Lindvall B, Bengtsson A, Ernerudh J, Eriksson P. Subclinical myositis is common in

[7] Eriksson P, Andersson C, Ekerfelt C, et al. Sjögren's syndrome with myalgia associated

[8] Ramos-Casals M, Anaya J-M, Garcia-Carrasco M, et al. Cutaneous vasculitis in primary

[9] Drake LA, Dinehart SM, Farma ER, Goltz RW, Graham GF, Hordinski MK, et al.: Guidlines of care of dermatomyositis, J Am Acad Dermatol 1996; 34: 824-829. [10] Molina R, Provost TT, Alexander EL. Two histopathologic prototypes of inflammatory

[11] Theander E, Anderson SI, Manthorpe R, Jacobson LT. Proposed core set of outcome

[12] Champey J, Corruble E, Gottenberg JE, et al. Quality of life and psychological status in

[13] Ringle SP, Forestot JZ, Tan EM, Wehling C, Griggs RC, Butcher D. Sjögren's syndrome and polymyositis or dermatomyosistis. Arch Neurol 1982; 39: 157-163. [14] Airio A, Pukkala E, Isomaki H. Elevated cancer incidence in patients with dermatomyositis: a population based study. J Rheumatol 1995; 22: 1300-1303. [15] Gerami P, Schope JM, McDonald L, Walling HW, Sontheimer RD. A systemic review of

[16] Mammen AL. Dermatomyositis and polymyositis: clinical presentation, autoantibodies

[17] Gherardi RK. Pathogenic aspects of dermatomyositis and overlap myositis. Press Med

[18] Shen H, Xia L, Lu J, Xiao W. Interleukin-17 and interleukin-23 in patients with polymyositis and dermatomyositis. Scand J Rheumatol 2011; 40: 217-220. [19] Ishii W, Matsuda M, Shimojima Y, Iyoh S, Sumida T, Ikeda S. Flow cytometric analysis

[20] Baka Z, Senolt L, Vencovsky J, Mann IT, Simon PS, Kiltel A, Buzas E, Nagy G. Increase

[21] Antiga E, Kretz CC, Klembt R, Massi D, Ruland V. Characterization of regulatory T cells in patients with dermatomyositis. J Autoimmun 2010; 35: 342-350.

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serum concentration of immune cell derived microparticles in polymyosisitis/

was also suggested that CD4+CD25+ cells, forkhead/winged helix transcription factor (Fox P3)+, transforming growth factor+ and IL-10+ cells were reduced in peripheral blood of patients with DM (21). Actually, CD4+CD8+ cells were significantly distributed around vessels in the interstitial tissues of the muscle bundles in our patients with DM. These cells might be CD4+CD 28+ (null) cells and CD8+CD28+ (null) cell infiltration, as reported, and it is of interest that circulating CD4+CD28+ cells and CD8+CD28+ cells were significantly increased in seropositive individuals, responded to human cytomegalovirus antigen stimulation and correlated with disease duration (22).

As to treatment for myopathy of patients with pSjS and DM, adequate doses of NSAIDs and/or oral steroids were mainly used in corresponding to their clinical severities and these were considered to be effective. However, in addition to these drugs, the combination with immunosuppressive agents such as azathioprine, cyclosporine, mycophenolate or methotrexate should be used for the autoimmune diseases, if they were not clinically controlled. The biological agent, rituximab, and tacrolimus may offer additional benefit to some patients and emerging agents against T cells, B cells, transmigration or transduction molecules may be discussed as New treatments (23).


*3.5. Ecological Health*

Contemporary 'ulu farmers demonstrate the ability to cultivate breadfruit in a way that protects the environment while remaining profitable. The majority of breadfruit farmers do not use fertilizers, herbicides, or irrigation, with a mere 7% of growers using pesticides (Table 2). Among the users of chemical and water inputs, only two had regular schedules, while the majority of growers applied fertilizers rarely with application times based on tree observations.

**Table 2.** Rates of various management practices associated with breadfruit cultivation among survey participants.

As mentioned previously, most 'ulu farmers surveyed intercropped their breadfruit trees. The diversification of crops in agroforestry systems increases the value of production, enhances profitability of traditional farms and reduces production costs due to a decreased need for inputs such as water, chemicals and energy [56]. Despite low inputs, farmers reported a 97% average success rate for all sapling establishment on average. When farmers added care to their trees, they largely chose environmentally friendly inputs such as recycled or green waste materials for mulch (e.g., clippings, woodchips, leaves, cardboard), organic or homemade fertilizers (e.g., fish emulsion, chicken manure) and natural compost (food scraps, mixed garden waste, human waste from composting toilets). "Farmer C" farmed without chemical inputs and only organic material as fertilizer yet reported exceptionally high yields. "Farmer H," utilized no inputs, also reported high yields and her tree's measurements showed the highest rates of photosynthesis of any producer measured. That breadfruit is highly productive without excessive inputs is captured by agricultural producer, "Farmer L," who said of his trees,

"Is it a superfood?! It's definitely a tree of abundance. I always joke around it's like a tree of life. Hundreds of pounds. With really minimal [inputs], I fertilize them once in a while but other than that they just go. It's definitely a tree that gives plenty. It's suited for here. I learned to not quite force anything. There's so many trees I've tried to make grow on a farm. It's the ones from here that can endure."

While we did not investigate the role of biodiversity, several farmers made mention of their breadfruit trees being home to the 'io (the Hawaiian hawk, *Buteo solitarius*) and the 'ope'ape'a ¯ (the Hawaiian hoary bat, *Lasiurus cinereus semotus*), an iconic, endemic and federally endangered species. While this is not an adequate proxy for biodiversity, the presence of these two rare animals nesting within breadfruit agroforestry clearly establishes the potential of increased habitat. Preserving biodiversity is an important concept for biocultural restoration, especially in agricultural development where the loss of biodiversity and environmental destruction are attributed to conventional agriculture methods such as monocropping [4].

Ultimately, breadfruit cultivation provides multiple environmental benefits under current production practices. Tree crops in general have shown to have multiple benefits and those impacts are enhanced in agroforestry settings. Forests are valuable systems needed to carry out ecosystem services essential to sustain the natural resource needs of society [5]. Agroforestry has the ability to replenish litter layer to the soil, increase and maintains soil quality, retain soil nutrients and prevent soil erosion [56,57]. For example, growers had, on average, very high levels of soil organic matter (mean 15.25%, standard error 1.38). These same functions improve water filtration and retention rates, increase water availability, reduce runoff and evapotranspiration, collectively improving water quality [56,57]. The shade and presence of multiple tree species also provide habitat, wind protection and essential microclimates that assure cooler summers and warmer winters [57]. The incorporation of increased biodiversity into farming systems at different trophic levels increases crop yields, wood production, yield stability, presence of natural pollinators and encourages weed and pest suppression [58]. The combination of low inputs, low labor, high productivity and ecosystem services benefits showcase the importance of breadfruit as a sustainable crop.

#### **4. Conclusions**

Breadfruit holds a unique position in the concept of biocultural restoration in Hawai'i by contributing to the five key themes described above. As a productive, low maintenance crop, 'ulu has the ability to be grown sustainably, which offers the dual role of food production and ecosystem restoration. Increasing availability of healthy foods in the market and through food sharing contributes to greater food security. The continued cultivation of breadfruit is reliant on the continuation of biocultural relationships, or passing knowledge on its growing needs and its importance as a food. Through these cycles, the cultivation of breadfruit in Hawai'i is supported by Hawaiian culture and Hawaiian culture is supported through breadfruit cultivation, where the restoration of one can affect the other (Figure 2).

Although we presented some of the key findings organized within reoccurring themes, these themes clearly overlap and interact with each other. The revival of customary values, for instance, brings forward practices that contribute to the development of community capital, which in turn provides direct benefits to food security through multiple direct and indirect effects. These interactive effects demonstrate, among other things, the intertwined nature of biocultural systems, in which the growth of one necessitates and requires the growth of the other. In multiple examples, we see both the positive and negative reinforcement that occurs within these systems. In a destructive loop, "Farmer F" was not able to receive cultural knowledge regarding breadfruit because the tree itself was not present in his mentors' lives. Conversely, through growing the tree he has recognized how it has contributed to the growth of his culture and identity within himself. As breadfruit cultivation continues to expand its inseverable connection to the Hawaiian culture will continue to fuel the growth of associated value systems. Scaling up, there is evidence that relatively small farms that practice diversified agriculture could increase food production globally, while simultaneously addressing sustainability issues regionally. Referring to Figure 2, traditional agriculture in Hawai'i is a major mediator between biological and cultural components and at the intersection of the three concepts of sustainability. This is where agriculture is significant in regard to sustainable socioecological systems. Breadfruit cultivation through traditional agriculture methods supports the needs of sustainable agriculture and can provide valuable ecosystem services while simultaneously producing food. From the social and cultural side, breadfruit is present in traditional practices that are necessary to sustain its cultivation long term.

Our findings suggest the support of breadfruit production has the potential to be a pivotal solution to sustainability issues in Hawai'i. Evaluating the experiences and lessons learned from discussions with farmers, we saw that breadfruit is making significant contributions to reconnecting people with place, revitalizing traditional agriculture and Hawaiian culture and that this phenomenon has practical implications for the future. While we cannot say if breadfruit itself promotes these activities or if individuals who engage in these practices are more prone to grow breadfruit, the biocultural restoration of breadfruit can be strongly linked to the furthering of a sustainable socioecological system regardless.

As stated by "Farmer H,"

"We have started calling breadfruit a 'solutionary' food. There are so many problems with our food system. I mean there is the health and nutrition, there are the environmental issues, there is food justice and food security. And there is just the loss of identity and enjoyment of food. So many issues that need to be fixed. We need a revolution of the way we deal with food. What is amazing about breadfruit is that it hits all of these. I mean, more than any other food I can think of. That it is a tree. That it is embedded in the culture. That it is a nutritious staple. It really has the potential to be a solution to many problems in our food system. So, it is the revolutionary solution that we need...'solutionary.' Get it?"

**Author Contributions:** Conceptualization, N.L.; Methodology, N.L.; Software, N.L.; Validation, N.L., B.L.; Formal Analysis, B.L., N.L.; Investigation, B.L., N.L.; Resources, N.L.; Data Curation, B.L., N.L.; Writing-Original Draft Preparation, B.L.; Writing-Review & Editing, N.L., B.L.; Visualization, B.L., N.L.; Supervision, N.L., B.L.; Project Administration, N.L., B.L.; Funding Acquisition, N.L.

**Funding:** This research was funded by the USDA Western SARE project, grant number SW17-050 and the USDA National Institute of Food and Agriculture, Hatch project HAW08035 administered by the College of Tropical Agriculture and Human Resources. The APC was funded by Hawai'i Community Foundation.

**Acknowledgments:** We would like to acknowledge all who offered their time, support and knowledge. This includes the breadfruit farmers who participated in the interviews, staff, interns and volunteers who helped with data collection, input, travel, sample collection, storage and processing including; Amber Au, Kat Hiu, Kimber Troumbley and Maxwell Bendes. We would like to thank the Hawai'i 'Ulu Producers Cooperative for connecting us with local 'ulu farmers. Thank you to Matthew Lucas for technical Geographic Information Systems Assistance. Thank you to friends and colleagues who edited and revised; Maxwell Bendes, Kalisi Mausio, Amber Au and Kawika Winter.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **Appendix A**

**Table A1.** List of survey question reported on in this paper as administered to 43 successful breadfruit cultivators in Hawai'i.

**Table A2.** List of general interview topics that were explored with willing participants.


**Crimmigration in the Age of COVID-19**

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**5. Sense of place and environmental change**

Sense of place is a social construction of place identity, dependence, and attachment that is mediated by physical attributes and conditions [4, 40]. For example, while many studies have found that long-term residents have higher place attachment, in Montana, McCool and Martin [41] surprisingly found that newer residents had higher place attachment. They explained this unusual finding as possibly reflecting the fact that many newer residents had moved to the area specifically because of the mountain access and environmental attributes of the area. Kibler et al. [42] highlighted the value of connecting human attachment to the condition of an ecosystem for evaluating the success of restoration projects. Specifically, they hypothesized that it is likely for ecosystem improvement in restoration projects to depend on the interaction between ecosystem function and sense of place. These interactions project that a restoration site where stakeholders have a high sense of place and where there is a highly functioning ecosystem will lead to emotionally invested stakeholders and iterative monitoring of the ecosystem. On the other end of the spectrum, they hypothesized that low sense of place and low ecosystem function would require enhancing stakeholder attachment for a restoration effort to be successful.

Minimal work has been conducted that moves beyond general attachment to the environment to directly connect sense of place to environmental attribute data. The connection of biophysical data with social data is often limited by the availability of the two types of data at the same meaningful scale. Many social scientists focus on survey respondents' or interview participants' environmental perceptions or landscape values for a location when biophysical data are unavailable [43]. For example, in Norway, Kaltenborn [44] found the most important contributing attribute to place attachment to be the perception of the quality of the natural environment. Brown and Raymond [37] investigated the relationships between landscape values which incorporate both ecological and social values, and sense of place in Australia. They found esthetic, spiritual, future generation, and wilderness values to be the best predictors of place attachment. Matarrita-Cascante et al. [45] found that

**27**

*\**

**Table 1.**

*integrity or water quality value.*

*Sense of Place and Water Quality: Applying Sense of Place Metrics to Better Understand…*

natural amenities increased place attachment for both seasonal and permanent residents. Larson et al. [46] applied nine natural environmental wellbeing factors to explain sense of place values, including general "environmental quality," "water quality," "fishing," "soil," and others. They found that coastal residents valued

were the best predictors of place identity, dependence, and attachment.

There has been some work connecting various water quality metrics to sense of place without using biophysical data. Brehm et al. [5] measured predictors of water quality concern and found place meanings to be linked to local environmental concern. They found that the level of water quality concern was predicted by the environmental values, gender (female > male), and assigned place meanings (how impacted they perceived the watershed was by environmental threats and how they perceived the watershed as a getaway). Smith et al. [10] connected perceived ecological integrity (along with a set of other place attachment indicators) with a set of desired social and ecological outcomes for lakes in Illinois. They found that the more people believed the lakes contributed to the ecological integrity of the area, the more they desired improved environmental outcomes and the less they

Stedman [20]\* Level of lake shoreline development (number of structures within a 100 m buffer of

2.This lake is important in providing habitat for wildlife 3.This lake is important in protecting water quality

Larson et al. [46] Environmental wellbeing factors for fishing, swimming, air quality, water quality, soil

the lake), water clarity, algal biomass, chlorophyll, color, alkalinity, and conductivity

1.Considering everything, how would you rate the overall condition of the following

quality, beauty of the landscape, condition of the landscape, access to the natural areas,

1.This lake is important in protecting the landscape from development

2.How would you rate the waterways in terms of the quality of the water? 3.How would you rate the waterways in terms of the vegetation along the shores? 4.How would you rate the waterways in terms of the number and variety of animals?

To date, very little work has connected sense of place with water quality assessments (see **Table 1**). In the only work that directly connects biophysical water quality data with sense-of-place meanings, Stedman [20] connected water quality to place attachment and satisfaction for property-owners in a lake-rich region of Wisconsin. He found that the construction of sense of place meanings was mediated by the level of shoreline development as well as the social influences of whether or not the lake felt like a wilderness escape place or a neighborhood of friends. In terms of place attachment, the two social influences (wilderness escape and sociability) that depend upon what the property owners were seeking essentially cancel one another out when considering shoreline development. More shoreline development leads to a more social environment, but also less wilderness, thereby differently affecting the experiences of each property owner. Jorgenson and Stedman [26] investigated the same dataset and found that perceptions of environmental features

*DOI: http://dx.doi.org/10.5772/intechopen.91480*

beauty and conditions of the environment.

**Author Water quality metric(s) used**

Smith et al. [10] Perceived ecological integrity Likert-scale questions:

Cox et al. [47] Perceived waterway condition Likert-scale questions:

biodiversity, overall-natural environment

*This is the only study that applied specific water quality data. The other studies applied perceived ecological*

waterways?

*Past research connecting water quality metrics and sense of place.*

Brehm et al. [5] Water quality concern

## *Sense of Place and Water Quality: Applying Sense of Place Metrics to Better Understand… DOI: http://dx.doi.org/10.5772/intechopen.91480*

natural amenities increased place attachment for both seasonal and permanent residents. Larson et al. [46] applied nine natural environmental wellbeing factors to explain sense of place values, including general "environmental quality," "water quality," "fishing," "soil," and others. They found that coastal residents valued beauty and conditions of the environment.

To date, very little work has connected sense of place with water quality assessments (see **Table 1**). In the only work that directly connects biophysical water quality data with sense-of-place meanings, Stedman [20] connected water quality to place attachment and satisfaction for property-owners in a lake-rich region of Wisconsin. He found that the construction of sense of place meanings was mediated by the level of shoreline development as well as the social influences of whether or not the lake felt like a wilderness escape place or a neighborhood of friends. In terms of place attachment, the two social influences (wilderness escape and sociability) that depend upon what the property owners were seeking essentially cancel one another out when considering shoreline development. More shoreline development leads to a more social environment, but also less wilderness, thereby differently affecting the experiences of each property owner. Jorgenson and Stedman [26] investigated the same dataset and found that perceptions of environmental features were the best predictors of place identity, dependence, and attachment.

There has been some work connecting various water quality metrics to sense of place without using biophysical data. Brehm et al. [5] measured predictors of water quality concern and found place meanings to be linked to local environmental concern. They found that the level of water quality concern was predicted by the environmental values, gender (female > male), and assigned place meanings (how impacted they perceived the watershed was by environmental threats and how they perceived the watershed as a getaway). Smith et al. [10] connected perceived ecological integrity (along with a set of other place attachment indicators) with a set of desired social and ecological outcomes for lakes in Illinois. They found that the more people believed the lakes contributed to the ecological integrity of the area, the more they desired improved environmental outcomes and the less they

#### **Table 1.**

*Past research connecting water quality metrics and sense of place.*

*Water Quality - Science, Assessments and Policy*

data to analyze relationships with sense of place.

**5. Sense of place and environmental change**

Applications of sense of place findings are primarily at a localized level but provide insights about the relative social value of different places. To increase the application of sense of place in environmental management, social data will need to be collected on a broader scale with more consistent means of data collection such as the use of standardized sense of place scales for use across locations. One promising advancement has been an increase in applying spatial techniques in place-based research (e.g., [37–39]). These techniques connect survey or interview data to places and landscapes which allows for the incorporation of spatially defined ecological

Beckley [4] calls for research that identifies the environmental attributes for which people develop place attachment; however, a big limitation in the use of sense of place, or many other place-based social indicators, for water quality assessment is the lack of localized biophysical water quality data. Place-based values are site specific and are not always generalizable past that location as testing and monitoring methods are not always consistent. Without corresponding localized water quality data—either perceptions or biophysical measurements—we may be able to capture the social value for that place as a whole but not for its environmental attributes. In the case of water quality valuation, this means that we are limited in our ability to explain changes in the social value for sense of place resulting from changes in water

Sense of place is a social construction of place identity, dependence, and attachment that is mediated by physical attributes and conditions [4, 40]. For example, while many studies have found that long-term residents have higher place attachment, in Montana, McCool and Martin [41] surprisingly found that newer residents had higher place attachment. They explained this unusual finding as possibly reflecting the fact that many newer residents had moved to the area specifically because of the mountain access and environmental attributes of the area. Kibler et al. [42] highlighted the value of connecting human attachment to the condition of an ecosystem for evaluating the success of restoration projects. Specifically, they hypothesized that it is likely for ecosystem improvement in restoration projects to depend on the interaction between ecosystem function and sense of place. These interactions project that a restoration site where stakeholders have a high sense of place and where there is a highly functioning ecosystem will lead to emotionally invested stakeholders and iterative monitoring of the ecosystem. On the other end of the spectrum, they hypothesized that low sense of place and low ecosystem function would require enhancing stakeholder attachment for a restoration effort to

Minimal work has been conducted that moves beyond general attachment to the environment to directly connect sense of place to environmental attribute data. The connection of biophysical data with social data is often limited by the availability of the two types of data at the same meaningful scale. Many social scientists focus on survey respondents' or interview participants' environmental perceptions or landscape values for a location when biophysical data are unavailable [43]. For example, in Norway, Kaltenborn [44] found the most important contributing attribute to place attachment to be the perception of the quality of the natural environment. Brown and Raymond [37] investigated the relationships between landscape values which incorporate both ecological and social values, and sense of place in Australia. They found esthetic, spiritual, future generation, and wilderness values to be the best predictors of place attachment. Matarrita-Cascante et al. [45] found that

quality due to gaps in our biophysical monitoring and understanding.

**26**

be successful.

desired competing economic outcomes. Cox et al. [47] also investigated water quality perceptions and found a weak connection with the number of visits, which then indirectly affected other quality of life indicators, including sense of place. In a qualitative investigation of sense of place, Lukacs and Ardoin [12] found that the perception of the environmental attributes and biophysical resources influenced sense of place and watershed group participation in Appalachia.


**Figure 9.** Rise in Global Railway Passenger Demand (World Bank Databank)

of such creation, and their fitness for the task in hand is now gaining momentum. In the face of such realisation and demands, our capacity to understand, characterise and evaluate human capabilities and latent potential has lagged significantly behind other technological advances.

We posit that human competence should be regarded as an integral facet of assuring designs, products and services, especially those with safety, security, sustainability or mission critical profile. The continual assurance processes advocated by modern standards need to comple‐ mented with focus on human competence to face the modern challenges of high risks and ever increasing complexity. The framework offered uses systems thinking to address assessment and management of competence within a coherent solution for enhancing quality, safety,

The statistics published by the Office of Rail Regulation (ORR) in the United Kingdom [17] is a timely reminder of the rise in passenger demand over the recent past that seems to illustrate

reliability and assuring integrity.

1987-88

with the airlines.

1989-90

1991-92

**Figure 8.** Rise in UK Railway Passenger Demand (ORR data)

especially in the developing economies (Figure 9).

1993-94

1995-96

1997-98

1999-00

2001-02

Data from the World Bank relating to a rather similar period [18] seem to pint to a rising trend

Overall, rise in global demand for rail transportation needs to be matched by increasing infrastructure investment, technology development and rising consciousness about the carbon foot print and global warming impact of transportation. Given the highly advantageous position of rail transportation with respect to sustainability, energy efficiency, carbon foot‐ print, convenience and the increasing speeds, this is a growth industry on a competition course

2003-04

2005-06

2007-08

2009-10

2011-12

2013-14

**8. General Trends and Emerging Issues**

74 Railway Research - Selected Topics on Development, Safety and Technology

a rising trend of roughly 50% per decade (Figure 8).

With the advancing technology, increasing automation, land speeds and demand for higher levels of safety, the key issues facing the industry from a safety and security perspective will be

Finally, with the maturity of the ICT technologies employed and improvement of safety performance, the concern will shift towards security as a more likely cause for incidents and accidents than the traditional concern over safety. Increasing levels of automation in train driving, traffic management and control would expose the future railway environment to a range of security threats that may take the operators, IMs and the authorities by surprise unless security, alongside safety is taken into account throughout the life cycle of products, systems and processes.

To this end, a similar reference portfolio as developed for the UK national railway's safety hazards is required to address security threats and vulnerabilities at railway system level. This will provide a rational, systematic and consistent support to the operators and the supply chain in the industry empowering them to effectively address the security requirements pertinent to the scope of their services, products, systems and processes.

## **9. Abbreviations**

CBRN Chemical, Biological, Radiological and Nuclear (attacks)

CDL Central Door Locking

CDM Construction, Design and Management (regulations)

Comms Communications

ConOps Concept of Operations

COTS Commercial-Off-the-Shelf

CRS Customer Requirements Specification

CSC Certificate of Safety Conformity

DRACAS Data Reporting and Corrective Action System

EMC Electro-Magnetic Compatibility

FMECA Failure Mode Effects and Criticality Analysis

FRACAS Failure Reporting and Corrective Actions System

FSaR Functional Safety Requirements

GDP Gross Domestic Product

HAZAN Hazard Analysis

Hazid Hazard Identification

HAZOP Hazard And Operability Study

HRC Human Resource Competence

HSE Health and Safety Executive (UK)

HW Hardware

IHA Interface Hazard Analysis

IP Intellectual Property

ISA Independent Safety Assessor

IT Information Technology

O&M Operation and Maintenance

OHA Operational Hazard Analysis

OHL Over-Head Line

Ops Operations

will provide a rational, systematic and consistent support to the operators and the supply chain in the industry empowering them to effectively address the security requirements pertinent

to the scope of their services, products, systems and processes.

76 Railway Research - Selected Topics on Development, Safety and Technology

CBRN Chemical, Biological, Radiological and Nuclear (attacks)

CDM Construction, Design and Management (regulations)

DRACAS Data Reporting and Corrective Action System

FMECA Failure Mode Effects and Criticality Analysis

FRACAS Failure Reporting and Corrective Actions System

**9. Abbreviations**

CDL Central Door Locking

Comms Communications

ConOps Concept of Operations

COTS Commercial-Off-the-Shelf

CRS Customer Requirements Specification

CSC Certificate of Safety Conformity

EMC Electro-Magnetic Compatibility

FSaR Functional Safety Requirements

HAZOP Hazard And Operability Study

HRC Human Resource Competence

IHA Interface Hazard Analysis

ISA Independent Safety Assessor

IP Intellectual Property

IT Information Technology

HSE Health and Safety Executive (UK)

GDP Gross Domestic Product

HAZAN Hazard Analysis

HW Hardware

Hazid Hazard Identification

OPSEC Operational Scenarios

OSHA Operation and System Hazard Analysis

PHA Preliminary Hazard Analysis

PSP Product, System or Process

PW People-ware, the human element in a control system

QMS Quality Management System

RAM Reliability, Availability, Maintainability

SDS System Design Specification

SDSS System Design Safety Specification

SHA System Hazard Analysis

SSHA Sub-system Hazard Analysis

SIL Safety Integrity Level

SLSR Railway System Level Safety Requirements

SMS Safety Management System

SMIS An old UK Safety Management Information System data base

SRS System Requirements Specification

SSHA Subsystem Hazard Analysis

SSRS Subsystem Requirements Specification

SW Software

THR Tolerable Hazard Rate

UML Unified Modelling Language

V&V Verification & Validation

VTR Validation Test Report

## **Author details**

Ali G. Hessami\*

Address all correspondence to<EMAIL_ADDRESS>
Vega Systems, London, UK

## **References**

**Author details**

Ali G. Hessami\*

**References**

Vega Systems, London, UK

Requirements.

9780717662234.

September 2002.

Address all correspondence to<EMAIL_ADDRESS>
78 Railway Research - Selected Topics on Development, Safety and Technology

[1] BS EN 50129:2003, Railway applications. Communication, signalling and processing

[3] CLC/TR 50451:2007, Railway applications. Systematic Allocation of Safety Integrity

[4] Hessami, A. (1999). Safety Assurance, A Systems Paradigm, Hazard Prevention.

[5] Risk Profiling of Railways Report (1997). Can access a copy that includes the Hazards Portfolio at: https://vegaglobalsystems.com/Resources.html. Look in Public Resour‐

[6] European Guide to Good Practice in Knowledge Management, Work Item 5: Culture

[8] Managing health and safety in construction, Construction (Design and Management) Regulations (2007). (CDM) Approved Code of Practice, HSE Books, ISBN

[9] Railway Safety Principles and Guidance: Part 3 Section A (2002). Developing and

[10] +Safe Version 1.2, A Safety Extension to CMMi-DEV Version 1.2, Defence Materials

[11] BS EN 50128:2011, Railway applications. Communications, signalling and processing

[12] Hessami, A. and Gray, R. (2002) Creativity, the Final Frontier? The 3rd. European Conference on Knowledge Management ECKM 2002, Trinity College Dublin, 24–25

Maintaining Staff Competence HSG197, HSE Books, ISBN 0 7176 1732 7.

Organisation, Australian Department of Defence, March 2007.

systems. Software for railway control and protection systems.

systems. Safety related electronic systems for signalling.

Journal of System Safety Society, Volume 35, No. 3, pp. 8–13.

ces/Safety Research online library for the file.

Working Draft 6.0, CEN-ISSS, July 2003.

[7] http://www.businessdictionary.com/definition/

[2] BS ISO/IEC 15288:2002, Systems engineering. System lifecycle processes.

**Chapter 3**


**4. Effect of cryopreservation on sea bass protein profile**

Defects in sperm proteins may compromise sperm motility, fertilization ability, and the early events after fertilization (Cao et al., 2003; Huanget al., 1999; Lessard et al., 2000). Protein screening has become an excellent approach with which to evaluate changes in expression due to different stresses. Using this method it has been demonstrated that the reduction in motility observed in boar and human spermatozoa following cryopreservation was associated with a decrease in heat shock protein 90 during cooling (Cao et al., 2003; Huanget al., 1999). Similarly, the loss of P25b (a protein associated with the plasma membrane covering the acrosome) may be responsible, at least in part, for the decrease in fertility following the freezing/thawing procedure of bull semen (Lessard et al., 2000). Cryoinjuries due to cryopreservation have been reported for thawed spermatozoa of many freshwater (Rana, 1995) and marine fish species (Gwo et al., 1992; Lahnsteiner et al., 2000). Shrinkage of the plasma membrane of the midpiece, breakage of mitochondria, and coiling of the axoneme have been observed.Cryopreserved sea bass sperm showed similar fertilization rates and class motility compared with fresh sperm, but also showed a decline in motility duration (Fauvel et al., 1998a), changes in metabolism (Zilli et al., 2004), and lower hatching rates (Fauvel et al., 1998b). For these reason we used (Zilli et al., 2005) the 2-DE to verify whether the cryopreservation procedure, applied to sea bass milt, affected the expression of proteins involved in the control of sperm functions and, in addition, matrix-associated laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry to identify some of these proteins.

Effect of Cryopreservation on Bio-Chemical Parameters, DNA Integrity,

al., 2003; Huanget al., 1999) and bull sperm (Lessard et al., 2000).

**fresh sperm (N=6)**

SPOT 1 17.8±2.1 7.0±1.1 SPOT 2 10.7±1.1 5.9±1.5 SPOT 3 48.0±9.1 20.2±5.1 SPOT 4 40.8±5.2 29.3±4.1 SPOT 5 54.9±11.3 20.6±3.0 SPOT 6 42.9±2.2 - SPOT 7 36.8±3.3 25.5±5.2 SPOT 8 110.0±23.0 33.4±6.3 SPOT 9 52.7±12.0 38.7±9.0 SPOT 10 31.9±8.0 4.7±2.0 SPOT 11 30.6±7.0 7.4±5.0 SPOT 12 34.9±15.2 - SPOT 13 44.3±13. - SPOT 14 34.3±11.3 - SPOT 15 11.4±3.2 - SPOT 16 8.4±2.8 - SPOT 17 6.2±3.5 - SPOT 18 11.8±4.2 - SPOT 19 30.6±7.2 10.6±4.2 SPOT 20 102.8±3.6 42.9±3.3 SPOT 21 46.3±6.6 29.2±7.3

Table 3. Differences in abundance of spots in fresh and cryopreserved sea bass sperm. Spot adundance is expressed as mean ± SD of normalized spot volume measured in six different

Five of the protein spots shown in Table 3 were analyzed by MALDI-TOF for protein identification. Three were selected among the spots that significantly decreased after cryopreservation (5, 8, and 20) and two (6 and 13) were taken from among those that were absent in the gel obtained with frozen-thawed sperm (Fig. 6 and Table 3). Protein identification was performed by three search programs (PeptIdent, Mascot, and MS-Fit).

gels. (This table was originally published in Zilli et al., Biol Reprod 2005).

**4.2 Identification of protein spots by MALDI-TOF**

**Spot Number Normalised spot volume in**

Protein Profile and Phosphorylation State of Proteins of Seawater Fish Spermatozoa 401

Differences were observed (by visual inspection and by using image analysis software) in the protein profiles of fresh and cryopreserved sperm samples. In fact, in the cryopreserved sperm samples, among the 163 spots considered, 13 were significantly (*P*<0.05) less expressed, and 8 completely disappeared. These 21 spots are highlighted in Figure 6 and the normalized spot volumes listed in Table 3. A decrease in protein abundance or spot disappearance in sperm after the cryopreservation procedure may be due to either leakage of proteins from spermatozoa to the extracellular medium or to degradation following freezing-thawing stress. The leakage of proteins is ruled out because we have previously demonstrated that the intracellular protein concentration and the seminal plasma protein concentrations do not change after cryopreservation (Zilli et al., 2004). Consequently, protein degradation seems to be responsible for the reduction in spot abundance (and disappearance). Similar results have been also reported in human and boar semen (Cao et

> **Normalised spot volume in cryopreserved sperm (N=6)**

### **4.1 Proteins expression in two-dimensional electrophoresis gels: Differences between fresh and frozen-thawed sea bass sperm samples**

To perform two-dimensional analysis sperm samples with similar fertilization rates (70%– 90%) and percentage of motility (80%–100%), before and after cryopreservation, were used to extract proteins. All the sperm samples used showed lower motility duration after the cryopreservation procedure. 163 spots were detected in all gels prepared from fresh samples (with molecular masses ranging between 190 and 10 kDa and isoelectric points between 3.5 and 8.0) and were used for comparative analysis. Results of a typical experiment performed on sperm samples before and after cryopreservation are showed in figure 6 (A and B).

Fig. 6. Two-dimensional Electrophoresis (2-DE) maps of fresh (A) and cryopreserved (B) sea bass sperm proteins. 2-DE was performed on an immobilized pH 3–10 strip, followed by the second-dimensional separation on 12.5% polyacrylamide gels. The separated proteins were stained with silver staining. Spots that are less expressed after cryopreservation are highlighted with a continuous line; spots that are entirely absent after cryopreservation are marked with a dotted line. (This figure was originally published in Zilli et al., Biol Reprod 2005).

**4.1 Proteins expression in two-dimensional electrophoresis gels: Differences between**

To perform two-dimensional analysis sperm samples with similar fertilization rates (70%– 90%) and percentage of motility (80%–100%), before and after cryopreservation, were used to extract proteins. All the sperm samples used showed lower motility duration after the cryopreservation procedure. 163 spots were detected in all gels prepared from fresh samples (with molecular masses ranging between 190 and 10 kDa and isoelectric points between 3.5 and 8.0) and were used for comparative analysis. Results of a typical experiment performed

Fig. 6. Two-dimensional Electrophoresis (2-DE) maps of fresh (A) and cryopreserved (B) sea bass sperm proteins. 2-DE was performed on an immobilized pH 3–10 strip, followed by the second-dimensional separation on 12.5% polyacrylamide gels. The separated proteins were

highlighted with a continuous line; spots that are entirely absent after cryopreservation are marked with a dotted line. (This figure was originally published in Zilli et al., Biol Reprod

stained with silver staining. Spots that are less expressed after cryopreservation are

2005).

on sperm samples before and after cryopreservation are showed in figure 6 (A and B).

**fresh and frozen-thawed sea bass sperm samples**

Differences were observed (by visual inspection and by using image analysis software) in the protein profiles of fresh and cryopreserved sperm samples. In fact, in the cryopreserved sperm samples, among the 163 spots considered, 13 were significantly (*P*<0.05) less expressed, and 8 completely disappeared. These 21 spots are highlighted in Figure 6 and the normalized spot volumes listed in Table 3. A decrease in protein abundance or spot disappearance in sperm after the cryopreservation procedure may be due to either leakage of proteins from spermatozoa to the extracellular medium or to degradation following freezing-thawing stress. The leakage of proteins is ruled out because we have previously demonstrated that the intracellular protein concentration and the seminal plasma protein concentrations do not change after cryopreservation (Zilli et al., 2004). Consequently, protein degradation seems to be responsible for the reduction in spot abundance (and disappearance). Similar results have been also reported in human and boar semen (Cao et al., 2003; Huanget al., 1999) and bull sperm (Lessard et al., 2000).

Table 3. Differences in abundance of spots in fresh and cryopreserved sea bass sperm. Spot adundance is expressed as mean ± SD of normalized spot volume measured in six different gels. (This table was originally published in Zilli et al., Biol Reprod 2005).

#### **4.2 Identification of protein spots by MALDI-TOF**

Five of the protein spots shown in Table 3 were analyzed by MALDI-TOF for protein identification. Three were selected among the spots that significantly decreased after cryopreservation (5, 8, and 20) and two (6 and 13) were taken from among those that were absent in the gel obtained with frozen-thawed sperm (Fig. 6 and Table 3). Protein identification was performed by three search programs (PeptIdent, Mascot, and MS-Fit).

Effect of Cryopreservation on Bio-Chemical Parameters, DNA Integrity,

**sperm**

*aurata*

tunicate (Nomura et al., 2000).

Protein Profile and Phosphorylation State of Proteins of Seawater Fish Spermatozoa 403

The results reported in figure 6 and tables 3 and 4 show that in sea bass spermatozoa the used cryopreservation procedure causes the degradation of 21 sperm proteins, and among these, 2 could be at least partially responsible for the observed decrease in sperm motility duration and the lower hatching rate of eggs fertilized with cryopreserved sperm. In addition, these observations suggest that two-dimensional electrophoresis coupled with MALDI-TOF analysis could be used as a tool to improve cryopreservation procedures.

**5. Effect of cryopreservation on proteins phosphorylation state of sea bream**

**5.1 Molecular mechanisms determining sperm motility initiation in sea bream** *sparus*

Most fish spermatozoa are quiescent in the testes, because the osmolality and composition of seminal plasma usually prevent motility in sperm ducts (Billard, 1986). During natural reproduction, fish sperm become motile after discharge into the aqueous environment (in oviparous species) or the female genital tract (in viviparous and ovoviviparous species) (Billard, 1986; Billard & Cosson, 1983; Stoss, 1983). Changes in the ionic and osmotic environment of the sperm cells have been identified as being critical external factors that may be responsible for initiating motility in fish spermatozoa (Morisawa, 1994). Several extracellular factors controlling sperm motility have been reported. In marine (Gwo et al, 1993; Krasznai et al, 2003a, 2003b; Morisawa & Suzuki, 1980; Oda & Morisawa, 1993) and freshwater (Billard, 1986; Morisawa et al., 1983; Stoss, 1983) teleosts, sperm motility is initiated by osmotic shock when sperm are ejaculated. In these species, spermatozoa are quiescent at the osmolality of seminal plasma (referred to as isotonic condition). In freshwater teleost sperm, flagellar motility is initiated by the hypo-osmotic shock, whereas in marine teleost sperm, flagellar motility is initiated by hyperosmotic shock. Furthermore, in medaka (Inoue & Takei, 2003) and tilapia (Linhart et al., 1999), motility regulatory mechanisms of sperm flagella are modulated to suit the spawning environment when they are in freshwater or acclimated to seawater. In herring sperm, motility initiation requires trypsin inhibitor-like sperm-activating peptide from the eggs (HSAPS), and the sperm exhibits chemotaxis when they are close to eggs (Oda et al., 1998; Yanagimachi et al., 1992).The extracellular factors controlling sperm motility (osmolality, ions, sperm-activating peptides, and chemoattractants) act on the flagellar motile apparatus, the axoneme, through signal transduction across the plasma membrane. Second messengers, such as cAMP and Ca, play key roles in the initiation of sperm motility in many animal groups, such as mammals (Lindemann, 1978; Okamura et al., 1985; Tash & Means, 1983), salmonid fish (Morisawa & Okuno, 1982), sea urchin (Cook et al., 1994), mussel (Stephens & Prior, 1992), and tunicate (Opresko & Brokaw, 1983). A cAMP-independent initiation of flagellar motility in sperm was observed in puffer fish (Morisawa, 1994) and striped bass (Shuyang et al., 2004). Second messengers (cAMP and Ca) determine the sperm motility initiation modifying dynein-mediated sliding of the axonemal outer-doublet microtubules through protein phosphorylation/dephosphorylation in different species, such as mammals (Lindemann & Kanous, 1989), rainbow trout, chum salmon, sea urchin (Inaba et al., 1999), and

In *Sparus aurata* osmolality is the key signal in sperm motility activation and motility initiation depends on a cAMP-dependent protein phosphorylation (Zilli et al., 2008). To elucidate which proteins are involved (phosphorilated/dephosphorilated) in the initiation

Three out of five sea bass proteins processed were found to have homologies with existing sequences in the databases used (Table 4). These proteins were identified from protein sequences already described in other teleost species and amphibians. In particular, two were from *Brachidanio rerio* (spots 5 and 20) and one was from *Xenopus laevis* (spot 13). Table 4 summarizes the data of the bio-informatics analysis for these proteins. For spot 5, the search engine PeptIdent found a homology with a protein of *Brachidanio rerio* (similar to SKB1 of human and mouse). This is a highly conserved cytoplasmic protein with methyltransferase activity that interacts with the members of the Janus family tyrosine kinases (JAK) (Pollack et al., 1999). Genome activation is one of the first critical events in the life of a new organism. Both the timing of genome activation and the array of genes activated must be controlled correctly, and these events depend on changes in chromatin structure and availability of transcription factors (Latham & Schultz, 2001).

In sea bass the observed reduction in SBK1 proteins in cryopreserved sperm could be responsible for abnormal early embryo development, which in turn, could determine the lower hatching rate observed (personal observation). The spot protein 13 matched in Mascot and MS-Fit with a G1/S-specific cyclin E2 protein, which is essential in the control of the cell cycle at the G1/S (start) transition (Moore et al., 2002). Cyclin E is involved in the activation of cyclin-dependent kinase 2 (cdk2). Recently, it has been demonstrated that cdk2 phosphorylates the protein phosphatase, PP1gamma2, a key enzyme in the development and regulation of sperm motility (Huang &Vijayaraghavan, 2004). The observed reduction in sea bass sperm motility duration in frozen-thawed spermatozoa could be a consequence of the cyclin E degradation. The protein spot 20 matched, in MS-Fit, with the hypothetical protein DKFZp566A1524 of unknown function.

Table 4. Results from peptide mass fingerprinting of protein spots excised from 2D gels. EWM: Experimental Weight Mass; EIP: Experimental Isoelectric Point; TWM: Theoretical Weight Mass; TIP: Theoretical Isoelectric Point. (This table was originally published in Zilli et al., Biol Reprod 2005).

Three out of five sea bass proteins processed were found to have homologies with existing sequences in the databases used (Table 4). These proteins were identified from protein sequences already described in other teleost species and amphibians. In particular, two were from *Brachidanio rerio* (spots 5 and 20) and one was from *Xenopus laevis* (spot 13). Table 4 summarizes the data of the bio-informatics analysis for these proteins. For spot 5, the search engine PeptIdent found a homology with a protein of *Brachidanio rerio* (similar to SKB1 of human and mouse). This is a highly conserved cytoplasmic protein with methyltransferase activity that interacts with the members of the Janus family tyrosine kinases (JAK) (Pollack et al., 1999). Genome activation is one of the first critical events in the life of a new organism. Both the timing of genome activation and the array of genes activated must be controlled correctly, and these events depend on changes in chromatin structure and availability of

In sea bass the observed reduction in SBK1 proteins in cryopreserved sperm could be responsible for abnormal early embryo development, which in turn, could determine the lower hatching rate observed (personal observation). The spot protein 13 matched in Mascot and MS-Fit with a G1/S-specific cyclin E2 protein, which is essential in the control of the cell cycle at the G1/S (start) transition (Moore et al., 2002). Cyclin E is involved in the activation of cyclin-dependent kinase 2 (cdk2). Recently, it has been demonstrated that cdk2 phosphorylates the protein phosphatase, PP1gamma2, a key enzyme in the development and regulation of sperm motility (Huang &Vijayaraghavan, 2004). The observed reduction in sea bass sperm motility duration in frozen-thawed spermatozoa could be a consequence of the cyclin E degradation. The protein spot 20 matched, in MS-Fit, with the hypothetical

> **SWISSPR-OT accession no.**

**Species ident-ified**

Q7ZZ07 *Brachidanio*

Q91780 *Xenopus*

Q96AZ5 *Brachidanio*

Table 4. Results from peptide mass fingerprinting of protein spots excised from 2D gels. EWM: Experimental Weight Mass; EIP: Experimental Isoelectric Point; TWM: Theoretical Weight Mass; TIP: Theoretical Isoelectric Point. (This table was originally published in Zilli

SPOT 6 110 6.0 — — *—* — — — — SPOT 8 100 5.2 — — *—* — — — —

**TWM (kDa) TIP** **Homology**

**Coverage %**

**Matched peptides**

*rerio* 71.8 5.98 8 22.0

*laevis* 47.78 6.3 6 20.0

*rerio* 37.13 5.6 4 21.0

transcription factors (Latham & Schultz, 2001).

protein DKFZp566A1524 of unknown function.

**EIP Identified protein**

> Novel protein similar to SKB1 human and mouse (PEPTIDENT)

> G1/S-specific cyclin E2 (MASCOT, MS-FIT)

Similar to hypothetical protein DKFZp566A1 524 (MS-FIT)

**Reference Spot**

**EWM (kDa)**

SPOT 5 80 6.5

SPOT 13 40 6.8

SPOT 20 30 4.5

et al., Biol Reprod 2005).

The results reported in figure 6 and tables 3 and 4 show that in sea bass spermatozoa the used cryopreservation procedure causes the degradation of 21 sperm proteins, and among these, 2 could be at least partially responsible for the observed decrease in sperm motility duration and the lower hatching rate of eggs fertilized with cryopreserved sperm. In addition, these observations suggest that two-dimensional electrophoresis coupled with MALDI-TOF analysis could be used as a tool to improve cryopreservation procedures.